EP0602044A4

EP0602044A4 - A cdna clone encoding a human norepineprhine transporter.

Info

Publication number: EP0602044A4
Application number: EP9292910856A
Authority: EP
Inventors: Susan G Amara; Tadeusz Pacholczyk; Randy D Blakely
Original assignee: Oregon State Board of Higher Education; Oregon State
Current assignee: Oregon State Board of Higher Education; Oregon State
Priority date: 1991-03-28
Filing date: 1992-02-20
Publication date: 1994-10-05
Also published as: AU1774292A; WO1992017568A1; DK107593A; EP0602044A1; FI934234A0; JPH07505040A; NO933378D0; CA2106190A1; FI934234A; DK107593D0; NO933378L

Abstract

A cDNA encoding human norepinephrine transporter isolated from human neuroblastoma cells is disclosed. Determination of the nucleotide sequence thereof permitted a putative amino-acid sequence of the transporter protein to be determined. Mammalian cells transfected with the cDNA produced an mRNA species similar in size to that produced by norepinephrine-producing neurons. Such transfected cells also became capable of norepinephrine uptake. The uptake was inhibitable by various uptake-inhibiting drugs, including cocaine, to a degree similar to the effect of such drugs on noradrenergic neurons. The cloned cDNA makes possible well-controlled studies of transporter function in non-neuronal cells without the obfuscating influence of other transporters in the same cells. Such studies include the relative effects of various drugs such as antidepressants.

Description

A CDNA CLONE ENCODING A HUMAN NOREPINEPHRINE TRANSPORTER

Field of the Invention This invention pertains to a cloned DNA sequence, encoding a mammalian protein, which can be transfected into other mammalian cells.

Background of the Invention Transmission of a nerve impulse across a 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 reaccumulation of neurotransmitter into presynaptic terminals. This reaccumulation is the result of reuptake of the neurotransmitter by the presynaptic neuron.

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. 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. 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 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.

Hence, it is an object of the present invention to provide a means to study the action of a single species of transporter on the cellular and molecular level without the interfering and obfuscating influence of the complex array of enzymes and other cellular processes of neuronal tissue. It is another object to provide a means by which the interactive behavior of psychotropic drugs on a single species of transporter can be studied in isolation from other transporters.

It is another object to provide a means whereby functioning transporters of a single species can be transferred to non-neuronal cells for study.

Summary of the Invention A cDNA encoding human norepinephrine transporter was isolated from human neuroblasto a cells using an expression-cloning strategy. After cloning the cDNA, the nucleotide sequence thereof was determined. From the nucleotide sequence data, the putative amino-acid sequence of the norepinephrine transporter protein was determined. The amino-acid sequence revealed multiple possible transmembrane domains, thus revealing the manner in which the protein is situated in the cell membrane.

The norepinephrine protein amino-acid sequence shared certain similarities with human -aminobutyric acid (GABA) transporter, although these two transporters were still distinct from each other. Nevertheless, it was possible to identify these transporters as representative of a new and distinctive gene family. When transfected into mammalian cells, the cDNA was transcribed into an mRNA species having a size similar to that found in neuronal cells that normally produce the norepinephrine transporter protein.

When transfected into non-neuronal mammalian cells that normally do not produce norepinephrine transporter, the cDNA rendered the cells capable of norepinephrine uptake. The norepinephrine uptake by the cells was inhibitable by a number of norepinephrine uptake-inhibiting drugs. Also, the degree of inhibition imparted by each drug closely matched the degree of uptake inhibition imparted by the drugs to normal noradrenergic neurons.

Included with the drugs tested herein was cocaine, which exhibited an effect on transfected cells comparable to that observed in prior studies using neuronal cells and tissues. Thus, the cloned cDNA disclosed herein is the first known cloned member of the "cocaine receptor" family.

The cDNA of the present invention makes possible, for the first time, we11-controlled studies of a specific neurotransmitter transporter conducted in the absence of competing transporters. Also possible for the first time are studies of the comparative effects of a wide variety of psychotropic and other drugs on a single species of transporter. These studies are now possible because cells transfected with an expression vector containing the cDNA are capable of producing functional norepinephrine transporter proteins that are apparently properly situated in the cell membrane. Brief Description of the Drawings

FIG. 1 shows the nucleotide sequence of the human norepinephrine transporter cDNA of the present invention, along with the deduced amino-acid sequence of the norepinephrine transporter protein encoded by the cDNA.

FIG. 2 is a schematic representation of the norepinephrine transporter protein in a putative orientation in a cell membrane.

FIG. 3 is a northern blot autoradiogram of RNAs from various cells and tissues to which radiolabeled cDNA- specific probes hybridized.

FIG. 4 is a plot of radiolabeled norepinephrine accumulation into HeLa (human non-neuronal) cells transfected with an expression vector containing the norepinephrine transporter cDNA of the present invention.

FIG. 5 is a plot of uptake velocity of radiolabeled norepinephrine into transfected HeLa cells in the presence of various concentrations of norepinephrine. FIGS. 6A and 6B are inhibition curves depicting the sensitivity to various drugs of norepinephrine uptake in transfected HeLa cells.

Detailed Description The noradrenaline transporter cDNA was isolated using an expression-cloning strategy. D'Andrea et al., Cell 5.7:277-285 (1989); Sims et al., Science 241:585-589 (1988); and Munro et al., Proc. Natl. Acad. Sci. USA _3j5:9248-9252 (1989) . The expression-cloning strategy differs substantially from the standard cloning method known in the art which is based on structure.

In the standard cloning method, the protein of interest is first isolated and purified from large amounts of cells or tissue. After the protein is sequenced, the sequence is used to predict the nucleotide sequence of the corresponding gene(s) that encodes the protein. The hypothetical nucleotide sequence is used to synthesize oligonucleotide probes which are used to search for the DNA region encoding the protein in actual cells. In the expression-cloning strategy, a cDNA encoding the protein(s) of interest is coupled to an expression vector which is introduced into a mammalian cell line. The recipient cells express the vector, thereby expressing the cDNA in a biologically active form. Thus, genes and gene products can be identified by (1) the appearance of a selectable or identifiable new phenotype in the cells (2) the production of a biologically active molecule such as a protein that can be isolated and/or assayed, or (3) the presentation on the cell surface of novel proteins encoded by the cDNA that can be identified by methods such as immunological methods or via binding to specific ligands. Thus, searching for cells that properly express the vector is relatively easy so long as a selective assay or other identification methods is employed. Expression cloning permits the isolation of genes coding for proteins for which there is no structural information and only, for example, a biological assay available.

In utilizing the expression-cloning strategy in the present instance, pools of clones from a human SK-N-SH cell (a human neuroblastoma cell line as disclosed in Bledler et al., Cancer Res. _3_3:2643-2657 (1973)) cDNA library were transfected into COS-1 cells. COS-1 cells are "immortal" African Green Monkey cells with an SV40 genome integrated into the cell genome. The transfected clones were in the form of expression vectors having an SV40 replication origin, which enabled the vectors to be amplified in these cells. Transfectants of these cells expressing the norepinephrine transporter were identified by employing an assay exploiting the fact that the norepinephrine analogue m-iodobenzylguanidine (_¹²⁵I]m-IBG) is accumulated intracellularly by SK-N-SH cells expressing an intact norepinephrine transporter. The accumulated radiolabel allowed autoradiographic visualization of transporter-expressing tranfectants. DNA was rescued from positive COS-1 transfectants by Hirt lysis. Hirt, J. Molec. Biol. 26:365-369 (1967). The resulting plasmid pools were rescreened and subdivided until a single clone was obtained.

Specifically, poly(A)-enriched mRNA was prepared from SK-N-SH neuroblastoma cells grown to confluence in 10-cm Petri dishes by the guanidinium isothiocyanate procedure. Chirgwin et al., Biochem. 18.:5294-5299 (1979). Double-stranded cDNA was generated using random hexanucleotide primers for first strand synthesis. Blunt- ended cDNA was ligated to semi-Xho adapters in a manner known in the art. Size-fractionated cDNA having a size of 1.5 kb or larger was ligated into the SV40-containing expression vector pXM. D'Andrea et al., Cell 57:277-285 (1989) . The ligation mixture was then electroporated into Escherichia coli DH10B bacteria to generate a library consisting of six pools ranging in size from about 20,000 to 50,000 independent clones, yielding a total library size of 250,000 colonies. Each pool was grown overnight and plasmid DNA was prepared by a double-banding caesium chloride procedure known in the art. A 25 μG sample from each plasmid pool was transfected into a 520 cm² plate of 15xl0⁶ COS-1 cells using DEAE-dextran (400 μg mL^"1) in Dulbecco's Minimal Essential Medium (DMEM) + 10% Nu-serum. After four hours, the cells were shocked with 10% dimethylsulfoxide in phosphate-buffered saline for 2.5 minutes. Lopata et al. , Nucl. Acids Res. 12:5707-5717 (1984) . The cells were then incubated in DMEM + 5% fetal calf serum containing 100 μM chloroquine for 4 hours. Luth an et al., Nucl. Acids Res. 1_1:1295-1308 (1983). Transfected cells were incubated for 48 hours before performi ■ng the [125I]m IBG uptake assay. lie

To perform the uptake assay, 20 μM of I]m IBG, pre-purified over an AG 1-X8 anion-exchange resin column (200-400 mesh; chloride form) to remove free iodide, were added to the cells in DMEM + 5% fetal bovine serum for 90 minutes with gentle agitation at 37°C. The cells were then washed three times with DMEM and frozen at -70°C. Plates of cells were exposed to photographic film for 48-72 hours. Single-cell positives were picked by scraping an area of about 7mιn² of frozen cells from the plate and Hirt lysates were prepared. Hirt, J. Molec. Biol. 26:365-369 (1967). DNA from the lysates was electroporated into E.coli HD10B bacteria and plated. Colony counts of the bacteria on the plates varied from 100 to 25,000; replica filters of the plates were stored at -70°C until use. DNA obtained from these pools was transfected into COS-1 cells and screened as before. Replica filters of positives were then sequentially subdivided until a single clone was ultimately obtained.

The nucleotide sequence of the complete cDNA for norepinephrine transporter was determined using the Sanger dideoxy sequencing method known in the art. Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977).

Specifically, the noradrenaline transporter cDNA was excised from the Xhol site of pXM and cloned into the Bluescript SKII(-) vector in both orientations. ("Bluescript" vectors are T7-promoter-containing expression vectors that are available from Stratagene, San Diego, CA.) Both strands were completely sequenced using Sequenase (US Biochem.) from a set of overlapping exonuclease Ill-digested Sacl, Hindlll fragments. Two regions of poor resolution on one strand (from nucleotides 75 to 85 and from 1,685 to 1,690) were unambiguously resolved on the other strand.

Sequence analysis of the cloned cDNA revealed a 1,851-base pair (bp) open reading frame within a l,983-bp insert, as shown in FIG. 1. Assignment of the first ATG in the nucleotide sequence as the translation initiation site is based on its resemblance to the consensus sequence described by Kozak, Nucl. Acids Res. 15:8125-8148 (1987). A protein of 617 amino acids, also shown in FIG. 1, was predicted. Based on the amino-acid sequence, the relative molecular mass of the protein was predicted to be about 69,000 daltons (l = 69 kD) . It is believed that the amino-acid sequence of FIG. 1 represents the actual norepinephrine transporter protein.

Hydrophobicity analysis (see Kyte and Doolittle, J. Molec. Biol. 157:105-132 (1982)) of the amino-acid sequence indicates that the norepinephrine transporter protein contains twelve to thirteen hydrophobic segments each containing 18-25 amino acids. These hydrophobic segments are of a type that would form transmembrane domains in the protein. These transmembrane domains are shown in FIG. 1 as bracketed and underlined regions. A motif resembling a "leucine zipper," possibly a structure that would mediate oligomerization of transporter molecules in the cell membrane, is shown in FIG. 1 as a doubly underlined region. White et al., Nature 340:103-104 (1989). Note the interspersed proline residues. Also, amino acids representing potential glycosylation sites on the putative extracellular loop are denoted in FIG. 1 by double underlining and the " " symbol. No signal sequence was identified. This implies that the N-terminus of the protein is located on the cytoplasmic face of the cell membrane.

A hypothetical structural model of the norepinephrine transporter protein is shown schematically in FIG. 2. This schematic shows a probable orientation of the protein in the cell membrane. As can be seen, the amino and carboxy termini are placed in the cytoplasm and a large loop containing the three amino acids representing potential glycosylation sites projects into the extracellular space. This structure is similar to the membrane topology proposed for the -aminobutyric acid (GABA) transporter. Guastella et al., Science 2_49:1303-1306 (1990). In FIG. 2, darkened circles represent residues conserved with the GABA transporter. Sequence database comparisons demonstrate an amino-acid sequence similarity between the norepinephrine transporter protein and GABA transporters in the human and rat. Nelson et al., FEES Lett. 269:181-184 (1990); Guastella et al., __________________ 24_9:1303-1306 (1990). In fact, the overall similarity between the amino-acid sequences of the human norepinephrine transporter and the human GABA transporter is about 46 percent. Allowing for conservative amino-acid substitutions, the amino-acid similarity between these two transporters can be increased to about 68 percent. A region of particular conservation occurs from amino acids 78 to 98, where 20 amino-acid residues are identical except for a single amino-acid difference at position 87. No other significant similarity was found between the norepinephrine transporter protein and other proteins in the Genbank and NBRF Protein Identification Resource Files, including facilitated glucose carriers, Na⁺/glucose and Na⁺/proline carriers, and the adrenergic receptors. These partial homologies between the norepinephrine and the GABA transporters indicate that these transporters represent a new and distinctive gene family. The homologies also suggest that certain domains and amino-acid residues in these transporter molecules are critical to their common mechanisms of neurotransmitter transport, such as the coupling of the downhill movement of sodium and chloride ions to the intracellular accumulation of neurotransmitter molecules against a concentration gradient. Regions of amino-acid sequence divergence may represent protein domains that are involved in more specific interactions with neurotransmitter substrates and antagonists.

Noradrenergic neurons (neurons that contain norepinephrine) project diffusely to most parts of the mammalian brain from cell bodies in various brainstem nuclei, particularly the locus coeruleus (Latin, "blue place") . The axons of these neurons mediate an excitatory modulation in the regions where they terminate. Production of norepinephrine by these neurons is predicated on the production of a corresponding messenger RNA (mRNA) . To evaluate norepinephrine transporter mRNA production, tissue and cell-line expression of norepinephrine transporter mRNA was examined by northern blot analysis. Cells and tissues examined were human neuroblastoma cells (SK-N-SH) , rat pheochromocytoma cells (PC-12 cells; Greene et al. , Brain Res. 129:247-263 (1977), various rat brain regions, and rat adrenal gland. Results of the northern blots are shown in FIG. 3. To perform the northern blots, the FAST-TRACK kit available from Promega was employed. To construct norepinephrine transporter probes, norepinephrine transporter cDNA (50 ng) excised from pXM vectors containing the cDNA was labeled by random-primed synthesis using ³²P-labeled dCIP (100 μCi) . Random oligonucleotides were used as primers. Poly(A)-enriched RNA was isolated from tissues. The RNA (5 mg) was size-fractionated on a denaturing formaldehyde agarose gel and transferred to a nylon membrane (ZETA PROBE, BioRad) by vacuum blotting. The blot was hybridized using the radiolabeled probe for 48 hours at 42°C in 50% formamide, 5x SSPE, lx Denhardt's solution, 10% dextran sulfate, 1% SDS, and 500 μg/mL salmon sperm DNA. The blot was washed at 65°C for l hour in O.lx SSPE buffer and 0.01% SDS. The SK-N-SH lane was exposed for 15 hours and the other lanes for 48 hours. As shown in FIG. 3, the SK-N-SH cells and the PC-12 cells each contain two RNAs, of 5.8 kilobases (kb) and 3.6 kb, which hybridize to the norepinephrine transporter probe. The 5.8-kb species is selectively expressed in a brainstem region containing the locus coeruleus and in the adrenal gland. Since noradrenergic neurons also radiate from the locus coeruleus, this result indicates that the 5.8-kb mRNA encodes a neuronal norepinephrine transporter protein. The 5.8-kb mRNA is also the predominant species expressed in SK-N-SH cells. This mRNA is also believed to correspond to the cloned cDNA.

The nature of the 3.6-kb RNA species is less certain. It could represent the transcript of a cross- hybridizing but distinct gene, or an alternatively processed transcript of the same norepinephrine transporter gene, encoding an identical or related transporter. Since desipri ine-sensitive, Na⁺-dependent norepinephrine transport activity has been observed in primary cultures of neonatal rat astrocytes, Kimelberg et al., J. Neurochem. 40:1265-1270 (1983), the 3.6-kb mRNA might represent a glial-specific form of the norepinephrine transporter.

To determine the extent to which the cloned cDNA reproduces the uptake characteristics and pharmacology of endogenous norepinephrine transporters, an expression vector containing the norepinephrine transporter cDNA was transfected into HeLa (a human non-neuronal cell line) cells. The uptake of [³H]-l-norepinephrine by these cells was monitored. Results are plotted in FIG. 4. Specifically, HeLa cells cultured in DMEM + 5% fetal bovine serum (FBS) and penicillin/streptomycin (100 U mL^'1) were plated at 2-3X10⁵ cells per well in 24-well plates. The cells were infected with T7 RNA polymerase-containing vaccinia virus at a multiplicity of about 10 PFU (plague-forming units) per cell. After 30 minutes, the cells were transfected with the norepinephrine transporter cDNA in Bluescript SKII vectors (100 ng) plus ordinary Bluescript vectors (900 ng) using 3 μg LIPOFECTIN (BRL) reagent. After twelve hours, the cells were assayed for uptake. [2,5,6,³H]-1- norepinephrine (43.7 Ci mmol^"1) at 10 nM (20 nM for competition curves) with 100 μM ascorbate and 50 μM pargyline were incubated with the cells for 15 minutes in Krebs-Ringer-HEPES (KRH) buffer containing either sodium chloride or choline chloride (120 mM) at 37°C.

Norepinephrine uptake was terminated by three ice-cold washes of the cells with appropriate KRH buffer. The cells were solubilized in 1% SDS. Radioactivity was determined by scintillation counting.

FIG. 4 shows a time course of [³H)]1- norepinephrine (NE) accumulation into HeLa cells transfected with an expression vector containing the noradrenaline transporter cDNA. The effects of choline substitution for sodium, and background labels with vector only are also shown. As can be seen, accumulation of the norepinephrine ligand hs a marked sodium dependence; replacement of extracellular sodium by choline ions reduces noradrenaline uptake to levels characteristic of ordinary vector-transfected controls.

FIG. 5 is a plot of norepinephrine uptake velocity observed in the presence of various concentrations of norepinephrine (NE) substrate in transected HeLa cells. The inset is an Eadie-Hofstee plot, as known in the art, of initial velocity data. As can be seen, norepinephrine uptake by these transfected cells is saturable, with a K_. of 457 nM. In FIGS. 6A and 6B, it can be seen that norepinephrine uptake by these cells is blocked by several uptake-inhibiting drugs known in the art. An important finding is that the rank order of potency was identical to that observed in SK-N-SH cells and cortical synaptosomes, as tabulated in Table 1.

TABLE 1 Inhibitor sensitivity of 1-norepinephrine uptake in HeLa cells transfected with noradrenaline transporter cDNA. itor

Nipecotic Acid >10,000

Phloridzen >10,000

Of the inhibitors listed in Table 1, reserpine is an inhibitor of vesicular uptake; nipecotic acid is an inhibitor of GABA transport; hemicholinium is an inhibitor of choline transport; pfloridzen is an inhibitor of Na⁺-dependent glucose transport; prazosin is an α-1 adrenergic antagonist; and yohimbine is an α-2 adrenergic antagonist. The listed K_j values reflect mean estimates from triplicate determinations of complete uptake inhibitions curves, adjusting for substrate concentration after Cheng and Prusoff, Biochem. Pharmacol. 22:3099-3108 (1973) . Hill coefficients obtained for these data do not deviate significantly from unity. Referring further to Table 1, of the tricyclic antidepressants examined, desipramine and nortriptyline were the most potent antagonists of norepinephrine transport. Two highly specific inhibitors of serotonin transport, citalopram and paroxetine, were weak inhibitors of norepinephrine uptake. GBR 12909 is a potent and specific inhibitor of the dopamine carrier, having K,=l nM for ³H-dopamine uptake into striatal synaptosomes. Anderson, Eur. J. Pharmacol. 166:493-504 (1930). This drug is notably less potent at inhibiting norepinephrine uptake (K^=133 nM) . These data permit the cloned norepinephrine transporter activity to be distinguished from catecholamine transporter activities characteristic of dopaminergic neurons.

Among the drugs with abuse potential, cocaine exhibited a _^ with these transfected cells of 140 nM

(Table 1) . This effect of cocaine is comparable to that observed in a variety of in vitro transport and binding studies. Thus, the cloned norepinephrine transporter cDNA of the present invention is the first known cloned member of the "cocaine receptor" family. The euphoric and addictive effects of cocaine seem to result from a blockade of the dopamine transporter in processes of ventral tegmental dopaminergic neurons. Ritz et al., Science 237:1219-1223 (1987). The norepinephrine transporter, however, mediates many systemic effects through an augmentation of sympathetic activity, resulting in acute changes in cardiac function and vascular tone. Hence, the studies summarized in FIGS. 4-6 and in

Table 1 demonstrate the exceptional utility of the cloned cDNA of the present invention. For the first time, it is possible to transfect a gene for a neurotransmitter transporter into "foreign" non-neural cells where the function of the transporter, as well as the effect on said function of a number of psychotropic drugs, can be studied without interference from competing transporters. These data also indicate that the norepinephrine transporter protein produced by the cDNA clone of the present invention is capable of normal incorporation into a cell membrane accompanied by preservation of normal transport activity similar to that seen in intact noradrenergic neurons.

Uptake of norepinephrine by these transfected cells was also inhibited by d-amphetamine (K^=56 nM; Cho, Science 24_.9:631-634 (1990)). Whereas cocaine is a non- transported inhibitor of the norepinephrine transporter, amphetamine is known in the art to be a substrate for intracellular accumulation by the transporter. Prolonged increases in intracellular concentrations of some transporter substrates can eventually cause neuronal death. Some substrates for the norepinephrine transporter such as MPP⁺ (l-methyl-4-phenylpyridinium ion) , DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) , and 6-OHDA (6-hydroxydopamine) are very potent neurotoxins. Javitch et al., Proc. Natl. Acad. Sci. USA 82:2173-2177 (1985); Ross et al. , J. Pharm. Pharmacol. 28:458-459 (1976); and Kostrzewa et al., Pharmacol. Re ., 26:199-288 (1974) . The anatomic specificity of their neurotoxicity in vivo is mediated by their relative affinities for different monoamine transporters. The availability of cDNAs encoding transporter proteins, such as that of the present invention, will facilitate the development of more sensitive screening techniques to identify environmental and other novel neurotoxins.

These results also demonstrate that the single cDNA clone of the present invention effectively reconstitutes many properties of the native norepinephrine transporter, including an appropriate ion dependence and pharmacology. The antagonism of monamine transport by anti-depressants and subsequent elevation of synaptic neurotransmitter concentrations is an important component of the biogenic amine hypothesis of affective disorders. Hence, it is possible that a deficiency of particular amines at functionally important synapses results in depression. Schildkraut, Am. J. Psvchiatr. 122:509-522 (1965) . In any event, the norepinephrine transporter is an important initial target for a number of tricyclic and other antidepressants. This transporter is also involved in the complex cascade of synaptic and neuronal biochemical alterations that ultimately ameliorates

> depression. The cDNA of the present invention is useful for determining the structural basis for antidepressant binding that may aid in the development of more selective therapeutic agents for the treatment of human depression. The human cDNA encoding the norepinephrine transporter also offers an opportunity to determine whether alterations in transporter genes could have actiological implications for major psychiatric affective disorders.

It will be appreciated by persons skilled in the art that the cDNA clone of 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 the cDNA clone is disclosed in FIG. 1. For studies of the expression of the cDNA clone, it is advantageous that the vector be expressible in mammalian cells. The specific vectors as used herein have at least a functioning origin of replication and an active promoter.

A Bluescript vector containing the cDNA clone is presently maintained in a permanent culture at the Howard Hughes Medical Institute, Yale University School of Medicine, Department of Neurology, New Haven, Connecticut.

Claims

1. A human norepinephrine transporter cDNA clone.

2. A cDNA clone encoding a human norepinephrine transporter.

3. A cDNA clone having a nucleotide sequence as disclosed in FIG. 1.

4. A cDNA clone as recited in claim 3 encoding a human norepinephrine transporter. 5. A human norepinephrine transporter DNA exhibiting substantial homology with a nucleotide sequence as disclosed in FIG. 1.

6. A probe which hybridizes to at least a portion of a nucleotide sequence as disclosed in FIG. 1. 7. A probe which hybridizes to at least a portion of a human norepinephrine transporter cDNA.

8. A polypeptide encoded by the cDNA clone of claim 1.

9. A norepinephrine transporter protein encoded by the cDNA clone of claim 1.

10. A polypeptide encoded by the cDNA clone of claim 2.

11. A norepinephrine transporter protein encoded by the cDNA clone of claim 2. 12. An expression vector containing the cDNA clone of claim 1.

13. An expression vector as recited in claim 12 that is capable of being expressed in mammalian cells.

14. The cDNA clone of claim 1 integrated into a Bluescript expression vector.

15. A line of mammalian cells transfected with the cDNA clone of claim 1.

16. A line of mammalian cells as recited in claim 15 which express the human norepinephrine transporter protein.

17. A line of mammalian cells as recited in claim 15 which are non-neuronal cells. AMENDED CLAIMS

[received by the International Bureau on 15 September 1992 (15.09.92); original claims 1-17 replaced by amended claims 1-33 (3 pages)]

1. An isolated DNA molecule encoding a norepinephrine transporter.

2. An isolated DNA molecule as recited in claim 1 encoding a human norepinephrine transporter.

3. An isolated DNA molecule comprising a nucleotide sequence as disclosed in FIG. 1. . A cloned DNA encoding a norepinephrine transporter. 5. A cloned DNA as recited in claim 4 encoding a human norepinephrine transporter.

6. A cloned DNA as recited in claim 4 wherein said cloned DNA comprises at least a portion of a DNA vector. 7. A cloned DNA as recited in claim 6 wherein the vector is an expression vector.

8. A cloned DNA comprising a nucleotide sequence as disclosed in FIG. 1.

9. A cloned DNA as recited in claim 8 wherein said cloned DNA comprises at least a portion of a DNA vector.

10. A cDNA encoding a norepinephrine transporter.

11. A cDNA as recited in claim 10 encoding a human norepinephrine transporter.

12. A cDNA comprising a nucleotide sequence as disclosed in FIG. 1.

13. A nucleotide sequence exhibiting substantial homology with either strand of a DNA sequence as disclosed in FIG. 1.

14. A nucleotide sequence as recited in claim 13 comprising RNA.

15. A nucleotide sequence as recited in claim 13 comprising single-stranded DNA. 16. A nucleotide sequence as recited in claim

13 including a label attached to the nucleotide sequence. 17. A nucleotide sequence that hybridizes under stringent conditions to either strand of a DNA sequence as disclosed in FIG. 1.

18. An isolated DNA molecule encoding a polypeptide as disclosed in FIG. 1.

19. A DNA sequence that hybridizes to at least a portion of a norepinephrine transporter gene.

20. A DNA sequence having the identifying characteristics of a gene encoding human norepinephrine transporter.

21. A polypeptide encoded by the DNA of claim 1.

22. A polypeptide encoded by the DNA of claim 3. 23. A polypeptide encoded by the cloned DNA of claim 8.

24. A polypeptide encoded by the cDNA of claim 10.

25. A polypeptide encoded by the cDNA of claim 12.

26. A cell transfected by the DNA of claim 1.

27. A cell transfected by the DNA of claim 3.

28. A cell transfected with the cloned DNA of claim 6. 29. A cell transfected with the cloned DNA of claim 9.

30. A procaryotic cell comprising the DNA of claim 6.

31. A procaryotic cell comprising the DNA of claim 8.

32. A method for rendering a cell incapable of expressing a norepinephrine transporter gene able to express said gene, comprising:

(a) providing an isolated DNA comprising a norepinephrine transporter-encoding sequence;

(b) coupling the isolated DNA to an expression vector comprising a functional promoter so as to render the vector able to transcribe, from the promoter, the norepinephrine transporter-encoding sequence in a correct norepinephrine-transporter reading frame;

(c) transfecting a host cell, susceptible to transfection by the expression vector, with the product of step (b) ; and

(d) culturing the transfected host cell so as to facilitate transcription and translation of the norepinephrine transporter-encoding sequence in the cell. 33. A method for rendering a eucaryotic cell normally incapable of transporting norepinephrine able to take up norepinephrine, the method comprising:

(a) providing an isolated DNA comprising a norepinephrine transporter-encoding sequence; (b) coupling the isolated DNA to an expression vector comprising a functional promoter so as to render the vector able to transcribe, from the promoter, the norepinephrine transporter-encoding sequence in a correct norepinephrine-transporter reading frame; (c) transfecting a host eucaryotic host cell, susceptible to transfection by the expression vector, with the product of step (b) ; and

(d) culturing the transfected host cell so as to facilitate transcription and translation of the norepinephrine transporter-encoding sequence in the cell, thereby causing the cell to synthesize norepinephrine transporter and take up norepinephrine.