AU707934B2 - DNA encoding taurine transporters and uses thereof - Google Patents

Description

1I DNA ENCODIG TA N TANSPOTE USES THEROF Background of the Invention S. Throughout this application various publications are referred to by partial citations within parenthesis.

Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Chemical neurotransmission is a multi-step process which Sinvolves release of neurotransmitter from the presynaptic terminal, diffusion across the synaptic cleft, and binding to receptors resulting in an alteration in the electrical properties of the postsynaptic neuron. For most neurotransmitters, transmission is terminated by the rapid uptake of neurotransmitter via specific, highaffinity transporters located in the presynaptic terminal and/or surrounding glial cells Since inhibition of uptake by pharmacologic agents increases the levels of neurotransmitter in the synapse, and thus enhances synaptic transmission, neurotransmitter transporters provide important targets for therapeutic intervention.

-2- The amino acid GABA is the major inhibitory neurotransmitter in the vertebrate central nervous system and is thought to serve as the neurotransmitter at approximately 40% of the synapses in the mammalian brain (13,28). GABAergic transmission is mediated by two classes of GABA receptors. The more prevalent is termed GABAA, which is a multi-subunit protein containing an intrinsic ligand-gated chloride channel in addition to binding sites for a variety of neuroactive drugs including benzodiazepines and barbiturates (35,73). In contrast, GABA, receptors couple to G-proteins and thereby activate potassium channels (2,35) and possible a. alter levels of the second messenger cyclic AMP Positive modulation of GABAA receptors by diazepam and related benzodiazepines has proven extremely useful in the treatment of generalized anxiety (77) and in certain forms of epilepsy (57).

a. Inhibition of GABA uptake provides a novel therapeutic 20 approach to enhance inhibitory GABAergic transmission in the central nervous system (36,62). Considerable evidence indicates that GABA can be taken up by both neurons and glial cells, and that the transporters on the two cell types are pharmacologically distinct (15,36,62).

A GABA transporter with neuronal-type pharmacology designated GAT-1 has previously been purified and cloned but the molecular properties of other GABA transporters including glial transporter(s) have not yet been elucidated. We now report the cloning of two additional GABA transporters (GAT-2 and GAT-3) with distinct pharmacology and localization, revealing previously unsuspected heterogeneity in GABA transporters.

-3- Taurine (2-aminoethane sulfonic acid) is a sulfurcontaining amino acid present in high concentrations in mammalian brain as well as various non-neural tissues.

Many functions have been ascribed to taurine in both the nervous system and peripheral tissues. The best understood (and phylogenetically oldest) function of taurine is as an osmoregulator (26,75). Osmoregulation is essential to normal brain function and may also play a critical role in various pathophysiological states such as epilepsy, migraine, and ischemia. The primary mechanism by which neurons and glial cells regulate osmolarity is via the selective accumulation and release of taurine. Taurine influx is mediated via specific, high-affinity transporters which may contribute to efflux 15 as well. Since taurine is slowly degraded, transport is an important means of regulating extracellular taurine levels.

Taurine is structurally related to the inhibitory amino 20 acid y-aminobutyric acid (GABA) and exerts inhibitory effects on the brain, suggesting a role as a neurotransmitter or neuromodulator. Taurine can be Sreleased from both neurons and glial cells by receptormediated mechanisms as well as in response to cell volume changes Its effects in the CNS may be mediated by GABA, and GABA B receptors (34,56) and by specific taurine receptors Additionally, taurine can also regulate calcium homeostasis in excitable tissues such as the brain and heart (26,41), via an intracellular site of action. Together, the inhibitory and osmoregulatory properties of taurine suggest that it acts as a cytoprotective agent in the brain. Depletion of taurine results in retinal degeneration in cats supporting a role in neuronal survival.

-4- Although most animals possess the ability to synthesize taurine, many are unable to generate sufficient quantities and therefore rely on dietary sources.

Taurine transport is thus critical to the maintenance of appropriate levels of taurine in the body. Highaffinity, sodium-dependent taurine uptake has been observed in brain and various peripheral tissues (27,64), but little is known about the molecular properties of the taurine transporter(s). Cloning of the taurine transporter will not only help elucidate the function of this important neuro-active molecule, but may also provide important insight into novel therapeutic approaches to treat neurological disorders.

cDNA clones (designated rB14b, rB8b, and rB16a) encoding transporters for two novel GABA transporters and a taurine transporter, respectively, have been isolated from rat brain, and their functional properties have been examined in mammalian cells. The transporters encoded by rB14b and rB8b display high-affinity for GABA (Ka=49M), and exhibit pharmacological properties distinct from the neuronal GABA transporter; the transporter encoded by rB16a displays high-affinity for taurine. All three are dependent on external sodium and chloride for transport activity. The nucleotide sequences of the three clones predict proteins of 602, 627, and 621 amino acids, respectively. Hydropathy analysis reveals stretches of hydrophobic amino acids suggestive of 12 transmembrane 15 domains, similar to that proposed for other cloned neurotransmitter transporters. The cloning of two additional GABA transporters and a taurine transporter from rat brain reveals previously undescribed heterogeneity in inhibitory amino acid transporter genes.

The use of human gene products in the process of drug development offers significant advantages over those of other species, which may not exhibit the same Spharmacological profiles. To facilitate this humantarget based approach to drug design in the area of inhibitory amino acid transporters, we used the nucleotide sequences of the rat GAT-2 and GAT-3 cDNAs to clone the human homologue of each gene. cDNA clones (designated hHE7a, hS3a, hFBl6a and hFB20a encoding the human homologue of the two novel GABA transporters GAT-2 and GAT-3 have been isolated.

Summary of the Invention This invention provides an isolated nucleic acid molecule encoding a mammalian GABA transporter. In one embodiment of this invention, the nucleic acid molecule comprises a plasmid designated EVJB-rB14b (ATCC Accession No. 75203). In another embodiment of this invention, the nucleic acid molecule comprises a plasmid designated EVJB-rB8b (ATCC Accession No. 75201).

This invention also provides an isolated nucleic acid molecule encoding a 10 mammalian taurine transporter. In one embodiment of this invention, the nucleic acid molecule comprises a plasmid designated EVJB-rB16a (ATCC Accession No.

75202).

This invention further provides isolated nucleic acid molecules encoding the human 15 homologue of the mammalian GABA transporters. In one embodiment of this invention, the nucleic acid molecule comprises a plasmid designated pcEXV-hGAT- 3 (ATCC Accession No. 75324). In another embodiment of this invention, the nucleic acid molecule comprises a plasmid designated pBluescript-hHE7a

(ATCC

Accession No. 75322). In another embodiment of this invention, the nucleic acid .i 20 molecule comprises the plasmid pBluescript-hS3a (ATCC Accession No. 75323).

This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a mammalian

GABA

transporter. This invention also provides a nucleic acid molecule of at least nucleotides capable of specifically hybridizing with a -7sequence included within the sequence of a nucleic acid molecule encoding a mammalian taurine transporter. This invention also provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human GABA transporter. This invention also provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human taurine transporter.

This invention further provides an antisense 15 oligonucleotide having a sequence capable of binding specifically to an mRNA molecule encoding a mammalian 6.

GABA transporter so as to prevent translation of the mRNA molecule. This invention also 'provides an antisense oligonucleotide having a sequence capable of binding 20 specifically to an mRNA molecule encoding a mammalian

S

taurine transporter so as to prevent translation of the mRNA molecule. This invention also provides an antisense oligonucleotide having a sequence capable of binding S" specifically to an aRNA molecule encoding a human GABA transporter so as to prevent translation of the mRNA molecule. This invention also provides an antisense oligonucleotide having a sequence capable of binding specifically to an mRNA molecule encoding a human taurine transporter so as to prevent translation of the mRNA molecule.

A monoclonal antibody directed to a mammalian GABA transporter is provided by this invention. A monoclonal antibody directed to a mammalian taurine transporter is also provided by this invention. A monoclonal antibody

I

-8directed to a human GABA transporter is also provided by this invention. A monoclonal antibody directed to a human taurine transporter is also provided by this invention.

This invention provides a pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a mammalian GABA transporter and a pharmaceutically acceptable carrier as well as a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of GABA transporter and a pharmaceutically acceptable carrier.

A pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a mammalian taurine transporter and a pharmaceutically acceptable carrier as well as a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a taurine transporter and a pharmaceutically acceptable carrier is also :provided by this invention.

A pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a human GABA transporter and a pharmaceutically acceptable carrier as well as a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a human GABA transporter and a pharmaceutically acceptable carrier is also provided by this invention.

-9- A pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a human taurine transporter and a pharmaceutically acceptable carrier as well as a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a human taurine transporter and a pharmaceutically acceptable carrier is also provided by this invention.

This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a mammalian GABA transporter so positioned within such genome as to be transcribed into antisense mRNA complementary to mRNA 15 encoding the GABA transporter and when hybridized to mRNA encoding the GABA transporter, the complementary mRNA reduces the translation of the mRNA encoding the GABA transporter.

This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a mammalian taurine transporter so positioned within such genome as to be transcribed into antisense mRNA complementary to mRNA encoding the taurine transporter and when hybridized 25 to mRNA encoding the taurine transporter, the complementary mRNA reduces the translation of the mRNA encoding the taurine transporter.

This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a human GABA transporter so positioned within such genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the human GABA transporter and when hybridized to mRNA encoding the human GABA transporter, the antisense mRNA thereby reduces the translation of mRNA encoding the human GABA transporter.

This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a human taurine transporter so positioned within such genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the human taurine transporter and when hybridized to mRNA encoding the human taurine transporter, the antisense mRNA thereby reduces the translation of mRNA encoding the human taurine transporter.

This invention also provides a transgenic, nonhuman 15 mammal whose genome comprises DNA encoding a mammalian GABA transporter so positioned within such genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the transporter and when hybridized to mRNA encoding the transporter, the antisense mRNA thereby prevents the translation of mRNA encoding -the transporter.

This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a mammalian 25 taurine transporter so positioned within such genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the transporter and when hybridized to mRNA encoding the transporter, the antisense mRNA thereby prevents the translation of mRNA encoding the transporter.

This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a human GABA transporter so positioned within such genome as to be transcribed into antisense mRNA which is complementary to -11mRNA encoding the transporter and when hybridized to mRNA encoding the human GABA transporter, the antisense mRNA thereby prevents the translation of mRNA encoding the human GABA transporter.

This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a human taurine transporter so positioned within such genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the human taurine transporter and when hybridized to mRNA encoding the human taurine transporter, the antisense mRNA thereby prevents the translation of mRNA encoding the human taurine transporter.

This invention provides a method of screening drugs to identify drugs which specifically interact with, and bind to, a mammalian GABA transporter on the surface of a cell which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a mammalian GABA transporter, the protein encoded thereby is expressed on the cell surface, with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and 25 bind to, a mammalian GABA transporter.

This invention provides a method of screening drugs to identify drugs which specifically interact with, and bind to, a mammalian taurine transporter on the surface of a cell which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a mammalian taurine transporter, the protein encoded thereby is expressed on the cell surface, with a plurality of drugs, determining those drugs which bind to the mammalian cell, -12and thereby identifying drugs which specifically interact with, and bind to, a mammalian taurine transporter.

This invention provides a method of screening drugs to identify drugs which specifically interact with, and bind to, a human GABA transporter on the surface of a cell which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human GABA transporter, the protein encoded thereby is expressed on the cell surface, with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, a human GABA transporter.

15 This invention provides a method of screening drugs to identify drugs which specifically interact with, and bind to, a human taurine transporter on the surface of a cell .*which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human taurine transporter, the protein encoded thereby is expressed on the cell surface, with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, a human taurine transporter.

This invention also provides a method of determining the physiological effects of expressing varying levels of mammalian GABA transporters which comprises producing a transgenic nonhuman animal whose levels of mammalian GABA transporter expression are varied by use of an inducible promoter which regulates mammalian GABA transporter expression.

This invention also provides a method of determining the physiological effects of expressing varying levels of -13mammalian taurine transporters which comprises producing a transgenic nonhuman animal whose levels of mammalian taurine transporter expression are varied by use of an inducible promoter which regulates mammalian taurine transporter expression.

This invention also provides a method of determining the physiological effects of expressing varying levels of human GABA transporters which comprises producing a transgenic nonhuman animal whose levels of human GABA transporter expression are varied by use of an inducible promoter which regulates human GABA transporter expression.

15 This invention also provides a method of determining the physiological effects of expressing varying levels of human taurine transporters which comprises producing a transgenic nonhuman animal whose levels of human taurine transporter expression are varied by use of an inducible promoter which regulates human taurine transporter expression.

This invention further provides a method of determining f the physiological effects of expressing varying levels of 25 mammalian GABA transporters which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of mammalian GABA transporter.

This invention further provides a method of determining the physiological effects of expressing varying levels of mammalian taurine transporters which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of mammalian taurine transporter.

-14- This invention further provides a method of determining the physiological effects of expressing varying levels of human GABA transporters which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human GABA transporter.

This invention further provides a method of determining the physiological effects of expressing varying levels of human taurine transporters which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human taurine transporter.

This invention provides a method for diagnosing a predisposition to a disorder associated with the 15 expression of a specific mammalian GABA transporter allele and a method for diagnosing a predisposition to a disorder associated with the expression of a specific mammalian taurine transporter allele which comprises: obtaining DNA of subjects suffering from the disorder; performing a restriction digest of the DNA with a panel of restriction enzymes; c.) electrophoretically separating the resulting

DNA

fragments on a sizing gel; contacting the resulting **"gel with a nucleic acid probe capable of specifically S. 25 hybridizing to DNA encoding a mammalian GABA or a mammalian taurine transporter and labelled with a detectable marker; detecting labelled bands which have hybridized to the DNA encoding a mammalian GABA or taurine transporter labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; preparing

DNA

obtained for diagnosis by steps a-e; and comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.

This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific human GABA transporter allele or a specific human taurine transporter allele which comprises: obtaining DNA of subjects suffering from the disorder; performing a restriction digest of the DNA with a panel of restriction enzymes; c.) electrophoretically separating the resulting DNA fragments on a sizing gel; contacting the resulting gel with a nucleic acid probe capable of specifically 15 hybridizing to DNA encoding a human GABA or human taurine transporter and labelled with a detectable marker; e.) detecting labelled bands which have hybridized to the DNA encoding a human GABA or human taurine transporter labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; preparing DNA obtained for diagnosis by steps a-e; and comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis 25 from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.

This invention provides a method for determining whether a substrate not known to be capable of binding to a mammalian transporter can bind to the mammalian GABA transporter which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding the GABA transporter with the substrate under conditions permitting binding of substrates known to bind to a -16transporter, detecting the presence of any of the substrate bound to the GABA transporter, and thereby determining whether the substrate binds to the GABA transporter.

This invention provides a method for determining whether a substrate not known to be capable of binding to a taurine transporter can bind to a taurine transporter which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding the taurine transporter with the substrate under conditions permitting binding of substrates known to bind to a transporter, detecting the presence of any of the substrate bound to the taurine transporter, and thereby determining whether the 15 substrate binds to the taurine transporter.

This invention provides a method for determining whether a substrate not known to be capable of binding to a human GABA transporter can bind to a human GABA transporter which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding the human GABA transporter with the substrate under conditions permitting binding of substrates known to bind to a transporter, detecting the :oe presence of any of the substrate bound to the human GABA 25 transporter, and thereby determining whether the substrate binds to the human GABA transporter.

This invention provides a method for determining whether a substrate not known to be capable of binding to a human taurine transporter can bind to a human taurine transporter which comprises contacting'a mammalian cell comprising an isolated DNA molecule encoding the human taurine transporter with the substrate under conditions permitting binding of substrates known to bind to a transporter, detecting the presence of any of the substrate bound to the human taurine transporter g and thereby determining whether the substrate binds to the human taurine transporter.

a.

a a a.

a. a a a.

a a a.

a 4 .a a.

a. a *0 a a a. a. a a a. a.

4a -18- Brief Description of the Picures Figure 1. Nucleotide Sequence, Deduced Amino Acid Sequence and Putative Membrane Topology of Two Novel Mammalian GABA Transporters and a Novel Mammalian Taurine Transporter. A. Mammalian GABA transporter encoded by GAT-2 (rBl4b)(Seq. I.D. Nos. 1 and Nucleotides are presented in the 5' to 3' orientation and the coding region is numbered starting from the putative initiating methionine and ending in the termination codon. Deduced amino acid sequence by translation of a long open reading frame is shown. B. Mammalian GABA transporter encoded by GAT-3 (rB8b) (Seq. I.D. Nos. 3, and Nucleotides are presented in the 5' to 3' orientation and the coding 15 region is numbered starting from the putative initiating methionine and ending in the termination codon. Deduced amino acid sequence by translation of a long open reading frame is shown. C. Taurine transporter encoded by rB16a S.' (Seq. I.D. Nos. 5 and Nucleotides are presented in the 5' to 3' orientation and the coding region is numbered starting from the putative initiating methionine and ending in the termination codon. Deduced amino acid sequence by translation of a long open reading frame is shown. D. Deduced amino acid sequence and putative 25 membrane topology of GABA tranporter GAT-2 (rBl4b).

Deduced amino acid sequence by translation of a long open reading frame in rB14b is shown. Residues which are identical to those of GAT-3 (rB8b) are shaded. Membrane topology is modeled after that proposed for GAT-I (21).

E. Deduced amino acid sequence and putative membrane topology of taurine transporter (rB16a). Deduced amino acid sequence by translation of a long open reading frame in rB16a is shown. Membrane topology is modeled after that proposed for GAT-1 (21).

-19- Figure 2. Alignment of the novel GABA transporters with the rat neuronal GABA transporter, the betaine transporter, and the glycine transporter. The twelve putative a-helical membrane spanning domains (I-XII) are bracketed. Residues identical to those of GAT-2 are shaded. GAT-2 is the GABA transporter encoded by rB14b; GAT-3 is the GABA transporter encoded by rB8b; GAT-1 is the rat neuronal GABA transporter Betaine is the dog betaine transporter and Glycine is the rat glycine transporter (68).

Figure 3. GABA transport by COS cells transfected with clone rB14b and rB8b. Non-transfected COS cells (control) or COS cells transfected with GAT-2 (panel A) 15 or GAT-3 (panel B) were incubated for 10 minutes (37*C) with 50nM 3 H]GABA in either HBS (150mM NaC1) or in a similar solution in which Na' was replaced by equimolar Li* (Na*-free), or Cl" was replaced by acetate (in some experiments, calcium gluconate was used instead of calcium acetate; C1l-free). Data show the specific uptake of GABA, expressed as pmoles/mg protein cellular protein. Data are from a single experiment that was repeated with similar results.

25 Figure 4. Concentration dependence of GABA transport.

COS cells transfected with GAT-2 (panel A) or GAT-3 (panel B) were incubated with the indicated concentrations of 3 H)GABA for 30 seconds and the accumulated radioactivity was determined. The specific activity of the 3 H]GABA was reduced with unlabeled GABA.

Data represent specific transport expressed as nmoles per minute per mg protein, and are from a single experiment that was repeated with similar results (see Text).

Figure Localization of GABA transporters.

A.

Northern blot analysis of mRNAs encoding GAT-2 (rB14b) and GAT-3 (rB8b). Total RNA (25 g) from rat brain and liver was separated by formaldehyde/agarose gel electrophoresis, blotted to nylon membranes, and hybridized at high stringency with 32 P-labeled GABA transporter cDNAs (rBl4b and rB8b, respectively). The autoradiogram was developed after a four day exposure.

The locations of ribosomal RNAs are indicated at the side. The hybridizing transcripts are =2.4kb (GAT-2) and =4.7kb (GAT-3). B. Tissue distribution of mRNAs encoding GAT-1, GAT-2, and GAT-3 as determined by PCR.

.Single-stranded cDNA converted from poly A+ RNA was used o for PCR amplification (30 cycles) of GABA transporter 15 cDNA sequences. Amplified products were detected by hybridization with specific oligonucleotide probes; autoradiograms of the Southern blots are shown. GAT-1 is the neuronal GABA transporter. GAT-2 is the transporter encoded by rB8b. GAT-3 is the transporter by rBl4b.

Equivalent samples of poly A+ RNA (not treated with reverse transcriptase) subjected to identical

PCR

conditions showed no hybridization with the three probes (not shown). Cyclophilin cDNA was amplified to an equal extent from all tissues examined (not shown). Each 25 experiment was repeated at least once with similar results.

Figure 6. Alignment of the taurine transporter with the GABA transporter GAT-1, the betaine transporter, and the glycine transporter. The twelve putative a-helical membrane spanning domains (I-XII) are bracketed.

Residues identical to those of the taurine transporter are shaded. Taurine is the taurine transporter encoded by rB16a; GAT-l is the rat brain GABA transporter (21); -21- Betaine is the dog betaine transporter Glycine is the rat glycine transporter (68).

Figure 7. Taurine transport by COS cells transfected with clone rB16a. Non-transfected COS cells (control) or COS cells transfected with rB16a were incubated for minutes (37*C) with 50nM 3 H)taurine in either HBS (150mM NaCl) or in a similar solution in which Na was replaced by equimolar Li* (Na*-free), or Cl1 was replaced by acetate (Cl1-free). Data show the specific uptake of taurine, expressed as of control cells. Each bar represents the mean±SEM of 3-7 experiments.

FFigure 8. Concentration dependence of taurine transport.

15 COS cells transfected with rB16a were incubated with the indicated concentrations of 3 H]taurine for 30 seconds and the accumulated radioactivity was determined. The -specific activity of 3 H]taurine was reduced with unlabeled taurine. Data represent specific transport expressed as nmoles per minute per mg protein, and are from a single experiment that was repeated with similar results (see Text).

Figure 9. Localization of the taurine transporter.

25 A. Tissue distribution of mRNA encoding the taurine transporter as determined by PCR. Single-stranded cDNA converted from poly A+ RNA was used for PCR amplification cycles) of taurine transporter cDNA from a variety of rat tissues. A plasmid containing the cloned taurine transporter was amplified under identical conditions as a control. Amplified products were detected by hybridization with an oligonucleotide probe specific to the taurine transporter; an autoradiogram of the Southern blot is shown. Equivalent samples of poly A+ RNA (not treated with reverse transcriptase) subjected to -22identical PCR conditions showed no hybridization with the transporter probe (not shown), indicating that the signals obtained with cDNA were not a result of genomic DNA contamination. The experiment was repeated with similar results. B. Northern blot analysis of mRNA encoding the taurine transporter. Poly A+ RNA (5mg) from a variety of rat tissues was separated by formaldehyde/agarose gel electrophoresis, blotted to a nylon membrane, and hybridized at high stringency with 32 P-labeled taurine transporter cDNA (rB16a). The autoradiogram was developed after an overnight exposure.

Size standards are indicated at the left in kilobases.

The hybridizing transcript is -6.2 kb.

0** 15 Figure 10. Nucleotide Sequence and Deduced Amino Acid of Human Transporters. A. Sequence of the Human GAT-2 GABA Transporter. Nucleotides are presented in the 5' to 3' orientation- and the Coding regi4-n- is- .umbered starting S-from the first nucleotide in a partial cDNA clone.

Deduced amino acid sequence by translation of a long open reading frame is shown. B. Sequence of the Human GAT-3 t* GABA Transporter. Nucleotides are presented in the 5' to 3' orientation and the coding region is numbered starting ;from the putative initiating methionine and ending in the terminating codon. Deduced amino acid sequence by translation of a long open reading frame is shown.

A

-23- Detailed escription of the Invention This invention provides an isolated nucleic acid molecule encoding a mammalian GABA transporter. This invention also provides an isolated nucleic acid molecule encoding a mammalian taurine transporter. This invention further provides an isolated nucleic acid molecule encoding a human GABA transporter. As used herein, the term "isolated nucleic acid molecule" means a non-naturally occurring nucleic acid molecule that is, a molecule in a form which does not occur in nature. Examples of such an isolated nucleic acid molecule are an RNA, cDNA, or 15 isolated genomic DNA molecule encoding a mammalian GABA, or mammalian taurine transporter. As used herein, "GABA transporter" means a molecule which, under physiologic conditions, is substantially specific for the a. neurotransmitter GABA, is saturable, of high affinity for GABA (Km-4AM), and exhibits pharmacological properties distinct from the neuronal GABA transporter. As used herein, "taurine transporter" means a molecule which, under physiologic conditions, is substantially specific for the neurotransmitter taurine, is saturable, and of .O 25 high affinity for taurine. One embodiment of this invention is an isolated murine nucleic acid molecule encoding a GABA or taurine transporter. Such a molecule may have coding sequences substantially the same as the coding sequences shown in Figure 1A, 1B or 1C. The DNA molecules of Figures 1A (Sequence I.D. No. 1) and IB (Seq I.D. No.3) encode the sequence of the mammalian GABA transporter genes. The DNA molecule of Figure 1C (Sequence I.D. No. 5) encodes the sequence of a mammalian taurine transporter gene. Another preferred embodiment of this invention is an isolated human nucleic acid molecule -24encoding a human GABA transporter. Such a molecule may have coding sequences substantially the same as the coding sequences shown in Figures 10A and 10B. The DNA molecules of Figures 10A (Sequence I.D. No.7) and (Sequence I.D. No.9) encode the sequences of human GABA transporter genes. Another preferred embodiment of this invention is an isolated nucleic acid molecule encoding a human taurine transporter. Such a molecule may have coding sequences substantially similar to the sequence shown in Figure 1C. One means of isolating a mammalian GABA or a mammalian taurine transporter is to probe a mammalian genomic library with a natural or artificially designed DNA probe, using methods well known in the art.

In the preferred embodiment of this invention, the 15 mammalian GABA and mammalian taurine transporter are human proteins and the nucleic acid molecules encoding them are isolated from a human cDNA library or a human genomic DNA library. DNA probes derived from the rat GABA transporter genes rB14b and rBBb, and DNA probes derived form the rat taurine transporter gene rB16a are useful probes for this purpose. DNA and cDNA molecules which encode mammalian GABA or mammalian taurine transporters are used to obtain complementary genomic DNA, cDNA or RNA from human, mammalian or other animal sources, or to 25 isolate related cDNA or genomic clones by the screening of cDNA or genomic DNA libraries, by methods described in more detail below. Transcriptional regulatory elements from the 5' untranslated region of the isolated clone, and other stability, processing, transcription, translation, and tissue specificity determining regions from the 3' and 5' untranslated regions of the isolated gene are thereby obtained.

This invention provides a method for obtaining an isolated nucleic acid molecule encoding a human taurine transporter which comprises using oligonucleotide primers based on the nucleic acid sequence coding for a mammalian taurine receptor and the polymerase chain reaction (PCR) to detect the presence of the nucleic acid molecule coding for the taurine transporter in a human cDNA library. PCR is carried out at reduced annealing temperatures to allow for mismatches between the nucleic acid sequences encoding the rat taurine transporter and nucleic acid sequences encoding the human taurine transporter. Amplified DNA sequences encoding a human taurine transporter are detected by hybridization at reduced hybridization stringency with radiolabelled cDNA encoding the mammalian taurine receptor. A human cDNA library identified by the above method to contain a 15 nucleic acid molecule encoding the human taurine transporter is then screened at low hybridization stringency with the same cDNA probe encoding the 4* mammalian taurine receptor to "isolate a cDNA clone encoding a human taurine transporter. A cDNA sequence from the resulting clone can then be used to screen additionally screen a human cDNA or human genomic DNA library to obtain the entire sequence of the human homologue of the mammalian taurine transporter. Primers used in the polymerase chain reaction to initially screen human cDNA libraries to identify human cDNA libraries containing clones encoding a human taurine receptor may be composed of a plurality of degenerate primers based on the.sequence of the mammalian taurine transporter. The methods of synthesizing primers, of screening cDNA libraries by PCR to identify libraries containing a cDNA clone encoding the protein of interest are well known by one of skill in the art and examples of this method for obtaining a cDNA clone encoding the human homologue of mammalian transporter are further given below. These same methods can be used to isolate cDNA and genomic DNAs -26encoding additional mammalian or human GABA transporter subtypes or taurine transporter subtypes encoded by different genes or encoded by the same gene and generated by alternative splicing of the RNA or rearrangement of the genomic DNA.

This invention provides an isolated nucleic acid molecule which has been so mutated as to be incapable of encoding a molecule having normal transporter activity, and not expressing native transporter. An example of a mutated nucleic acid molecule provided by this invention is an isolated nucleic acid molecule which has an in-frame stop codon inserted into the coding sequence such that the transcribed RNA is not translated into a protein having 15 normal transporter activity.

This invention further provides a cDNA molecule encoding a mammalian GABA transporter, wherein the cDNA molecule has a coding sequence substantially the same as the coding sequence shown in Figure 1A or IB. (Sequence I.D.

Nos. 1 or This invention also provides a cDNA molecule encoding a mammalian taurine transporter, wherein the cDNA molecule has a coding sequence substantially the same as the coding sequence shown in Figure IC. (Sequence I.D. No. This invention also provides a cDNA molecule encoding a human GABA transporter, wherein the CDNA molecule has a coding sequence substantially the same as the coding sequence shown in Figures 10A (Sequence I.D. No. 7) and (Sequence I.D. No. These molecules and their equivalents were obtained by the means described above.

This invention also provides an isolated protein which is a mammalian GABA transporter. This invention further provides an isolated protein which is a mammalian taurine -27transporter. In one embodiment of this invention, the protein is a murine GABA transporter protein having an amino acid sequence substantially similar to the amino acid sequence shown in Figures 1A (Seq. I.D. Nos. 1 and 2) or 1B (Seq. I.D. Nos. 3 and In another embodiment of this invention, the protein is a murine taurine transporter protein having an amino acid sequence substantially similar to the amino acid sequence shown in Figure 1C (Seq. I.D. Nos. 5 and In a preferred embodiment of this invention, the protein is a human GABA transporter protein having an amino acid sequence substantially the same as the sequence shown in Figure 10A (Sequence I.D. Nos. 7 and 8) and Figure 10B (Sequence I.D. Nos. 9 and 10). Another preferred embodiment of 15 this invention, the protein is a human taurine transporter protein having an amino acid sequence substantially similar to the amino acid sequence shown in Figure 1C (Seq. I.D. Nos. 5 and As used herein, the term "isolated protein" is intended to encompass a protein molecule free of other cellular components. One means for obtaining an isolated GABA or taurine transporter is to express DNA encoding the transporter in a suitable host, such as a bacterial, yeast, or mammalian cell, using methods well known to those skilled in the 25 art, and recovering the transporter protein after it has been expressed in such a host, again using methods well known in the art. The transporter may also be isolated from cells which express it, in particular from cells which have been transfected with the expression vectors described below in more detail.

This invention also provides a vector comprising an isolated nucleic acid molecule such as DNA, RNA, or cDNA, encoding a mammalian GABA transporte r. This invention also provides a vector comprising an isolated nucleic -28acid molecule such as DNA, RNA, or cDNA, encoding a mammalian taurine transporter. This invention also provides a vector comprising an isolated nucleic acid molecule such as DNA, RNA, or cDNA, encoding a human GABA transporter. This invention also provides a vector comprising an isolated nucleic acid molecule such as DNA, RNA, or cDNA, encoding a human taurine transporter.

Examples of vectors are viruses such as bacteriophages (such as phage lambda), cosmids, plasmids (such as pUC18, available from Pharmacia, Piscataway, NJ), and other recombination vectors. Nucleic acid molecules are inserted into vector genomes by methods well known to those skilled in the art. Examples of such plasmids are plasmids comprising cDNA having a coding sequence 15 substantially the same as: the coding sequence shown in Figure 1A (Seq. I.D. No. 1) and designated clone pEVJBrB14b deposited under ATCC Accession No. 75203, the coding sequence shown in Figure'lB (Seq. I.D. No. 3) and designated clone pEVJB-rB8b deposited under ATCC Accession No. 75201, the coding sequence shown in Figure IC (Seq. I.D. No. 5) and designated pEVJB-rB16a deposited under ATCC Accession No. 75202, the coding sequence shown in Figure 10A, (Sequence I.D. No. 7) designated .pBluescript-hHE7a and pBluescript-hS3a and deposited under ATCC Accession Nos. and respectively, or the coding sequence shown in Figure 10B (SEQ. I.D. No.

9) and designated pcEXV-hGAT-3 and deposited under ATCC Accession No. Alternatively, to obtain these vectors, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules which base pair with each other and are then ligated together with a ligase. Alternatively, linkers can be ligated to the insert DNA which correspond to a restriction site in the vector DNA, which is then -29digested with the restriction enzyme which cuts at that site. Other means are also available.

This invention also provides vectors comprising a DNA molecule encoding a mammalian GABA transporter and vectors comprising a DNA molecule encoding a mammalian taurine transporter, adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cells so located relative to the DNA encoding a mammalian GABA transporter or to the DNA encoding a mammalian taurine transporter as to permit expression thereof. DNA having coding sequences substantially the 15 same as the coding sequence shown in Figure 1A or Figure 1B may usefully be inserted into the vectors to express mammalian GABA transporters. DNA having coding sequences substantially the same as the coding sequence shown in Figure IC may usefully be inserted into the vectors to express mammalian taurine transporters. This invention also provides vectors comprising a DNA molecule encoding a human GABA transporter adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cells so located relative to the DNA encoding a human GABA transporter as to permit expression thereof.

DNA having coding sequences substantially the same as the coding sequence shown in Figures 10A and 10B may usefully be inserted into the vectors to express human GABA transporters. This invention also provides vectors comprising a DNA molecule encoding a human taurine transporter adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cells so located relative to the DNA encoding a human taurine transporter as to permit expression thereof. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982). Similarly, a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained .e .commercially or assembled from the sequences described by methods well known in the art, for example the methods described above for constructing vectors in general.

Expression vectors are useful to produce cells that express the transporter. Certain uses for such cells are described in more detail below.

In one embodiment of this invention a plasmid is adapted for expression in a bacterial, yeast, or, in particular, a mammalian cell wherein the plasmid comprises a DNA molecule encoding a mammalian GABA transporter or a DNA molecule encoding a mammalian taurine transporter and the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cell so located relative to the DNA encoding a mammalian GABA transporter or to the DNA encoding a mammalian taurine transporter as to permit expression thereof. In another embodiment of this invention a plasmid is adapted for expression in a bacterial, yeast, or, in particular, a mammalian cell wherein the plasmid comprises a DNA molecule encoding a -31human GABA transporter or human taurine transporter and the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cell so located relative to the DNA encoding a human GABA transporter or human taurine transporter as to permit expression thereof. Suitable plasmids may include, but are not limited to plasmids adapted for expression in a mammalian cell, EVJB or EXV. Examples of such plasmids adapted for expression in a mammalian cell are plasmids comprising cDNA having coding sequences substantially the same as the coding sequence shown in Figures 1A, 1B, 1C, 10OA and 10B and the regulatory elements necessary for expression of the DNA in the mammalian cell. These plasmids have been designated pEVJB-rB14b deposited under ATCC Accession No.75203, pEVJB-rBSb deposited under ATCC Accession No.75201, pEVJB-rB16a deposited under ATCC Accession No.75202, pBluescript-hHE7a and pBluescripthS3a deposited under ATCC Accession Nos.75322 and 75323 and pcEXV-hGAT-3 deposited under ATCC accession No.

75324 respectively. Those skilled in the art will readily appreciate that numerous plasmids adapted for expression in a mammalian cell which comprise DNA encoding a mammalian GABA transporter, a mammalian taurine transporter, a human GABA transporter or human taurine transporter and the regulatory elements necessary to express such DNA in the mammalian cell may be constructed utilizing existing plasmids and adapted as appropriate to contain the regulatory elements necessary to express the DNA in the mammalian cell. The plasmids may be constructed by the methods described above for expression vectors and vectors in general, and by other methods well known in the art.

The deposits discussed supra were made pursuant to, and in satisfaction of, the provisions of the Budapest Treaty 32 on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852. The deposits deposited under ATCC Accession Nos. 75201, 75202 and 75203 were made on 7 February, 1992. The deposits deposited under ATCC Accession Nos. 75322, 75323 and 75324 were made on 8 October, 1992.

This invention provides a mammalian cell comprising a DNA molecule encoding a mammalian GABA transporter or a DNA molecule encoding a mammalian taurine transporter, such as a mammalian cell comprising a plasmid adapted for expression in a mammalian cell, which comprises a DNA molecule encoding a "::."mammalian GABA transporter or a DNA encoding a mammalian taurine transporter and the regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding a mammalian transporter as to permit expression thereof. This invention also provides a mammalian cell "*'.."comprising a DNA molecule encoding a human GABA transporter or a human taurine transporter, such as a mammalian cell comprising a plasmid adapted for expression in a mammalian cell, which comprises a DNA molecule encoding a human GABA transporter or DNA encoding a human taurine transporter and the regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding a human transporter as to permit expression thereof. Numerous mammalian cells may be used as hosts, including, but not limited to, the mouse fibroblast cell NIH3T3, CHO cells, HeLa cells, Ltk" cells, Cos cells, etc. Expression plasmids such as that described supra may be used to transfect mammalian cells by methods well known in the art such as calcium phosphate precipitation, or DNA encoding these transporters may be otherwise introduced into mammalian cells, by microinjection, to obtain mammalian cells which comprise DNA, cDNA or a plasmid, encoding a mammalian

GABA

transporter, -33encoding a mammalian taurine transporter or encoding a human GABA trasnporter.

This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a mammalian GABA transporter, for example with a coding sequence included within the sequences shown in Figures lA and 1B. This invention also provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a taurine 15 transporter, for example with a coding sequence included within the sequence shown in Figure IC. This invention also provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human GABA transporter, for example with a coding sequence t*oo* included within the sequence shown in Figures 10A and This invention also provides a nucleic acid probe comprising a nucleic acid molecule of at least 25 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human taurine transporter, for example with a coding sequence substantially similar to the coding sequence included within the sequence shown in Figure IC. As used herein, the phrase "specifically hybridizing" means the ability of a nucleic acid molecule to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs. Nucleic acid probe technology is well known to those skilled in the V ^I 1 JV -34art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe. Detection of nucleic acid encoding a mammalian GABA transporter, mammalian taurine transporter, human GABA transporter or human taurine transporter is useful as a diagnostic test for any disease process in which levels of expression of the corresponding GABA or taurine transporter are altered.

DNA probe molecules are produced by insertion of a DNA molecule which encodes the mammalian GABA transporter, the mammalian taurine transporter, the human GABA o.

transporter or the human taurine transporter or fragments thereof into suitable vectors, such as plasmids or 15 bacteriophages, followed by insertion into suitable bacterial host cells and replication and harvesting of the DNA probes, all using methods well known in the art.

For example, the DNA may be extracted from a cell lysate using phenol and ethanol, digested with restriction enzymes corresponding to the insertion sites of the DNA into the vector (discussed above), electrophoresed, and cut out of the resulting gel. Examples of such DNA molecules are shown in Figures A, 1B, 1C, 10A and 0 The probes are useful for 'in situ' hybridization or in order to locate tissues which express this gene family, or for other hybridization assays for the presence of these genes or their mRNA in various biological tissues.

In addition, synthesized oligonucleotides (produced by a DNA synthesizer) complementary to the sequence of a DNA molecule which encodes a mammalian GABA transporter or a mammalian taurine transporter or complementary to the sequence of a DNA molecule which encodes a human GABA transporter or human taurine transporter, are useful as probes for these genes, for their associated mRNA, or for the isolation of related genes by homology screening of VTL 71I a0z*, genomic or cDNA libraries, or by the use of amplification techniques such as the Polymerase Chain Reaction.

This invention also provides a method of detecting expression of a GABA transporter on the surface of a cell by detecting the presence of mRNA coding for a GABA transporter. This invention also provides a method of detecting expression of a taurine transporter on the surface of the cell by detecting the presence of mRNA coding for a taurine transporter. This invention further provides a method of detecting the expression of a human GABA or human taurine transporter on the surface of the cell by detecting the presence of mRNA coding for the corresponding GABA or taurine transporter. These methods 15 comprise obtaining total aRNA from the cell using methods well known in the art and contacting the mRNA so obtained with a nucleic acid probe as described hereinabove, under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the transporter by the cell. Hybridization of probes to target nucleic acid molecules such as mRNA molecules employs techniques well known in the art.

However, in one embodiment of this invention, nucleic eg acids are extracted by precipitation from lysed cells and 25 the mRNA is isolated from the extract using a column which binds the poly-A tails of the mRNA molecules (48).

The mRNA is then exposed to radioactively labelled probe on a nitrocellulose membrane, and the probe hybridizes to and thereby labels complementary mRNA sequences. Binding may be detected by autoradiography or scintillation counting. However, other methods for performing these steps are well known to those skilled in the art, and the discussion above is merely an example.

WU 7 al1".3

W-

-36- 5 *5 S

S

S

This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a mammalian GABA transporter so as to prevent translation of the mammalian GABA transporter. This invention also provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a mammalian taurine transporter so as to prevent translation of the mammalian taurine transporter. This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a human GABA transporter so as to prevent translation of the human GABA transporter. This invention 15 also provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a human taurine transporter so as to prevent translation of the human taurine transporter. As used herein, the phrase 20 "binding specifically" means the ability of an antisense oligonucleotide to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs. The antisense oligonucleotide may have a sequence capable of binding specifically with any sequences of the cDNA molecules whose sequences are shown in Figures 1A, IB, 1C. 10A and 10B. A particular example of an antisense oligonucleotide is an antisense oligonucleotide comprising chemical analogues of nucleotides.

This invention also provides a pharmaceutical composition comprising an effective amount of the oligonucleotide described above effective to reduce expression of a mammalian GABA transporter by passing through a cell -37membrane and binding specifically with mRNA encoding a mammalian GABA transporter in the cell so as to prevent its translation and a pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane. This invention provides a pharmaceutical composition comprising an effective amount of the oligonucleotide described above effective to reduce expression of a mammalian taurine transporter by passing through a cell membrane and binding specifically with mRNA encoding a mammalian taurine transporter in the cell so as to prevent its translation and a pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane. This invention also provides a pharmaceutical composition comprising an effective amount S 15 of the oligonucleotide described above effective to reduce expression of a human GABA transporter by passing through a cell membrane and binding specifically with mRNA encoding a human GABA transporter in the cell so as to prevent its translation and a pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane. This invention also provides a pharmaceutical composition comprising an effective amount of the oligonucleotide described above effective to reduce expression of a human taurine transporter by passing through a cell membrane and binding specifically with mRNA encoding a human taurine transporter in the cell so as to prevent its translation and a pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The oligonucleotide may be coupled to a substance which inactivates mRNA, such as a -38ribozyme. The pharmaceutically acceptable hydrophobic carrier capable of passing through cell membranes may also comprise a structure which binds to a transporter specific f or a selected cell type and is thereby taken up by cells of the selected cell type. The structure may be part of a protein known to bind a cell-type specific transporter, for example an insulin molecule, which would target pacetccells. DNIA molecules having coding sequences substantially the same as the coding sequence shown in Figures 1A, 1B, 1C, lOA or lOB may be used as the oligonucleotides of the pharmaceutical composition.

*This invention also provides a method of treating abnormalities which are alleviated by reduction of expression of a GABA transporter. This method comprises administering to a subject an effective amount of the pharmaceutical composition described above effective to reduce expression of the GABA transporter by the subject.

This invention further provides a method of treating an abnormal condition related to GABA transporter activity which comprises administering to a subject an amount of pharmaceutical composition described above effective to reduce expression of the GABA transporter by the asubject. Examples of such abnormal conditions are 25 epilepsy and generalized anxiety. This invention also provides a method of treating abnormalities which are alleviated' by reduction of expression of a taurine transporter. This method comprises administering to a subject an effective amount of the pharmaceutical composition described above effective to reduce expression of the taurine transporter by the subject.

This invention further provides a method of treating an abnormal condition related to taurine transporter activity which comprises administering to a subject an amount of the pharmaceutical composition described above -39effective to reduce expression of the taurine transporter by the subject. Examples of such abnormal conditions are epilepsy, migraine, and ischemia.

Antisense oligonucleotide drugs inhibit translation of mRNA encoding these transporters. Synthetic antisense oligonucleotides, or other antisense chemical structures are designed to bind to mRNA encoding a GABA transporter or to mRNA encoding a taurine transporter and inhibit translation of mRNA and are useful as drugs to inhibit expression of GABA transporter genes or taurine transporter genes in patients. This invention provides a means to therapeutically alter levels of expression of mammalian GABA or taurine transporters by the use of a 15 synthetic antisense oligonucleotide drug (SAOD) which inhibits translation of mRNA encoding these transporters.

Synthetic antisense oligonucleotides, or other antisense chemical structures designed to recognize and selectively ~bind to mRNA, are constructed to be complementary to portions of the nucleotide sequences shown in Figures lA, 1B, IC, 10A or 10B of DNA, RNA or of chemically modified, artificial nucleic acids. The SAOD is designed to be stable in the blood stream for administration to patients by injection, or in laboratory cell culture 25 conditions, for administration to cells removed from the patient. The SAOD is designed to be capable of passing through cell membranes in order to enter the cytoplasm of the cell by virtue of physical and chemical properties of the SAOD which render it capable of passing through cell membranes by designing small, hydrophobic SAOD chemical structures) or by virtue of specific transport systems in the cell which recognize and transport the SAOD into the cell. In addition, the SAOD can be designed for administration only to certain selected cell populations by targeting the SAOD to be recognized by specific cellular uptake mechanisms which bind and take up the SAOD only within certain selected cell populations. For example, the SAOD may be designed to bind to a transporter found only in a certain cell type, as discussed above. The SAOD is also designed to recognize and selectively bind to the target mRNA sequence, which may correspond to a sequence contained within the sequences shown in Figures IA, 1B, 1C, 10A or by virtue of complementary base pairing to the mRNA.

Finally, the SAOD is designed to inactivate the target mRNA sequence by any of three mechanisms: 1) by binding to the target mRNA and thus inducing degradation of the mRNA by intrinsic cellular mechanisms such as RNAse I digestion, 2) by inhibiting translation of the mRNA 15 target by interfering with the binding of translationregulating factors or of ribosomes, or 3) by inclusion of other chemical structures, such as ribozyme sequences or reactive chemical groups, which_ either degrade--or chemically modify the target mRNA. Synthetic antisense oligonucleotide drugs have been shown to be capable of the properties described above when directed against mRNA targets (11,76). In addition, coupling of ribozymes to antisense oligonucleotides is a promising strategy for inactivating target mRNA An SAOD serves as an 25 effective therapeutic agent if it is designed to be administered to a patient by injection, or if the patient's target cells are removed, treated with the SAOD in the laboratory, and replaced in the patient. In this manner, an SAOD serves as a therapy to reduce transporter expression in particular target cells of a patient, in any clinical condition which may benefit from reduced expression of GABA or taurine transporters.

This invention provides an antibody directed to the mammalian GABA transporter. This antibody may comprise, -41for example, a monoclonal antibody directed to an epitope of a mammalian GABA transporter present on the surface of a cell, the epitope having an amino acid sequence substantially the same as an amino acid sequence for a cell surface epitope of the mammalian GABA transporter included in the amino acid sequence shown in Figures A or 1B. This invention provides an antibody directed to the mammalian taurine transporter. This antibody may comprise, for example, a monoclonal antibody directed to an epitope of a mammalian taurine transporter present on the surface of a cell, the epitope having an amino acid sequence substantially the same as an amino acid sequence for a cell surface epitope of the mammalian taurine transporter included in the amino acid sequence shown in Figure 1C. This invention provides an antibody directed to a human GABA transporter. This antibody may comprise, for example, a monoclonal antibody directed to an epitope of a human GABA transporter present on the surface of a cell, the epitope having an amino acid sequence substantially the same as an amino acid sequence for a cell surface epitope of the human GABA transporter included in the amino acid sequence shown in Figures and 10B. This invention provides an antibody directed to a human taurine transporter. This antibody may comprise, 25 for example, a monoclonal antibody directed to an epitope of a human taurine transporter present on the surface of a cell, the epitope having an amino acid sequence substantially similar to the amino acid sequence for a cell surface epitope of the mammalian taurine transporter shown in Figure IC. Amino acid sequences may be analyzed by methods well known to those skilled in the art to determine whether they produce hydrophobic or hydrophilic regions in the proteins which they build. In the case of cell membrane proteins, hydrophobic regions are well known to form the part of the protein that is inserted WLU Y73 16143 -42into the lipid bilayer which forms the cell membrane, while hydrophilic regions are located on the cell surface, in an aqueous environment. Therefore antibodies to the hydrophilic amino acid sequences shown in Figures 1A or 1B will bind to a surface epitope of a mammalian GABA transporter, and antibodies to the hydrophilic amino acid sequences shown in Figure 1C will bind to a surface epitope of a mammalian taurine transporter, as described.

Antibodies to the hydrophilic amino acid sequences shown in Figures 10A or 10B will bind to a surface epitope of a human GABA transporter. Antibodies directed to conserved hydrophilic amino acid sequences specific to a mammalian taurine transporter will bind to a surface epitope of a human taurine transporter. Antibodies directed to mammalian or human transporters may be serum-derived or monoclonal and are prepared using methods well known in the art. For example, monoclonal antibodies are prepared using hybridoma technology by fusing antibody producing B cells from immunized animals with myeloma cells and selecting the resulting hybridoma cell line producing the desired antibody. Cells such as NIH3T3 cells or Ltk" cells may be used as immunogens to raise such an antibody. Alternatively, synthetic peptides may be prepared using commercially available machines and the amino acid sequences shown in Figures 1A, 1B, 1C, 10A and 10B. As a still further alternative, DNA, such as a cDNA or a fragment thereof, may be cloned and expressed and the resulting polypeptide recovered and used as an immunogen. These antibodies are useful to detect the presence of mammalian transporters encoded by the isolated DNA, or to inhibit the function of the transporters in living animals, in humans, or in biological tissues or fluids isolated from animals or humans.

TVL 7..I -43- This invention also provides a pharmaceutical composition which comprises an effective amount of an antibody directed to an epitope of the mammalian transporter, effective to block binding of naturally occurring substrates to the transporter, and a pharmaceutically acceptable carrier. A monoclonal antibody directed to an epitope of a mammalian GABA transporter present on the surface of a cell which has an amino acid sequence substantially the same as an amino acid sequence for a cell surface epitope of the mammalian GABA transporter included in the amino acid sequences shown in Figures A and IB is useful for this purpose. A monoclonal antibody directed to an epitope of a mammalian taurine transporter present on the surface of a cell which has an amino acid 15 sequence substantially the same as an amino acid sequence for a cell surface epitope of the mammalian taurine transporter included in the amino acid sequence shown in Figure 1C is also useful for this purpose.

This invention also provides a pharmaceutical composition which comprises an effective amount of an antibody directed to an epitope of the human transporter, effective to block binding of naturally occurring substrates to the transporter, and a pharmaceutically 25 acceptable carrier. A monoclonal antibody directed to an epitope of a human GABA transporter present on the surface of a cell which has an arino acid sequence substantially the same as an amino acid sequence for a cell surface epitope of the human GABA transporter included in the amino acid sequences shown in Figures or 10B is useful for this purpose.

This invention also provides a pharmaceutical composition which comprises an effective amount of an antibody directed to an epitope of a human taurine transporter, WLU 70110144J -44effective to block binding of naturally occurring substrates to the human taurine transporter, and a phurmaceutically acceptable carrier. A monoclonal antibody directed to a conserved epitope specific to a mammalian taurine transporter present on the surface of a cell which has an amino acid sequence substantially the same as an amino acid sequence f or a cell surface epitope of the mammalian taurine transporter included in the amino acid sequence shown in Figure lC is useful for this purpose.

This invention also provides a method of treating abnormalities in a subject which are alleviated by S'.reduction of expression of a mammalian transporter which comprises administering to the subject an effective -amount of the pharmaceutical composition described above effective to block binding of naturally occurring substrates to the transporter and thereby alleviate abnormalities resulting from overexpression of a mammalian transporter. Binding of the antibody to the transporter prevents the transporter from functioning, thereby neutralizing the effects of overexpression. The monoclonal antibodies described above are both useful for this purpose. This invention additionally provides a a. 25 method of treating an abnormal condition related to an excess of transporter activity which comprises administering to a subject an amount of the pharmaceutical composition described above effective to block binding of naturally occurring substrates to the transporter and thereby alleviate the abnormal condition.

Some examples of abnormal conditions associated with excess GABA transporter activity are epilepsy and generalized anxiety. Excess taurine transporter activity associated disorders are epilepsy, migraine, and ischemia.

V 7.J1 a101J This invention provides methods of detecting the presence of a GABA or a taurine transporter on the surface of a.

cell which comprises contacting the cell with an antibody directed to the mammalian GABA transporter or an antibody directed to the mammalian taurine transporter, under conditions permitting binding of the antibody to the transporter, detecting the presence of the antibody bound to the cell, and thereby the presence of the mammalian GABA transporter or the presence of the taurine transporter on the surface of the cell. Such methods are useful for determining whether a given cell is defective in expression of GABA transporters or is defective in expression of taurine transporters on the surface of the S- cell. Bound antibodies are detected by methods well known in the art, for example by binding fluorescent markers to the antibodies and examining the cell sample under a fluorescence microscope to detect fluorescence on a cell indicative of antibody binding. The monoclonal antibodies described above are useful for this purpose.

This invention provides a transgenic nonhuman mammal expressing DNA encoding a mammalian GABA transporter and a transgenic nonhuman mammal expressing DNA encoding a .:mammalian taurine transporter. This invention further provides a transgenic nonhuman mammal expressing DNA encoding a human CABA transporter and a transgenic nonhuman mammal expressing DNA encoding a human taurine transporter. This invention also provides a transgenic nonhuman mammal expressing DNA encoding a mammalian GABA transporter so mutated as to be incapable of normal transporter activity, and not expressing native GABA transporter and a transgenic nonhuman mammal expressing DNA encoding a mammalian taurine transporter so mutated as to be incapable of normal transporter activity, and not expressing native taurine transporter. This invention WO Y3/15143 -46further provides a transgenic nonhuman mammal expressing DNA encoding a human GABA transporter so mutated as to be incapable of normal transporter activity, and not expressing native GABA transporter and a transgenic nonhuman mammal expressing DNA encoding a human taurine transporter so mutated as to be incapable of normal transporter activity, and not expressing native taurine transporter.

This invention provides a transgenic nonhuman mammal whose genome comprises DNA encoding a mammalian GABA transporter so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a GABA transporter and which hybridizes to mRNA encoding a GABA 15 transporter thereby reducing its translation and a transgenic nonhuman mammal whose genome comprises DNA encoding a mammalian taurine transporter so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a taurine transporter and which hybridizes to mRNA encoding a taurine transporter thereby reducing its translation. This invention further 0* provides a transgenic nonhuman mammal whose genome comprises DNA encoding a human GABA transporter so placed as to be transcribed into antisense mRNA which is 25 complementary to mRNA encoding a GABA transporter and which hybridizes to mRNA encoding a GABA transporter thereby reducing its translation and a transgenic nonhuman mammal whose genome comprises DNA encoding a human taurine transporter so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a taurine transporter and which hybridizes to mRNA encoding a taurine transporter thereby reducing its translation. The DNA may additionally comprise an inducible promoter or additionally comprise tissue specific regulatory elements, so that expression can be WU V3/ lOMJ3 -47induced, or restricted to specific cell types. Examples of DNA are DNA or cDNA molecules having a coding sequence substantially the same as the coding sequences shown in Figures IA, 1B, 1C, 10A and 10B. An example of a transgenic animal is a transgenic mouse. Examples of tissue specificity-determining regions are the metallothionein promotor (46,83) and the L7 promotor (84).

Animal model systems which elucidate the physiological and behavioral roles of mammalian transporters are produced by creating transgenic animals in which the expression of a transporter is either increased or decreased, or the amino acid sequence of the expressed transporter protein is altered, by a variety of techniques. Examples of these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a mammalian transporter or homologous animal versions of these genes, by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embryos in order to produce a transgenic animal (24) or 2) Homologous recombination (7,82) of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these transporters. The technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and so is useful for producing an animal that cannot express native transporter but does express, for example, an inserted mutant transporter, which has replaced the native transporter in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and so is useful for TVLW 7.J A OJ.F -48producing an animal which expresses its own and added transporters, resulting in overexpression of the transporter.

One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as M2 medium DNA or cDNA encoding a mammalian transporter is purified from a vector (such as plasmids EVJB-rB14b, EVJB-rBBb, or EVJBrB16a described above) by methods well known in the art.

Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the trans-gene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the trans-gene. The DNA, in an appropriately buffered solution, is put into a 20 microinjection needle (which may be made from capillary tubing using a pipet puller) and the egg to be injected is put in a depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is injected. The injected egg is then transferred into the oviduct of a pseudopregnant mouse (a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant), where it proceeds to the uterus, implants, and develops to term. As noted above, microinjection is not the only method for inserting DNA into the egg cell, and is used here only for exemplary purposes.

Since the normal action of transporter-specific drugs is to activate or to inhibit the transporter, the transgenic animal model systems described above are useful for -49testing the biological activity of drugs directed against these transporters even before such drugs become available. These animal model systems are useful f or predicting or evaluating .possible therapeutic applications of drugs which activate or inhibit these transporters by inducing or inhibiting expression of the native or trans-gene and thus increasing or decreasing expression of normal or mutant transporters in the living animal. Thus, a model system is produced in which the biological activity of drugs directed against these transporters are evaluated before such drugs become *'*available. The tranagenic animals which over or under produce the transporter indicate by their physiological *state whether over or under production of the transporter is therapeutically useful. It is there fore useful to 2.evaluate drug action based on the transgenic model system. One use is based on the f act that it is well known in the art that a drug such as an antidepressant acts by blocking neurotransmitter uptake, and thereby increases the amount of neurotransmitter in the synaptic *449cleft. The physiological result of this action is to stimulate the production of less transporter by the affected cells, leading eventually to underexpression.

Therefore, an animal which underexpresses transporter is useful as a test system to investigate whether the actions of such drugs which result in under expression are in fact therapeutic. Another use is that if overexpression is found to lead to abnormalities, then a drug which down-regulates or acts as an antagonist to the transporter is indicated as worth developing, and if a promising therapeutic application is uncovered by these animal model systems, activation or inhibition of the GABA transporter is achieved therapeutically either by producing agonist or antagonist drugs directed against YIj 7JIf 1O Z-' these GABA transporters or by any method which increases or decreases the expression of these transporters in man.

Further provided by this invention is a method of determining the physiological effects of expressing varying levels of mammalian transporters which comprises producing a transgenic nonhuman animal whose levels of mammalian transporter expression are varied by use of an inducible promoter which regulates mammalian transporter expression. This invention also provides a method of determining the physiological effects of expressing varying levels of mammalian transporters which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of mammalian transporter.

15 Such animals may be produced by introducing different amounts of DNA encoding a mammalian transporter into the oocytes from which the transgenic animals are developed.

This invention provides a method of determining the physiological effects of expressing varying levels of 2 human transporters which comprises producing a transgenic nonhuman animal whose levels of human transporter expression are varied by use of an inducible promoter which regulates transporter expression. This invention also provides a method of determining the physiological effects of expressing varying levels of human transporters which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of the human transporter. Such animals may be produced by introducing different amounts of DNA encoding a human transporter into the oocytes from which the transgenic animals are developed.

This invention also provides a method for identifying a substance capable of alleviating abnormalities resulting Vj .7vf &oCA7r -51from overexpression of a mammalian transporter comprising administering the substance to a transgenic nonhuman mammal expressing at least one artificially introduced DNA molecule encoding a mammalian transporter and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of overexpression of a mammalian transporter. This invention also provides a method for identifying a substance capable of alleviating abnormalities resulting from overexpression of a human transporter comprising administering the substance to a transgenic nonhuman mammal expressing at least one artificially introduced DNA molecule encoding a human transporter and determining whether the substance 15 alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of overexpression of a human transporter. As used herein, the term "substance" means a compound or composition which may be natural, synthetic, or a product derived from screening. Examples of DNA molecules are DNA or cDNA molecules having a coding sequence substantially the same as the coding sequences shown in Figures 1A, 1B, 1C, 10A or 25 This invention provides a pharmaceutical composition comprising an amount of the substance described sura effective to alleviate the abnormalities resulting from overexpression of GABA transporter and a pharmaceutically acceptable carrier. This invention also provides a pharmaceutical composition comprising an amount of the substance described supra effective to alleviate the abnormalities resulting from overexpression of taurine transporter and a pharmaceutically acceptable carrier.

This invention further provides a pharmaceutical composition comprising an amount of the substance WVJ 731 101J -52described supra effective to alleviate the abnormalities resulting from overexpression of a human GABA or human taurine transporter and a pharmaceutically acceptable carrier.

This invention also provides a method for treating the abnormalities resulting from overexpression of a mammalian transporter which comprises administering to a subject an amount of the pharmaceutical composition described above effective to alleviate the abnormalities resulting from overexpression of a mammalian transporter.

This invention further provides a method for treating the abnormalities resulting from overexpression of a human "GABA or human taurine transporter which comprises administering to a subject an amount of the pharmaceutical composition described above effective to alleviate the abnormalities resulting from overexpression of a human GABA or taurine transporter.

This invention provides a method for identifying a substance capable of alleviating the abnormalities resulting from underexpression of a mammalian transporter comprising administering the substance to the transgenic nonhuman mammal described above which expresses only nonfunctional mammalian transporter and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of underexpression of a mammalian transporter. This invention further provides a method for identifying a substance capable of alleviating the abnormalities resulting from underexpression of a human GABA or human taurine transporter comprising administering the substance to the transgenic nonhuman mammal described above which expresses only nonfunctional human GABA or human taurine transporter and determining vvx -C -53whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of underexpression of a human GABA or human taurine transporter.

This invention also provides a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of transporter and a pharmaceutically acceptable carrier.

This invention also provides a pharmaceutical composition comprising an amount of a substance effective to 'alleviate abnormalities resulting from underexpression of a human GABA or human taurine transporter and a "pharmaceutically acceptable carrier.

This invention provides a method for treating the abnormalities resulting from underexpression of a mammalian transporter which comprises administering to a subject an amount of the pharmaceutical composition 20 described above effective to alleviate the abnormalities resulting from underexpression of a mammalian transporter. This invention further provides a method for treating the abnormalities resulting from underexpression of a human GABA or human taurine transporter which comprises administering to a subject an amount of the pharmaceutical composition described above effective to alleviate the abnormalities resulting from underexpression of a human GABA or human taurine transporter.

This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific mammalian transporter allele which comprises: a) obtaining DNA of subjects suffering from the disorder; b) performing a restriction digest of WU 7J3/1 jld -54the DNA with a panel of restriction enzymes; c) electrophoretically separating the resulting DNA fragments on a sizing gel; d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a mammalian transporter and labelled with a detectable marker; e) detecting labelled bands which have hybridized to the DNA encoding a mammalian transporter labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; f) preparing DNA obtained for diagnosis by steps a-e; and g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether 15 the patterns are the same or different and thereby to diagnose predisposition to the disorder if the patterns are the same. This method may also be used to diagnose a disorder associated with the expression of a specific mammalian transporter allele.

This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific human GABA or human taurine transporter allele which comprises: a) obtaining DNA of subjects suffering from the disorder; b) performing a restriction digest of the DNA with a panel of restriction enzymes; c) electrophoretically separating the resulting DNA fragments on a sizing gel; d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a human GABA or human taurine transporter and labelled with a detectable marker; e) detecting labelled bands which have hybridized to the DNA encoding a human GABA or human taurine transporter labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects YVTJ 7.3I AOA.J suffering from the disorder; f) preparing DNA obtained for diagnosis by steps a-e; and g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and thereby to diagnose predisposition to the disorder if the patterns are the same. This method may also be used to diagnose a disorder associated with the expression of a specific human GABA or human taurine transporter allele.

This invention provides a method of preparing the oo.: isolated transporter which comprises inducing cells to express transporter, recovering the transporter from the 15 resulting cells, and purifying the transporter so recovered. An example of an isolated GABA transporter is an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in Figures 1A or 1B. An example of an isolated taurine transporter 20 is an isolated protein having substantially the same amino acid sequence shown in Figure 1C. This invention further provides a method for preparing an isolated human GABA transporter which comprises inducing cells to express the human GABA transporter, recovering the human GABA transporter from the resulting cells, and purifying the human GABA transporter so recovered. An example of an isolated human GABA transporter is an isolated protein having substantially the same aarno acid sequence as the amino acid sequence shown in Figures 10A or 10B. This invention further provides a method for preparing an isolated human taurine transporter which comprises inducing cells to express the human taurine transporter, recovering the human taurine transporter from the resulting cells, and purifying the human taurine transporter so recovered. An example of an isolated V. -56human taurine transporter is an isolated protein having an amino acid sequence substantially similar to the amino acid sequence of a mammalian taurine transporter shown in Figure 1C. For example, cells can be induced to express transporters by exposure to substances such as hormones. The cells can then be homogenized and the transporter isolated from the homogenate using an affinity column comprising, for example, GABA, taurine, or another substance which is known to bind to the transporter. The resulting fractions can then be purified by contacting them with an ion exchange column, and determining which fraction contains transporter activity or binds anti-transporter antibodies.

C

15 This invention provides a method of preparing the isolated mammalian GABA transporter which comprisesinserting nucleic acid encoding the mammalian GABA transporter in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the transporter produced by the resulting cell, and purifying the transporter so recovered. An example of an isolated GABA transporter is an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in Figures 1A or 1B. This invention also provides a method of preparing the isolated mammalian taurine transporter which comprises inserting nucleic acid encoding a mammalian taurine transporter in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the transporter produced by the resulting cell, and purifying the transporter so recovered. This invention also provides a method of preparing the isolated human GABA transporter which comprises inserting nucleic acid encoding the human GABA transporter in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the human GABA -57transporter produced by the resulting cell, and purifying the human GABA transporter so recovered. These methods for preparing GABA or taurine transporters uses recombinant DNA technology methods well known in the art.

For example, isolated nucleic acid encoding GABA or taurine transporter is inserted in a suitable vector, such as an expression vector. A suitable host cell, such as a bacterial cell, or a eukaryotic cell such as a yeast cell, is transfeocted with the vector. GA.EA or taurine transporter is isolated from the culture medium by affinity purification or by chromatography or by other *methods well known in the art.

This invention provides a method f or determining whether a substrate not known to be capable of binding to a GABA transporter can bind to the mammalian GABA transporter which comprises. contacting a mammalian cell comprising a DNA molecule encoding a mammalian GABA transporter with the substrate under conditions permitting binding of substrates known to bind to the transporter, detecting the presence of any' of the substrate bound to the transporter, and thereby determining whether the substrate binds to the transporter. The DNA in the cell may have a coding sequence substantially the same as the coding sequences shown in Figures IA, or lB. This invention provides a method for determining whether a substrate not known to be capable of binding to a mammalian taurine transporter can bind to the mammalian GABA transporter which comprises contacting a mammalian cell comprising a DNA molecule encoding a mammalian taurine transporter with the substrate under conditions permitting binding of substrates known to. bind to the transporter, detecting the presence of any of the substrate bound to the transporter, and thereby determining whether the WyV r-re eve we -58substrate binds to the transporter. The DNA in the cell may have a coding sequence substantially the same as the coding sequences shown in Figure 1C.

This invention also provides a method for determining whether a substrate not known to be capable of binding to a human GABA transporter can bind to a human GABA transporter which comprises contacting a mammalian cell comprising a DNA molecule encoding a human GABA transporter with the substrate under conditions permitting binding of substrates known to bind to the transporter, detecting the presence of any of the substrate bound to the transporter, and thereby determining whether the substrate binds to the 15 transporter. The DNA in the cell may have a coding sequence substantially the same as the coding sequences shown in Figures 10A or 10B. This invention also provides a method for determining whether a substrate not known to be capable of binding to a human taurine 20 transporter can bind to a human taurine transporter which comprises contacting a mammalian cell comprising a DNA molecule encoding a human taurine transporter with the substrate under conditions permitting binding of substrates known to bind to the transporter, detecting the presence of any of the substrate bound to the transporter, and thereby determining whether the substrate binds to the transporter. Preferably, the mammalian cell is nonneuronal in origin. An example of a nonneuronal mammalian cell is a Cos7 cell. The preferred method for determining whether a substrate is capable of binding to the mammalian transporter comprises contacting a transfected nonneuronal mammalian cell (i.e.

a cell that does not naturally express any type of transporter, thus will only express such a transporter if it is transfected into the cell) expressing a transporter -59on its surface, or contacting a membrane preparation derived from such a transfected cell, with the substrate under conditions which are known to prevail, and thus to be associated with, in yviv binding of the substrates to a transporter, detecting the presence of any of the substrate being tested bound to the transporter on the surface of the cell, and thereby determining whether the substrate binds to the transporter. This response system is obtained by transfection of isolated DNA into a suitable host cell. Such a host system might be isolated from pre-existing cell lines, or can be generated by inserting appropriate components into existing cell lines. Such a transfection system provides a complete response system for investigation or assay of the 15 functional activity of mammalian transporters with substrates as described above. Transfection systems are useful as living cell cultures for competitive binding assays between known or candidate drugs and substrates o which bind to the transporter and which are labeled by radioactive, spectroscopic or other reagents. Membrane preparations containing the transporter isolated from transfected cells are also useful for these competitive binding assays. A transfection system constitutes a "drug discovery system" useful for the identification of natural or synthetic compounds with potential for drug development that can be further modified or used directly as therapeutic compounds to activate or inhibit the natural functions of the mammalian transporter and/or the human transporter. The transfection system is also useful for determining the affinity and efficacy of known drugs at the mammalian transporter sites and human transporter sites.

This invention provides a method for isolating membranes which comprise GABA or taurine transporters. In a T V Ikp .vw-m« preferred embodiment of the invention, membranes comprising a GABA or taurine transporter are isolated.

from transfected cells comprising a plasmid vector which further comprises the regulatory elements necessary for the expression of the DNA encoding a GABA or taurine transporter so located relative to the DNA encoding the GABA or taurine transporter as to permit expression thereof. The DNA may have the coding sequence substantially the same as the sequence shown in Figure 1A, 1B, 1C, 10A or 10B. The host cell may be a bacterial, yeast, or a mammalian cell. Examples of such cells include the mouse fibroblast cell line NIH3T3, CHO cells, HELA cells, Ltk- cells and Y1 cells. A method for isolating membranes which contain a GABA or taurine 9 transporter comprises preparing a cell lysate from cells expressing the GABA or taurine transporter and isolating membranes from the cell lysate. Methods for the isolation of membranes are well known by one of skill in the art. A method for the isolation of membranes from transfected cells is further described by Branchek et al.

(1990). Membranes isolated from transfected cells expressing a GABA or taurine transporter are useful for identifying compounds which may include substrates, drugs or other molecules that specifically bind to a GABA or taurine transporter using radioligand binding methods (Branchek et al. 1990) or other methods described herein.

The specificity of the binding of the compound to the transporter may be identified by its high affinity for a particular transporter.

This invention further provides a method for the isolation of vesicles from cells expressing a GABA or taurine transporter. In a preferred embodiment of the invention, vesicles comprising a GABA or taurine transporter are isolated from transfected cells -61comprising a plasmid vector which further comprises the regulatory elements necessary for the expression of the DNA encoding a GABA or taurine transporter so located relative to the DNA encoding the GABA or taurine transporter as to permit expression thereof. The DNA may have the coding sequence substantially the same as the sequence shown in Figure lA, 1B, 1C, 10A or 10B. A method for the isolation of vesicles is described by Barber and Jamieson (1970) and by Mabjeesh et al. (1992).

Vesicles comprising a GABA or taurine transporter are useful for assaying and identifying compounds, which may include substrates, drugs or other molecules that enhance or decrease GABA or taurine transporter activity. The compounds may modulate transporter activity by 15 interacting directly with the transporter or by interacting with other cellular components that modulate transporter activity. Vesicles provide an advantage over whole cells in that the vesicles permit one to choose the ionic compositions on both sides of the membrane such 0. 20 that transporter activity and its modulation by can be studied under a variety of controlled physiological or non-physiological conditions. Methods for the assay of transporter activity are well known by one of skill in the art and are described herein below and by Kannner (1978) and Rudnick (1977).

This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the mammalian GABA transporter on the surface of a cell which comprises contacting a mammalian cell comprising a DNA molecule encoding a mammalian GABA transporter on the surface of a cell with a plurality of drugs, detecting those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, the mammalian GABA -62transporter. The DNA in the cell may have a coding sequence substantially the same as the coding sequences shown in Figure 1A or 1B. This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the mammalian taurine transporter on the surface of a cell which comprises contacting a mammalian cell comprising a DNA molecule encoding a mammalian taurine transporter on the surface of a cell with a plurality of drugs, detecting those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, the mammalian taurine transporter. The DNA in the cell may have a coding sequence substantially the same as the coding sequences shown in Figure 1C. This 15 invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to, a human GABA transporter on the surface of a cell which comprises contacting a mammalian cell comprising a DNA molecule encoding a human GABA transporter on the surface of a cell with a plurality of drugs, detecting those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, the human GABA transporter. The DNA in the cell :may have a coding sequence substantially the same as the coding sequences shown in Figures 10A or 10B. This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to,.a human taurine transporter on the surface of a cell which comprises contacting a mammalian cell comprising a DNA molecule encoding a human taurine transporter on the surface of a cell with a plurality of drugs, detecting those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, the human taurine transporter. Various methods of detection may be employed. The drugs may be "labeled" -63by association with a detectable marker substance radiolabel or a non-isotopic label such as biotin).

Preferably, the mammalian cell is nonneuronal in origin.

An example of a nonneuronal mammalian cell is a Cos7 cell. Drug candidates are identified by choosing chemical compounds which bind with high affinity to the expressed transporter protein in transfected cells, using radioligand binding methods well known in the art, examples of which are shown in the binding assays described herein. Drug candidates are also screened for selectivity by identifying compounds which bind with high affinity to one particular transporter subtype but do not bind with high affinity to any other transporter subtype or to any other known transporter site. Because 15 selective, high affinity compounds interact primarily with the target transporter site after administration to the patient, the chances of producing a drug with unwanted side effects are minimized by this approach.

This invention provides a pharmaceutical composition comprising a drug identified by the method described above and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, *"such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. Once the candidate drug has been shown to be adequately bio-available following a particular route of administration, for example orally or by injection (adequate therapeutic concentrations must be maintained at the site of action for an adequate period to gain the desired therapeutic benefit), and has been shown to be non-toxic and therapeutically effective in appropriate disease models, the drag may be administered to patients by that route of administration determined to make the drug bio-available, -64 in an appropriate solid or solution formulation, to gain the desired therapeutic benefit.

Applicants have identified individual transporter subtype proteins and have described methods for the identification of pharmacological compounds f or therapeutic treatments. Pharmacological compounds which are directed against specific transporter subtypes provide effective new therapies with minimal side effects.

Elucidation of the molecular structures of the neuronal goes 0 GABA and taurine transporters is an important step in the .0r.understanding of GABAergic neurotransmissionl. This' 0015 disclosure reports the isolation, amino acid sequence, 0 ~and functional expression of a cDNA clones from- rat brain 6*which encode a GABA transporters. and a cDNA clone from 0*00 rat brain which encodes a taurine transporter. This disclosure reports the isolation, amino acid sequence, 0 0* 20 and functional expression of cDNA clones which encode human GABA transporters. The identification of these transporters will play a pivotal role in elucidating the molecular mechanisms underlying GABAergic transmission, :00 :and should also aid in the development of novel therapeutic agents.

Complementary DNA clones (designated rB14b, rB~b, and rBl6a) encoding two GABA transporters and a taurine transporter, respectively, have been isolated from rat brain, and their functional properties have been examined in mammalian cells. The nucleotide sequence of rB14b predicts a protein of 602 amino acids, rB~b predicts a protein of 627 amino acids, and rB16a predicts a protein of 621 amino acids, with 12 highly hydrophobic regions compatible with membrane-spanning domains. When incubated with 50 nM 3 H]GABA, COS cells transiently transfected with rB14b or rB8b accumulated greater than as much radioactivity as non-transfected control cells. The transporters encoded by rB14b and rB8b display high-affinity for GABA(Km-4AM) and are dependent on external sodium and chloride. Similarly, when incubated with 50nM [3H]taurine, Cos cells transiently transfected with rB21a accumulated approximately 7-fold as much radioactivity as non-transfected control cells.

The pattern of expression of mRNA encoding two GAB.

transporters has been examined in the rat brain.

Additionally, complementary DNA clones (designated hGAT- 3, hHE7a, hS3a) and a genomic DNA clone encoding human GABA transporters have been isolated and their functional 15 properties examined in mammalian cells.

Analysis of the GABA and taurine transporter structure and function provides a model for the development of drugs useful for the treatment of epilepsy, generalized anxiety, migraine, ischemia and other neurological disorders.

This invention identifies for the first time three new mammalian transporter proteins, their amino acid 25 sequences, and their mammalian genes. The invention further identifies the human homologues of two mammalian GABA transporter proteins, their amino acid sequence and their human genes. The information and experimental tools provided by this discovery are useful to generate new therapeutic agents, and new therapeutic or diagnostic assays for these new transporter proteins, their associated mRNA molecules or their associated genomic DNAs. The information and experimental tools provided by this discovery will be useful to generate new therapeutic agents, and new therapeutic or diagnostic assays for WUJ y.J/ o0sJ -66these new transporter proteins, their associated mRNA molecules, or their associated genomic DNAs.

Specifically, this invention relates to the first isolation of three mammalian cDNAs and genomic clones encoding GABA and taurine transporters and the first isolation of cDNAs and a genomic clone encoding the human homologues of two mammalian GABA transporters. The new mammalian genes for these transporters identified herein as rBl4b, rB8b, and rB16a have been identified and characterized, and a series of related cDNA and genomic clones have been isolated. In addition, the mammalian GABA and mammalian taurine transporters have been expressed in Cos7 cells by transfecting the cells with 15 the plasmids EVJB-rB14b, EVJB-rB8b, and EVJB-rB16a. The pharmacological binding properties of the proteins encoded have been determined, and these binding properties classify these proteins as GABA transporters Sand a taurine transporter. Mammalian cell lines expressing the mammalian and human GABA transporters and the mammalian taurine transporter on the cell surface have been constructed, thus establishing the first well-defined, cultured cell lines with which to study the GABA and taurine transporters.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative, and are not meant to limit the invention as described herein, which is defined by the claims which follow thereafter.

MATERAL muG

METHOD

Materials f or Mammalian GAB)' transporter Studies: 3

H)GABA

3 (98.9Ci/mmolS) was obtained from New England Nuclear (Boston, KA). p-alanile, betaine and L-DABA (LdiaminobutyriC acid) were from Sigma Chemical Company (St. Louis, MO); guvacine, nipecotic acid, OHnipecotic (hydroxyfipcotic acid), and THPO (4,5,6,7tetrahydroisoxazolo (4,5-cjpyridin3-ol) were from RBI (Natick, MA) ACHC (cis-;3-aminocyclohexanecarboxCylic acid) was kindly provided by Drs. Richard Milius and William White of the NIMH Chemical Synthesis Program.

Materials for MammalianI Taurine Transpor~ter Studies: 3 H~taurine (25.6Ci/mmole) was from New England Nuclear S(Boston, MA); taurine,

GABA

2 hypotaurine, AEPA, ANSA, .15 APSA, CSA, KEA, and P-alanine were f rom Sigma Chemical corporation (St. Louis, NO); GES was a ki nd gift of Dr.

J. Barry Lombardini (Department of Pharmacology, Texas Tech University).

Cloning and Sequencing of Mammalian GAZA Transporters:

A

rat brain cDNA library in the Lambda ZAP II vector (Stratagene, La Jolla, CA) was screened at reduced stringency using probes representing the complete coding region of the rat GABA transporter cDNA (GAT-l Exact primers derived from the nucleotide sequence of GAT-1 were used to generate GAT-I PCR products from randomly-primed rat brains cDNA; the GAT-l probes were ,then labeled and used to screen the library under reduced stringency as previously described Lambda phage hybridizing with the probes at low stringency were plaque purified and rescreened at high stringency to eliminate clones which were identical to GAT-l. One of the clones hybridizing at high stringency was subsequently confirmed by sequence analysis to encode GAT-l Clones hybridizing only at low stringency were converted to vV se, &J--W -68phagemids by in vivo excision with fl helper phage.

Nucleotide sequences of double-stranded cDNAs in pBluescript were analyzed by the Sanger dideoxy nucleotide chain-termination method (59) using Sequence Biochemical Corp., Cleveland, Ohio).

Expression of Mammalian GAB Transporters: cDNA clones (designated rB14b and rB8b) representing the complete coding regions of two putative transporters were cloned into the eukaryotic expression vector pEVJB (modified from pcEXV-3; Utilizing restriction enzyme sites present in pBluescript, rB14b was subcloned as a 2.0 kb HindIII/Xbal fragment which contained 126 base pairs of 5-untranslated sequence and 94 base pairs of 3'- 15 untranslated sequence. Similarly, rB8b was subcloned as a 2.1 kb XbaI/SalI fragment containing 0.3 kb of 3'untranslated sequence. Transient transfections of COS cells were carried out using DEAE-dextran with DMSO "**:according to the method of Lopata et al. (44) with minor modifications. COS cells were grown 5%C0 2 in high glucose Dulbecco's modified Eagle medium supplemented with 10% bovine calf serum, 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate. Cells were routinely used two days after transfection for transport studies.

Transport Studies of Mammalian GABA Transporters: To measure transport, COS cells grown in 6-well (well diameter 35mm) or 24-well (well diameter 18mm) plates were washed 3X with HEPES-buffered saline (HBS, in mM: NaCl, 150; HEPES, 20; CaCl 2 1; glucose, 10; KC1, HgCl 2 1; pH 7.4) and allowed to equilibrate in a 37*C water bath. After 10 minutes the medium was removed and a solution containing 3 H]GABA (New England Nuclear, sp.

activity. 89.8Ci/mmole) and required drugs in HBS was -69added (1.5 ml/35mm well; 0.5ml/18mm well). Non-specific uptake was defined in parallel wells with ImM unlabeled substrate, and was subtracted from total uptake (no competitor) to yield specific uptake; all data represent specific uptake. Plates were incubated at 370C for minutes unless indicated otherwise, then washed rapidly 3x with ice-cold HBS. Cells were solubilized with 0.05% sodium deoxycholate/0.lN NaOH, an aliquot neutralized with 1N HC1, and radioactivity was determined by scintillation counting. Protein was quantified in an aliquot of the solubilized cells using a BIO-RAD protein assay kit, according to the manufacturers directions.

Northern Blot Analysis of RUA Encoding Mammalian '15 Transporters Total cellular RNA was isolated from rat brain and liver using RNazol (Cinna/Biotecx Laboratories Inc.; Houston, TX) as outlined by the manufacturer. Denatured RNA samples (25Mg) were separated in a 1.0% agarose gel containing 3.3% formaldehyde. RNAs were transferred to nylon membranes (Genescreen Plus; New England Nuclear, Boston, MA) by overnight capillary blotting in 10X SSC.

Northern blots were rinsed and then baked for 2 hours at 80"C under vacuum. Prehybridization was for 2 hours at 25 65 C in a solution containing 50% formamide, 1M NaCl, dextran sulfate, and 1% sodium dodecyl sulfate. Blots were hybridized overnight at 65"C with 3 2 P-labeled DNA probes (randomly primed CAT-2 or GAT-3 full-length cDNA clones) in prehybridization mixture containing 100 pg/ml sonicated salmon sperm DNA. The blots were washed successively in 2X SSC/2% SDS, 1X SSC/2% SDS, and 0.2X SSC/2% SDS at 65 0 C, then exposed to Kodak XAR-5 film with one intensifying screen at -90 0 C for four days.

Tissue 'Localization Studies: To identify tissues expressing mRNAs for the novel GABA transporters and the previously cloned GABA transporter GAT-2 specific PCR primers (25mers) were designed such that =700 base pair f ragments encoding TMs 1 through 5 of each transporter could be amplified and detected by hybridization with 3 2 p-labeled oligonucleotides. For rBl4b, the sequences of the sense and anti-sense oligonucleotides were derived from amino acids 36 to 43 (51-GACCAACAAGATGGAGTCGTACTG) and 247 to 254 TGTTACTCCTCGGATCAACAGGACC); for rB~b, the oligonucleotides were derived from amino acids 52 to (59-GGAGTCGTGTTGAGCGTAGGAGAG) and 271 to 279 GAACTTGATGCCTLrCCGAGGCACCC); and for GAT-I the is oligonucleotide sequences were derived from amino acids to 57 (51-ACGCTCGACTCCTCATGTCCTGT) and 274 to 282 '-GAATCAGACAGCTXTL'CGGAAGTTGG). Primers were also designed to amplify the cONA encoding cyclophilin, a constitutively expressed gene, as a control 0 o20 GTCTGCTTCGAGCTGTTTGCAGACA, sense; TTAGAGTTGTCCACAGTCGGAGATG, anti-sense) To detect amplif ied sequences, oligonucleotide probes were synthesized for GAT-1, rBl4b, and rB8b which corresponded to amino acids 196 to 219, 161 to 183, and 207 to 229, respectively. Each probe was shown to hybridize with its respective transporter cDNA and not with any other transporter cONA under study.

Poly A+ RNA (1 mg, Clonetech, Palo Alto, CA) from each of seven rat tissues was converted to single-stranded cDNA by random priming using Superscript reverse transcriptase (BRL, Gaithersburg, MD). PCR reactions were carried out in a buffer containing 20mN Tris (pH 50 mM KCl, NgC1 2 0.001% gelatin, 2mM dJNTP's, lUN each primer, and Taq polymerase with either cDNA, RNA, water,, or a WU Y3f *OZgJ -71control plasmid for 30 cycles of 94*c./2 min., 68*C./2 min., 72*C./3 min. PCR products were separated by electrophoresis in 1.2% agarose gels, blotted to nylon membranes (Genescreen Plus; New England Nuclear, Boston, MA), and hybridized at 40'C. overnight with 32 P-labeled oligonucleotide probes in a solution containing formamide, 10% dextran sulfate, 5X SSC, IX Denhardt's, and 100 gg/ml sonicated salmon sperm DNA. Blots were washed successively in 2 X SSC at room temperature and 0.1 X SSC at 500C., and exposed to Kodak XAR film for to 4 hours with an intensifying screen at -700C.

Cloning and sequencing of Manalian Taurine Receptor: A t*oo .rat brain cDNA library in the Lambda ZAP II vector (Stratagene, La Jolla, CA) was screened at low stringency "to with the complete coding region of the rat GABA transporter cDNA (GAT-1; Exact primers were used to generate PCR products from randomly-primed rat brain cDNA; the products were labeled and used to screen the 20 library under reduced stringency (25% formamide, hybridization; 0.1X SSC, 40*C. wash) as previously described Lambda phage hybridizing at low stringency with the GAT-1 sequence were plaque purified and rescreened with the same probes at high stringency (50% formamide, 40*C. hybridization; 0.1X SSC, wash) to eliminate clones identical to GAT-1. Clones hybridizing only at low stringency were converted to phagemids by in vivo excision with fl helper phage.

Nucleotide sequences of double-stranded cDNAs in pBluescript were analyzed by the Sanger dideoxy nucleotide chain-termination method using Sequenase Biochemical Corp., Cleveland, Ohio).

Expression of Mammalian Taurine Transporter: A complementary DNA (designated rB16a) containing the V -IU -72complete coding region of a putative transporter was cloned into the eukaryotic expression vector pEVJB.

(modified from pcEXV-3; as a 2.5 kb XbaI\SalI fragment using restriction enzyme sites within the vector. In addition to the coding region, 0.1 kb of untranslated sequence and 0.5 kb of 3 -untranslated sequence were included in the construct. Transient transfections of COS cells with the plasmid pEVJB-rB16a were carried out using DEAE-dextran with DMSO according to the method of Lopata et al. (44) with minor modifications. COS cells were grown (37°C.,5%CO 2 in high glucose Dulbecco's modified Eagle medium supplemented with 10% bovine calf serum, 100 U/ml penicillin G, and 100 gsg/ml streptomycin sulfate. Cells were routinely 15 used two days after transfection for transport studies.

Transport Studies of Masmalian Taurine Transporter: To measure transport, COS cells grown in 6-well (well diameter 35mm) or 24-well (well diameter 18mm) plates were washed 3X with HEPES-buffered saline (HBS, in mM: NaCl, 150; HEPES, 20; CaCl 2 1; glucose, 10; KC1, MgCl 2 1; pH 7.4) and allowed to equilibrate in a 37*C water bath. After 10 minutes the medium was removed and a solution containing 3 H]taurine (New England Nuclear, sp. activity 25.6 Ci/mmole) and required drugs in HBS was added (1.5 ml/35mm well; 0.5ml/18mm well). Nonspecific uptake was defined in parallel wells with ImM unlabeled taurine and was subtracted from total uptake (no competitor) to yield specific uptake; all data represent specific uptake. Plates were incubated at 37*C for 10 minutes unless indicated otherwise, then washed rapidly 3X with ice-cold HBS. Cells were solubilized with 0.05% sodium deoxycholate/0.1N NaOH), an aliquot was neutralized with 1N HC1, and rcdioactivity was determined by scintillation counting. Protein was quantified in an -73aliquot of the solubilized cells using a BIO-RAD protein assay kit, according to the manufacturer's directions.

PCR Tissue LocalizatiRU Studies of Nammalian Taurin.

Transporter: To identify tissues expressing mRNA f or the tauxine transporter, exact primers (25mers) were designed such that a 707 base pair fragment of rBi6a could be amplified from cONA and detected by Southern blot analysis. The sequaences of the sense and anti-sense primers were derived from amino acids 40 to 47 TCAGAGGGAGAAGTGGTCCAGCAAG) and 268 to 275 ATTTCATGCCTTCACCAGCACC'GG), respectively. Primers were also designed to amplify thae cDNA encoding cyclophilin a constituitively expressed gene, as control is. 1 ACGCTTCGACTTCCTCATGTCCTGT, s en se; 51 T'rAGAGTIGTCCACAGTCGGAGATG, antisense). To detect amplified sequences, an oligonucleotide probe was synthesized (corresponding to amino acids 249 to 271) which was specific for rBl6a. Poly A+ RNA (1 g, Clontech, Palo Alto, CA) from each of seven rat tissues 9*was converted to single-stranded cDNA by random priming using Superscript reverse transcriptase

(BRL,

Gaithersburg, MD). PCR reactions were carried out in a buffer containing 20mM Tris (pH 50 mM lC1, MgCl 2 0.001% gelatin, 2mM dNTP's, 1gM each primer, Taq polymerase, and either cDNA, RNA, water, or a control plasmid containing rBl6a for 30 cycles of 94'C./2 min., 68*C./2 min., 72 0C./3 min. PCR products were-separated by electrophoresis in 1.2% agarose gels, blotted to nylon membranes (Genescreen Plus; New England Nuclear, Boston, and hybridized at 40*C. overnight with specific 3 2

P_

labeled oligonucleotideS in a solution containing formamide, 10% dextran sulfate, 5X SSC, IX Denhardt's, and 100 pg/ml of sonicated salmon sperm DNA. Blots were washed at high-stringency (0.1X SSC, 500*C.) and exposed 1%0 a %P A -74to Kodak xAR film for 0.5 to 4 hours with one intensifying screen at -70 0 C.

northern Blot Analysis of mRill encoding mammalian Taurine Transporter: Samples of poly A+ RNA isolated from each of eight rat tissues (5 msg, Clontech; Palo Alto, CA) were separated in a 1.0% agarose: gel containing 3.3% formaldehyde and transferred to a nylon membrane (Genescreen Plus; New England Nuclear, Boston, M(A) by overnight capillary blotting in lOX SSC. Prior to hybridization, the Northern blot was incubated for 2 hours at 428C. in a solution containing 50% formamide, IM Hadl, 10% dextran sulfate, and 1% sodium dodecyl sulfate (SDS). The blot was hybridized overnight at 420C. with 3 2 P-labeled DNA probe (randomly-primed HindIII/Kpnl fragment of rBl6a representing amino acids 6-336) in the prehybridization solution containing 100 pg/ml sonicated salmon sperm DNA. The blot wai washed successively in 2X SSC2% DS,1X SC/% SS, nd .2XSSC/2% SDS at 650C.

9.*20 and exposed to Kodak XAR-5 f ilm with one itniyn screen at -700C. for 1-4 days. To confirm that equal *amounts of RNA were present in each lane, the same blot was rehybridized with a probe encoding cyclophilin (12).

.925 Ue of PCR to ldentify human oDJIl Libraries for screening: For hGAT-2, the sequences of the rat PCR primers were 51-GACCAACAAGATGGAGTT (sense) and TGYTACTCCTCGGATCAA (ant isense). PCR reactions were carried out in a buffer containing 20mM Tris (pH mM KCl, 1.5mM MgCl 2 0.002% gelatin, 2mM dNTP's, lg&M each primer, Taq polymerase, and an aliquot of a lambda phage library, water, or a control plasmid for 40 cycles of 94 0C. for 2 min., 50*C. for 2 min., and 72*C. for 3 min.

For hGAT-3, the sequences of the degenerate primers were 5' -TGGAATTCG(G/C)CAA(C/T)GTITGG(C/A)GITT(C/T)CCITA (sense) and (antisense). PCR reactions were carried out as described above for 40 cycles of 940C. for 2 min., 40*C. for 2 min., and 72 C. for 3 min. PCR products were separated by electrophoresis in 1.2% agarose gels, blotted to nylon membranes (Genescreen Plus; New England Nuclear, Boston, MA), and hybridized at 40"C. overnight with 32 P-labeled probes in a solution containing 25% formamide, dextran sulfate, 5X SSC, 1X Denhardt's, and 100 ug/ml of sonicated salmon sperm DNA. Blots were washed at low stringency (0.1X SSC, 400C.) and exposed to Kodak XAR film for up to three days with one intensifying screen at Isolation and Sequencing of Human Clones: Human cDNA libraries in the Lambda ZAP II vector (Stratagene, La Jolla, CA) that were identified as containing hGAT-2 or hGAT-3 were screened under either reduced stringency formamide, 40C. hybridization; 0.1X SSC, 400C. wash) or 20 high stringency (50% formamide, 40C. hybridization; 0.1X SSC, 50*C. wash). Hybridizing lambda phage were plaque purified and converted to phagemids by in vivo excision with fl helper phage. Nucleotide sequences of doublestranded cDNAs in pBluescript were analyzed by the Sanger 25 dideoxy nucleotide chain-termination method (59) using Sequenase Biochemical Corp., Cleveland, Ohio).

Fragments of genomic clones in the lambda FIX II vector were subcloned into pUC18 prior to double-stranded sequencing.

Preparation of Primary Brain Cell Cultures: Astrocytes, neurons and meningeal fibroblasts were prepared from the brains of E19 embryonic rats. Briefly, the brains were removed, dissected free of meninges, and trypsinized.

Cells were dissociated mechanically by passage through a a I^Jf r J- -76- Pasteur pipet, and resuspended in DMEM containing fetal bovine serum and antibiotics. The cells were added to tissue culture dishes that had been previously coated with 10M poly-D-lysine.

For astrocytes, the cells were plated at a density of approximately 3x10 6 cells per 100mm dish. The astrocytes were allowed to reach confluence, then passaged 1 or 2 times prior to harvesting. For neurons, a plating density of 15x10 6 cells per 100mm dish was employed; the medium was supplemented with insulin. Cytosine arabinoside (ara-C) was added to a final concentration of on day 2 or 3 to inhibit the proliferation of nonneuronal cells. The neurons were harvested 1 week after plating. To obtain meningeal fibroblasts the meninges were trypsinized, then mechanically dissociated as described above. The cells recovered from a single embryo were plated into a 100mm dish, grown to confluence, and passaged 1-2 times prior to harvesting.

Isolation of RMA from Cell Cultures: Plates were placed on ice and quickly rinsed twice with ice-cold phosphatebuffered saline (PBS). Cells were then dissolved in lysis solution (7M urea, 350mM NaCl, 2% sodium 25 dodecyl sulfate (SDS), IBM EDTA, and 10 mM Tris-HC1, pH 8.0) and transferred to a sterile tube. Lysates were homogenized (Virtis, lowest speed, 5 seconds) and then digested with proteinase K (0.lag/ml) at 37*C. for minutes. Samples were extracted twice with phenol/chloroform and once with chloroform before ethanol precipitation. Total RNA was collected by centrifugation, resuspended in diethylpyrocarbonate (DEPC)-treated water, and stored at -20*c. until use.

-77- Detection of Transporter inRZ~A Using PCR: To identify cell types expressing iDRNAs f or the GABA transporters GAT-l, GAT-2, and GAT-3, specif ic PCR primers were designed-such that =700 base pair fragments encoding transembrane domains 1 through 5 of each transporter could be amplified and detected by hybridization with 3 2

P-

labeled oligonucleotides. For rBl4b (GAT-2), the sequences of the sense and anti-sense oligonucleotides were derived from amino acids 36 to 43 GACCAACAAGATGGAGTTCGTACTG) and 247 to 254 TGTTACTCCTCGGATCAACAGGACC); for rB~b (GAT-3), the oligonucleotides were derived from amino acids 52 to CQ-LCTGTTGAGCGTAGGAGAG) and 271 to 279 GAACTGATGCCTTCCGAGGCACCC); and for GAT-1 the oligonucleotide sequences were derived from amino acids to 57 (5'-ACGCTCGACTCCTCATGTCCTGT) and 274 to 282 '-GAATCAGACAGCTTTCGGAAGTTGG). To detect amplified sequences, oligonucleotide prc~bes, were synthesized for GAT-1, GAT-2, and GAT-3 which corresponded to amino acids :>20 196 to 219, 161 to 183, and 207 to 229, respectively.

Each probe was shown to hybridize with its respective transporter cDNA and not with the other transporter cDNAs.

Total RNA isolated from cultured neurons, astrocytes, and fibroblasts was converted to singlestranded cDNA by random priming using Superscript reverse transcriptase (BRL, Gaithersburg, MD). PCR reactions were carried out in a buffer containing 20mM Tris (pH 50 mM KCl, 1.5mM MgCl 2 0.001% gelatin, 2mM dNTP's, 1MuM each primer, and Taq polymerase With either cDNA, RNA, water, or a control plasmid f or 30 cycles of 94 0C./2 min., 68BaC.12 min., 72*C./3 min. PCR products were separated by electrophoresis in 1.2t agarose gels, blotted to nylon membranes (Genescreen Plus; New England -V -78- Nuclear, Boston, MA), and hybridized at 40*C. overnight With 32 p-labeled oligonucleotide probes in a solution containing 50% formamide, 10%. dextran sulfate, 5X SSC, 3lX Denhardt's, and 100 uq/mi sonicated salmon sperm DNA.

Blots were washed successively in 2X SSC, 0.1% SDS at room temperature and 0.1X SSC, 0.1% SDS at 509C., and exposed to Kodak XAR f ilm f or 0. 5 to 4 hours with an intensifying screen at -700C.

In Situ Hybridization: Male Sprague-Dawley rats (Charles River) were decapitated and the brains rapidly frozen in isopentane. Sections were cut on a cryostat, thawmounted onto poly-L-lysine coated coverslips., and stored at -806C until use. Tissue was fixed in 4% paraf ormaldehyde, treated with 5mM dithiothreitol (DTT) acetylated (0.25% acetic anhydride in 0.214 triethanolamine), and dehydrated. Tissue was prehybridized (1 hour, 400C) in ar solution containing foruaamide, 4X SSC (0.6M4 NaCl/0.06N sodium citrate), lX Denhardt's solution polyvinylpyrrolidine, 0.2% Ficoll, 0.2% bovine serum albumin), 50mX DTT, 500Mg/mi salmon sperm DNA, 500Mig/mi yeast tRNA, 10% dextran sulfate, then hybridized overnight with 3 5 S-labeled antisense ol igonuc loot ides (45mers) in the same solution.

~.25 After washing and dehydration, sections were apposed to Kodak X-OMAT AR film for 4 days at -200C. To verify the specificity of the hybridization signal, parallel tissues were pretreated with 100 smg/ml RNase A (370, 30 minutes) prior thyrdzto.Two different olgnclcie designed to separate regions of the GABA transporters (loop region between transmembrane domains III and IV, 3 untranslated region) showed identical patterns or hybridization.

-79- 1. GABA Transporters

RESULT

Cloning of Novew ammalian GABA Transporter Sequences: We screened a rat brain cDNA library at low stringency with probes encoding the rat neuronal GABA transporter (GAT-1; in order to identify additional inhibitory amino acid transporter genes. Two clones were identified which hybridized at low but not at high stringency with the GABA transporter probes. DNA sequence analysis revealed that the clones encoded putative transporters which were structurally related to GAT-1. The first clone, rB14b, contained a 2.0 kb sequence with an open reading frame of 1806 base pairs which could encode a protein of 602 amino acids (Figure lA). The second clone, rB8b, contained a 2.1 kb sequence which had an open reading frame of 1881 base pairs encoding a protein of 627 amino'acids (Figure 1B). rB14b and rB8b exhibited 59% nucleotide identity throughout the coding region with 20 the neuronal rat GABA transporter (GAT-1) and nucleotide identity with each other. Comparison to sequences in Genbank and EMBL data bases demonstrated that both nucleotide sequences were novel and that the most homologous sequence was the rat GABA transporter 25 GAT-1 Subsequent comparisons which included recently cloned transporters revealed that the most closely related sequence is the canine betaine transporter (79) which exhibits 69% nucleotide identity with both rB14b and rB8b. The taurine transporter (66) and the glycine transporter (68) are also significantly related, exhibiting "64% and "56% nucleotide identity, respectively, to both rB14b and rB8b.

The amino acid sequence deduced from the nucleotide sequence of rB14b is shown in Figure 1D modeled after the proposed membrane topology of GAT-1 Residues identical to those in rB8b are shaded and represent 67% amino acid identity between the two clones. The translation products of both rB14b and rB8b are predicted to have relative molecular masses of =68,000 Daltons.

Hydropathy analyses indicate the presence of 12 hydrophobic domains in both proteins which may represent membrane spanning segments. For each transporter, several potential sites for Asn-linked glycosylation are found in the extracellular loop between the third and fourth transmembrane domains. Comparison and alignment of the deduced amino acid sequences of rB14b (GAT-2) and rB8b (GAT-3) with the neuronal GABA transporter (GAT-1) (Figure 2) revealed 52.5% and 52% amino acid identities, respectively. The betaine transporter (Figure which can also transport GABA (79) exhibited a significantly higher degree of homology-- 68% and 65% amino acid identities to rB14b and rB8b, respectively. Similarly, the transporter for taurine (66) an inhibitory amino 20 acid, is 61% homologous to both. In contrast, comparison of the new transporters with the rat glycine transporter (Figure 2 and Ref.(68)) or the human norepinephrine transporter (55) showed a lower degree of amino acid identity similar to that between the neuronal 25 GABA and norepinephrine transporters These data suggested that the new sequences might encode additional amino acid transporters expressed in the brain. To explore this possibility, the sequences were each placed C" in a mammalian expression vector, transfected into COS cells, and screened for transport of a variety of radioloabeled neurotransmitters and amino acids. These studies revealed (see below) that rB14b and rB8b encode novel GABA transporters with pharmacological properties distinct from the neuronal GABA transporter.

-81- Pharmacological CharacterixatiOn of Mammalian GADPI TZ'angloorters: COS cells transiently transfected with rB14b or rB8b (COS/rBl4b and COS/rBBB, respectively) accumulated more 3 H]GABA than non-transfected control cells; representative experiments are shown in Figure 3. During a 10 minute incubation (37*C) with a low concentration of 3 H]GABA, specific uptake was increased 52±11l-fold (meani±SDI, n=6) and 64±1l2-told over control for rBl4b and rB~b, respectively. In contrast, the uptake of 3 Hjglutanate, 3 H]glycine, I 3 H]5-HT, 3 H]dopamine, and 3 H~taurine was unaltered. Specific uptake represented greater than 95% of total uptake in transfected cells.

Uptake of 3 H]GABA was not observed following mock transfection or transfection with an irrelevant insert, indicating that the enhanced uptake was not the result of non-specif ic perturbation of the membrane. The transport C of 3 H]GABA by both COS/rBl4b and COS/rB~b was decreased when Na" was replaced by Li* (Table similar 20 results were obtained with COS cells expressing GAT-l (COS/GAT-1), which we re-cloned (see Materials and Methods). When C1- was replaced by acetate, 3 ijcGAB transport by COS/GAT-1 was nearly completely eliminated (Table consistent with previous results obtained with this transporter (21,29). In contrast, transport by COS/rB14b and COS/rB~b was decreased to 43 and 20% of control, respectively (Table The difference in 9*****sensitivity to removal of chloride exhibited by the three a. transporters was statistically significant (GAT-l vs.

COS/rBl4b,, p<0.001; GAT-2 vs. rBab, p<0.05; rBl4b vs.

rB~b, p<0.05).

To determine the affinity of GABA for the cloned transporters, COS/rB14b and COS/rB~b were incubated with various concentrations of 3 H]GABA and the specific -82accumulation of radioactivity was determined.

Accumulation of 3 H]GABA was dose-dependent and reached saturation at higher concentrations (Figure Nonlinear regression analysis of the data yielded the following values: K 8±3#M and 12±6MM, and V=X= 2.5±1.2 and 3.0±0.9 nmoles/mg protein for COS/rB14b and COS/rB8b, respectively (mean SEM, n-4 experiments). Taken together, these data indicate that both rB14b and rB8b encode saturable, high-affinity, sodium- and chloridedependent GABA transporters. Accordingly, we propose the terms GAT-2 and GAT-3 for the transporters encoded by rBl4b and rB8b, respectively, according to the nomenclature proposed by Guastella et al. (21).

To determine the pharmacological properties of the cloned GABA transporters, we examined the ability of various drugs to inhibit the accumulation of 3 H]GABA by GAT-2 and GAT-3; for comparison, we also examined the pharmacology of GAT-1. As shown in Table 2, the 20 pharmacological properties of GAT-2 and GAT-3 are similar to one another, but differ considerably from GAT-1. For example, P-alanine, a ligand reported to be selective for glial GABA transport is more potent at the new cloned transporters than at GAT-1. In contrast, ACHC, 25 guvacine, nipecotic acid, and hydroxynipecotic acid are more potent at GAT-1 than at GAT-2 and GAT-3.

Interestingly, the two newly cloned tranporters can be distinguished by L-DABA which displays high affinity for GAT-2 as well as GAT-1, but is less potent at GAT-3.

To further chararacterize the pharmacological properties of GAT-2 and GAT-3, we examined the ability of Tiagabine and CI-966 to inhibit the uptake of [3H]GABA; for comparison, we also examined these compounds at GAT- 1. These compounds are lipophilic derivatives of -83nipecotic acid and guvacine, respectively. As shown in Table 2, (R)-Tiagabine at a concentration of 00~mM completely inhibits uptake at GAT-1 but has no effect at GAT-2 and GAT-3. Tiagabine is reported to have high potency at both neuronal and glial GABA transporters and has demonstrated efficacy as an anticonvulsant in early clinical trials The finding that Tiagabine has very low affinity f or GAT-2 and GAT-3 underscores the potential of these transporters as unique drug targets.

Similar to Tiagabine, the GABA uptake blocker CI-966 (72) displays far greater potency at GAT-l than at GAT-2 and GAT-3 (Table 2) CI-966 was developed as an anticonvulsant but was withdrawn due to severe side effects observed in Phase 1 clinical trials (63).

is -84- Table 1. Ion DependenceS of E 3 HIMIB Uptake Uttakea Condition GAT-1 GAT-2 GAT-3 Na*-free 0.5±0.3 0.1±0.06 0.3±0.03 (3) Cl1-free 5±2 43.2±4.0 20.2±5.8 aCOS-.7 cells transfected with rB46a, rBl4b, or rB~b were incubated for 10 minutes (376C) With 5OnM 3 HJGABA in either HBS, or in MBS in which Li 4 was substituted for Na+ (Na+-free), or in which acetate was substituted for Cl- (C1--free). Non-specific uptake was determined with 1mM GABA. Data represent specific uptake, expressed as percent. or uptake in HBS (mean values in parentheses indicate number of experiments).

Table 2. pharmacological Specificity of 3 g]GAIA Uptake Inhibitora concentration GAT-1 GAT-2 GAT-3 ACHCb' P-alanine betaine

L-DABA

gjuvacine OH-nipecotic nipecotic

THPO

-Tiagabine CI-966 100M 100M 500;M 100M 10M 101M 1 OUM 100M 100M 100M 49±10 (3) 11±1 (8) 0(2) 49±8 (7) 41±3 (4) 34±5 (3) 51±5 (3) 10(2) 100±1 (3) 91±2 (3) 3±3 (3) 86±1 (8) 9(2) 43±8 (7) 13±1(3) 9±7 (3) 5±5(3) 9(2) 0±1(3) 9±6 (3) 0±0 (3) 70±1 (7) 1(2) 4±1(5) 8±5 (3) 5±2 (3) 12±6 (3) 4(2) 0±1 (3) 10±6 (3) or rB~b were 0* *0 0 *000 *0*0 ~0 00 0 0000 00 0 0 *0 *0 *0 *000 *0 00 *0 ~00 aC0S-7 cells transfected with rB46a, rBl4b, 20 incubated for 10 minutes (376C) with 5OnM 3 HJGABA and the indicated compounds. Non-specific uptake was determined with 1mM GABA. Data show percent displacement of specific 3 H)GABA uptake, mean tSEM (values in parentheses indicate number of experiments).

b L-DABA L-(2,4)diaminobutyric acid THPO 4,5,6,7-tetrahydroisoxazolo(4 ,5-c]pyridin-3-ol ACHC cis-3-aminocyclohexanecarboxylic acid C I -9 66 1 [2-b is 4- (trif luoromethyl) phenyljmethoxy) ethyl)]- 1 2 5 6tetrahydro-3-pyridinecarboxylic acid Tiagabine- ,4-bis (3 -methyl-2 -thienyl) but- 3-en-1yl]nipecotic acid -86- Tissue Localization Studies of Mammalian GABP.

TransDorters: To define the tissue distribution patterns of the novel GABA transporters, polymerase chain reaction (PCR) was used to detect each sequence in cDNA from seven different rat tissues. For comparison, the distribution of GAT-1 was also studied. Radiolabeled probes were used to detect individual PCR products by hybridization; each of the probes was highly specific for the transporter under study (data not shown). As shown in Figure 5B, GAT-1 was detectable in brain and retina but not liver, kidney, heart, spleen, or pancreas after 30 cycles of PCR. GAT-2 was present not only in brain and retina, but also in liver, kidney, and heart. Levels of GAT-2 mRNA were also detectable in spleen with overexposure of the autoradiogram (data not shown). Similar to GAT-1, the distribution of GAT-3 was limited to brain and retina.

Cyclophilin was amplified to a similar extent from all the tissues (data not shown), indicating that adequate 20 cDNA was present in each sample. Samples of poly A+ RNA not treated with reverse transcriptase and subjected to identical PCR conditions showed no hybridization with the transporter probes (not shown), indicating that the signals obtained with cDNA could not be accounted for by genomic DNA contamination. Thus, among the tissues examined, the distribution of GAT-3 is limited to the CNS, while GAT-2 has a wide peripheral distribution as well. These results are supported by Northern blot .o analyses of total RNA isolated from rat brain and liver; a single =2.4kb transcript hybridizing with GAT-2 is present in both liver and brain, while a =4.7kb transcript hybridizing with GAT-3 is detectable only in brain (Figure -87- Cellular Localization of GABA Transporter mRNAs: Prior to the recent cloning of GABA transporters (4,21), pharmacological evidence suggested that multiple transporters contributed to the high-affinity GABA uptake observed in rat brain Both neuronal and glial elements transport GABA, and preparations enriched in each cell type display differential sensitivities to inhibitors of GABA transport 53, 61), suggesting the presence of distinct neuronal and glial GABA transporters. The ability to design neuronal- or glial- selective GABA uptake inhibitors would be a major advantage in the design of effective therapeutic agents.

The GABA transporter cloned from rat brain, designated GAT-1 displays a pharmacological profile consistent with a "neuronal"-type carrier. Our cloning of two additional GABA transporters from rat brain, GAT-2 and GAT-3 (previously termed Ggabal and Ggaba2, respectively), confirms the principle of heterogeneity in high-affinity GABA transporters. Further, the sensitivity of GAT-2 and GAT-3 to inhibition by B-alanine distinguishes them from GAT-1, and raises the possibility that one or both represent "glial"-type transporters. The availability of three cloned high-affinity GABA transporters now provides the opportunity to begin to 25 examine the relationship between the pharmacologically defined neuronal and glial subtypes, and the transporters encoded by the cloned genes.

The presence of mRNAs representing each of the three GABA transporters was investigated in primary cultures of embryonic rat brain neurons, astrocytes, and meningeal fibroblasts. Polymerase chain reaction (PCR) was used to amplify each sequence for detection with specific probes.

As shown in Table 3, the messenger RNAs encoding each GABA transporter had a unique pattern of distribution.

-88- GAT-l uRNA was present in all threc culture types, whereas GAT-3 mRNA was restricted to neuronal cultures.

GAT-2 i.RNA was present in both astrocyte and fibroblast cultures, but not in neuronal cultures. Thus, GAT-2 and GAT-3, which exhibit extremely similar pharmacological profiles, display non-overlapping cellular distribution patterns. GAT-1, which displays a Oneuronal"-type pharmacology, is apparently not restricted to a neuronal distribution.

Table 3. Cellular Localization of GABII Transporters by

PCR.

S

S

S

S.

a S. S a *5*S

S

S.

S.

1.5 Neuronal Astrocyte Fibroblast Cultures Cultures Cultures- CAT-i GAT-2 GAT-3 Total RNA isolated from cultured embryonic rat neurons, 20 astrocytes, or fibroblasts was converted to cDNA and subjected to PCR for detection of mRNAs encoding GAT-l, GAT-2, and GAT-3 as described in Experimental Procedures.

Amplified products were separated on agarose gels, blotted to nylon membranes, and hybridized with 25 radiolabeled oligonucleotides specific for each transporter cDNA. The blot was exposed to film and the autoradiogram developed after several hours. A sign signifies that a positive signal was detected on the autoradiogram; a signifies that no signal was detectable. The same results were observed in two independent experiments.

-89- It is important to note that primary cultures, while enriched for a specific population of cells, may contain a small proportion of additional cell types. The sensitivity of PCR is sufficient to amplify a sequence contributed by a small number of cells; therefore, an unequivocal assignment of neuronal vs. glial localization would require combined in situ hybridization/ immunocytochemistry. However, the presence of GAT-3 mRNA only in neuronal cultures suggests that detection of GAT-1 mRNA in astrocyte cultures is not due to the presence of contaminating neurons, and that GAT-1 is probably present in astrocytes in addition to neurons.

The presence of GAT-1 and GAT-2 in fibroblast as well as astrocyte cultures may be explained by our recent finding that meningeal fibroblast cultures contain a large proportion of astrocytes as defined by staining with antibodies to glial fibrillary acidic protein (GFAP) (data. not shown); thus, GAT-1 and GAT-2 signals in meningeal fibroblasts probably result from contaminating 20 astrocytes.

These studies suggest that multiple high-affinity GABA transporter subtypes are present in different functional compartments, with at least two subtypes present in neurons (GAT-1 and GAT-3) and in glia (GAT-1 and GAT-2).

Further, they indicate that pharmacologic agents selective for each subtype may have different therapeutic applications.

Localization of GAT-1 and GAT-3 aRMA by in sit Bybridisation: In situ hybridization of GAT-1 and GAT-3 was carried out using antisense probes to the 3' untranslated region and the 3,4 extracellular loop of each clone. Hybridization wU YJ/ lei*of sense probes (control) to the same regions were also studied.

GAT-1 mRNA was observed in all rat brain areas examined (Table In the telencephalon, the highest levels were observed in the glomerular layer of the olfactory bulb, the orbital cortex, the lateral septal nucleus, the ventral pallidum, the globus pallidus, amygdaloid area, and layer 4 of the cerebral cortex. Moderate levels were observed in the islands of Calleja, the internal and external plexiform layers, and the piriform, retrospenial, and cingulate cortices, as well as in all regions of the hippocampal formation.

In the diencephalon, the highest levels were found in the paraventricular and reticular thalamic nuclei, and in the dorsal lateral geniculate. Lower levels were seen in the reuniens and rhomboid thalamic nuclei. In the hypothalamus, moderate levels were seen in the 20 suprachiasmatic and paraventricular nuclei, and in the medial preoptic area. Lower levels were seen in the supraoptic and anterior hypothalamic nuclei.

9. 99 In the midbrain, high levels were seen in the substantia 25 nigra (pars compacta and pars reticulata), median raphe, and the olivary pretectal nucleus. Lower levels were observed in the superior colliculus.

9 No label was seen in the pontine nuclei, nor in the cerebellar Purkinje cells.

GAT-3 mRNA was observed throughout the neuraxis (Table Within the telencephalon, the highest levels were detected in the medial septal nucleus, the nucleus of the diagonal band, and the ventral pallidum. Lower levels V ~Y1 -91were found in the amygdala and the shell of the nucleus accumbens. Low levels were observed in the hippocampus.

No labeling above background was observed in the neocortex.

In the thalamus, many nuclear groups were labeled. The areas with the highest labeling were the xiphoid, paraventricular, and rhomboid nuclei, and the zona incerta. Lower levels were observed in the following nuclei: reuniens, reticular, medial and lateral ventral posterior, and the medial geniculate. In the hypothalamus, moderate labeling was found in the lateral and ventromedial regions. Lower levels were observed in the arcuate nucleus and median eminence.

In the midbrain, the highest levels were observed in the dorsal tegmentum.

In the metencephalon, the highest levels were found in 20 the medial vestibular and deep cerebellar nuclei, and lower levels in the lateral superior olivary nucleus. No "label was observed in the cerebellar cortex.

A comparison of the localization of GAT-1 and GAT-3 mRNAs 25 indicates that both are widely distributed in the brain, and while GAT-1 is more abundant on a per cell basis, the two tend to have overlapping distributions. Notable exceptions are cortex and hippocampus which contain large numbers of neurons containing GAT-1 mRNA but few cells with GAT-3 mRNA. On the other hand, GAT-3 mRNA levels appear to be higher than GAT-1 in the superficial layers of the superior colliculus and in the deep cerebellar nuclei.

-92- Table 4. In situ localization of GAT-1 in the Rat CNS Areal a 1 icr Probe 191 Probe 179 AS 3trA 3. loo BREGMA 6. mitral cells glomerular layer ext.plexiform layer +4 +4 ant. alt nerve BREGMA 52m ext.plexiform layer int.plexiform layer ant.coma.intrabulb AOM,D,V orbital cortex a,v,l +j +j frontal. cortex BREGMA 1,60m tenia tecta lat.septal nucleus lat.septal interm. +4 ICjm 4caudate-putamen 1 AcbSh 4 AcbC

J+

vent.pallidum +4+ S. olf.tubercle- ICj +4 pirif or. ctx. cingulate ctx *indusium griseum BREGMA-1,40mm retrosplen.ctx 4+ 94cortex I. a @s@V reticular thal.rw. +j woo*: 40 globus pallidus caudate-putamen ant.dor thal.nu.

paraventr. thai. flu 44 supraoptic nu. k+ 4 suprachiasmatic flu. med.preoptic area +j -93- Table 4 (Continued) ArealLbln2 Probe 191 Probe 179 AS31TA W. lo perivent. hypoth.- nu. anter. hypoth. flu. paravent. hypoth. flu.+44 nlu. horizontal. limb diag. band ant. amygd. area +k BREGMA -1.80 reuniens thal.nu.4++ rhomboid thal.nu.4+4 ::.retrochiasmatic area BREGMA -4.52.

choroid plexus- PMCO AHiA Basolateral Auygdaloid flu. dorsal endopirifor. nlu. hippocampus (all levels) polymorphic dendate qyrus olivary pretectal flu. dorsal lateral genicul. flu. BREGMA -5.30.

substantia nigra pars reticulata pars compacta 4++ red nucleus parvocellular retrospenial cortex occipital cortex nucleus Darkschewitsch nucleus posterior commis., magnocel1lular BREGMA -7.64m superior colliculus central greydorsal grey median Raphe 4+ pontine nuclei-- Purkinje cells 1 abbreviations as in Paxinos, G. and Watson, C. (1986) The Rat Brain in Stereotactic coordinates, second edition. Academic Press.

-94- Table 4 (continued) 2 Antisense probes 191 and 179 were to 3' Untranslated region and to the 3,4 extracellular loop, respectively.

Control data using sense probes to the sane regions shoved no labeling.

Labeling scale: -,no labeling; very weak, weakc; moderate; s A heavy. Note that the scale is based on maximal labeling obtained with GAT-1 probes and should not be compared to results for GAT-3.

Table S. In situ Localization of GAT-3 in the Rat CNB &real La±lina telencephalon: cortex piriform ctx 4+ nu. accumbens core shell olf. tubercle 4+ med. septal fu. 44 nu. horiz.limb diag. band ventral pallidum ant. cortical amygdaloid fu. medial amygdaloid fu. +4 i e.

Diencephalon: paraventricular thalamic fu. ++I reticular thalamic nu. +j VPM +4 VPL zona incerta

++I

rhomboid thalamic fu. ++4 reuniens thalamic nu. xiphoid thalamic nu. medial geniculate nu. arcuate hypoth. nu. 4+ ventroedial hypoth.nu. lateral hypoth. nu.

median eminence 44 hippocampus fee: Mesencepha on: superior colliculus ++j central gray, dorsal 4+ central gray substantia nigra not examined interpeduncular nu.

caudal dorsal raph. cuneiform nu. lateral dorsal teguen. nu.

dorsal tegmental fu., pericentral WI~u 701 11q43 -96- Table S. (ContiziUed) Areal Label i=2 Metencephalon: medial vestibular nu...

lateral superior olive 4+ inferior olive not examined cerebral cortex deep cerebellar nuclei+4 1 abbreviations as in Paxinos, G. and Watson, C. (1986) The Rat Brain in Stereotactic Coordinates, second edition. Academic Press.

2 Data are pooled from antisense probes to the 3' *.untranslated region and to the 3,4 extracellular loop.

control data using sense probes to the same regions showed no labeling.

Labeling scale: no labeling; very weak, weak; moderate; heavy. Note that the scale is based on maximal labeling obtained with GAT-3 probes and should not be compared to results for GAT-l.

-97- The recent cloning of transporters for GABA (21), norepinephrine dopamine (33,65), serotonin (3,23), glycine and taurine (66) has helped to define the structural properties of this class of membrane proteins.

In contrast with neurotransmitter receptors, however, it has not been determined for neurotransmitter transporters whether multiple subtypes exist and/or play a role in synaptic transmission. Our identification of two cDNA clones from rat brain encoding novel GABA transporters (designated GAT-2 and GAT-3) provides the first molecular evidence for heterogeneity within the neurotransmitter transporter gene family, and raises the possibility that 15 multiple GABA transporters participate in the regulation of GABAergic neurotransmission.

Both proteins have 12 putative transmembrane domains and can be modeled with a similar topology to the neuronal 20 GABA transporter (GAT-1; including a large glycosylated extracellular loop between TMs 3 and 4.

Analysis of amino acid homologies of the various transporters reveals some unexpected relationships. For example, GAT-2 and GAT-3 exhibit greater amino acid 25 sequence identity to each other than to GAT-1 S" despite all three transporters displaying nearly identical affinities for GABA. Surprisingly, the sequence closest to GAT-2 and GAT-3 is the dog betaine transporter (79) which, in fact, is as homologous to GAT- 2 and GAT-3 as they are to one another. Significantly, the cloned betaine transporter has also been reported to transport GABA although the affinity of GABA at the betaine transporter is nearly 10-fold lower than at GAT-2 and GAT-3. Conversely, the betaine transporter displays at least 10-fold higher affinity for betaine than do GAT- W. %P I- -98- 2 and GAT-3 (see Table Thus, transporters with as little as 53% amino acid homology can display high affinity for the same substrate (eg. GAT-1 vs. GAT-2 and GAT-3), whereas transporters only slightly more divergent can demonstrate markedly different substrate specificities GAT-1 vs. glycine, 45% homology; Pharmacologically distinct GABA transporters have previously been identified in neuronal and glial cell cultures (15, 36 and 62). Thus, it was of interest to examine the sensitivity of GAT-2 and GAT-3 to a variety of inhibitors and to compare this to published values for endogenous transporters in primary cell cultures, as well S 15 as to GAT-1. It is noteworthy that GAT-2 and GAT-3 display greater sensitivity to the glial-selective drug -alanine than does the previously cloned GAT-1, suggesting similarity to the tranporter(s) characterized in glial cell cultures. However, a lack of identity with 20 the pharmacologically defined glial-type transporter is demonstrated by the finding that guvacine, nipecotic acid, Tiagabine, and hydroxynipecotic acid are much less potent inhibitors of GABA uptake at GAT-2 and GAT-3 than at the transporter present in glial cultures 15, 36, 25 62). Additionally, these compounds are more potent in neuronal cultures (and at the previously cloned GAT-1) than at GAT-2 and GAT-3, which also distinguishes the newly cloned transporters from the neuronal GABA transporter 15, 21, 36 and 62). Lastly, although GAT-2 and GAT-3 display similar sensitivity to a number of the inhibitors examined and show similar affinity for GABA itself, they can be distinguished by L-DABA, which displays higher potency at GAT-2 than at GAT-3.

Interestingly, the potency of L-DABA at GAT-2 is similar to that of GAT-1 (Table blurring the distinction -99between 'the newly cloned tranporters and the neuronaltype transporter. This finding may indicate that a spectrum of GABA transport activities underlie the neuronal and glial profiles observed in tissue preparations. Lastly, the three cloned GABA transporters can also be distinguished by their differential dependence on external chloride: GAT-1 is the most chloride dependent, GAT-2 the least, and GAT-3 is intermediate in its sensitivity. The finding that GABA transport by GAT-2 and GAT-3 is not completely eliminated in chloride-free medium suggests that their mechanism of transport is fundamentally different from that of GAT-1.

9* It is somewhat surprising that the pharmacological 15 profiles of GAT-2 and GAT-3 differ from those of previously characterized transporters in neuronal and glial cultures. One possible explanation is that the unique pharmacology of GAT-2 and GAT-3 reflects species differences, as the cloned transporters were obtained 20 from a rat cDNA library, while mouse tissue was employed in many of the earlier studies (15, 36 and 62). This hypothesis gains validity from the finding that certain GABA uptake blockers are potent anticonvulsants in rats, but are ineffective in mice although differences in 25 drug metabolism or distribution have not been ruled out.

A second possibility is that since neuronal and glial cultures are prepared from fatal or newborn animals, the discrepant results may reflect developmental changes in GABA transporters or peculiarities of glia and neurons when maintained in cell culture. Alternatively, the two newly cloned transporters may in fact represent members of a novel class of transporters that have not been previously identified, perhaps due to their low abundance in cultured cells. This would suggest that further GAB% transporters with pharmacological profiles consistent -100with those seen in neuronal and glial cultures remain to be cloned. Lastly, it should be pointed out that the pharmacological profiles of cloned transporters for serotonin dopamine (33,65), and norepinephrine as well as GAT-1 are similar to those observed in brain homogenates, thus arguing that the unique properties of GAT-2 and GAT-3 are not the result of the heterologous expression system.

Despite the generally similar pharmacology of GAT-2 and GAT-3, their patterns of distribution are distinct. All three high-affinity GABA transporters are present in brain and retina, while only GAT-2 was detected in peripheral tissues. This finding is consistent with 15 recent studies suggesting a role for GABA in liver (52), kidney (1,19) and other peripheral tissues (for review, ref. 14). Further distribution studies of GAT-2 and GAT- 3 by in situ localization of transporter mRNAs in conjunction with immunocytochemistry will help to define 20 the roles of these transporters in GABAergic transmission.

In conclusion, we now report the identification in mammalian brain of two novel high-affinity GABA 25 transporters with unique pharmacological properties.

These studies indicate previously unsuspected complexity in the regulation of GABAergic transmission, and provide the opportunity for the development of selective therapeutic agents to treat neurological and psychiatric disorders.

Cloning of Human High-Affinity GABA Transporters: The use of human gene products in the process of drug development offers significant advantages over those of other species, which may not exhibit the same -101pharmacologic profiles. To facilitate this human-target based approach to drug design in the area of inhibitory amino acid transporters, we used the nucleotide sequences of the rat GAT-2 and GAT-3 cDNAs to clone the human homologues of each gene.

To obtain a cDNA clone encoding the human GAT-2 GABA transporter (hGAT-2) we used PCR primers based on the rat GAT-2 sequence to detect the presence of hGAT-2 in human cDNA libraries. PCR was carried out at a reduced annealing temperature to allow mismatches between rat and human sequences (see Experimental Procedures); amplified hGAT-2 sequences were detected by hybridization at low stringency with radiolabeled (randomly primed) rat GAT-2 15 cDNA. A human heart cDNA library (Stratagene) was identified and screened at low stringency with the same probe, resulting in isolation of a partial cDNA clone (hHE7a) containing the C-terminal portion of the coding region of hGAT-2. Using human sequence derived from this 20 clone, a partial cDNA clone (hS3a) was isolated from a human striatum cDNA library (Stratagene) that provided additional sequence in the coding region. The hGAT-2 nucleotide sequence from these two clones and the deduced amino acid sequence based on translation of a long open 25 reading frame is shown in Figure 10A. The sequence includes 738 base pairs of coding region (246 amino acids) and 313 base pairs of 3' untranslated region.

Comparison with the rat GAT-2 amino acid sequence reveals identity over the region encoded by the clones, which includes predicted transmembrane domains 8-12 and the carboxy terminus of hGAT-2.

To obtain the nucleotide sequence of the human GAT-3 GABA transporter (hGAT-3), degenerate PCR primers were used to amplify transporter sequences from human cDNA libraries.

-102- Amplified hGAT-3 sequences were detected in the library by hybridization at low stringency with radiolabeled oligonucleotides representing the region of the rat GAT-3 cDNA that encodes a portion of the second extracellular loop. The human fetal brain library (Stratagene) identified by this approach was screened at highstringency with the same probes; positive plaques were purified by successive screening at low stringency. Two cDNA clones were isolated (hFB16a, hFB20a) which together comprise nearly the entire coding region of hGAT-3; the sequence of the remaining 7 base pairs was supplied by a genomic clone (hp28a) isolated from a human placental library. A vector comprising the complete coding sequence of hGAT-3 was constructed using appropriate oo* 15 fragments of these three clones, and is designated pcEXVhGAT-3. The complete nucleotide sequence and predicted e amino acid sequence of hGAT-3 are shown in Figure In addition to 1896 base pairs of coding region, the sequence includes 5' and 3' untranslated sequence (34 and 20 61 base pairs, respectively). Translation of a long open reading frame predicts a protein of 632 amino acids that is 95% identical to the rat GAT-3 and contains 12 putative transmembrane domains. Methods similar to *e methods used to clone the human homologues of the 25 mammalian GABA transporters can similarly be used to clone the human homologues of the mammalian taurine transporter.

The cloning and expression of the human GAT-2 and GAT-3 will allow comparison of pharmacological profiles with those of rat GABA transporters, and also provide a means for understanding and predicting the mechanism of action of GABA uptake inhibitors as human therapeutics.

Recently, several additional transporters have been cloned which exhibit significant sequence homology with -1 -103previously cloned neurotransmitter transporters. cDNA and genomic clones representing the mouse homologues of GAT-1 were recently reported In addition, a glycine transporter cDNA that is similar but not identical to that cloned by Smith et al. (68) was cloned from both rat (22) and mouse A high-affinity Lproline transporter was reported by Fremeau et al.(18), supporting a role for L-proline in excitatory neurotransmission. A rat cDNA identified as a choline transporter was reported by Mayser et al. A taurine transporter cDNA was recently cloned from dog kidney cells (74) which is 90% identical to the rat taurine transporter amino acid sequence reported by Smith et al. A cDNA encoding a mouse GABA transporter 15 was recently cloned by Lopez-Corcuera et al. the transporter encoded by this cDNA is 88% identical to the dog betaine transporter and may represent the mouse homologue of that gene. Finally, a P-alanine-sensitive GABA transporter from rat brain has been cloned (10) that 20 exhibits 100% amino acid identity with the rat GAT-3 sequence reported by Borden et al. Taurine Results and Discussion Clonina of NMmali an Taurine Transporter: We screened a rat brain cDNA library at low stringency with probes encoding the rat brain GABA transporter GAT-1 (21) in order to identify additional inhibitory amino acid transporter genes. Several clones were isolated which hybridized at low but not at high stringency with the GABA transporter probes. Characterization of the clones by DNA sequence analysis revealed that they represented a novel transporter sequence related to GAT- 1. None of the clones contained the complete coding region of the putative transporter, and thus the library -104was rescreened at high stringency using oligonucleotides designed from the new sequence. A 2.5 kb cDNA clone (designated rB16a) was isolated which contained an open reading frame of 1863 base pairs encoding a protein of 621 amino acids (Figure 1C). Comparison of this sequence with the rat GABA transporter cDNA revealed 58% nucleotide identity within the coding region. Comparison with sequences in Genbank and EMBL data bases demonstrated that the sequence was novel and that the most closely related sequence was the rat GABA transporter (21) followed by the human norepinephrine transporter Subsequent comparisons to recently cloned transporters indicate that the most homologous sequences are two novel GABA transporters designated GAT- 15 2 and GAT-3 and the betaine transporter which exhibit 62-64% nucleotide identity with rB16a.

The amino acid sequence deduced from the nucleotide sequence of rB16a is shown in Figure 1E with a membrane topology similar to that proposed for the rat GABA transporter (21) and other cloned neurotransmitter transporters 23, 33, 55 and 65). The translation product of rB16a is predicted to have a relative molecular mass of -70,000 Daltons. Hydropathy analysis 25 indicates the presence of 12 hydrophobic domains which may represent membrane spanning segments. Three potential sites for Asn-linked glycosylation are found in the extracellular loop between the third and fourth transmembrane domains. Alignment of the deduced amino acid sequence of rB16a with the rat GABA transporter (GAT-1; and the dog betaine transporter (79) revealed 50% and 58% amino acid identities, respectively (Figure Comparison of rB16a with the glycine transporter (Figure 6; and the human norepinephrine transporter (55) also showed significant amino acid -105homology similar to that between GAT-1 and the norepinephrine transporter As predicted from nucleotide comparisons, the strongest amino acid homology is with the GABA transporters GAT-2 and GAT-3 recently cloned from rat brain In contrast, the sodium/glucose cotransporter which shows a low degree of homology with cloned neurotransmitter transporters, displays only 21% amino acid identity with rB16a. These data suggested that the new sequence might encode an inhibitory amino acid transporter expressed in the brain. To explore this possibility, rB16a was placed in a mammalian expression vector, transfected into COS cells, and screened for transport of a variety of radiolabeled neurotransmitters and amino acids.

Pharmacoloiial Characterization of a-maliam Taurine Transporter: COS cells transiently transfected with rB16a (COS/rB16a) accumulated approximately 6-fold more [3H]taurine than 20 control, non-transfected cells (Figure Specific uptake represented greater than 95% of total uptake in transfected cells. In contrast, the uptake of 3 Hglutamate, 3 H]glycine, 3 H)5-HT, 3 H]dopamine, and [3H]GABA was unaltered. Uptake of 3 H]taurine was not observed following mock transfection, indicating that the enhanced uptake was not the result of non-specific perturbation of the membrane. The transport of 3 H]taurine by COS/rB16a was decreased >95% when Na* was replaced by Li*, or when C"1 was replace by acetate (Figure In the absence of sodium or chloride, taurine transport in COS/rB21a decreased to levels below that of non-transfected controls, demonstrating that endogenous taurine transporter activity present in COS cells is also dependent on these ions. A similar ion dependence has been observed for taurine transport in -106vivo as well as for the activity of other cloned neurotransmitter transporters such as those for GABA glycine and norepinephrine To determine the affinity of taurine for the cloned transporter, COS/rB16a was incubated with various concentrations of 3 H]taurine and the specific accumulation of radioactivity was determined.

Accumulation of 3 H]taurine was dose-dependent and reached saturation at higher concentrations (Figure 8).

Non-linear regression analysis of the data yielded the following values: K. 43±6 MM, and VMax 0.96±0.27 nmoles/mg protein (mean SEM, n-4 experiments). The affinity of the cloned transporter for taurine is similar 15 to that of high-affinity taurine transporters in both the central nervous system (42,80) and peripheral tissues (37) which exhibit KM values from 10 to 60. MM. Taken together, these data indicate that rB16a encodes a saturable, high-affinity, sodium- and chloride-dependent taurine transporter.

To determine the pharmacological specificity of the cloned transporter, various agents were examined for their ability to inhibit the transport of 3 H]taurine by 25 COS/rB16a (Table As the endogenous taurine transporter in COS cells accounted for, on average, 16% of the total transport activity observed in transfected cells, we were concerned that this could influence results. Accordingly, we also examined the sensitivity of the endogenous taurine transporter present in nontransfected cells. As shown in Table 6, the pharmacologic properties of the cloned taurine transporter closely matched those of the endogenous transporter and thus did not lead to erroneous results.

-107- The most potent inhibitors were taurine and hypotaurine, each of which inhibited specific [3H]taurine uptake approximately 30-40% at 10pM, 90% at 100pM, and 100% at ImM. P-alanine was slightly less potent, inhibiting specific uptake 15%, 51%, and 96% at 10M, 100M, and ImM, respectively; the high potency of 0-alanine as an inhibitor of taurine uptake is consistent with the finding that COS/rBl6a showed a 6-fold increase in the specific uptake of 3 H]P-alanine (data not shown), essentially identical to the fold-increase observed with [3H]taurine. The taurine analogue GES was also quite potent, inhibiting specific uptake of 3 H]taurine 11%, 45% and 92% at 10M, 100pM and ImM, respectively.

APSA

and GABA both inhibited uptake approximately 10% and at 100aM and ImM, respectively. The observations that GABA is a poor inhibitor of taurine uptake, and that transfection with rB16a did not result in enhanced uptake of 3 H]GABA (see above), are consistent with the previous report (38) that GABA is a weak non-competitive inhibitor of taurine uptake. Less than 10% inhibition of H]taurine uptake was observed for the following compounds (each tested at ImM): the structural analogues AEPA and MEA as well as the sulfur-containing amino acids cysteine and methionine (Table and (data not shown) 25 norepinephrine, dopamine, glutamate, glycine, serine, betaine, L-methionine, and a-methylaminoisobutyric acid (a substrate for amino acid transporter designated system A; Taken together, these results indicate that the taurine transporter encoded by rB16a is similar to the endogenous taurine transporter in COS cells (Table as well as the endogenous taurine transporter(s) present in neural tissue (25),(see also ref. 27 and references therein).

-108- It is of interest that sensitivity to P-alanine is shared by the two high-affinity GABA transporters recently cloned from rat brain (GAT-2 and GAT-3 which are even more closely related to the taurine transporter (62% amino acid identity) than to the neuronal-type GABA transporter GAT-1 p-alanine has been shown to activate an inward chloride current in spinal neurons (9,49) and is released in a calcium-dependent manner from several brain areas (31,58), suggesting a role as an inhibitory neurotransmitter in the CNS. The similar sensitivities of the newly cloned GABA transporters (4) and the taurine transporter to p-alanine, combined with their sequence homologies, suggest that they represent a ;subfamily of inhibitory neurotransmitter transporters.

15 Despite these similarities, these transporters unexpectedly exhibit widely divergent affinities for GABA: GAT-2 and GAT-3 show the highest affinity while the affinity of the taurine transporter is extremely low (-ImM, Table Interestingly, the dog betaine transporter which displays a similar degree of homology to the members of this subfamily (ca. exhibits an intermediate affinity for GABA (-100M). The finding that four structurally related transporters display overlapping substrate specificities for the neuroactive amino acids GABA and P-alanine suggests that multiple transporters may regulate the synaptic levels of these substances. This crossreactivity underscores the importance of understanding the action of therapeutic agents at both GABA and taurine transporters.

Table 6. Phar Uptake Inhibitora,

AEPA

AMSA

APSA

-109- .acooogical SP 'ecificity of 3 31taurin.

*r p.

p p. a a a *5 P-alanine

CSA

cystein.

GABA

GES

Concentration 1mM laM 100CM luM 10Cm 100M laM laM laM 1AM 1ooM laM loM 100CMM 1oom 100Cm lm laM laM Inhibition cofntrol rBl 0±0 3±3 1±1 7±3 (4) 7±3 8±4 (4) 45±3 36±4 9±2 15±6(6) 63±3 51±4(10) 97±1 96±1 (8) 2±1 .7±5 (3) 4±3 2±2 (3) 1±1 9±6 (4) 9±4 10±4 49±2 44±6(8) 6t3 11±4 (4) 47±3 45±5 89±1 92±1 (6) 41±3 26±7 (7) 91±1 84±3 (4) 99±1 100±1 (4) 1±0 3±3 (4) 1±1 1±1 (3) hypotaurine

MEA

methionine -110- -&able 6 (continued) taurine 10AM 38±5 29±8 1001AM 89±2 83±2 1BM134 100 b a Non-transfected COS-7 cells (control),* or COS-7 cells transf ected with rBl6a were incubated f or 10 minutes (370C) with 50nM34 3 Hjtaurine and the indicated compounds.

Data show percent displacement of specific 3 Hitaurine uptake (mean±SEK; values in parentheses indicate number of experiments).

bNon-specific uptake defined with 1mM taurine.

Abbreviations: AEPA, 2-aminoethylphospholic acid; AMSA, aminomethanesulfonic acid; .APSA, 3-amino-lpropanesulfonic acid; CSA, cysteinesulfinic acid; GABA, gamma -aminobutyric acid; GES, guanidinoethaflesulfonic acid; KEA, 2-mercaptoethylamife.

-111- Tissue .LocaliatioR Studies of Mammalian Taurine Transporter: To define the tissue distribution patterns of the taurine transporter, polymerase chain reaction (PCR) was used to detect the rB16a sequence in cDNA representing mRNA from seven different rat tissues. As a control, the distribution of the constitutively expressed protein cyclophilin was also examined. Radiolabeled oligonucleotides specific for rB16a were used to detect PCR products by hybridization. As shown in Figure 9A, the taurine transporter was detectable in all tissues examined, including brain, retina, liver, kidney, heart, spleen, and pancreas, after 30 cycles of PCR.

Cyclophilin was amplified to a similar extent from all 15 the tissues (data not shown), demonstrating that adequate cDNA was present in each sample.

To evaluate both the abundance and the size of the mRNA encoding the taurine transporter, Northern blot analysis was carried out on poly A+ RNA isolated from the same rat tissues used for PCR analysis, with the addition of lung.

As shown in Figure 98, a single -6.2 kb transcript which hybridized with the taurine transporter cDNA probe was detected in brain, kidney, heart, spleen, and lung after 25 an overnight exposure of the autoradiogram. After a 3day exposure, bands of the same size were also visible in liver and pancreas (data not shown). Rehybridization of the blot with the cDNA encoding cyclophilin (12) confirmed that roughly equal amounts of RNA were present in each sample except that of retina, which was significantly degraded (data not shown). Thus, taurine transporter mRNA levels were highest in brain and lung, intermediate in kidney, heart, and spleen, and lowest in liver and pancreas. The abundance and pattern of distribution of the taurine transporter mRNA by Northern 112blot are consistent with data obtained using PCR (Figure further, the same size transcript is present in all tissues evaluated. These findings suggest that a single taurine transporter functions in both the brain and peripheral tissues; however, we can not exclude the existence of additional taurine transporters.

Taurine is abundant in the central nervous system and is involved in a variety of neural activities. Unlike classical neurotransmitters, the effects of taurine are mediated both intra- and extracellularly. A major regulator of taurine levels, both within cells and in the synaptic cleft, is the transport of taurine across the plasma membrane. Our cloning of a high-affinity taurine transporter represents a critical step in defining the role of taurine in both neural and non-neural tissues, and in the development of therapeutic agents that alter taurine and GABA neurotransmission. In addition, the identification of a new member of the set of inhibitory amino acid transporters will aid in elucidating the molecular structure-function relationships within the transporter family.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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-123- SEQUENCE

LISTING

GENERAL

INFORMATION:

APPLICANT: Smith, E. Kelli Borden, A. Laurence Hartig, R. Paul Weinshank, L. Richard (ii) TITLE OF INVENTION: DNA ENCODING TAURINE AND GABA TRANSPORTERS

AAD

USES THEREOF (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Cooper Dunham STREET: 30 Rockefeller Plaza CITY: New York STATE: New York COUNTRY: USA ZIP: 10112 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release #1.24 (vi) CURRENT APPLICATION

DATA:

APPLICATION

NUMBER:

FILING DATE:

CLASSIFICATION:

(vi.i) ATTORNEY/AGENT INFORMATION: NAME: White, John REGISTRATION NUMBER: 28,678 REFERENCE/DOCKET NUMBER: 40558A (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 212-977-9550 TELEFAX: 212-664-0525 TELEX: 422523 COOP UI INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2028 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

N

(iv) ANTI-SENSE:

N

FRAGMENT TYPE: N-terminal (vii) IMMEDIATE SOURCE: LIBRARY: rat brain -124- CLONE: rBl4b (ix) FEATURE: NAME/KEY: CDS LOCATION: 126. .1932 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGCAGCGAAC ACAAGCGCAT CCGOTAGAAC GGAAAOAACA GGAATTGCAG AGTGACTTCA AGTCTCCATA CGATTTACTA CCCGGGTGAC GGCAGTGACT CGACAGAGTA GCGGCTGCAG GTGGG ATG GAT AAC AGG GTC TOG OGA AG ACC AGT AAT GGA GAG ACA Met Asp Aun Arg Val Ser Gly Thr Thr Ser Asn Gly Giu Thr 1 5 120 167 A.AG CCA GTG TGT CCA GTC ATG GAG AAG 0 *0 0 0 Oe 0 0009 0* 0* 0 0* 0.0.

Lys

GAA

Giu

GGA

Gly

TAC

Tyr

TTT

Phe

TAC

Tyr

TTC

Ph.

GTC

Val1

AGC

Sex

ACA

Thr Pro

CGG

Arg

GAG

Glu

AAG

LysB

ACC

Thr s0

ACC

Thr

GAG

Glu

TAC

Tyr

TTC

Ph.

GAA

Glu 160 Val

GAG

Glu

ATC

Ile

A.AC

Asn 65

TGT

Cy.

AAC

Asn

GGC

Gly

TAC

Tyr

ACC

Thr 145

AAC

Asn Cys

CAA

Gin

ATT

Ile 50

GGG

Gly

GCC

Gly

CAG

Gin

ATC

Ile

ATC

Ile 130

ACT

Thr

TGT

cys Pro Val 20 TGO ACC Trp Thr GCC TTA Gly Lou GGA GOT Gly Gly ATT CC? Ile Pro GOA GOC Gly Gly 100 GGC TAT Gly Tyr 115 OTT GTC Val Val GAC CTC Asp Lou GTG GAG Val Olu met

AAC

Asn

GOC

Gly

GCC

Ala

OTC

Val 85

ATC

Ile 0CC Ala

CTG

Lou

CCC

Pro

TTC

Phe 165 Glu

AAG

Lys

AAC

Aen

TTC

Ph.

70

TTC

Ph.

AcA Thr

TCA

Ser

CC

Ala

TGG

Trp 150

CAG

Gin Lys

ATG

Met

OTC

Val 55

TTT

Ph.

TTC

Ph., 0CC Ala

CAG

Gin

TGO

Trp 135

GOT

Gly

A

Lys

GTG

Vol

GAG

Giu 40

TGO

Trp

AT?

Ile

CTO

Lou

TG

Trp

ATG

Hot 120

GCC

Ala

AGC

Ser

ACC

Thr Giu 25

TTC

Ph.

AGG

Arg

CCC

Pro

GAG

Glu

AGG

Arg 105

ATC

Ile

CTC

Lou

TGC

Cy.

MAC

Asn Glu

GTA

Val

TTT

Phe

TAC

Tyr

ACA

AAA

Lys

GTC

Val

TTC

Ph.

AGC

Ser

ART

Asn 170 Asp

CTG

Lou

CCC

Pro

CTC

Lou 0CC Ala

ATC

Ile

AGC

Ser

TAC

Tyr

CAC

His 155

TCC

Ser Gly

TCA

Ser

TAT

Tyr

ATC

Ile

CT?

Lou

TOT

Gym

CT?

Lou

CTC

Lou 140

GAG

Glu

CTG

Lou Thr

GTG

Vol

CTC

Lou

TTC

Phe

C

Gly

CCC

Pro

CTC

Lou 125

TTC

Ph.

TGG

Trp

MAT

Aun GAG CMA GAG GOT ACC TTG Lou

GCG

Ala

TOC

Gys

CTA

Lou

CAG

Gin

ATC

Ile 110

MAT

Amn

AGC

Ser

MAT

Amn

GTG

Val 551 599 ACT TCT GAG MAT GCC ACA TCC CC? GTC ATC GAG TTC TOG GAG AGO CGA Thr 175 hSer Glu Asn Ala Thr Ser Pro Vol Ile Glu Ph. Trp Glu Arg 175O 18018 Arg 190 -125- *9 a a.

a a a a a a *0W a a a. a a a a a a a.

GTC

Val1

GAG

Giu

ATC

Ile

ACT

Thr

CTG

Lou 255

ACA

Thr

TTC

Phe

TAC

Tyr

ATT

Ile

ATC

Ile 335

GCT

Ala

GTG

Val

GTG

Val

GTG

Val1

CTG

Leu

CTG

Lou

TGG

Trp

TTC

Phe 240

CCT

Pro

CGT

Ar;

TTC

Phe

AAC

crc Leu 320

CTG

Leu

GAA

Glu

ATG

Met

OTT

Val1

ACA

Thr 400

AAG

Lys

GTC

Val

AAA'

Lys 22S

CCT

Pro

GGA

Gly

CTO

Leu

TCC

Se r

AAG

Lys 305

AAC

Ann

GGC

G-'y

TCA

Ser

TTA

Lou

CTC

Lou 385

GCG

Ala

ATC

Ile

CTG

Lou 210 GG0 Gly

TAC

Tyr

GCA

Ala

TGG

Trp

TI?

Phe 290

TAC

Tyr

AGC

Ser

TTC

Ph.

GGC

Gly

CC?

Pro 370

CTG

Lou

CTG

Lou ?CA4 Ser 195

?GC

Cys

GTC

Val

CTC

Lou

CC

Ala4 GAT4 Asp 275

GCC

Ala

CAC

His

AC

Scr

ATG

Met

CCT

Pro 3S5

TTC

Phe

GGA

Gly

GO

Val1

GAT

Ap Lou vA Lys k.TG '40t

CAG

3iri 260 ccc Pro

ATC

Ile

AAC

Asri

ACC

Thr

TC?

Ser 340

C

Gly

TCG

Ser

CTA

Lou

GAC

Asp

GGC

Giy CG4 Lou

TCC

Ser

CTC(

Lou 245

GGA

Gly CAG4 Gin

TOC

Cys

AAC

Asn

AGC

5cr 325

CAG

Gin

CG

LOU

CCT

Pro

GAC

Asp

ATG

met 405 ATC4 Ile

CTT(

Lou

WCA

rhr 230

ATT

Ile

GTG

Val CTG4 Lou4

TOC

Cy.

310

TC

Ph.

GAG

Glu

GCA

Ala

TTG

LOU

AGC

Ser 390

TAT

Tyr

CAG

Gln kia 215 1ly

TC

I

:AG

Trp

~GG

fly 295

TAC

Tyr

GTG

Val

CAG

Gin

TTC

Ph&

TG

Trp 375

CAG

Gin

CCC

Pro

CAC

His 200

TOG

Trp

AAG

Lys

CTG

Lou

TI?

Phe

ATG

Met 280

TOC

Cys

AGG

Arg Ala Oly

ATC

Ile 360 0CC Ala Ph.

*CGG

CTG

Lou

ATC

Ile

GTG

Val

TTG

Lou

TAC

Tyr 265

GAT

Asp

CTC

Lou

CAC

Asp 000 Gly

OTA

Val 345

CC

Ala

TGC

Cys

GTO

Val

OTG

CG

Gly

AC

Ii.

OTO

Val1

ATC

Ile 250

CTG

Lou

GCG

Ala

ACG

Thr

TOC

CyC

TTT

Ph.

330

CCC

Pro

TAC

Tyr

TG?

Cym

TGT

Cys

TTC

TCC

Ser

TGC

Cys

TAC

Tyr 235

CCA

Arg

TAC

Tyr

C

O ly

CC

Ala

GTC

Val 315 0CC Ala

ATA

Ile

CC?

Pro

TTC

Phe

GTA

Val 395

CGT

C -G Lou

TAT

Tyr 220

TTC

Ph.

GGA

Gly

CCC

Pro

ACC

Thr

CTG

Lou 300 0CC Ala

ATC

Ile

TCT

Ser

CGA

Arg

TTC

Pho 380

GAA

O iu

AAG

CGC

Arg 205

TTC

Ph.

ACA

Thr

GTA

Val

AAC

Acm

CAG

Gin 285

C

Gly

C??

Lou

TTC

Phe

GAG

Giu

GCT

Ala 365

TIC

Ph.

AGC

5cr

AAG

TG

Trp

TGC

Cy s

GC?

Ala

ACA

Thr

ATC

Ile 270

ATC

Ile

AGC

Scr

TOC

CyC

TCC

Ser

OTI

Val 350

CTG

Val

ATG

Met

CTC

Lou

AAC

743 791 839 887 935 983 1031 1079 1127 1175 1223 1271 1319 1367 1415 Ar; Val Phe Ar; Lys Lys Acm 410

CG

Arg 415 AGO GAG ATI CTC Arg Giu Ile Lou

ATC

Ile 420 CC ATC GTO ?C? Lou Ile Val Ser

GTC

Val1 425 GC TC? TIC TIC Val Ser Phe Ph.

ATC

Ile 430 -126- GGG CTC ATT ATG CTC ACA GAG GGC GGC ATG TAC GTG TTC CALO CTC TTC Gly Lou Ile Met Lou Thr Glu Gly Gly Met Tyr Val Ph* Gln Lou Phe 43S

S.

Sn.

S.

S

S *5

S.

S

S.

S S

S.

S

GAC

Asp

GAG

Glu

AAC

Aun

TGT

Cys 495

T'CC

Ser

CCA

Pro

CTC

Val.

CCA

Pro

CCC

Pro 575

TCC

TAC

Tyr

TCC

Sec

AT?

Ile 480

TG

Trp

CTG

Lou

TG

Trp

TGC

Cys

CTC

Lou 560

CAG

Gln

CTC

TAT

Tyr

CTC

Lou 465

GAA

Glu

CTC

Lou

ATC

Ile

TG

Trp

AT?

II.

545

AGA

Arg

AAG

Lysa

CTC

C

Ala 450

TOT

Cys

CA?

Asp

TTT

Ph.

AAA

Lys

GG

Gly 530

CCT

Pro

GAG

C lu

AGC

Ser

AGO

GCC ACT Ala Ser GTG OCT Val Ala ATG AT? met Ile TC ACG Ph* Thr 500 TAC ACG Tyr Thr 515 GAT GCC Asp Ala GCC TG Ala Trp AGA CT? Arg Lou CAA CCA Gin Pro 580 CTC ACA

GCC

Gly

TOG

Trp,

CG

dly 485

CCA

Pro

CCA

Pro

CTG

LOU

AGC

Ser

CGC

Arg 565

GAG

Glu

GAA

TOT

Cys '55

TAC

Tyr

AAG

Lys

GTO

Val

ACC

Thr

TOG

Trp S35

TAC

Tyr

CTC

Lou

ACT

Thr

GAG

440

CT?

Lou

GGA

Gly cco Pro

TGC

Cys

TAC

Tyr 520

CTC

Lou

AAG

Lys

GTG

Val

TCT

Ser

TCT

Ser 600 CTC TTT Lou Phe CCC AC Ala Ser TOG CC? Try, Pro 490 CTG OCA Lou Ala

SOS

AAC AAG Asn Lys CTA C Lou Ala CTC AGO Lou Arg TGC CCG Cy. Pro 570 CCA oco Pro Ala 585 MAC TOC Asn Cys

GTG

Val

COC

Arg 475

CTT

Lou

ACC

Thr

AAG

Lys

CTG

Lou

ACT

Thr 555

OCT

Ala

ACA

GCC

Ala 460

TTC

Ph@

ATC

I10

TTC

Ph*

TAC

Tyr

TCC

Ser 540

CTC

Lou

GMA

Glu

CO

445

ATC

Ile

TAT

Tyr

AM

Lys

CTO

Lou

ACA

Thr 525

TCC

Ser

MAG

Lys

GAC

Asp

ATO

TT?

Phe

GAC

Asp

TAC

Tyr

TTC

Ph*

TAT

Tyr net

GC

Gly

CT?

Lou 1463 1511 1559 1607 16S5 1703 1751 1799 1847 1895 1942 2002 2028 Thr Pro Not Th: 590 T AGGGAOGAGG Ser Lou Lou Ac; Lou Thr Glu Lou Glu 595 CCrTTACAC ACCTGCGAGT CTGTCTGTGG GGACAGCTAC A CCCCTCCGTG CTGOGGCAGA GAGACA INFORMATION FOR SEQ ID 30:2: SEQUENCE CHARACTERISTICS: LENGTH: 602 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: ~ACACAGAG GGCAGMACCA -127- Net Ap Ann Arg 1 Sec Gly Thr Thr Ser hAn Gly Glu Thr 10 Lys Pro

S

a.

S

S

*5

S.

*5*e a.

S. a

S

a a. a a Sc Val Giu hAn Cys Ann Gly Tyr Thr 145 Asn Giu Lys Val Lys 225 Pro Gly Lou Ser Lys 305 Ann cys Gin Ile so Gly Gly Gin Ile Ile 130 Thr Cyrn An Ile Lou 210 Gly Tyr Ala Trp Ph.

290 Tyr Ser Pro Trp Gly Gly Ii.

Gly Giy 115 Val Asp Val Ala 5cr 195 Cya Val1 Lou Ala Asp 275 Ala His Ser Val Thr Lou Gly Pro Gly 100 Tyr Vali Lou Giu Thr 180 Asp Lou Lys Met Gin4 260 Pro4 Ile Ann Thr Not Ann Gly Ala Val Ile Ala Lou Pro Phe 165 5cr Gly Lou 5cr Lou 245

G

1 y Gin cye Asn Sor 325 Giu Lys Ann Ph.

70 Ph.

Thr Ser Ala Trp, 150 Gin Pro Lou Thr 230 Val1 Ile Val1 Lou 310 Ph* ILys not Val 55 Ph.

Pho Ala Gin Trp 135 Gly Lys Val Gin Ala 215 Gly .Val1 Gin Trp Gly 295 Tyr Val Val Giu 40 Trp Ii.

Lou Trp Met 120 Ala Sicr Thr Ile His 200 Trp Lys Lou Phe Met 280 Cys Arg Ala diii 25 Ph.

Arg Pro Giu Arg 105 Ii.

Lou cys ^on Glu 185 Lou Ii.

Val Lou Tyr 265 Ap Lou Asp Gly Giu Val Ph.

Tyr Thr 90 Lys Val1 Ph& Ser Ann 170 Phe Gly 110 Val Ile 250 Lou Ala Thr Cys Ph.

330 Lou PCo Lou 75 Ala Ile Ser Tyr His 155 Sec Trp Ser Cys Tyr 235 hrg Tyr Gly Ala Val1 325 Ala 5cr Tyr Ilie Lou eye Lou Lou 140 Glu Lou Glu Lou Tyr 220 Phe Gly Pro Thr Lou 300 Ala Ile Val Lou Ph., dly Pro Lou 125 Ph.

Trp Ann Arg hrq 205 Ph., Thr Val Ann Gin 285 Giy Lou Ph.

Ala eys Lou Gin 110 110 Ann Ser Ann Val hrg 190 Trp eye Ala Thr 110 270 Ile 5cr eye Soc Gly Tyr Ph.

Tyr Ph* Val Soc Thr 175 Val Giu Ile Lou 255 Thr Ph* Tyr Ile Ile 33S clu Lys Thr s0 Thr Glu Tyr Phe clii 160 Ser Lou Lou Trp Ph.

240 Pro Arg Ph.

Ann Lou 320

LOU

Ap Gly Thc Lou Giu Arg -128- Gly Ph* Not Ser Gin Giu Gin Gly Val Pro Ile Ser Glu Val Ala Giu 340 345 350 Scr Gly Pro Gly Lou Ala Ph. xIe Ala Tyr Pro Arg Ala Va1 Va1 Met 355 360 365 Lou Pro Ph* Ser Pro Lou Trp Ala CYs CYs Ph. Ph. Ph. Hot Val Val.

370 375 380 Lou Lou Gly Lou Asp 8cr Gin Ph* Val Cys Val Giu Ser Lou Val Thr 385 390 395 400 Ala Lou Va1 Asp Met Tyr Pro Arg Va1 Ph. Ar; Lys Lys hen Ar; Ar; 405 410 415 Glu Ile Lou Ile Lou Ile Val ser Val Va1 Ser Ph. Ph. le Gly Lou 420 425 430 Ile Met Lou Thr Giu Gly Gly Met Tyr Va1 Ph. Gin Lou Ph* Asp Tyr 435 440 445 *Tyr Ala Ala Ser Gly Met Cys Lou Lou Ph* Val Ala Ile Ph. Glu Ser 450 455 460 Lou Cys Val Ala Trp Val Tyr Gly Ala Ser Ar; Ph. Tyr Asp Ann Ile 465 470 475 480 Glu Asp Met ile Gly Tyr Lys Pro Trp Pro Lou Ile Lys Tyr Cys Trp .485 490 .495 Leu Phe Phe Thr Pro Aia.V.1 Cys Lou Aia Thr Ph. Lou Ph. Ser Lou Sle Lys Tyr Thr Pro Lou Thr Tyr Ann Lys Lys Tyr Thr Tyr Pro Trp 515 520 525 Trp Cly Asp Ala Lou Gly Try, Lou Lou Ala Lou Ser Ser net Val Cys 530 535 Ile Pro Ala Trp Ser Ile Tyr Lys Lou Ar; Thr Lou Lys Gly Pro Lou 545 550 555 Ar; Giu Ar; Lou Ar; Gin Loeu Val Cys Pro Ala Glu Asp Lou Pro Gin c 65 570 575 Lys Scr Gin Pro Glu Lou Thr Ser Pro Ala Thr Pro met Thr Ser Lou 580 585 590 Lou Ar; Lou Thr Glu Lou Glu Ser Ann Cys 595 600 INFORMATION FOR SEQ ID NO:3: SEQUENCE

CHARACTERISTICS:

LENGTH: 1938 base pairs TYPE: nucleic acid STRANDEDWESSI both TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL:

N

-129- (iv) ANTI-SENSE:

N

FRAGMENT TYPE: N-tergainl (vii) IMMEDIATE SOURCE: LIBRARY: rat brain CLONE: rBSb (ix) FEATURE: NA.HZ/TX CDS LOCATION: 16..1897 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGCGGCAGGG CGGCC ATG ACT GCG GAG CAA CC CTG CCC CTrC CCC ARC C Met Thr Ala Clu Gin Ala Lou Pro Lou dly Aen Gly 1 S

C

C

C

C.

C a a a a.

CCC.

C

a a

CC

CC..

C

C

C

CC

Lye

CG

Gly

CGC

Arg

GAG

Giu

A.AG

Lys

TGC

Cyg

ACG

Thr Glu 125

TAC

Tyr

TTC

Ph.

GCGI

Ala

GOC

Gly 30

OCT

Gly

ATC

Ile

ARC

Acn

TGT

Cye

AGC

Ser 110

GC

Gly

TAC

Tyr

ACC

Thr

GCC

Ala 15

GC

Ala

CAC

His

ATC

Ile Gcc Gly

GGA

Gly 95

GAG

Glu

ATC

Ile

ATC

Ile

ACC

Thr

GAG

Glu

CCC

Ala

TGG

Trp

GGT

Gly

GGA

Gly so

ATC

Ile

GCC

Gly

GC

Gly

ATC

Ile

GAG

Glu 160 Glu

CCC

Gly

ARC

Aen

CTG

Lou 65

GGG

Cly

CCC

Pro

GC

Gly

TAT

Tyr

ATC

Ile 145

CTC

Lou Ala

ACC

Thr

ARC

Asn 5o

CCCC

Gly

GCA

Ala

GTC

Val

ATC

Ile

GCA

Ala 130

CTG

Lau

CCC

Pro Arc) Arq 35

ARG

Lys

ARC

Ken

TTC

Phe

TTC

Ph.

ACO

Thr 115

ACA

Thr

GCG

Al a

TG

Trp Cly 20

GAG

Glu Val1

OTO

Val

CTG

LOU

TTC

Ph.

100

TGC

Cys

CAG

Gin

TCC

Trp

GCC

Ala

CIC

Ala

GAG

C lu

TGG

Trp AlT Ile 35

CTC

Leu

TG

Trp

CTG

Val1

CC

Ala

ACC

Thr 165 GAG GC CGA GGC TCC

GAG

Glu Cac Arg

TTC

Phim 0CC Avg 70

CCT

Pro

CAR

Glu

ACC

Ar;

ATC

Ile

ATC

Ile 150

TGT

Cyc

C

Ala

CAC

Asp

GTG

Val 55

TTC

Ph*

TAC

Tyr Thr

AGA

Arg

GAG

Giu 135

TTC

Phe

GGG

Gly CTC, CC 0CC 0CC C Lou Gly Gly Gly Gly ARC C GTC CAC GAG Lye Ala Val His Glu TTG, ACC GTA 0CG GGA Lou Sor Val Ala Gly CCC TAC CTC TOC TAC Pro Tyr Lou Cys; Tyr 7S GTC GTG TT? TTC ATC Val Val Ph* Ph. Ile OCT CTG, COG ChG TTC Ala Lou Gly Gin Ph* 105 GTC TGT CCT TTA TTT Val Cye Pro Lou Phe, 120 C CAT CTC ART GTC Ala isi Lou Ken Val 140 TAC TTA ACC ARC TOC Tyr Lou Ser Aen Cys CAT GAG TOG ARC ACA His Glu Trp Ken Thr 170 99 147 195 243 291 339 387 43S 483 531 GAG AAA TOT Clu Lye Cyc 17S CTG GAG TTC Val Glu Phe

CAC

Gin ARC CTG ARC TTC ACC ARC TAC AGT CAT 579 Tyr Ser His L80 Lou han Ph* Ser 185 -130- GTG TCC CTG CAG Val Ser Lo*u Gin 190 AMC GCA ACC hAn Ala Thr 295 TCC CCG GTC ATG GAG TTC Ser Pro Val Met Glu Phe 200

COG

Akrg 205

TOG

Trp

TOC

cys,

GCA

Ala Thr S.28S 0* ACT 9.r

ATC

AT

Mer

GTC

Val

GAG

Glu

ATC

Ile

ACC

Thr 770 Lou 270

TCC

Sor

TTT

Phe

TAC

Tyr

TOT

Cys

GTC

Val1 350

GCA

Ala

ACT

Thr

CTC

Lou

TTC

Lou CrC Lou

TG

Trp

TTC

Ph.

255 ccC Pro

COG

Arg

TTC

Ph*

MAC

hAn

CTG

Lou 335

CTG

Lou

GMA

Glu

ATO

met

ATC

Ile

GC

Ala

GCA

Ala

MCG

Lys 240

CCC

Pro

GOT

Cly

CTC

Lou

TCC

Ser

MAC

hun 320

MAC

Agm

GCC

Gly

TCA

Ser

ATO

Hot

TTC

Phe 400

ATA

Ii.

CTG

Lou 225

OCT

Cly

TAC

Tyr Gcc Ala

TCT

Ser

TAT

Tyr 305

TAT

Tyr

ACT

Ser Ph&

GOCT

Cly Pro 38S

CTO

Lou

TCT

Ser 210

TOT

Cys

ACO

Thr

ATC

Ile cC Ser

GAT

Asp 290

CCC

Ala

MAC

hAn

C

Gly

ATG

met

CCT

Pro 370 Cr0 Lou

GCC

Cly

CAT

Asp

CTC

Lou

MAG

Lys

ATO

met

CMA

Clu 275

CCA

Pro

ATC

Ile MhC hon

AC=

Thr 0CC Ala 355

OGA

Cly

TCC

Ser

CTC

Lou

GCC

Gly

CTO

Lou

TCA

Ser Cr0 Lou 260

GCC

Cly

CAG

Gln

C

Cys

MAC

Aen hOC Ser 340

TAC

Tyr

CTO

Lou

CCA

Pro

CAC

Asp

ATT

Il1e oco Ala

ACT

Thr 245 CT0 Lou

ATC

Ile 070 Val

CTO

Lou 7C Cys 325

TTC

Ph.

GAG

G3.u

OCT

Ala

TTG

Lou

ACT

Ser 40S

CMA

Glu

OCT

Ala 230

GGA

Cly

ATC

Ile

MAG

Lys

TOG

Trp

GCC

Cly 310

TAC

Tyr 070 Val

CAC

Gin

TTC

Ph.

TOG

Trp 390

CAG

Gln

CAC

His 215

TOG

Trp

MGC

Lys

CTC

Lou Phe Val 295 7c Cym

AGO

Ar;

OCT

Ala

GCC

Gly

ATC

Ile 375 Gcc Ala TTy Phe

ATC

I1e

A=C

Thr

GTC

Va1

CTC

Lou

TAC

Tyr 280

GAT

Asp

CTO

Lou

GAC

ASP

COG

Gly 070 Val 360 Gcc Ala

ACC

Thr 070 Val

COG

Cly

ATC

Ile Val

ATC

Ile 265

CTO

Lou

OCT

Ala Acc Thr

TOT

Cys Ph.

345

CCT

Pro

TAC

Tyr

CTO

Lou

TOT

Cys

TOG

Trp

MAC

hen TaC Cy.

TAT

Tyr 250

COA

Arg

TAC

Tyr

COG

Gly

OCT

Ala

ATT

Ile 330

OCT

Ala

AT?

Ile

CCC

Pro

TTC

Ph.

070 Val 410

GMA

Clu CrC Lou

TAC

Tyr 235

CTC

Val

COG

Cly

CCT

Pro

AC

Thr Cr0 Lou 315

ATO

met

ATC

Ile

OCT

Ala

MAG

Lys

TTC

Phe 395

GAG

Glu

CC

Arg

CGA

Ar; 220

TTC

Ph*

ACT

Thr

OTC

Val

GAC

Asp

CAC

O ln 300

OCC

Cly

CTC

Lou

TTC

Ph.s

GAG

Glu

OCT

Ala 380

ATO

met

AOC

Ser 627 675 723 771 819 867 915 963 1011 1059 1107 1155 1203 1251 1299 CTT 070 ACA CCC G70 OTT CAC ATG Lou Val Thr Ala Val Val ASP Met 415 420 TAC CCC MCG Tyr Pro Lys CTC TC Val Phe 425 COG COG GCC Arg Arg Oly -131p p p.

p p p.

p p p.

p tap...

p p. a.

TAC COG OA OAA Tyr Ar; Ar; Glu 430 CTA wcC CTG GOTC Lou Gly Lau Val 445 TTT GAC TCA TAC ph* Asp Ser Tyr TTT GAG TOT OTC Ph* Glu CY8 Val 's0 CAC AAT ATT GAG Asp Asfi Glu 495 TGG TOC TOG AA Trp, Cys Trp Lys 510 T~c TTT C70G OTC Ph* Ph* Lou Val 525 TAT CCT GCT TOG Tyr Pro Ala Trp ATC CTG TOC ATC Met LOU C-Is 110 GGC ACC C TG CCC Gly Thr Lou Pro 575 cTC, AAA ATO AGO Lou Lys Not Ax; 590 AAT GAC TOT GAG Ann Asp Cys Glu 605 ACA GAG AAG GAG Thr Glu Lys Glu AGCCTTCCT*. C CTO CTC ATC Lou Lou Ile 435 ATO CTG ACA met Lou Thr 450 Gcc OCC MOT Ala Alt Sur 465 TOC ATC GCC Cys Ile Oly GAC ATOW AT? Asp Hot 1ie OTT OTO ACC Val Val Thr 515 AAG TAC AMG Lys Tyr Lys 530 CCC TAC CGC Cly Tyr Gly 545 CCC CTC TOG Pro Lou Trp GAG "AA TTA Glu Lys Lou GCC AAG Oly LYS LOU 5,5 GCC AAG GTC Ala Lys Val 610 ACG CAC TTC Thr His Phe 625

CTO

LOU

GAG

Glu

OCC

Oly Too ?rp

OA

Cly

CCT

Pro Pro

ATT

Ile

ATC

Ile CAd Gin 580

GOO

Gly

AAA

GCC

Ala

GA

Oly

ATO

het

OTO

Val 485

TAO

Tyr

CG

dly

CTC

LOU

Occ dly

TTC

Phe, 56S

AAG

Lys

OCC

Ala

OC

CTO

LOU

C

Gly

TGO

Cys 470

TAT

Tyr

COG

Arg

ATC

Ile hAM Lys Too Trp 550

ATC

Ile

TTO

Lou

AC

Sur

GAC

TCC AT? Sur Ile 440 ATO TAC Hot Tyr 455 TTO CTC Lou Lou GOA ACT Gly Sur CCA CTG Pro Lou TOT 0CO Cys Ala 520 TAC AAC Tyr Ann 535 CTC ATO Lou Not AAO CTG Lys Lou ACA OTC Thr Val CCA COG Pro Ar; 600 GGT ACC Gly Tb: 615

AT?

Ii.

TTC

Phe

AAC

Ann TCh Sur 505

GCC

Gly

ART?

Asn

OCT

Ala Too Trp,

CCC

Pro 585

ATO

Not

ATC

Ile

TTC

Phe 070

AGO

Arg 490

CTC

Lou

ATC

Ile Val

CTC

LoMU

AMG

Lys 570

AGC

Val

TCT

Ser

CAG

Gln

CC

Ala 475

TC

Ph.

ATC

Ile

TTC

Phu

TAC

Tyr

TCC

Sur 555

ACA

Tb:

OCT

Ala

ACC

Thr

CCC

Ala

CT?

Lou 460

ATC

Ile

TAT

Tyr

AMG

Lys

ATC

110

ACA

Thr 540

TCC

Sur

GAG

Glu

CAT

Asp

OTT

Val

ATC

Ile 620 OTC TCT TAT TTC Val Ser Tyr Ph* 1347 1395 1443 1491 1539 1587 1635 1683 1731 1779 1827 1875 1927 1938 Lys Gly Asp T GATCCCCGCC AGCCACTTGG ATGTGTCTCC INFORMATION FOR SEQ ID NO:4t SEQUENCE CHARACTERISTICS:, LzNGTH: 627 amino acids TYPE: amino acid TOPOLOGY: linear -132- (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:4: Met Thr Ala Giu Gin Ala Lou Pro Lou 9 9 9* 9 99**

S

90..

*9 S .9 9* 9 9999 0* S. 9* 59

S

*5055*

S

99 5 9* 5* Glu Gly Ann Lou Gly Pro Gly Tyr Ile 145 Lou Giu Ann Ile Lou 225 Gly Tyr Ala Ala Thr Asn Cly Ala Val Ile Ala 130 Lou Pro Phe Ala Ser 210 cys Thr Ile Ser Arg Arg Lys Ann Ph.

Ph* Thr 115 Thr Ala T rp Gln Thr 195 Asp Lou Lys Met Glu Gly Glu Val1 Val Lou Ph.

100 Cys Gin Trp Ala Lys 180 Ser Gly Lou Ser Lou 260 Gly Ser Ala Glu Trp Ile 85 Lou Trp, Val1 Ala Thr 165 Lou Pro Ile Ala Thr 24S Lou Ile Glu Ar; Ph.

Arg 70 Pro Glu Ar; Ile Ile 150 Cys Ann Val1 Glu Ala 230 Gly Ile Lys Ala Asp Val1 55 Ph* Tyr Thr Ar; Glu 135 Ph.

Gly Phe Met His 215 Trp Lys Lou Phe Lou Lys 40 Lou Pro Val Ala Val 120 Al a Tyr His Ser Glu 200 Ile Thr Val1 Lou Tyr 280 dly 25 Ala Ser Tyr Val1 Lou 105 Cyu His Lou Glu An 185 Phs, Cly Ile Val Ile 265 Lou 10 Gly Va1 Val Lou Phs 90 Gly Pro Lou Ser Trp 170 Tyr Trp Ann Cys Tyr 250 Arg Tyr Gly Gly Kis Glu Ala Gly cys Tyr 75 Ph* Ile Gin Ph* Lou Ph* Ann Val 140 hen Cym 155 Ann Thr 5cr His Giu Ar; Lou Ar; 220 Tyr Phe 235 Val Thr Gly Va1 Pro Asp Oly Gly Ar; Gly Glu lie Lys Ann Cyn eye Thr Ser 110 Glu Gly 125 Tyr Tyr Ph. Thr Glu, Lys Val 5cr 190 Ar; Val 205 Trp Giu Cys Ile Ala Thr Thr Lou 270 Lou Ser 285 Ann Gly Lys Ala Al a Al a His Ile Gly Gly Giu Ile Ile Thr Cys 175 Lou Lou Lou Trp Ph.

255 Pro Ar; Glu Ala Trp Gly Gly Ile Gly Gly 110 Giu 160 Val Gin Ala Ala Lys 240 Pro Gly Lou 275 Ser Asp Pro 290 Gin Val Trp Va1 Asp Ala Gly Thr Gin Ile Phe Phe Ser -133- 9 9**9 9 99 S 99 99 9 9.

9 9 9 99 Tyr 305 Tyr Ser Phe Gly Pro 385 Leu Va1 Leu Met Ala 465 Cys Asp Val Lys Gly 545 Pro Giu Gly Ala Thr 625 Ala Agn dly Met Pro 370 Leu Gly Val Leu Leu 450 Ala Ile Met Val Tyr 530 Tyr Leu Lys Lys Ile Ann Thr Ala 355 Giy Ser Leu Asp Ile 435 Thr Ser Gly Ile Thr 515 Lys Gly Trp Leu Lou 595 Cys Ann Ser 340 Tyr Lou Pro Asp Met 420 Leu Clu Gly Trp Gly 500 Pro Pro Ile Ile Gin 580 Gly Leu Cys 325 Ph.

Glu Ala Lou Ser 405 Tyr Ala Gly Met Val 485 Tyr Gly Leu Gly Phe 565 Lys Ala GlY 310 Tyr Val Gln Phe Trp 390 Gln Pro Lou Gly Cys 470 Tyr Arg Ile Lys Trp.

550 Il10 Lou Ser Cys Ar; Ala Gly Ile 375 Ala Phe Lys Ser met 455 Lou Gly Pro cys Tyr 535 Leu Lys Thr Pro Gly 615 Leu Asp Gly Val 360 Ala Thr Val Val Ile 440 Tyr Lou Ser Lou Ala 520 Asn Met Lou Val Ar; 600 Thr Cy.

Phe 345 pro Tyr Lou Cys Ph* 425 Val Ile Ph.

Ann Ser 505 Giy Aun Ala Trp Pro 585 Met Ala le 330 Ala Ile Pro Ph* Val 410 Arg Set Ph* Val Arg 490 Lou Ile Val Lou Lys 570 Ser Val Leu 315 Met 110 Ala Lye Phe 395 Glu Arg Tyr Gln Ala 475 Phe Ile Phe Tyr Ser 555 Thr Ala Thr Gly Lou Ph* Glu Ala 380 met Set Gly Ph* Lou 460 Ile Tyr Lys Ile Thr 540 Set Glu Asp Val Ile 620 Ser Cys Set Val 365 Ji 46t Lou Tyr Lou 445 Phe Phe Asp Trp Ph.

525 Tyr Met Gly Lou Ann 605 Tyr Cys Val 350 Ala 4 Thr I Lou Val Arg; 430 Gly Asp Glu Asn Cys 510 Phe Pro Lou Thr Lys 590 Asp Asn Lou 335 Lou Glu Met Ile Thr 415 Arg Lou Ser Cys Ii 495 Trp Lou Ala Cym Lou 575 Met Cym Ann 320 Ann Gly Set net Phe 400 Al a Glu Val Tyr Val 480 Glu Lys Val Trp Ile 560 Pro Ar; Glu Lys Val 610 His Ph.

Lys Gly Asp Thr Ile Set Ala Thr Giu Lys Giu -134- INFORMATION FOR SEQ ID NO:S: SEQUENCE CHARACTERISTICS: LENGTH: 2093 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: ORGANISM: Taurine (vii) IMMEDIATE SOURCE: LIBRARY: rat brain CLONE: rBl6a (ix) FEATURE: NAME/KEY: CDS oo~e(B) LOCATION: 127. .1989 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID CCCA.ACGCCG CCATCGCCCC CAATCCCGCC AGCCTCGCCC CGGGCCATCC GCTGTCGGCT TAGCCACCCA GATGCAGAGC CACTGCCACA GCCTCTTCAG AGGAGCCTCT CAAGCAAAAC 120 GAGGAG ATG GCC ACC AAG GAG AAG CTT CAA TOT CTG AAA GAC TTC CAC 168 Met Ala Thr Lys Glu Lys Lou Gin Cys Lou Lys Asp Ph. His 1 5 AAA GAC ATC CTG AAG CCT TOT CCA GCC MAG AGC CCA CCC ACG CGG COT 216 Lys Asp Ile Leu Lys Pro 5cr Pro Gly Lys Ser Pro Gly Thr Arq Pro 1520 25 GAG GAT GAGGOCT GAT GGGAAG CCC CCT CAG AGOGGAG AAG TGGCTCC AGC 264 Glu Asp Glu Ala Asp Gly Lys Pro Pro Gin Arg Glu Lys Trp Sor 35 40 MAG ATC GAC TTT GTG CTG TCT GTG 0CC GGA CCC TTC TG OT TTG CCC 312 Lys Ile Asp Ph. Val Lou 5cr Val Ala Gly Oly Ph. Val Gly Lou Cly 55 MAT OTC TOO COT TTC CCG TAC OTC TGC TAC AMA MT OT OGA OCT CCA 360 Aen Val Trp Arg Ph. Pro Tyr Leu Cys Tyr Lys Aun Gly Oly Oly Ala 70 TTC OTC ATA CCG TAT TTT ATT TTC CTG TTT COG AGC GCC CTG COT GTG 408 Ph. Lou Ile Pro Tyr Ph. Ile Ph. Lou Ph. Gly 5cr Cly Leu Pro Val 85 TTT TTC CTG GAG. GTC ATC ATA GGC CAG TAC ACC TCA GAM GCC 0CC ATC 456 Ph. Phe Lou Glu Val Ile Ile Gly Gin Tyr Thr Ser.Clu Gly Giy Ile 100 105 110 -135- ACC TGC TGO GAG AAG ATC TGC CCC TTG TTC TCTr GGC ATT GGC TAC GCG Thr Cys Trp Giu Lys Ile Cys Pro Lou Pile Ser Gly Ile Gly Tyr Ala 115120 12S TCC ATC GTC ATC OTO TCC CTC CTG AAT =T TAC TAC ATC OTC ATC CTG Ser Ile Val Ile Val Ser Lou Lou Ann Val Tyr Tyr Ile Val Ile Lou 130 135 140 GCC TGC GCC ACA TAC Ala Trp Ala Thr Tyr 145 9*

C

999* 9* C 9999

C

CCCC

eq C 9 *9 9* 9 9* 99 C. 'C

C

99*9

C

9* G 9*

TG

Trp

ACC

Thr 175

TTC

Phe

TCC

Ser

TGC

Cys

OTT

Val1 ccc Ala 255

OCT

Gly

GAC

Asp

OCT

Ala

AAO

Lys

GCC

Ala 160

CTG

Leu

ACT

Thr

TCC

Ser

CTC

Leu

COO

Arg 240

ATG

Met

GAA

Glu

CCA

Pro

ATC

Ile

TAT

Tyr 320

CAC

His

COT

Arg

TCO

Ser Gly

CTC

Lou 225

TCC

Se r

CTT

Lou

GC

Gly

CAO

Gin

TGC

Cys 305

AAC

Asn

TOC

Cys

AGG

Ar;

CCT

Pro

ATC

Ile 210

TTA

Lou

ACA

Thr

CTG

Lou

ATC

Ile

OTG

Val 290

CTG

Lou

TCG

Ser

AAC

Ann

AAC

Aim

GTG

Val1 195

GAC

Asp

GTC

Val1

C

Gly

GTG

Val

AAA

Lys 275

TG

Trp

GGG

Oly

TAC

Tyr TAC CTA TTC CAG Tyr Lou Ph@ Gin 150 CAT ACC TOG AAC His Ser Trp Aen 165 GAG AGT CAC TG Olu Ser His Trp 180 ATC GAG TTC TG Ile Glu Ph. Trp, CAC CCA GOC MGT His Pro Gly Ser 215 TGG CTC GTC TOT Trp Lou Val Cys 230 AAG OTT GTC TAC Lys Val Val Tyr 245 CTG CTG GTC COT Lou Lou Val Ar; 260 TTC TAC CTG TAC Ph. Tyr Lou Tyr ATC GAC OCT OGA Ile Asp Ala Oly 295 GCC ATG ACC TCA Ala Met Thr Ser 310 AGG GAC TOT ATG Ar; Asp Cys Met 325

TCT

Ser

AG

Thr

GTC

Val1

GAG

Glu 200

CTO

Lou

TTT

Ph.

TTC

Ph.

OCA

Gly

CCT

Pro 280

ACT

Thr

CTO

Lou

CTO

Lou

TTC

Ph.

CCA

Pro

TCC

Ser 185

COC

Ar;

AAA

Lys

TTC

Ph.

ACT

Thr

CTG

Lou 265

AAC

Ann

CAC

Gin

OCA

Gly

CTO

Lou

CAG

Gin

CAG

Gin 170

CTT

Lou

AAC

Ann

TOG

Try,

TOC

Cys

OCT

Ala 250

ACC

Thr

ATC

Ile

ATA

Ile

AOC

Ser

OGA

Gly 330

AAG

Lys 155

TOC

cys

AGC

5cr

GTG

Val

GAC

Asp

ATC

Ile 235

ACT

Thr

CTG

Lou

AGC

Ser

TTC

Ph.

TAT

Tyr 315

TGC

Cys CAT CTT CCC 600 Asp

ATO

Met 0CC Ala

CTC

Lou

CTC

Lou 220

TG

Trp

TTC

Ph.

CCA

Pro

COC

Arg

TTT'

Ph* 300

AAC

Ann

CTG

Lou Lou

GAG

Glu

GCC

Ala

AGC

Ser 205

GCG

Ala

AAO

Lys

CCC

Pro

OCT

Gly

CT?

Lou 285

TCC

Ser

AAG

Lys

AAC

Ann Pro

GAC

Asp

AAC

Asn 190

CTO

Lou

CTC

Lou

OCT

Gly

TTT

Ph.

OCT

Ala 270

GAG

Glu

TAC

Tyr

TAC

Tyr

AG?

Ser 648 696 744 792 840 s88 936 984 1032 1080 1128 1176

OCT

Gly 335 ACC ACT TTT GTG TCT 0CC TTC OCA AT? TTT TCC ATC CTO 0CC TTC Thr Ser Ph. Val 5cr Gly Ph. Ala Ile Ph. 5cr Ile Lou Gly Ph.

340 345 350 -136- ATG OGCA CAA GAG CAM GGG GTG GAC ATT GCT GAT Met Ala Gln Glu *0

CCT

Pro

CTG

Lou

GGA

Gly

OTT

Val1 415

TTC

Phe

GTG

Val

GCT

Ala

ATT

Ile

ATG

Met 495

ATC

Ile

TAT

Tyr

ATC

Ile TTc Lou

GC

Oiy

CCC

Pro

CTG

Lou 400

GAT

Asp

AT?

Ile

ACG

Thr

ACT

Ser Gcc Ala 480

ATC

Ile

ACT

Thr

GTA

Val

GCC

Oly

GTC

Val1 560

TTG

Lou

ACC

Thr 385

GAC

Asp

CT?

Lou

GCC

Ala

GAG

Giu

OCT

Gly 465

TG

Trp

C

Gly

CCA

Pro

CCC

Pro

CTG

Lou 545

ATT

Ile 0CC Ala 370 77? Ph.

AOC

Ser

TAC

Tyr

ATC

Ile

GOT

Gly 450

GTA

Val1

ATA

Ile

TAT

Tyr

GCT

Ala

CTG

Lou 530

C

Giy

GTC

Val Gin 355

TTC

Phe,

TGO

Trp

CAG

Gin

CCG

Pro

OTO

Val1 435

C

Gly

TOC

Cys

TAT

Tyr

CG

Arg

CTC

Lou 515

ACC

Thr

TOG

Trp

ATC

Ile

ATT

Ii.

TCC

Ser 77? Ph* 7cc Ser 420

TOC

Cy.

ATO

Met

CT?

Lou

C

Giy

CCT

Pro 500

TOT

Cys

TAC

Tyr

C

Oly

CTC

Lou

ATA

Ile 0CC Ala

ATT

Ile Val 405 TrC Ph.

A=C

Scr

TAT

Tyr

TG

Lou

GT

Oly 485

GGA

Gly

GTT

Val

AAC

Ann

CTG

Lou

CTC

Lou 565

TAC

Tyr

CTO

Lou 390

GMA

Glu

CTA

Lou

ATC

Ile 070 Val

TGO

Trp, 470

GAT

Ap

CCC

Pro

GGA

Giy

AAA

Lys 0CC Ala 550 7C Cys

CCA

Pro 375 Ph.

GTC

Val1

AGO

Arg

AGC

Scr Ph.

455

OTC

Val1

MAC

Ann

TGO

Trp

TOT

Cys

GTC

Val1 535 Leu

CG

Arg Oly Val Asp Ala 360

AAA

Lys 7?? Phs,

GMA

Glu

MAG

Lys

TAC

Tyr 440

CMA

Gin

GCA

Ala

TTA

Lou

ATO

Met

TTC

Ph.

520

TAC

Tyr

TCC

Ser

ACG

Thr

GAG

Glu Asp

OCT

Ala

AT?

Ile

OGA

Oly

OCT

Gly 425

CTG

Lou

CTC

Lou

TTC

Phis

TAT

Tyr

MAG

Lys 50S

ATC

Ile

COG

Ar; 7CC Ser

GAO

Glu

OTG

Val1 Val1

ATO

met CRd Gin 410

TAT

Tyr

CTO

Lou Ph.

Ph.

GAC

Asp 490

TAC

Tyr

T~C

Ph*

TAC

Tyr

ATO

Met

GGA

Giy 570 ACC ATO Thr Met 380 CC CTC Lou Lou 395 ATC ACA Ile Thr COT OG Ar; Ar; GG CTG Gly Lou GAC TAC Asp Tyr 460 GMA TOT Glu Cys 475 GGT A?? Gly Ile AOC TOO Ser Trp TC7 CTC Ser Lou CC? GAT Pro Asp 540 GTG TOT Val Cys 555 *CCO CTC Pro Lou ATO CCG Met Pro 770 CTT Lou Lou 7CC 770 Ser Lou GMA ATC Giu Ile 430 ACO ATO Thr Met 445 TAT OCA Tyr Ala 777 0?? Phe Val GAG GAC Giu Asp GCT GTC Ala Val 510 OTC MOG Vai Lys 525 TG GCA Trp Ala ATC CCC Ile Pro COC 070 Ar; Vai GCT GAG TCA OCT Ala Olu 5cr Oly 365 1224 1272 1320 1368 1416 1464 1512 1560 1608 1656 1704 1752 1800 1848 1896 AGA ATC Ar; Ile 575 AMA TAC CTG Lys Tyr Lou ACC CCC AGG Thr Pro Ar; CCC MAC CGC TOG OCT 070 Pro Ann Ar; Trp Ala Val 590 -137- GAG CGT GAA GGG GCT ACG CCC TTT cAC TCC AGA GCA ACC CTC ATG AAC 1944 Giu Arg Giu Gly Ala Thr Pro Ph. His Ser Arg Ala Thr Lou Met Ann 595 600 605 GGT GCA CTC ATG AAA CCC ACT CAC GTC ATT GTG GAG ACC ATG ATG 1989 Gly Ala Lou Met Lys Pro Ser His Val lie Val Giu Thr Met Met 610 615 620 TGAGGTCCGG GCTGTGTGAC CGGCGCCGCT L&-.CTGCCGT TTACTAACCT TAGATTCTCC 2049 TAGGACCAGG TTTACAGAGC TTTATATTTG TACTAGGATT -4T17T 2093 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 621 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Ala Thr Lys Glu Lys Lou Gin Cys Lou Lys Asp Ph. His Lys Asp 1 5 10 Ile Lou Lys Pro Ser Pro Gly Lys Scr Pro Gly Thr Arg Pro Glu Asp 25 Glu Ala Asp Gly Lys Pro Pro Gin*Arg Glu Lys Trp Ser Ser Lys Ile 40 Asp Phe Val Lou Ser Vai Ala Gly Gly Ph. Val Gly Lou Gly Ann Val 55 *Trp Arg Phe Pro Tyr Lou Cys Tyr Lys Ann Gly Gly Gly Ala Ph. Lou 70 75 Ile Pro Tyr Ph. Ile Ph. Lou Ph. Gly Ser Giy Lou Pro Val Ph* Phe 90 Lou Glu Val Ile Ile Gly Gin Tyr Thr 5cr Giu Gly Gly Ilie Thr Cys *:100 105 110 Trp Glu Lys Ilie Cys Pro Lou Ph. Ser Gly Ile Gly Tyr Ala Ser Ii.

115 120 125 Val Ile Val Ser Lou Lou Ann Val Tyr Tyr Ile Val Ile Lou Ala Trp 130 135 140 Ala Thr Tyr Tyr Lou Ph. Gin Ser Ph. Gin Lys Asp Lou Pro Trp Ala 145 150 155 160 His Cys Ann His Ser Trp Ann Thr Pro Gin Cys Met Glu Asp Thr Lou 165 170 175 Arq Arg Ann Glu Ser His Trp Val Ser Lou Ser Ala Ala Asn Phe Thr 180 185 190 Ser Pro Val Ilie Giu Phe Trp Giu Arg Asn Val Lou Ser Lou Ser Ser 195 200 205 -138- Gly lie Asp His Pro Gly Ser Lou Lys Trp Asp Leu Ala Leu Cys Lou 220 210 215

C

C

C

CC..

C

C

C.

Lou 225 Ser Lou Gly GIn Cy 305 Asn Ser Gln Leu Thr 385 Asp Lou Ala Glu Gly 465 Trp Gly Pro Leu Thr Lou Ile Val 290 Leu Ser Phe Glu Ala 370 Phe Ser Tyr lIle Gly 450 Va1 Ile Tyr Ala Va1 Gly Va1 Lys 275 Trp Gly Tyr Val Gln 355 Phe Trp Gin Pro Val 435 Gly Cy Tyr Arg Lou 515 Trp Lys Lou 260 Ph.

Ile Ala Arg Ser 340 Gly Ile Ser Phe Ser 420 Cys Met Lou Gly Pro 500 cys Lou Val 245 Lou Tyr Asp Met Asp 325 Gly Val Ala Ile Val 405 Phe Ser Tyr Leu Gly 485 Gly Val Val 230 Val Val Lou Ala Thr 310 Cys Ph.

Asp Tyr Lou 390 Glu Lou lie Val Trp 470 Asp Pro Gly Cys Tyr Arg Tyr Gly 295 Ser met Ala Ile Pro 375 Phe Val Ar; Ser Ph.

455 Val Asn Trp Cys Phe Ph.

Cly Pro 280 Thr Lou Lou Ile Ala 360 Lys Phe Glu Lys Tyr 440 Gin Ala Lou Met Ph.

520 Ph.

Thr Lou 265 Asn Gln Gly Lou Ph* 345 Asp Ala Ile Gly Gly 425 Lou Lou Phe Tyr Lys 505 I le Cys Ala 250 Thr Ile 110 Ser Cly 330 Ser Val Val Met Gln 410 Tyr Lou Ph.

Ph.

Asp 490 Tyr Phe Ile 235 Thr Lou Ser Ph.

Tyr 315 Cys Ile Ala Thr Lou 395 Ile Arg Gly Asp Glu 475 Gly Ser Ser Trp Phe Pro Ar; Ph.

300 Asn Lou Lou Clu Met 380 Lou Thr Arg Lou Tyr 460 cys Ile Trp Lou Lys Pro Gly Lou 285 Ser Lys Asn Gly Ser 365 Met Lou Ser Glu Thr 445 Tyr Phe Glu Ala Val 525 Cly Ph.

Ala I 270 Glu Tyr Tyr Ser Ph.

350 Gly Pro Lou Lou Ile 430 Met Al a Va1 Asp Val 510 Lys Va1 Ala 255 Gly Asp Ala Lys G1y 335 Met Pro Lou Gly Val 415 Ph.

Val Ala Ile Met 495 Ile Tyr Ar; 240 Met Clu Pro Ile Tyr 320 Thr Ala Gly Pro Lou 400 Asp Ile Thr Ser Ala 480 Thr Val Pro Lou 530 Thr Tyr Asn Lys Val 535 Tyr Ar; Tyr Pro Asp 540 Trp Ala lie Gly -139- 0s a. a a a *aaaa.

a. a Le 54 Ill Lys Gil Lou (2)

CTG

Leu

I

CCT

Pro

GAT

Asp

ATG

Met

CTT

Lou u Gly Trp Gly Leu Ala Leu Ser Ser Met Val cys 5 550 555 e Val Ile Lou Lou Cys Arg Thr Glu Gly Pro Leu 565 570 s Tyr Lou Ile Thr Pro Ar; Glu Pro Asn Ar; Trp 580 SOS Gly Ala Thr Pro Ph. His Ser Ar; Ala Thr Lou 595 600 IMet Lys Pro Ser His Val Ile Val Glu Thr Met 610 615 620 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1051 base pairs TYPE: nuclesic acid STRANDEDNESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (vii) IMMEDIATE SOURCE: LIBRARY: human heart, human brain CLONE: hHE7a,hS3a (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..739 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: OCT TTC ATC GCT TAC CCG CGG GCT 070 GTG ATG Ala Phe Ile Ala Tyr Pro Ar; Ala Val Val Met 5 CTC TGG GCC TGC TOT TTC TTC TTC ATG GTC OTT Leu Trp Ala Cys Cys Ph.e Ph. Phe Met Val Val 25 AGC CAG TTT GTG TOT 0TA GAA AGC CTG GTG ACA Ser Gin Phe Val Cys Val Glu Ser Lou Val Thr 40 TAC CCT CAC GTG TTC CGC AAG AAC AAC COG AGG Tyr Pro His Val Phe Ar; Lys Lys Asn Ar; Arg 55 GGA GTA TCT OTC GTC TCC TTC CTT GTG G CTG Gly Val Ser Val Val Ser Ph. Lou Val Gly Lou 70 75 Ile Pro Leu Val 560 IAr; Val Ar; Ile 575 Ala Val Glu Ar; 590 Met Asn Gly Ala 605 met

CTG

Lou

CTC

Lou

C

Ala

GAA

Glu

ATC

Ile

CCC

Pro

CTG

Lou

CTG

Lou

GTC

Val1

ATG

Met

TCT

CTG

Lou

GAC

Asp

ATC

Ilec

ACA

Thr 48 96 144 192 240 -140- GAG GCC GGA ATG TAC GTG TTC CAG CTC 77? GAC TAC TAT GCG GCC ACT GJlu Gly GJy mot Tyr Val Ph. Gin Lou Ph. Asp Tyr Tyr Ala Ala Ser 0CC ATG TGC CTC CTG S.

S.

S

*5 S S

S.

S *5 S S *5 Oly

TOG

Trp

OG

Gly

CCA

Pro 145

CCG

Pro

CTG

Leu

AGC

Ser

CT

Arg

GGA

Gly 225

GAG

Met

GTT

Val

TAC

Tyr 130

GC?

Ala

CTG

Lou

GC

Gly

CTC

Leu

CAG

Gin 210

CCC

Pro

CTA

Cys

TAC

Tyr 115

AGG

Arg

GTG

Val

ACC

Thr

TG

Trp

TAC

Tyr 195

CTC

Leu

TCG

Ser

GAG

Lou 100

OGA

Gly

CCA

Pro

TOC

Cys

TAC

Tyr

CTC

Lou 180

AGA

Arg

ATG

Met

GCT

Ala

TCT

Lou

CC

Ala

TG

Trp

ACA

Thr

AAC

Asn 165

CTG

Lou

CTC

Lou

TGC

Cys

CCC

Pro

CAC

TTC

Ph&

PAOG

Lys

CCT

Pro

GCC

Ala 150

PAO

Lys

OCT

Ala

GCA

Oly

CCA

Pro 0CC Ala 230

TGC

OTG 0CC ATC TTC GAG TCC Val Ala Ile Phe Glu Ser 105 COC TTC TAC GAC AAC ATC Arg Phe Tyr Asp Asn 110 120 CTT ATC AMA TAC TOT TOG Lou Ile Lys Tyr Cys Trp, 135 140 ACC TTT CTC TTC TCC CTO Thr Ph. Lou Phe Ser Lou 1S5 AAG TAC ACO TAC CCG TOO Lys Tyr Thr Tyr Pro Trp 170 CTG TCC TCC ATO GTC TC Lou Ser 5cr Mot Val Cys 185 ACC CTC AAO 0CC CCC TTC Thr Lou Lys Oly Pro Ph.

200 0CC GAG GAC CTG CCC CAG Ala Olu Asp Lou Pro Gin 215 220 ACC CCC AOG ACC TCA CTG Thr Pro Arg Thr Ser Lou 235 T AGGCGGCAGC CCCTTGGATG CTC TOT GTG Leu Cys Val 110 GMA GAC ATO Glu Asp Met 125 CTC TTC CTC Lou Ph. Lou ATA AAG TAC Ile Lys Tyr TGG 0CC OAT Trp Gly Asp 175 ATT CC? GCC Ile Pro Ala 190 AGA GAO AGA Arg Glu Arg 205 CCC AAC CCA Arg Asn Pro CTC AGA CTC Lou Arg Lou

GTGCCTGTGT

OCT

Ala

ATT

Ile

ACA

Thr

ACT

Thr 160

CC

Ala

TOC

Trp

ATC

Ile

C

Ala

ACA

Thr 240 336 384 432 480 528 576 672 Olu Lou Glu Ser His Cys 245 GCCTGO CCT? GGGCATGOCT GTGGAGCGAA CGTGGCAGLA GCCCCCGACC TGGAOTOGAT AAOACAAGAG GCCTATTTTG AGGCCTCCCA CTCCAACTTT TCAOCTCACG CTTOTTOAA CAAGAGTGTC CCTCTGAGAC CCTTOGGAAG CTGCGTGCCC TTGCTGCCTT CGCCCCCTCT CATCCTTCAT TCCATTAAAT INFORM(ATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 246 amino acids TYPE: amino acid TOPOLOGY: linear GCAGCCCCAT GTGCTTCCCT GAGTCCACCT GCTGAOCTGG CAGATGTGAA AGGCCAGTOC GCTGCTAGCT OGGGCOAGAC cc 829 889 949 1009 1051 -141- (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Ala Phe Ile Ala Tyr Pro Ar; Ala Val 1 5 10 NO: 8: Val Met Leu Pro Phe Ser Pro Lou Trp Ala Cys Cys Ph. Phe hop Ser Gin 0 at 6.* 0..

0:C Met Lou Glu Gly Trp Gly Pro 145 Pro Leu Ser Arg Gly 225 Glu Tyr Pro Gly Val Gly Gly Met Cye Val Tyr 115 Tyr Arg 130 Ala Val Leu Thr Gly Trp Lou Tyr 295 Gin Lou 210 Pro Ser Lou Glu Ph. Val His Val Ser Val Met Tyr Leu Lou 100 Gly Ala Pro Trp Cys Thr Tyr Asn 165 Lou Lou 180 Ar; Lou Met Cys Ala Pro Ser His 245 Cys, Phe Val 70 Val Ph.

Lys Pro Ala 150 Lys Ala Gly Pro Ala 230 Cys Val Arg Ser Ph.

Val Ar; Lou 135 Thr Lys Lou Thr Ala 215 Thr Glu 40 Lys Ph.

Gin Ala Ph.

120 Ile Ph.

Tyr Ser Lou 200 Glu Pro Ph.

25 Ser Lys Lou Lou Ile Tyr Lys Lou Thr Ser Lys Asp Arg Lou Amn Val Ph., 90 Ph* Asp Tyr Ph.

Tyr 170 Met Gly Lou Thr Val1 Arg Gly 75 Asp Glu Asn Cys 5Cr 155 Pro Val1 Pro Pro Ser 235 Thr Ar; Lou Tyr Ser Ile Trp 140 Leu Trp Cys Ph.

Gin 220 Leu Met Val Val Lou Lou Gly Lou Ala Glu Ile Tyr Lou Glu 125 Lou Ile Trp Ile Ar; 205 Ar; Lou Lou Val Met Ala Cys 110 Asp Ph.

Lys Gly Pro 190 Glu Asn Ar; Val Lou Lou Ala Val1 Met Lou Tyr Asp 175 Ala Ar; Pro Lou Asp Ile Thr so Ala Ile Thr Thr 160 Ala Trp I le Ala Thr 240 INFORMATION FOR SEQ ID 140:9: SEQUENCE CHARACTERISTICS: LENGTH: 1991 base pairs TYPE: nucleic acid STRANDEDNESS: both TOPOLOGY: linear MOLECULE TYPE: cDNA -142- (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N (vii) IMMEDIATE SOURCE: LIBRARY: human brain CLONE: hGAT-3 (ix) FEATURE: NAME/KEY: CDS LOCATION: 35..1930 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AGCCGGGCCG GCGCACGAGC CAGCCAGCGC GGCC ATG ACG GCG GAG AAG GCG Met Thr Ala Giu Lys Ala 9* S a.

S S a a a.

a a. a a a.

CTG

Leu

GCG

Ala

CCG

Pro

AAC

Ann 55

GC

Gly

GCA

Ala

GTT

Val1

ATT

Ile

GCA

Ala 135

CCC

Pro

CCG

Pro

CGC

Arg

AAG

Ly s

AAC

Asn

TTC

Phe

TTT

Phe

ACG

Thr 120

ACA

Thr

CTG

Leu

GGT

Gly 25

GTC

Val1

GTG

Val1

GTG

Val1

CTG

Leu

TTC

Phe 105

TOT

Cys

CAG

Gin

GGC

Gly

GCC

Gly

MAG

Lys

GAG

Glu

TGG

Trp

ATT

Ile 90

CTG

Leu

TG

Trp

GTG

Val

AAT

Ann

CC

Gly

CC

Arg

TTC

Phe

CGC

Arg 75

CCC

Pro

GAG

Giu

AGG

Arg

AT?

Ile

GGG

Gly

TC

Cys

GAC

Asp

GTG

Val1 60

TTC

Ph*

TAC

Tyr

ACA

Thr

AAA

Ly s

GAG

Glu 140 hAG Lys

AGC

Ser

AAG

Lys 45

CTG

Lou

CCC

Pro

GTG

Val1

GCT

Ala

OTT

Val1 125

CC

Ala

OCT

Ala

AGC

Scr 30

C

Ala

AGC

Ser

TAC

Tyr

CTC

Val1

CTG

Lau 110

TGC

Cys

CAT

His

GCT

Ala i5

GGG

Cly

CTC

Val

GTC

Val

CTG

Lou

TTT

Phe 95

GCC

Gly

CCT

Pro

CTG

Lau GAG GAG Giu Clu GGC GCG Cly Ala CAC GAG His Giu CCC CCC Ala Gly 65 TGC TAC Cyc Tyr 80 TTT ATT Ph& Ile CAC TTC Gin Ph.

TTA 7?? Lou Ph.

MAT CTG Amn Val 145 Ala

GCC

Ala

CC

Arg 50

GAG

Giu

AAG

Lysu

TGC

Cym

ACA

Thr

GMA

Giu 130

TAC

Tyr Arg

CCC

Pro 35

C

Gly

ATC

Ile

MAC

Ann

TGT

Cys

AG?

5cr 115

C

Giy

TAC

Tyr Glu

GCC

Ala

CAC

His

ATT

Ile

GGA

Gly

GGA

Gly 100

GMA

Giu

AT?

Ile

ATC

Ile Ser

CC

Arg

TOG

Trp

GGG

Oly

GGA

Cly

ATT

Ile

GT

Gly

GCC

Gly

ATC

Ile Glu

CAC

His

AAC

Ann

CTG

Lou 000 Gly

CC?

Pro

GCC

Cly

TAT

Tyr

ATC

Ile 150 GCG CGG GAG TCC GAG 148 196 244 292 340 388 436 484 CTC GCA TOG 0CC AT? TTT TAC CTC AGC AAC TGC TTC ACT ACT GAG CTA Glu Lou Lou Ala Trp Ala Ile 155 Phe Tyr Lou 5cr Ann Cys Ph. Thr Thr 160 -143- CCC TG CT ACC TOT Pro Trp Ala Thr Cys 170 GG CAT GAd TGG AAC ACA GAG AAT TOT GTG GAG Gly His GiU

S

*5 S S

S

*5 S S *5eS

S

*SSS*S

5* S S

S.

TTC CAG Phe Gin GCC ACC Ala Thr 200 TCT GAC Ser Asp 215 TOT CTC Cys Lou ACC AAG Thr Lys ATC ATG Ile met TCA GAG Ser Giu 280 GAC CCC Asp Pro 295 GCC A?? Ala Ile AAC AAC Ann Asn GCC ACC Giy Thr ATG CCG Met Ala 360 CCC GOC Pro Gly 375 AAA CTC Lys LOU 185 7CC CC? Ser Pro COG ATC G2.y Ile TTG GCA Leu Ala TCT ACA Ser Thr 250 C--G CG Leu Lou 265 GGC ATC Cly Ile CAG GTC Gin Val TGC CTC Cys Leu AAC TGC Asn Cys 330 AGC TTC Ser Ph.

345 TAC GAG Tyr Clu crc GCC Lou Ala

AAT

Ann

OTC

Val1

GAG

G iu

GC

Ala 235

GOA

Gly

ATC

Ile

AAG

Lys

TG

Trp

C

Gly 315

TAC

Tyr

GTG

Val1

CAG

Gin

TTT

Ph.

CTG

Val

ATG

Met

CAC

His 220

TOG

Trp

AAG

Lys

CTC

Lou

TTC

Ph.

GTA

Val1 300

TOT

Cys

AGO

Arg

CT

Ala

CCC

Gly

ATT

Ile 380

AGC

Ser

GAG

Glu 205

ATC

Ilo

ACC

Thr

OTT

Val1

CTG

Lou

TAC

Tyr 285

GAT

Asp

CO

Lou

GAC

Asp

CCC

Cly

OTA

Val 365

C

Ala

AAC

Ann 190 7?? Phe

G

Gly

ATC

Ile

GTA

Val1

ATA

Ile 270

TTG

Lou

CT

Ala

ACC

Thr

C

Cys 7?? Phe 350

CCC

Pro

TAG

Tyr Trp 175

TAC

Tyr

TOG

Trp

AAC

Asfl

TOT

Gys

TAC

Tyr 255

CGA

Arg

TAC

Tyr

OGA

Giy

CT

Al a

ATC

Ii.

33S Cc Ala

AT?

Ilie

CCC

Pro Asn Thr Giu Ann Gym Val (G±u 180

AGC

Ser

GAG

Glii

C??

Lou

TAC

Tyr 240

OTO

Val1 000 Gly

CC?

Pro

ACG

Thr C?0 Lou 320

ATO

Hot

ATC

Ile

CT

Ala

AAG

Lys

CAT

His

CAC

His coc Arg 225

TTC

Phe

ACT

Thr

GC

Val1

GAG

Asp

CAC

Gin 305

GGA

Gly

CTC

Lou

?TC

Ph.

GAG

Ciu 0CG Ala 385

GTG

Val1

COG

Ar; 210

TOG

Trp

TOT

Cys

GG

Ala

AG

Thr

.CC

Lou 290

ATC

Ile

ACT

Ser

TOT

Gys

TCA

Ser

GTG

Val1 370

GTC

Val

TC?

Ser 195

OTC

Val

GAO

Glu

ATC

Ile

ACA

Thr Tro Lou 275

TCC

Ser

.TT

Ph.

TAT

Tyr

TOC

Cys

GTC

Val1 355

OCA

Ala

ACC

Thr CC CAG Lou Gin CO CC Lou Ala CG GCC Lou Ala TOG AAG ?rp Lys 245 TC* CCC Ph. Pro 260 CCC CG Pro Gly CCC CC Arg Lou TTC TCC Ph. 5cr AAC AAT Ann Ann 325 CTO AAC Lou Ann 340 CG OT Lou Cly GAG TCA Clu Ser ATG ATG Met Met AC TTC Ile Ph.

AAT

Ann

ATC

Ile 770 Lou 230 000 Cly

TAC

Tyr Ala

TCC

Ser

TAT

Tyr 310

TAT

Tyr

AOC

Ser

TTT

Phe

GCC

Gly

CCT

Pro 390

CTO

Lou 628 676 724 772 820 868 916 964 1012 1 060 1108 1156 1204 1252 CC TCC CCG CTG TOG CCC ACC 770 TTC TTC ATO ATC CTC Lou Ser Pro Lou Tro Ala Thr Leu Phe Ph. Met Met Leu .395 400 -144- GOC CTG GAC AOC CAG 777 070 707 Gly Lou Asp Ser Gin Ph. Val Cys 070 GAA Val Giu AGC CTG CTG ACC GCC 070 Ser Lou Val Thr Ala Val 4 4 4* 4* 4 4U U 4 U U

U.

U U.

U

9*

U

4U***U

U

U.

GTG

Val1

CTC

Lou

TTA

Leu 455

GCC

Ala

ATC

Ile A7G Met

ATG

Met

TAC

Ty r 535

TAT

Ty r

CTC

Lou

AAA

Lys

AAG

Lys

AAA

Lys 615

GAC

Asp

ATC

Ile 440

ACA

Thr

AGT

Ser

GOC

Gly

ATT

Ile

ACC

Thr 520

AAG

Ly s CGCc Gly

TG

Trp

CTC

Lou

CTT

Lou 600

CTC

ATG

Met 425

CTA

Lou

GAG

Glu

GGC

Gly

TG

Trp

GCC

Gly 505

CCT

Pro

CCA

Pro

ATT

Ile

ATC

Ile

CAG

Gin 585

GGG

Gly

AAG

TAC

Tyr

GCC

Ala

GGT

Gly

ATG

Met 070 Val1 490

TAC

Tyr

GG

Gly

CTC

Lou

GC

Gly

GC

Cys 570

AAG

Lys 070 Val

ACT

CCC

Pro 770 Lou

GCC

Gly 7C Cys 475

TAT

Tyr

CGG

Ar;

ATC

Ile

AAG

Lys

TG

Trp 555

ATC

Ile

TTC

Lou

AGC

Ser

GAC

KMG

Lys

TCT

Ser

ATG

met 460

CTT

Lou

GGA

Gly

CCA

Pro

C

Cys

TAC

Tyr 540

CTC

Lou

ACA

Thr

ACG

Thr

CCA

Pro

GGG

Gly 620 077 Val1

OTT

Val 445

TAC

Tyr

CTC

Lou

AGC

Ser

CCC

Pro

GCC

Ala 525

AAC

Ann

ATC

met

CTC

Val1

ACC

Thr

CG

Ar; 605

ACC

TTC

Ph* 430

ATC

Ile

ATC

Ile

TTC

Ph.

AAC

An 7CC Ser 510

CCC

Cly

AAC

An

CC

Ala 7CC Trp

CCC

Pro 590

ATG

Met

ATC

415

CG

Ar;

TCC

Ser

TTC

Ph.

Val1

CCC

Arg 495

CTC

Lou

ATC

110

ATC

Ile

CTC

Lou

AAC

Lys 575

AGC

Ser

CTG

Val

GCA

ACC

Ar;

TAT

Tyr

CAC

Gin

GCC

Ala 480

TTC

Ph* A77 Ile

TTC

Ph.

TAC

Tyr

TCC

Ser 560

ACC

Thr

ACA

Thr

ACA

Thr

GCC

GGT

Gly 777 Ph*

CTC

Lou 465

ATC

Ile

TAT

Tyr hAG Lys

ATC

110

ACC

Thr 545 7CC Ser

GAG

Giu

CAT

Asp 077 Val1

ATC

TAC

Tyr

CTG

Lou 450 777 Ph.

777 Ph.

GAT

Asp

TCC

Trp

TTC

Ph.

530

TAC

Tyr

ATG

Met

GGC

Gly

CTG

Lou

AAT

Ann 610

ACA

CCC

Ar; 435

GCC

Gly

GAC

Asp

GAG

Clu

AIC

Ann

C

Cy.

515

TTC

Ph.

CCA

Pro

CTC

Lou

ACA

Thr

AAA

Lys 595

GAC

Asp

GAG

CCC

Arg

CTC

Lou 7CC Ser 7C Cys

ATT

11e 500 7CC Trp 770 Lou

CC

Ala 7C Cys Cro Lou 580

ATG

met

TGT

Cym

ARG

GAG

Glu 070 Val

TAT

Tyr

ATC

Ile 485

GAA

Giu

ATG

Met

ATC

Ile

TG

Trp

ATC

Ile 565

CCC

Pro

CCC

Arg

GAT

Asp

GAG

CTG

Lou

ATG

Met

CC

Ala 470

C

Cys

CAC

Asp

ATC

110

AAG

Lys

GCC

Cly 550

CCC

Pro

GAG

C iu

GCC

Cly

CC

Ala

ACG

1300 1348 1396 1444 1492 1540 1588 1636 1684 1732 1780 1828 1876 1924 1980 1991 Lou Lys Ser Asp Thr Ile Ala Ala Ile Thr Giu Lys Giu Thr 625 630 His Ph.

ATGTAAATGA A -145- INFORMATION FOR SEQ ID 140:10: SEQUENCE CHARACTERISTICS: LENGTH: 632 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Thr Ala Glu Lys Ala Leu Pro Lou Gly Amn Gly Lye Ala Ala Glu 1 5 10 Glu Ala Arg Glu Ser Glu Ala Pro Gly Gly Gly Cys Ser Ser Gly Gly 25 Ala Ala Pro Ala Arg His Pro Arg Val Lye Arg Asp Lys Ala Val His 40 Glu Arg Gly His Trp Ann Asn Lys Val Glu Phe Val Lou Ser Val Ala 55 y Glu Ile Ile Gly Lou Gly Aen Val Trp Arg Phe Pro Tyr Lou eye 70 75 s0 Tyr Lys Ann Gly Gly Gly Ala Phe Lou Ile Pro Tyr Val Val Phe Ph.

90 Ile Cys Cys Gly Ile Pro Val Ph. Ph. Lou Glu Thr Ala Lou Gly Gin 100 105 110 Phe Thr Ser Glu Gly Gly Ile Thr Cys Trp, Arg Lye Val Cys Pro Lou Se.115 120 125 Phe Glu Gly Ile Gly Tyr Ala Thr Gin Val Ile Glu Ala His Lou Ann 130 135 140 Val Tyr Tyr Ile Ile I'le Lou Ala Trp Ala Ile Phe Tyr Lou Scr Ann 145 150 155 160 Cys Phe Thr Thr Glu Lou Pro Trp Ala Thr Cys Gly His Glu Trp Ann 165 170 175 Thr Glu Asn Cys Val Glu Phe Gin Lye Lou Ann Val Ser Aen Tyr Ser 180 185 190 His Val Ser Lou Gin Asn Ala Thr 5cr Pro Val Met Glu Ph. Trp Glu 195 200 205 His Arg Val Lou Ala Ile Ser Asp Gly Ile Glu His Ile Gly Ann Lou 210 215 220 Arg Trp Glu Leu Ala Lou Cys Lou Lou Ala Ala Trp Thr Ile Cys Tyr 225 230 235 240 Ph. Cys Ile Trp Lys Gly Thr Lye 5cr Thr Gly Lye Val Val Tyr Val 245 250 255 Thr Ala Thr Ph. Pro Tyr Ile Met Lou Lou Ile Lou Leu Ile Arg Gly 260 265 270 -146- Val Thr Leo Pro Gly Ala Ser 275 Glu 280 Gly Ile Lys Phe Leu Tyr Pro

S

S

S.

*0e* *0 0

S

S.

Asp Gln 305 Gly Lou Phe Glu Ala 385 Met Ser Gly Phe Leu 465 lle Tyr Lys Ile Thr 545 Ser Glu Asp Leu 290 Ile Ser cys Ser Val 370 Val Met Leu Tyr Leo 450 Phe Phe Ap Trp Phe 530 Tyr Met Gly Lau Ser Ph.

Tyr Cys Val 355 Ala Thr Lou Val Arg 435 Gly Asp Glu Asn Cys 515 Ph.

Pro Lau Thr Lys 595 Arg Phe Asn Lou 340 Lou Glu met Ile Thr 420 Arg Lou Ser CyS Ile 500 Trp Leu Ala Cys Lou 580 Met Lou ser Asn 325 Asn Gly Ser Met Ph.

405 Ala Glu Val Tyr Ile 485 Giu Met Ile Trp Ile 565 Pro Arg Ser Tyr 310 Tyr Ser Ph* Gly Pro 390 Lou Val Leu Met Ala 470 ys Asp Ile Lys Gly 550 Pro Glu Gly Asp 295 Ala An Gly Met Pro 375 Lau Gly Va1 Lou Lou 455 Ala Ile Met Met Tyr 535 Tyr Leu Lys Lys Ile Asn Thr Ala 360 Gly Ser Lou Asp Ile 440 Thr Ser Gly Ile Thr 520 Lys Gly Trp Lou Lou 600 Cys Asn Ser 345 Tyr Lou Pro Asp Met 425 Lau Glu Gly Trp Giy

SOS

Pro Pro Ile Ile Gln 585 Gly Lou Cys 330 Ph.

Glu Ala Lou Ser 410 Tyr Ala Gly Met Val 490 Tyr Gly Lou Gly Cys 570 Lys Val Gly 315 Tyr Val Gln Ph.

Trp 395 Gln Pro Lau Gly Cys 475 Tyr Ar; lIle Lys Trp 555 Ile Lau Ser Pro Gin Val Trp Va1 300 Cys Ar; Ala Gly Ile 380 Ala Ph.

Lys Ser Met 460 Lou Gly Pro Cys Tyr 540 Lou Thr Thr Pro Asp Lou Asp Gly Val 365 Ala Thr Val Va1 Val 445 Tyr Lou Ser Pro Ala 525 An Met Val Thr Arg 605 Ala Thr Cys Phe 350 Pro Tyr Leu Cys Ph.

430 Ile Ile Ph.

Asn Sor 510 Gly Asn Ala Trp Pro 590 Met Gly Ala Ile 335 Ala Ile Pro Phe Va1 415 Arc Ser Phe Val Arg 495 Lou Ile Ile Lou Lys 575 5cr Val Thr Leu 320 Met Ile Ala Lys Ph.

400 Glu Ar; Tyr Gln Ala 480 Ph.

Ile Ph.

Tyr Ser 560 Thr Thr Thr 147 Val Asn Aup Cym Amp Ala Lys Leu Lys Ser Asp Gly Thr I3.e Ua Ala 610 615 620 lie Thr Glu Lys Glu Thr His Phe 625 630 4 4 4* .4 4 4 4444 .4 4 4 .4.

4 4 .4 4* 44 4 .4 4 4 4.

44*444 4 44 4 4 ~4 4 44 -148- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An isolated nucleic acid molecule encoding a rat taurine transporter having an amino acid sequence substantially similar to the amino acid sequence shown in Seq. I.D.

Nos. 5 and 6.

2. An isolated nuclei acid molecule encoding a human taurine transporter having an amino acid sequence substantially similar to the amino acid sequence shown in Seq. I.D.

Nos. 5 and 6.

3. An isolated nucleic acid molecule of either of claims 1 or 2, wherein the nucleic acid molecule is a DNA molecule.

4. A DNA molecule of claim 3 wherein the DNA molecule is a cDNA molecule.

5.

6 OS.6.

S 5

S

*SS 7 *5 7

S

5 A vector comprising the DNA molecule of claim 3.

A vector of claim 5, wherein the vector is a plasmid.

A vector of claim 5 adapted for expression in a bacterial cell which comprises regulatory elements necessary for expression of the DNA in the bacterial cell so located relative to the DNA encoding the transporter as to permit expression thereof.

A vector of claim 5 adapted for expression in a yeast cell which comprises regulatory elements necessary for expression of the DNA in the yeast cell so located relative to the DNA encoding the transporter as to permit expression thereof.

0005 0 55 0*

S.

S.

S

0@

S.

S

500 0 0 0

SO

Claims (21)

1. 9. A vector of claim 5 adapted for expression in a mammalian cell which comprises regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding the transporter as to permit expression thereof. A plasmid of claim 6 adapted for expression in a mammalian cell which comprises regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding the transporter as to permit expression thereof.
2. 11. A plasmid designated pEVJB-rB16a (ATCC Accession No. 75202).
3. 12. A mammalian cell comprising the plasmid of claim
4. 13. The mammalian cell of claim 12, wherein the mammalian cell is a Cos7 cell.
5. 14. A Cos7 cell comprising the plasmid of claim
6. 15. A membrane preparation obtained from a mammalian cell of claim 12.
7. 16. A method for determining whether a compound not known to be capable of specifically binding to a taurine transporter can specifically bind to the taurine transporter on the surface of a mammalian cell, which comprises contacting a mammalian cell of claim 12 which comprises a plasmid adapted for expression in the mammalian cell, wherein the plasmid comprises DNA which expresses a taurine transporter on the cell's surface, with the compound under conditions permitting binding of ligands known to bind to the taurine transporter, and detecting whether the compound binds to the taurine transporter so as to thereby determine that the compound -150- is capable of specifically binding to the taurine transporter.
8. 17. The method of claim 16, wherein the mammalian cell is a non-neuronal cell.
9. 18. The method of claim 17, wherein the non-neuronal cell is a Cos7 cell.
10. 19. A method for identifying a compound which enhances or decreases taurine transporter activity which comprises contacting a mammalian cell of claim 12, which comprises a plasmid adapted for expression in the mammalian cell wherein the plasmid comprises DNA which expresses a taurine transporter on the cell's surface, with the compound under suitable conditions to permit binding of the compound, and detecting an increase or decrease in taurine transporter activity.
11. 20. The method of claim 19, wherein the mammalian cell is a non-neuronal cell.
12. 21. The method of claim 20, wherein the non-neuronal cell is a Cos7 cell.
13. 22. A method of screening to identify compounds which interact with, and specifically bind to, a taurine transporter on the surface of a mammalian cell, which comprises contacting a mammalian cell of claim 12 which comprises a plasmid adapted for expression in the mammalian cell, wherein the plasmid comprises DNA which expresses a taurine transporter on the cell's surface, with a plurality of compounds under conditions permitting binding of ligands known to bind to the taurine transporter, determining which compounds specifically bind to the taurine transporter so as to thereby identify -151- compounds which interact with, and specifically bind to, the taurine transporter.
14. 23. The method of claim 22, wherein the mammalian cell is a non-neuronal cell.
15. 24. The method of claim 23, wherein the non-neuronal cell is a Cos7 cell. A method for determining whether a compound not known to be capable of specifically binding to a taurine transporter can specifically bind to the taurine transporter on the surface of a mammalian cell, which comprises contacting a membrane preparation according to claim 15 from a mammalian cell which comprises a plasmid adapted for expression in the mammalian cell, wherein the plasmid comprises DNA which expresses a taurine transporter on the cell's surface, with the compound under conditions permitting binding of ligands known to bind to the taurine transporter, and detecting whether the compound binds to the taurine transporter so as to thereby determine that the compound is capable of specifically binding to the taurine transporter.
16. 26. The method of claim 25, wherein the mammalian cell is a non-neuronal cell.
17. 27. The method of claim 26, wherein the non-neuronal cell is a Cos7 cell. S 28. A method for identifying a compound which enhances or decreases taurine transporter activity which comprises contacting a membrane preparation according to claim from a mammalian cell, which comprises a plasmid adapted for expression in the mammalian cell wherein the plasmid comprises DNA which expresses a taurine transporter on Sthe cell's surface, with the compound under suitable -152- conditions to permit binding of the compound, and detecting an increase or decrease in taurine transporter activity.
18. 29. The method of claim 28, wherein the mammalian cell is a non-neuronal cell. The method of claim 29, wherein the non-neuronal cell is a Cos7 cell.
19. 31. A method of screening to identify compounds which interact with, and specifically bind to, a taurine transporter on the surface of a mammalian cell, which comprises contacting a membrane preparation according to claim 15 from a mammalian cell which comprises a plasmid adapted for expression in the mammalian cell, wherein the plasmid comprises DNA which expresses a taurine transporter on the cell's surface, with a plurality of compounds under conditions permitting binding of ligands known to bind to the taurine transporter, determining which compounds specifically bind to the taurine transporter so as to thereby identify compounds which interact with, and specifically bind to, the taurine transporter. 05
20. 32. The method of claim 31, wherein the mammalian cell is a non-neuronal cell.
21. 33. The method of claim 32, wherein the non-neuronal cell is a Cos7 cell. DATED this 25th day of February 1999 Synaptic Pharmaceutical Corporation by their Patent Attorneys DAVIES COLLISON CAVE S. P:OPER\MROTAUpJIflABS 21/8/98 ABSTRACT The present invention provides isolated nucleic acid molecules, proteins, monoclonal antibodies, pharmaceutical compositions, transgenic animals, methods of treatment, methods of screening, and methods of diagnosis in respect of the GABA transporter and taurine transporter. More particularly, the present invention relates to the taurine transporter. 00 *0 0 0000 0000 0 0000 00 00 0 0000 9 09.. 00 0 00 9e 0 S 00 OeOO 00 00 0 0* 00 0 0 09 0000 0 0000 S 000000 0 £0 0 CO 00