Pharmacophore-based discovery of 3,4-disubstituted pyrrolidines as a novel class of monoamine transporter inhibitors

3,4-Disubstituted pyrrolidines were discovered as a novel class of monoamine transporter inhibitors through 3-D database pharmacophore searching using a new pharmacophore model. The most potent analogue 12 has Ki values of 0.084 microM in [3H]mazindol binding, 0.20, 0.23, and 0.031 microM in inhibition of dopamine (DA), serotonin (SER), and norepinephrine (NE) reuptake, respectively. Functional antagonism testing in vitro showed that 11 and 12 are weak cocaine antagonists.     

Characterization of Xylamine Binding to Proteins of PC12 Pheochromocytoma Cells

PC12 pheochromocytoma cells take up 3,4-dihydroxyphenylethylamine (dopamine) and norepinephrine by a Na+-dependent, cocaine-sensitive system. The kinetics suggest that the same transporter functions for both substrates. Xylamine, a nitrogen mustard that blocks catecholamine uptake into neurons, irreversibly inhibited norepinephrine uptake into PC12 (IC50 = 15 microM). Pretreatment with 10 microM xylamine did not inhibit norepinephrine transport if 10 microM cocaine or 100 microM norepinephrine was also present during the pretreatment period or if Na+ was absent. These results indicate that xylamine must interact with the norepinephrine transporter to inhibit norepinephrine uptake. PC12 accumulated [3H]xylamine; this uptake had Na+-dependent and Na+-independent components. The Na+-dependent uptake was saturable (Km = 13 microM), and it was inhibited by cocaine (IC50 = 0.6 microM), desipramine (IC50 less than 1 nM), and norepinephrine (IC50 = 1 microM). Several proteins became prominently labeled when intact PC12 cells were incubated with [3H]xylamine; these proteins were enriched in a plasma membrane fraction and have molecular weights of 17,000, 24,000, 31,000, 33,000, 41,000, 42,000, 52,000, and 80,000. Other proteins were labeled less prominently. The labeling of all proteins was markedly decreased when the incubation with [3H]xylamine occurred in the presence of cocaine, desipramine, gramicidin D, or in a Na+-free buffer. These results indicate that xylamine must be transported into the cells for covalent binding to proteins to occur. [3H]Xylamine labeled essentially the same proteins when incubated with cell homogenates, but competition experiments with bretylium, desipramine, and cocaine failed to reveal which of the [3H]xylamine-labeled proteins is associated with the norepinephrine transporter.     

The role of zinc ions in reverse transport mediated by monoamine transporters

The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.     

Expression of a Human Cocaine-Sensitive Dopamine Transporter in Xenopus laevis Oocytes

The human dopamine transporter was expressed in Xenopus laevis oocytes following injection of mRNA isolated from human brain substantia nigra. The specific accumulation of [3H]dopamine into these oocytes was time and Na+ dependent. Furthermore, [3H]dopamine accumulation was prevented by coincubation of oocytes with dopamine (100 microM) or with the dopamine uptake inhibitors GBR 12909 (1 microM) or cocaine (3 microM). In contrast, oocyte injection of mRNA isolated from human globus pallidus, an area devoid of dopamine neuron perikarya, did not elicit expression of the dopamine transporter. Oocyte expression of the human dopamine transporter can be used for the further characterization and cloning of this low-abundance membrane protein.     

High affinity stereospecific binding of [3H] cocaine in striatum and its relationship to the dopamine transporter

A high affinity (KD 35 nM) binding site for [3H]cocaine is detected in rat brain striatum present at 2-3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [3H]dopamine ([3H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [3H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [3H]cocaine binding stereospecifically, but with lower potency (IC50 approximately equal to 1 microM) than does cocaine. It is suggested that the DA transporter in striatum is the putative "cocaine receptor." Binding of [3H]cocaine, measured in 10 mM Na2HPO4-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na+ and K+ and by biogenic amines. DA and Na+ reduce the affinity of the putative "cocaine receptor" for [3H]cocaine without changing the Bmax, suggesting that inhibition may be competitive. However, TRIS reduces [3H]cocaine binding noncompetitively while Na+ potentiates it in TRIS buffer. Binding of [3H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [3H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na+ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

Temperature Dependence of Drug Interaction with the Platelet 5-Hydroxytryptamine Transporter: A Clue to the Imipramine Selectivity Paradox

Although [3H]imipramine is a selective radioligand for the 5-hydroxytryptamine (5-HT) transporter in human platelets, its affinity for binding to the 5-HT transporter complex at 0 degrees C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5-HT uptake at the physiological temperature of 37 degrees C (Ki = 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5-HT transporter at assay temperatures between 0 degrees C and 37 degrees C, using as radioligands [3H]imipramine (0 degrees C and 20 degrees C) and [3H]paroxetine (20 degrees C and 37 degrees C), a newly available probe for the 5-HT transporter. At 20 degrees C, Ki values of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p less than 0.001), validating the use of these two radioligands to study the 5-HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5-HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5-HT transporter at 0 degrees C. At 37 degrees C, the Ki of imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals its Ki value for inhibition of 5-HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5-HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5-HT transporter similar to that of imipramine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modeling of the interaction of Na+ and K+ with the binding of dopamine and [3H]WIN 35,428 to the human dopamine transporter

Although much is known about the effects of Na+, K+, and Cl- on the functional activity of the neuronal dopamine transporter, little information is available on their role in the initial event in dopamine uptake, i.e., the recognition step. This was addressed here by studying the inhibition by dopamine of the binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a phenyltropane analogue of cocaine, to the cloned human dopamine transporter expressed in HEK-293 cells. The decrease in the affinity of dopamine (or WIN 35,428) binding affinity with increasing [K+] could be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K+ IC50 for inhibiting dopamine or WIN 35,428 binding increased linearly with [Na+], indicating a K(D,Na+) of 30-44 mM and a K(D,K+) of 13-16 mM for this cation site. A second Na+ site (2), distal from the WIN 35,428 domain but linked by positive allosterism, was indicated by model fitting of the WIN 35,428 binding affinities as a function of [Na+]. No strong evidence for this second site was obtained for dopamine binding in the absence or presence of low (20 mM) Cl- and could not be acquired for high [Cl-] because of the lack of a suitable substitute ion for Na+. The K(D) but not Bmax of [3H]WIN 35,428 binding increased as a function of the [K+]/[Na+] ratio regardless of total [Cl-] or ion tonicity. A similar plot was obtained for the Ki of dopamine binding, with Cl- at > or = 140 mM decreasing the Ki. At 290 mM Cl- and 300 mM Na+ the potency of K+ in inhibiting dopamine binding was enhanced as compared with the absence of Cl- in contrast to the lack of effect of Cl- up to 140 mM (Na up to 150 mM). The results indicate that Cl- at its extracellular level enhances dopamine binding through a mechanism not involving site 1. The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na+ and K+ on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step.     

Functional importance of the C-terminus of the human norepinephrine transporter

Three C-terminal variants of the human norepinephrine transporter (hNET) are known: the wild-type hNET in which exon 14 encodes the last seven amino acids and two variants with either three or 18 amino acids encoded by an alternatively spliced exon 15. In transfected HEK293 cells we compared by means of [(3)H]norepinephrine ([(3)H]NE) uptake and [(3)H]nisoxetine ([(3)H]NIS) binding the functional properties of the wild-type hNET with those of the more abundant long splice variant containing exon 15 (hNET-Ex15L) and of two artificial hNET mutants lacking either the last three (hNET-Ex14-4) or all seven (hNET-Ex14-0) C-terminal amino acids of exon 14. No differences among the NET isoforms were observed concerning the K(m) for uptake of NE and the K(D) for binding of NIS. However, compared with the wild-type hNET, the three isoforms (hNET-Ex15L, hNET-Ex14-4 and hNET-Ex14-0) showed a pronounced decrease in V(max) of [(3)H]NE uptake and B(max) of [(3)H]NIS binding which correlated with strongly reduced surface expression of the transporter isoforms. The decrease in surface expression of the hNET isoforms is probably a consequence of the lack of the three amino acids leucine, alanine and isoleucine at the C-terminal end which may represent a motif facilitating cell surface expression of the hNET. Expression of hNET-Ex15L exerted a dominant negative effect on plasma membrane expression of the wild-type hNET and thus may represent a novel mechanism for regulation of noradrenergic neurotransmission.     

Identification and characterization of the catecholamine transporter in bovine chromaffin granules using [3H]reserpine

Characterization of the catecholamine transporter in chromaffin granule membranes has been hampered by the lack of a radioligand with high specific activity which binds selectively to the carrier with high affinity. We report here the identification of a high affinity binding site for [3H]reserpine on chromaffin granule membranes isolated from bovine adrenal gland which has the characteristics expected of the catecholamine transporter. [3H]Reserpine bound predominately to a high affinity site with a Kd for [3H]reserpine of 9 nM and a binding site density of 7.8 pmol/mg of protein. Comparison of the characteristics of the high affinity reserpine binding site to the characteristics of catecholamine transport indicated that (a) the Ki and rank order of potency for inhibition of [3H]reserpine binding by various biogenic amines was similar to their Ki for inhibition of catecholamine transport (b) both the inhibition of (-)-[3H]norepinephrine transport and inhibition of [3H]reserpine binding showed similar stereo-specificity, and (c) Kd for binding of reserpine to chromaffin granule membranes was similar to the Ki for reserpine inhibition of catecholamine transport. These results demonstrate that the high affinity binding site for [3H]reserpine on chromaffin granule membranes is associated with the catecholamine transporter.     

Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells

The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.     

Styrene and styrene oxide affect the transport of dopamine in purified rat striatal synaptic vesicles

Animal and human studies suggest a dopamine-mediated effect of styrene neurotoxicity. To date, mechanisms of cerebral membrane transport of neurotransmitter amines in the presence of styrene in relation to its neurotoxicity have not been addressed properly. So, the present study has examined to test the hypothesis that dopaminergic malfunction in vesicular transport is a critical component in styrene-induced neurotoxicity in rats. Both styrene and its intermediate reactive metabolite, styrene oxide antagonized the in vitro striatal binding of [3H] tyramine, a putative marker of the vesicular transporter for dopamine. Both styrene and styrene oxide potently inhibited the uptake of [3H] dopamine in purified synaptic vesicles prepared from rat brain striata, in a dose-related manner, with inhibitory constants (Ki) 2.5 and 2.2 microM respectively. However, neither styrene nor styrene oxide significantly increased the basal efflux of [3H] dopamine that has been preloaded into striatal vesicles in vitro. On the other hand, both styrene and styrene oxide have failed to significantly inhibit the uptake of either [3H] norepinephrine, or [3H] serotonin into striatal synaptic vesicles. It is concluded that both styrene and styrene oxide are capable of producing impairments in dopaminergic transport in purified striatal synaptic vesicles, an effect which may be a critical component in styrene-induced neurotoxicity.     

21-Aminosteroids Interact with the Dopamine Transporter to Protect Against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

U-78518F, a 21-aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U-78518F up to 30 microM. Non-specific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U-78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of gamma-[3H]aminobutyric acid. In human striatal membrane preparations, U-78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 microM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21-aminosteroids in MPP(+)-induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter.     

Inhibition of the norepinephrine transporter by the venom peptide chi-MrIA. Site of action,Na+ dependence,and structure-activity relationship

chi-Conopeptide MrIA (chi-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether chi-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 microM) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [3H]nisoxetine and [3H]mazindol, to the expressed rat and human NET was inhibited by chi-MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by chi-MrIA was competitive in nature. It has previously been demonstrated that chi-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for chi-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of chi-MrIA was found to be dependent on Na+ with the conopeptide becoming a less effective blocker of [3H]norepinephrine by the NET under the conditions of reduced extracellular Na+. In this respect, chi-MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of chi-MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of chi-MrIA and a His in loop 2 played a dominant role in the interaction between chi-MrIA and the NET. H alpha chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of chi-MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET.     

Active uptake of MPP+, a metabolite of MPTP, by brain synaptosomes

Mouse brain synaptosomal preparations were used to study uptake of N-methyl-4-phenylpyridine (MPP+), a metabolite of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). The uptake of [3H]-MPP+ by striatal synaptosomes was approximately 25 X greater than that of [3H]-MPTP, with a KM of 0.48 microM and a Vmax of 5.3 nmoles/g tissue/min. Uptake was Na+ dependent and inhibited by ouabain, cocaine and dopamine (Ki 0.12 microM). Synaptosomes prepared from the corpus striatum accumulated [3H]-MPP+ at a rate 5-10 times higher than preparations from other brain regions. This selective uptake of MPP+ may contribute to the specificity of the toxic effects of MPTP on nigrostriatal dopaminergic neurons.     

The dopamine transporter and cytochrome P45OIID1 (debrisoquine 4-hydroxylase) in brain: resolution and identification of two distinct [3H]GBR-12935 binding proteins

Two [3H]GBR-12935 binding proteins, identified as the dopamine transporter and cytochrome P45OIID1, were solubilized in digitonin from canine striatal membranes, and were resolved following wheat germ agglutinin (WGA)-lectin column chromatography. Protein adsorbed to and specifically eluted from WGA-lectin with N-acetylglucosamine displayed saturable, high affinity (KD approximately 3 nM), and sodium-dependent binding of [3H]GBR-12935, which was inhibited in a concentration-dependent and stereoselective manner by dopamine uptake blockers and substrates with a pharmacological profile indicative of the dopamine uptake site. Protein not adsorbed to WGA-lectin also bound [3H]-GBR-12935 with high affinity (approximately 7 nM), in a sodium-independent manner, and was insensitive to classical dopamine uptake blockers and substrates such as mazindol or dopamine, corresponding to the so-called "piperazine acceptor" site seen in native membranes. [3H]GBR-12935 binding to this latter protein was, however, inhibited by various compounds with a pharmacological profile indicative of a form of cytochrome P450 designated P45OIID1 (debrisoquine/sparteine monooxygenase) with the following rank order of inhibitory potency: GBR-12909 greater than budipine greater than alpha-lobeline greater than quinidine greater than alpha flupenthixol greater than SKF-525A greater than sparteine greater than quinine. Ki values obtained for inhibition of [3H]-GBR-12935 binding to neuronal WGA passthrough fractions by these drugs correlate well with their respective Ki values for liver P45OIID1 activity. Western blotting and immunoprecipitation analysis with rabbit anti-rat P45OIID1 antibody also supported the identity of the mazindol-insensitive [3H]GBR-12935 binding site (or piperazine acceptor site) as P45OIID1. Furthermore, a [3H]GBR-12935 binding protein with pharmacological and immunological characteristics similar to those of P45OIID1 was solubilized from both bovine and human liver membranes, and GBR-12909 was found to be a potent competitive inhibitor (Ki approximately 100 nM) of sparteine monooxygenase activity in human liver microsomes. These data clearly indicate that [3H]GBR-12935 and its analogs display similar affinities for both the dopamine transporter and neuronal P45OIID1, and that this radioligand may be a useful probe of P45OIID1 activity in brain and liver. The exact molecular and functional association (if any) between these two distinct binding protein populations remains to be established; however, it is tempting to speculate that P45OIID1 is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells.     

The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors.

It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.     

The Role of Cysteines and Histidins of the Norepinephrine Transporter

The thiol reagent N-ethylmaleimide (NEM) is known to inhibit irreversibly ligand binding by the norepinephrine transporter (NET), while the simultaneous presence of NET substrates or ligands protects from this inhibition. Therefore, cysteine residues located within the substrate binding pocket of the NET were assumed to play an important role in ligand binding. To examine which (if any) of the 10 cysteines (Cys) of the human (h) NET might be involved in transport and/or binding function, we mutated all hNET cysteines to alanine. Using transfected HEK293 cells we studied NEM effects on the hNET with respect to [³H]nisoxetine binding. Two cysteines (Cys176 and Cys185) within the extracellular loop of the NET have been proposed to form a disulfide bond. We could demonstrate that this is of crucial importance as corresponding hNET mutants, in which these cysteines have been replaced, showed a lack of plasma membrane expression. However, due to their oxidized state in the native NET protein, Cys176 and Cys185 may not be targets for NEM. All other Cys-to-Ala hNET mutants were fully active and showed no change in inhibition of [³H]nisoxetine binding by NEM. These observations clearly exclude cysteines as being involved in hNET ligand binding. Since NEM also interacts with histidin (His), we mutated all 13 histidins of the hNET to alanine and examined the NET mutants in functional and binding assays. His222 within the large extracellular loop of the transporter was identified as an interaction partner of NEM since in the corresponding hNET mutant NEM exhibited a significantly reduced inhibitory potency. Furthermore, we could show that histidins in position 296, 370 and 372 are important for nisoxetine binding, while His220, 441, 598 and 599 are crucial for plasma membrane expression of the hNET.     

Putative cocaine receptor in striatum is a glycoprotein with active thiol function

Dopamine transporters of bovine and rat striata were identified by their specific [3H]cocaine binding and cocaine-sensitive [3H]dopamine [( 3H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes. Inorganic and organic mercury reagents inhibited both [3H]DA uptake and [3H]cocaine binding, though they were all more potent inhibitors of the former. n-Ethylmaleimide inhibited [3H]DA uptake totally but [3H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [3H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [3H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (less than 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (greater than 10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation.     

Syntheses of 3-carbomethoxy-4-(aryl)piperidines and in vitro and in vivo pharmacological evaluation: identification of inhibitors of the human dopamine transporter

A series of 3-carbomethoxy-4-(aryl-substituted)piperidines with various aryl groups were synthesized and examined for binding and reuptake inhibition at the human dopamine transporter, the human serotonin transporter, and the human norepinephrine transporter. The binding potency and reuptake inhibition efficacy was compared with that of (-)-cocaine to determine the significance of removing the two-carbon bridge of the cocaine nucleus on the inhibition of transporter binding and reuptake. Of the transporters examined, the substituted piperidines were relatively selective for the human dopamine transporter. In all cases examined, the cis-diastereomer of the 3-carbomethoxy-4-(aryl-substituted)piperidine was observed to be a more potent inhibitor of the human dopamine transporter than the trans diastereomer. Based on the K(i) (binding) and IC(50) (reuptake inhibition) values obtained, the most potent inhibitor of the series was cis-3-carbomethoxy-4-(4'-chlorophenyl)piperidine, and this compound suppressed spontaneous- and cocaine-induced stimulation in non-habituated male Swiss-Webster mice. The conclusion is that substantial portions of the cocaine structure can be dissected away to provide compounds with significant binding and reuptake inhibition of the human dopamine transporter.     

Cocaine increases dopamine uptake and cell surface expression of dopamine transporters

In HEK 293 cells expressing the human dopamine transporter (DAT), a 10-min incubation with 10 microM cocaine followed by extensive washing resulted in a 30% increase in [3H]dopamine (DA) uptake as well as an increase in cell surface DAT in biotinylation experiments. Consistent with this novel regulation, [3H]DA uptake into synaptosomes prepared from the nucleus accumbens of rats sacrificed 30 min after a single cocaine injection (30 mg/kg) was significantly increased compared to controls (56% increase in V(max), no change in K(m)). In addition, DA clearance in the striatum of anesthetized rats was increased after local application of a low (3 pmol) but not high (65 pmol) dose of cocaine, presumably as a result of mobilization of DAT to the cell surface. Cocaine-induced increases in cell surface expression of DAT and associated changes in DA clearance represent a novel mechanism that may play a role in its addictive properties.