WO1993008262A1

WO1993008262A1 - A cDNA CLONE ENCODING AN EXPRESSIBLE DOPAMINE TRANSPORTER

Info

Publication number: WO1993008262A1
Application number: PCT/US1992/008874
Authority: WO
Inventors: Susan G. Amara; John E. Kilty; Dominique Lorang
Original assignee: Oregon Health and Science University; Oregon State
Current assignee: Oregon Health and Science University; Oregon State
Priority date: 1991-10-25
Filing date: 1992-10-16
Publication date: 1993-04-29
Anticipated expiration: 1994-04-25
Also published as: AU2919292A

Abstract

A cloned cDNA encoding an expressible and functional dopamine transporter is disclosed. Also disclosed is a ca. 700-base pair portion of the dopamine transporter cDNA which is adaptable for use as a dopamine-transporter-specific probe of polynucleotides from various animal cells. Non-neural cells transfected with the cloned cDNA acquire dopamine uptake ability. The uptake by such transfected cells was inhibitable by various uptake-inhibiting drugs. The drugs exhibited a relative dopamine-uptake inhibition that was in accord with the relative effects of such drugs on dopaminergic neurons, thereby confirming that the cDNA encodes a functional dopamine transporter protein. The cDNA clone makes possible well-controlled studies of dopamine transport in cellular systems. The 700 base-pair portion is particularly suitable for high-stringency screening of cells and tissues from a variety of animal species for dopamine transporter-specific polynucleotides.

Description

A CDNA CLONE ENCODING AN EXPRESSIBLE

DOPAMINE TRANSPORTER

Field of the Invention

This invention pertains to the cloning of a. DNA sequence. In particular, this invention pertains to the cloning of a complete complementary DNA (cDNA) sequence for a neurotransmitter transporter protein and the cloning of a portion of said cDNA sequence usable for high-stringency screening of such genes in animal cells. Background of the Invention

Transmission of a nerve impulse across a chemical synapse involves the secretion of neurotransmitter substances by the presynaptic neuron into the synaptic cleft. This facilitates the transmission of a chemical signal across the synaptic cleft to the postsynaptic neuron. Transmission of the chemical signal is normally transient. Otherwise, if the neurotransmitter substances persisted in the synaptic cleft, a new signal would not get through. Nervous tissue normally disposes of soluble or unbound neurotransmitter in the synaptic cleft by various mechanisms, including diffusion and enzymatic degradation. In addition, at most synapses, chemical signaling is terminated by a rapid reaccu ulation of neurotransmitter into presynaptic terminals. This reaccumulation is the result of reuptake of the neurotransmitter by the presynaptic neuron. Of the various known disposal mechanisms for neurotransmitters, reuptake of the neurotransmitter from the synaptic cleft is probably the most common mechanism used for terminating the chemical signal.

At presynaptic terminals, the various molecular apparatuses for reuptake are highly specific for such neurotransmitters as choline and the biogenic amines (low molecular-weight neurotransmitter substances such as dopamine, norepinephrine, epinephrine, serotonin, and histamine) . These apparatuses are termed "transporters" because they transport the corresponding neurotransmitter from the synaptic cleft back across the cell membrane of the presynaptic neuron into the cytoplasm of the presynaptic terminus.

Certain psychotropic drugs such as cocaine and imipramine are effective because they block these reuptake processes by, for example, interfering with action of one or more transporters. The administration of such drugs to block reuptake prolongs and enhances the action of neurotransmitters such as the biogenic amines. These drugs also include therapeutic antidepressants and amphetamines.

Different aspects of cocaine's effects can be ascribed to inhibition of specific biogenic a ine transporters. For example, the elevations of blood pressure and increased cardiac excitability are largely the consequences of increased sympathetic tone caused by reuptake blockade at both peripheral noradrenergic synapses and central noradrenergic terminals of fibers originating from cell bodies in discrete brainstem nuclei. However, the addictive properties of cocaine have been attributed to inhibition of dopamine reuptake in targets of the esolimbic dopamine system, such as the nucleus accumbens. Ritz et al., Science 237:1219 (1987) . Determining whether these dopamine transporters are structurally or functionally distinct from dopamine transporters in other dopaminergic systems such as the nigro-striatal pathway, the retina, the hypothalamus, or the olfactory bulb is an important step in understanding the neurobiologic basis of cocaine abuse. Also, elucidating the structural and functional distinctions between different types of transporters, including non- dopaminergic transporters, is important in understanding the cellular and molecular bases of behavior.

Study of the action of psychotropic drugs on the cellular and molecular level has heretofore been hindered by the inability of researchers to isolate cells having only a single species of transporter. Neural cells typically have multiple species of transporters and/or produce interfering enzymes. Studies with such cells require complicated kinetic studies and/or blocking protocols in an attempt to isolate the behavior of the transporter of interest. Such studies have also been hindered by the unavailability of a variety of cloned transporter genes. While several transporters have recently been cloned (see. Guastella et al., "Cloning and Expression of a Rat Brain GABA Transporter," Science 249:1303 (1990); and Pacholczyk et^'al., "Expression Cloning of a Cocaine- and Antidepressant-Sensitive Human Norepinephrine Transporter," Nature 350:350 (1991)), it would be advantageous for rapid progress in the field to have • available cloned genes encoding other transporters such as the dopamine transporter. In particular, the availability of a cloned dopamine transporter gene would permit a wide variety of heretofore impossible experiments aimed at elucidation of the molecular mechanisms of psychotropic drug action on dopamine uptake, such as the action of cocaine.

The genomes of multicellular animals are very large, and specific transporter genes (which are expressed in a relatively small number of specialized cells in, for example, the mammalian body) apparently represent a very small portion of the animal genome. Consequently, and as a result of other limitations in currently available technology, isolation of transporter gene sequences (particularly intact transporter genes) is very difficult. It would be advantageous to have transporter-specific hybridization probes to permit accurate screening of gene libraries from various animal species for the transporter gene of interest.

Hence, there is a need for a cloned, intact, expressible dopamine transporter gene. There is also a need for a hybridization probe usable for screening of animal genomes to permit identification and isolation of gene sequences specific for dopamine transporters. There is also a need for a means for studying the action of dopamine transporters on the cellular and molecular level without the interfering and obfuscating influence of the complex array of enzymes in other cellular tissue.

There is also a need for a means by which the interactive behavior of psychotropic drugs on dopamine transporters can be studied in isolation from other transporters. There is also a need for a means whereby functioning transporters of an animal species can be transferred to non-neuronal cells for study.

Summary of the Invention The above-listed needs are met by one aspect of the present invention which provides a complementary DNA (cDNA) encoding a complete dopamine transporter gene. The cDNA was maintained in a clone named "pDAT". According to another aspect of the present invention, another clone is provided, named "KW27", which comprises a ca. 700 base-pair (bp) portion of the dopamine transporter cDNA. We show herein that KW27 is usable as a high-stringency probe to identify and isolate DAT-specific polynucleotide sequences from cells, including the entire coding sequence for the dopamine transporter.

To prepare pDAT and KW27, degenerate oligonucleotides were synthesized corresponding to regions of high sequence identity between the norepinephrine transporter (NET) and the γ-aminobutyric acid transporter (GABAT) . The oligonucleotides were used in polymerase chain reactions (PCR) with mRNA from mid-brain. The amplified DNA sequences were cut with a restriction endonuclease, electrophoresed, precipitated, and ligated into expression vectors to create a library.

One clone, designated "KW27", from the library had a 700-bp nucleotide sequence that was similar but not identical to a corresponding region in the NET gene sequence. KW27, when radiolabeled and used as a probe in Northern blots of RNA isolated from rat brain tissues, hybridized specifically to a 3.6 kilobase (kb) mRNA isolated from the substantia nigra, a brain region known to be especially rich in dopaminergic neurons.

Radiolabeled KW27 was used to probe cDNA libraries from idbrain and substantia nigra at high-stringency. Selected "KW27-positive" clones having DAT-specific sequences at least 1.5 kb long were sequenced. One clone, named "pDAT", had a complete open reading frame encoding a putative protein having DAT characteristics and exhibiting substantial amino-acid conservation with NET and GABAT.

The DAT-gene specificity of KW27 was demonstrated by in situ hybridization to sections of brain tissue. KW27 hybridized intensely and selectively to the substantia nigra, ventral tegmental areas, and the periphery of the olfactory bulb, all of which are known to be rich in dopaminergic neurons. When pDAT was transfected interspecifically into HeLa (non-neuronal) cells that normally do not accumulate dopamine, the cells acquired the ability to accumulate the neurotransmitter with accumulation kinetics that agreed substantially with the kinetics of dopamine accumulation by striatal synaptosomes.

Dopamine accumulation in these, cells also exhibited the same relative sensitivity to three uptake antagonists. Brief Description of the Drawings FIG. 1 shows the complete amino sequences of the norepinephrine and GABA transporters as disclosed in the prior art, and the putative dopamine transporter protein encoded by pDAT dopamine transporter cDNA according to the present invention.

FIG. 2 shows the complete nucleotide sequences of pDAT and KW27.

FIG. 3 shows cumulative dopamine uptake by pDAT- transfected HeLa cells; the inset is an Eadie-Hofstee plot of the uptake. FIG. 4 shows the effect of desipramine, cocaine, and azindol on dopamine uptake by pDAT-transfected HeLa cells .

Detailed Description A dopamine-transporter cDNA (DNA sequence complementary to a messenger RNA (mRNA) produced by the dopamine transporter gene) according to the present invention was isolated and cloned using a strategy that exploited certain similarities in the amino-acid sequences of other transporter proteins, namely the norepinephrine transporter (NET, responsible for uptake of norepinephrine, one of the "biogenic amine" group of neurotransmitters) and the γ-aminobutyric acid transporter (GABAT, responsible for uptake of γ- aminobutyric acid, one of the "amino acid" group of neurotransmitters) . It was hoped when we began these studies that the dopamine transporter (DAT) would exhibit a similar degree of amino-acid sequence conservation, which would provide a way to selectively isolate and clone cDNA encoding the DAT.

The NET amino-acid sequence is shown in FIG. 1, as disclosed- in Pacholczyk et al., Nature 350:350 (1991) and U.S. Patent application Serial No. 07/676,980, filed on March 28, 1991, by the same authors as the Nature paper. The '980 application, of which one of the inventors (Amara) is an inventor of the present invention, is incorporated herein by reference. The amino-acid sequence of the GABAT is also shown in FIG. 1, as obtained from Guastella et al. , Science 249:1303- 1306 (1990) and Nelson et al. , FEBS Lett. 269:181-184

(1990) . In FIG. 1, the amino-acid sequences of NET and GABAT are shown in register to illustrate highly conserved regions (shaded areas) . The brackets labeled with roman numerals designate regions believed to be transmembrane domains.

Two highly conserved regions between NET and GABAT were selected and used to design oligonucleotide primers to be used in a polymerase chain reaction (PCR) protocol to selectively isolate DAT cDNA from cells known to exhibit a high degree of DAT expression. Referring to FIG. 1, the first conserved region that was selected resides near the beginning of the transmembrane .region designated "II" and comprises the amino acids

KNGGGAFLIPY. This first conserved region is referred to herein as the "KNG" region. The second conserved region that was selected resides near the beginning of the transmembrane region designated "VI" and comprises the amino acids WIDAATQIFF. This second conserved region is referred to herein as the "WID" region. In FIG. l, abbreviations for the amino acids are as indicated below:

A ala D asp

F phe

H his

K lys

M met P pro

R arg

T thr

W trp

Our cloning strategy was to prepare oligonucleotide. primers corresponding to the KNG and WID regions to be used to find and amplify genomic DNA regions encoding DAT.

The amino-acid sequences of the KNG and WID regions enabled oligonucleotide primers for PCR to be designed comprising codons that would encode either the KNG or

WID amino-acid sequence. Because of the degeneracy of the genetic code, more than one oligonucleotide could encode each region. As a result, for each KNG and WID region, we prepared a "pool" comprised of all possible oligonucleotides capable of encoding the respective region. The KNG pool comprised the PCR "sense" primers each having the following coding sequence:

CCGCTCGAGAAGAACGG(C/T)GG(C/T)GG(C/T)GC(C/T)TTC(C/T)T(G/A )AT(C/T)CC(A/G)TA, wherein the eight portions residing within parentheses reflect the degeneracy of the code. As a result, the KNG pool comprised oligonucleotides having 2⁸ = 256 different, yet similar, coding sequences. The WID pool comprised the PCR "antisense" primers each having the following coding sequence:

GCTCTAGAAA(G/A)AAGATCTG(G/A)GT(G/T)GC(G/A)GC(G/A)TC(G/A/ C)A(G/T)CCA, wherein the seven portions residing within parentheses also reflect the degeneracy of the cod. As a result, the WID pool comprised oligonucleotides having 3x2⁶ = 192 different, yet similar, coding sequences. The oligonucleotides comprising the KNG and WID pools were prepared using a conventional automated nucleotide synthesizer apparatus. In addition to the coding sequences shown, eight additional nucleotides were added to the 5' ends of the KNG oligonucleotides to form an Xhol cleavage site and eight additional nucleotides were added to the 5' ends of the WID oligonucleotides to form an Xbal cleavage site. These cleavage sites were added to aid subsequent cloning. Tissue from midbrain (including the substantia nigra and ventral teg ental areas) was dissected from rats. The midbrain tissues were selected because they were known to be relatively rich in DAT. Poly-A selected mRNA was isolated from these tissues using conventional methods. Chirgwin et al., Biochem. 18:5294 (1979) . "First-strand" (single-stranded) cDNA was prepared using RAV reverse transcriptase (Amersham

Corp., Arlington Heights, Illinois), wherein the mRNA was used as a template and random hexanucleotides were used as primers. The first-strand DNA was used as a template in PCR reactions using the KNG and WID primer pools.

The PCR reactions were performed using conventional techniques, Mullis and Faloona, Methods in Enzymol. 155:335 (1987), and the thermostable DNA polymerase from Thermus aquaticus. Saiki, et al., Science 239:487 (1988) . Each PCR reaction contained cDNA synthesized from 50 ng of RNA. Thermal cycling was carried out for 25 cycles, wherein each cycle comprised a regimen of 94°C for one min. , 47°C for 2 min. , and 72°C for 3 min. , followed by a soak at 72°C for 12 min. The PCR reactions produced amplified DNA sequences presumably including a number of transporter sequences and, it was hoped, DAT sequences. The amplified DNA sequences were cleaved using

EcoRI. This endonuclease enzyme was known to cleave the GABAT gene but was otherwise known as an "infrequent cutter". Thus, we were able to readily remove GABAT- specific sequences from the population of amplified DNA molecules (as' confirmed electrophoretically) . We hoped, however, that cleaving with EcoRI did not cleave DAT- specific sequences. The remaining "uncut" DNA was then cleaved with Xhol and Xbal to create sticky ends. The resulting fragments were electrophoresed on a 5% polyacrylamide gel. Selected bands were eluted from the gel, precipitated, and ligated into "Bluescript" SKII(-) vectors previously cut using Xhol and Xbal. (Bluescript vectors are available from Stratagene, La Jolla, California. They contain the T7 promoter and are popular expression vectors.) The recombinant vectors were used to create a library of bacterial clones each containing a length of PCR-amplified DNA.

We sequenced a number of clones using the conventional "dideoxy" sequencing method. Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977). We soon found that most of the clones shared the same sequence. Because available evidence indicated that the dopamine transporter was relatively rare among transporters, we surmised that clones containing commonly occurring sequences did not contain DAT-gene sequences. Using one of the commonly occurring sequences as a probe, we screened a large number of clones to eliminate from further consideration those clones containing the commonly occurring sequences, thereby leaving a relatively small number of clones that contained less common sequences. We hoped that at least one of these remaining clones contained a DAT gene sequence. Upon sequencing the remaining clones, we found that one, which we designated "KW27", had a sequence about 700 base pairs (bp) long that was relatively similar to a corresponding region in the NET gene but less similar to a corresponding region in the GABAT gene. This preliminary result was in accordance with other research data indicating that DAT may be more similar to NET than to GABAT. Therefore, we strongly suspected that KW27 contained a DAT-specific sequence. If so, then the ca. 700-bp length of KW27 would be highly discriminating for the DAT gene if a DAT-specific probe could be made from KW27.

We performed an experiment designed to confirm that KW27 indeed contained DAT gene sequences. We prepared [³²P]-CTP-labeled KW27 adapted for use as a hybridization probe of mRNA produced by various brain tissues. (Adapting KW27 for use as a hybridization probe also included denaturation. ) We similarly prepared radiolabeled probes based on corresponding sequences from other selected clones. The probes were prepared using the "random priming" method known in the art. Feinberg and Vogelstein, Anal. Bioche . 132:6 (1983). (We used the "Random Prime DNA-Labeling Kit" available from Boehringer Mannheim Biochemicals, Indianapolis, Indiana) . RNA isolated from various brain tissues was separated by size on denaturing agarose electrophoretic gels and transferred to nylon membranes according to the conventional "Northern" hybridization blotting technique. See, Alwine et al., Proc. Natl. Acad. Sci. USA 74:5350 (1977); Alwine et al. , Methods in Enzvmol. £8_.:220 (1979). The membranes were incubated with the radiolabeled probes. Hybridization of the ca. 700 bp KW27 fragment was very stringent. Subsequent autoradiography revealed that a 4-kilobase (kb) mRNA band originally isolated from the substantia nigra was strongly labeled with the KW27 probe. The length of this mRNA was subsequently more accurately measured to be 3.6 kb. Bands isolated from other brain tissues were poorly labeled with KW27. (The substantia nigra is a midbrain tissue known to have many dopaminergic neurons; therefore, the substantia nigra produces a relatively high amount of DAT mRNA.) The other probes did not produce such a marked labeling contrast of the substantia nigra versus other areas of the brain. These results indicate that KW27 is a highly discriminating probe for DAT-specific nucleotide sequences and that the strongly labeled ca. 4-kb RNA band contained a DAT gene sequence. These results also indicate that KW27 can be reliably used to isolate DAT-specific nucleotide sequences (RNA or DNA polynucleotides) from cells.

KW27 enabled us to determine that there is a single dopamine transporter gene product encoded by the ca. 4- kb mRNA. The same mRNA appears to be expressed in all dopaminergic cell bodies including those of the nigro- striatal and meso-limbic systems.

Now that we had found KW27 and had confirmed its specificity for DAT-specific sequences, radiolabeled KW27 was used to perform high-stringency screening of midbrain and substantia nigra cDNA libraries in an effort to find a full-length cDNA encoding DAT. In particular, first-strand cDNA was generated from poly(A)-selected mRNA (Chirgwin et al., Bioche . 18:5294 (1979) ) isolated from substantia nigra and midbrain using RAV reverse transcriptase (Amersham Corp. , Arlington Heights, Illinois) and random hexanucleotide primers. The first-strand DNA was then used to produce double-stranded cDNA by conventional methods. cDNA having blunt ends was ligated to semi-Xho adaptors

(synthetic oligonucleotide linkers known in the art) and size-fractionated on an agarose gel. cDNA having a length greater than 1.5 kb was ligated into Bluescript SKII(-) vectors and electroporated into competent E_^_ coli bacteria to create a library. (The 1.5-kb lower size limit was imposed because we wanted to isolate an entire coding sequence for DAT. We knew that the NET and GABAT polypeptides were about 600 amino acids long, requiring a minimal coding sequence length of 1.8 kb since each codon comprises three bases.)

Colony lifts (replica plates) of the resulting libraries were screened at high stringency using the radiolabeled KW27 probe. (As used herein, "high stringency" means that an unusually high "melting" temperature was required to denature the hybrids, indicating that the hybrids comprised at least very nearly perfectly homologous sequences.) cDNAs from selected "KW27-positive" colonies were completely sequenced in both strands with Sequenase (U.S. Biochemical Corp., Cleveland, Ohio) using a set of overlapping exonuclease Ill-digested unidirectional deletions. A clone, designated "pDAT", containing the complete DAT coding region was generated by ligating two overlapping clones at a Pfl MI site in the cDNAs. pDAT comprised a single open reading frame encoding a putative protein having an amino-acid sequence and other characteristics strongly suggestive that it was a dopamine transporter.

The complete nucleotide sequence of pDAT is shown in FIG. 2. (and in Seq. ID No:l). In FIG. 2 (and in Seq. ID No:l), the probable amino-acid sequence of the DAT is also shown, as predicted by deciphering the nucleotide sequence of pDAT. Nucleotides are counted starting from the first residue in the insert. Stars are placed above every twentieth nucleotide, and every hundredth nucleotide is numbered. Numbering of amino-acid residues begins at the putative translational start site (ATG) with cumulative amino-acid counts shown in the right margin. Putative transmembrane domains are square-bracketed and underlined. Potential glycosylation sites on a putative extracellular loop are denoted by double underlining (beneath four separate amino-acid triplets) . Two potential sites for phosphorylation by protein kinase C in the N-terminal cytoplasmic domain and one potential site for phosphorylation by either protein kinase C or calmodulin-dependent protein kinase II .in the C-terminal cytoplasmic domain are indicated by solid triangles. Abbreviations of the amino-acid residues are as tabulated above. Referring further to FIG. 2, of the two potential translational start codons at the 5' end of the cDNA, the nucleotide sequence surrounding the upstream methionine (M) codon at nucleotide 90 has the best match to the Kozak consensus sequence, Kozak, Nucl. Acids Res. .15:8125 (1987), and therefore probably represents the translational start site. However, as also observed in the norepinephrine transporter cDNA sequence, a pyrimidine is present at the^" third position upstream from the ATG. This has only been observed in three percent of mRNAs included in the Kozak study and may suggest that such mRNAs are less efficiently translated.

The PCR oligonucleotides used to isolate the KW27 probe hybridize to pDAT sequences indicated in FIG. 2 (and in Seq. ID No:l) by double underlining. The sequence of the intervening nucleotides is identical to the sequence of the cloned PCR product, KW27. It will be appreciated by persons skilled in the art that the specific PCR oligonucleotides shown in FIG. 2 could have been any of the corresponding "sense" or "antisense" primers, as appropriate, encoding the "KNG" or "WID" amino acid sequences, respectively.

Hydrophobicity analysis of the amino-acid sequence of FIG. 2 (and in Seq. ID No:l) revealed twelve hydrophobic segments. (See. Kyte and Doolittle, J_^ Molec. Biol. 157:105 (1982) for general information on hydrophobicity analysis.) Thus, the same topological model suggested for GABAT and NET (Pacholczyk et al., Nature 350:350 (1991); Guastella et al. , Science 249:1303 (1990); and Nelson et al., FEBS Lett. 269:181 (1990)) seems to apply to DAT, wherein all three transporters appear to have twelve transmembrane domains. Also, as in GABAT and NET, the N- and C- termini of DAT appear to be located cytoplasmically. As shown in FIG. 1, there is a high degree of amino-acid sequence conservation between the putative transporter protein encoded by pDAT and the NET and GABAT proteins. In particular, the pDAT protein and NET have about 64 percent amino-acid identity and the pDAT protein and GABAT have about 40 percent amino-acid identity. The degree of similarity increases to 75 percent and 50 percent, respectively, if conservative amino-acid substitutions are made. Therefore, in accordance with data in the research literature pertaining to DAT function, the pDAT protein shares more similarity to the NET than the GABAT.

As further illustrated in FIG. 1, the amino-acid conservation between these three transporters is distributed fairly evenly throughout the amino-acid sequences thereof except for the cytoplasmic N-termini which are relatively transporter-specific. Even in the large extracellular loop between transmembrane domains III and IV, where our PCR analysis has identified transporter-specific sequences in other members of this transporter family, pDAT has 57 percent and 33 percent amino-acid identity to the NET and GABAT, respectively.

One potentially significant difference seen with the pDAT protein versus other transporters is the presence of four predicted N-glycosylation sites in pDAT (FIG. 2) , while the other two transporters have only three sites. Also, within the N-and C-terminal cytoplasmic domains ^"of pDAT, there are amino-acid sequence motifs characteristic of calmodulin-dependent kinase II and protein kinase C substrate phosphorylation sites. Kemp and Pearson, TIBS 15:342 (1990). (Since phosphorylation is an important way in which proteins are regulated, the presence of potential phosphorylation sites in an amino-acid sequence provides some insight into how the putative protein might be regulated in living cells.) The presence of four N-linked glycosidation sites in the large extracellular loop of the dopamine transporter compared to the three sites conserved between the norepinephrine and GABA transporters raises the possibility that variations in glycosylation patterns could contribute to dopamine transporter heterogeneity. The high degree of amino-acid identity between the dopamine and norepinephrine transporters seems to reflect the similar substrate specificities and overlapping pharmacologic sensitivities. However, this increased degree of homology relative to other members of the transporter gene family is distributed throughout the protein, precluding the unambiguous identification of a localized catacholamine or cocaine binding domain with the secondary structure of the transporter. It is possible that the multiple discrete amino acids common to the catecholamine transporters but absent from the GABA transporter combine to form a similar three- dimensional catecholamine binding site. However, the subtle differences in substrate and antagonist affinities suggest that there may be fundamental structural differences in the active sites of these two transporters. In fact, prior detailed pharmacological binding studies have indicated significant differences in the conformation of the cocaine binding sites. Ritz et al. , Life Sciences 4j5:635 (1990) . The pDAT nucleotide sequence has been reported to Genbank, with accession number M80233.

Thus, according to the present invention, a cloned expressible dopamine transporter cDNA is provided for the first time. As described in the Examples hereinbelow, pDAT can be readily incorporated into vectors and transfected into other cells, including non- neuronal cells and cells from different animal species. Such pDAT-transformed cells produce functional dopamine transporter that appears to integrate functionally into the cell membrane. The activity of pDAT-encoded dopamine transporter in such cells is sensitive to antagonists of transporter function, including cocaine. Thus, it will be readily appreciated that these features of the present invention offer unlimited opportunities for the study of transporter structure and function, including mechanisms of drug addiction, on the molecular level. The present invention also provides, for the first time, a cloned cDNA segment (KW27) capable of hybridizing to a portion of the DAT gene with high stringency. KW27 makes possible, for the first time, the rapid screening of central nervous system tissues so as to isolate DAT gene sequences therefrom. Screening methodology would be as described in detail herein or other methodology familiar to skilled artisans. Since research data strongly indicate that similar transporters are highly conserved among different animal species, particularly mammalian species, it will be readily appreciated by persons skilled in the art that KW27 can be used to isolate dopamine-transporter gene sequences in a large variety of animal species.

Of course, it will be appreciated by persons skilled in the art that pDAT itself can be used in its own right as a DAT-specific probe. Example 1

The marked amino-acid similarity of the deduced DAT protein to the NET protein and the relative abundance in midbrain of a 4-kb mRNA that hybridized to the KW27 probe indicates that pDAT comprises a DAT cDNA. In situ hybridization studies provided further support for this, wherein autoradiograms were obtained after hybridizing KW27 to sections of rat midbrain. Cox et al., Dev. Biol. 101:485 (1984); Simerly et al. , J. Comp. Neurol. 294:76 (1990) .

Briefly, ³⁵S-labeled sense and antisense cRNA transcripts of KW27 were synthesized using T7 and T3 RNA poly erases and used to probe cryostat sections of perfusion-fixed rat brain. Anesthetized Sprague-Dawley rats were initially perfused transcardially with ice- cold 4% paraformaldehyde solution followed by a second solution of 4% paraformaldehyde and 0.05% glutaraldehyde in borate buffer at pH 9.5. Twenty-micron cryostat sections mounted on slides were processed through a mild proteinase K treatment (i.e. 10 μg/mL at 37°C for 30 min.), followed by acetylation and dehydration steps. Sections were hybridized with radiolabeled denatured KW27 probe (1.5xlθ⁷ dpm/mL in a hybridization buffer containing 50% formamide, 0.25 M NaCl, lx Denhardt's solution, and 10% dextran sulfate.) Hybridization time was about 20 hours at 60°C. Sections were subsequently washed in decreasing concentrations of SSC, digested with RNase A (20 μg/mL at 37°C for 30 min.) and washed at a final stringency of O.lx SSC (wherein "SSC" denotes standard sodium citrate solution as known in the art) at 65-75°C in O.l SSC. In rostral sections of in situ hybridized tissue, two bands of intense hybridization were seen just dorsally to the cerebral peduncles corresponding exactly to the substantia nigra. More caudal sections that contain the ventral tegmental area contained an additional region of hybridization reflective of the presence of additional dopaminergic cell bodies present in this region. In situ hybridization of these sections using a "sense" cRNA probe (a negative control which does not hybridize selectively to DAT-rich regions) revealed only background hybridization. Hybridization was also seen in tissues obtained from the periphery of the olfactory bulb and discrete regions of the hypothalamus, both of which are sites of dopaminergic cell bodies. No hybridization was seen to noradrenergic cell bodies in the locus coeruleus or regions of the brainste containing serotonergic cell bodies. These results provide further support that pDAT specifically encodes a DAT protein and not another transporter. Example 2 To further confirm that pDAT represents the actual dopamine transporter and not a related gene product coincidentally expressed in dopaminergic neurons, pDAT was introduced into HeLa cells. HeLa cells, which are derived from a human cervical carcinoma, are non-neural cells widely used in the art. These cells are normally incapable of transporting dopamine. pDAT was introduced into HeLa cells by an infection/transfection technique employing the T7 promoter present in the Bluescript SKII(-) vector and T7 polymerase encoded by a vaccinia virus vector as described by Blakely et al. , Anal. Biochem. 194:302 (1991); and Fuerst et al., Proc. Natl. Acad. Sci. USA 83.:8122 (1986).

Briefly, HeLa cells were plated in DMEM (Dulbecco's Minimal Essential Medium) , 5% FBS (Fetal Bovine Serum) in 24-well plates at l-2xl0⁵ cells per well and infected with a T7 RNA polymerase-encoding vaccinia virus at a multiplicity of infection of 10 pfu/cell (wherein "pfu" denotes plaque-forming units, a measure of virus "concentration") . After 30 minutes, cells were transfected with 3 μg of lipofectin reagent (Bethesda Research Laboratories, Gaithersburg, Maryland) and 1 μg Bluescript SKII(-) vectors containing either the pDAT or pNET cDNAs inserted downstream of the T7 promoter. After 10 'to 12 hours, cells were assayed for uptake of dopamine using 10 nM radiolabeled dopamine, i.e., 3,4- [Ring-2,5,6 ³H]-dopamine (45.4 Ci/mmol) , and increasing concentrations of unlabeled dopamine for 20 minutes.

Cells were incubated in Krebs-Ringers-HEPES buffer (KRH buffer) which included 100 μM ascorbate. Assays were terminated by three ice-cold washes in KRH buffer. The cells were solubilized in 1% SDS (sodium dodecyl sulfate) for scintillation counting.

FIG. 3 comprises plots of the kinetics of dopamine uptake into pDAT-transfected HeLa cells. The larger plot shows a time-course of labeled dopamine accumulation in HeLa cells transfected with the pDAT- containing vector. As can be seen, transfected HeLa cells demonstrated saturable dopamine accumulation. The inset plot is an Eadie-Hofstee plot of initial velocity data. The Eadie-Hofstee plot enabled us to determine the value of K_M, the Michaelis constant. of dopamine uptake by the dopamine transporter. In the Eadie- Hofstee plot, -K_M is equal to the slope of the line. Although a K_M value of 885 nM is slightly lower than reported K_M ranges for dopamine uptake in striatal synaptosomes, Horn, "Mechanisms of Neuronal and Extraneuronal Transport of Catecholamines," pp. 195, in Paton (ed.), Raven Press, NY (1976); Horn, Progress in Neurobiol. 3_.4:382 (1990) , these results definitely show that pDAT, when transfected into cells normally incapable of transporting dopamine, renders the transformed cells capable of accumulating dopamine.

These results provide strong supporting evidence that pDAT encodes the dopamine transporter and that non- neural cells transformed with pDAT acquire dopamine- transporting capability. Thus, it is now possible for the first time to study dopamine transport in living non-neuronal cells free from the influences of other transporters normally present in neural cells. Example 3

Although the dopamine and norepinephrine transporters have similar substrate specificities, we attempted to distinguish them pharmacologically. That is, this Example was aimed at distinguishing the cloned pDAT activity from other transporter activities such as norepinephrine. This Example was performed similar to Example 2 in which pNET- and pDAT-transfected HeLa cells were incubated with 50 nM ³H-dopamine. However, the cells were also exposed to various concentrations of three well-characterized antagonists of catecholamine transport in synaptosome preparations: disipramine (a tricyclic antidepressant which is a norepinephrine transporter antagonist) , cocaine (a known antagonist of reuptake of biogenic amines such as dopamine, serotonin, and norepinephrine) , and mazindol (which blocks catecholamine transporters with high affinity) .

FIG. 4 shows representative dose-response curves for the inhibition of dopamine transport in pDAT- transfected HeLa cells by the three antagonists. Parallel experiments (curves not shown) were performed wherein dopamine transport was determined in pNET- transfected HeLa cells in the presence of various doses of the three antagonists. The ordinate of FIG. 4 is specific dopamine uptake expressed as a percent of dopamine transport by the pDAT-transfected cells in the absence of inhibitor. Each data point reflects the mean +/- one standard error of the mean. From these plots, inhibition constants (K_r values) were determined by ascertaining the antagonist concentration that caused a fifty-percent level of specific dopamine uptake.

Inhibition constants of dopamine transport in pNET- transfected cells were 2 nM, 4 nM, and 200 nM for mazindol, desipramine, and cocaine, respectively. From the data of FIG. 4, inhibition constants of dopamine transport in pDAT-transfected cells were 70 nM, 2 μM, and 4 μM, for mazindol, cocaine, and desipramine, respectively. This rank K_τ order for pDAT-transfected cells agrees with the ranked order of potency that would be expected for the effect of mazindol, cocaine, and desipramine on dopamine transport. Thus, the pharmacological sensitivity of dopamine transport in pDAT-transfected cells, including cocaine sensitivity, was distinguishable from the inhibition of dopamine transport in pNET-transfected cells. These results are compatible with pDAT encoding a dopamine transporter and not a norepinephrine or other transporter.

The results.of these Examples illustrate that pDAT encodes a protein exhibiting various properties of the native dopamine transporter, including an expected rank order of pharmacological inhibition by transporter antagonists.

It will be appreciated by persons skilled in the art that a cDNA clone according to the present invention can be inserted into_.any of a number of expression vectors, particularly in view of the fact that the entire nucleotide sequence of pDAT is disclosed. It will also be appreciated that suitable vectors will have at least a functional promoter. For studies of the expression of pDAT, it is advantageous that the vector be expressible in the target cells of interest. The availability of a DAT cDNA clone according to the present invention now allows pharmacologic studies of dopamine transport to be undertaken in transfected cells devoid of vesicular storage compartments and free from the obfuscating influences of other transport pathways. Thus, it will be appreciated by persons skilled in the art that pDAT permits high-level expression of the dopamine transporter to be attained in a variety of mammalian and other animal systems which heretofore has not been possible. It will also be appreciated that pDAT allows the development of far more sensitive assay systems for ascertaining the effects of various drugs on transport function than has heretofore been possible.

The pDAT clone can also be used in conjunction with various animal-cell expression systems (transfected by pDAT) to provide a screen for identifying more selective pharmacologic agents that affect dopamine transport function.

The pDAT clone can be used to ascertain potential side effects of various pharmacologic agents related to their ability to interact with the dopamine transporter.

Also, since various compounds are accumulated intracellularly by dopamine transporter, pDAT can be used diagnostically for imaging studies involving such compounds and other potential agents. The ability to assess the relative affinities of various agents for the transporter in pDAT-transfected cell lines can serve as a useful screen for other potentially neurotoxic agents and can suggest therapeutic options for acute intoxications. Whereas previous studies have relied upon laboratory animals as sources of membranes with transport activities, the use of animal cell lines transfected with pDAT for this and other research offers substantial advantages.

Having illustrated and described the principles of our invention, with the inclusion herein of illustrative examples and drawings, it should be apparent to persons of ordinary skill in the art that the specific examples described herein may be modified in detail without departing from such principles. We claim as our invention all such modifications as come within the true spirit and scope of the following claims.

Claims

1. A cloned cDNA encoding a dopamine transporter.

2. A cloned cDNA comprising a nucleotide sequence as disclosed in FIG. 2 and in Seq. ID No:l.

3. A cloned cDNA as recited in claim 2 encoding a dopamine transporter.

4. A cloned cDNA as recited in claim 1 encoding a mammalian dopamine transporter.

5. A cloned cDNA as recited in claim 4 encoding a rat dopamine transporter.

6. A nucleotide sequence exhibiting substantial homology with a nucleotide sequence as disclosed in FIG. 2 and in Seq. ID No:l.

7. A nucleotide sequence as recited in claim 6 exhibiting said homology under stringent conditions.

8. A polynucleotide probe that hybridizes to at least a portion of a nucleotide sequence as disclosed in FIG. 2 and in Seq. ID No:l.

9. A probe as recited in claim 8 that hybridizes under stringent conditions to said sequence.

10. A polynucleotide probe comprising a single strand of KW27.

11. A probe as recited in claim 10 further comprising a label.

12. A polynucleotide probe comprising a single strand of pDAT.

13. A DNA sequence having the identifying characteristics of KW27.

14. A DNA sequence as recited in claim 13 that hybridizes to at least a portion of a dopamine transporter gene.

15. A DNA sequence as recited in claim 14 further comprising a label.

16. A nucleotide sequence substantially homologous to KW27.

17. A DNA sequence having a 5' end and a 3' end, the sequence comprising a region that hybridizes to at least a portion of a dopamine transporter and having coupled to the 5' end an oligonucleotide sequence selected from a group consisting of the "KNG" pool of oligonucleotide primers and having coupled to the 3' end an oligonucleotide sequence selected from a group consisting of the "WID" pool of oligonucleotide primers.

18. A polypeptide encoded by the cDNA of claim 1.

19. A polypeptide encoded by the cDNA of claim 2.

20. A polypeptide encoded by the cDNA of claim 4.

21. An expression vector comprising the cDNA of claim 1.

22. An expression vector comprising the cDNA of claim 2.

23. A cell transfected with the cDNA of claim 1.

24. A cell transfected with the cDNA of claim 2.

25. A cell as recited in claim 23 that is a non- neuronal cell.

26. A cell as recited in claim 25 that synthesizes functional dopamine transporter.

27. A procaryotic cell comprising the expression vector of claim 21.

28. A procaryotic cell comprising the expression vector of claim 22.

29..A method for isolating DAT-specific polynucleotides from cells, comprising: removing polynucleotide material from the cells; separating the polynucleotide material into fractions; hybridizing the polynucleotide fractions with labeled denatured KW27; identifying polynucleotide fractions to which the labeled denatured KW27 hybridized; and separating the polynucleotide fractions to which KW27 hybridized from polynucleotide fractions to which KW27 did not hybridize.

30. A method for finding, in a population of cells, DAT gene-expressing cells, comprising: synthesizing labeled transcripts of KW27; rendering the population of cells capable of taking up the labeled KW27 transcripts; adding the labeled KW27 transcripts to the population of cells to allow the cells to take up the labeled KW27 transcripts; and identifying cells that have acquired the label.

31. A method for rendering a non-DAT-producing cell capable of producing DAT, comprising: coupling pDAT to a functional promoter in an expression vector such that transcription of pDAT can be initiated by the promoter in a susceptible non-pDAT-. producing cell; transfecting the pDAT-containing vector into the susceptible non-pDAT-producing cell; and culturing the transfected cell so as to facilitate transcription and translation of the pDAT-containing vector in the cell.

32. A method for rendering a cell normally incapable of dopamine uptake able to take up dopamine, comprising: coupling pDAT to a functional promoter in an expression vector such that transcription of pDAT can be initiated by the promoter in a susceptible cell incapable of dopamine uptake; transfecting the pDAT-containing vector into the susceptible cell; and culturing the transfected cell so as to facilitate transcription and translation of the pDAT-containing vector in the cell so as to cause the cell to synthesize DAT and take up dopamine.