FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2047143254> ?p ?o ?g. }

• W2047143254 endingPage "29018" @default.
• W2047143254 startingPage "29012" @default.
• W2047143254 abstract "The binding affinity of the cocaine analog [3H]2β-carbomethoxy-3β-(4-fluorophenyl) tropane (WIN) for the dopamine transporter (DAT) is increased by the reaction of Cys-90, at the extracellular end of the first transmembrane segment, with methanethiosulfonate (MTS) reagents. Cocaine enhances the reaction of Cys-90 with the sulfhydryl reagents, thereby augmenting the increase in binding. In contrast, cocaine decreases the reaction of Cys-135 and Cys-342, endogenous cysteines in cytoplasmic loops, with MTS reagents. Because this reaction inhibits [3H]WIN binding, cocaine protects against the loss of binding caused by reaction of these cysteines. In the present work, we compare the abilities of DAT inhibitors and substrates to affect the reaction of Cys-90, Cys-135, and Cys-342 with MTS ethyltrimethylammonium (MTSET). The results indicate that the different abilities of compounds to protect against the MTSET-induced inhibition of binding are attributable to differences in their abilities to attenuate the inhibitory effects of modification of Cys-135 and Cys-342 as well as to enhance the reaction with Cys-90 and the resulting potentiation of binding. The inhibitor benztropine was unique in its inability to protect Cys-135. Moreover, whereas cocaine, WIN, mazindol, and dopamine enhanced the reaction of Cys-90 with MTSET, benztropine had no effect on this reaction. These two features combine to give benztropine its weak potency in protecting ligand binding to wild-type DAT from MTSET. These results indicate that different inhibitors of DAT, such as cocaine and benztropine, produce different conformational changes in the transporter. There are differences in the psychomotor stimulant-like effects of these compounds, and it is possible that the different behavioral effects of these DAT inhibitors stem from their different molecular actions on DAT. The binding affinity of the cocaine analog [3H]2β-carbomethoxy-3β-(4-fluorophenyl) tropane (WIN) for the dopamine transporter (DAT) is increased by the reaction of Cys-90, at the extracellular end of the first transmembrane segment, with methanethiosulfonate (MTS) reagents. Cocaine enhances the reaction of Cys-90 with the sulfhydryl reagents, thereby augmenting the increase in binding. In contrast, cocaine decreases the reaction of Cys-135 and Cys-342, endogenous cysteines in cytoplasmic loops, with MTS reagents. Because this reaction inhibits [3H]WIN binding, cocaine protects against the loss of binding caused by reaction of these cysteines. In the present work, we compare the abilities of DAT inhibitors and substrates to affect the reaction of Cys-90, Cys-135, and Cys-342 with MTS ethyltrimethylammonium (MTSET). The results indicate that the different abilities of compounds to protect against the MTSET-induced inhibition of binding are attributable to differences in their abilities to attenuate the inhibitory effects of modification of Cys-135 and Cys-342 as well as to enhance the reaction with Cys-90 and the resulting potentiation of binding. The inhibitor benztropine was unique in its inability to protect Cys-135. Moreover, whereas cocaine, WIN, mazindol, and dopamine enhanced the reaction of Cys-90 with MTSET, benztropine had no effect on this reaction. These two features combine to give benztropine its weak potency in protecting ligand binding to wild-type DAT from MTSET. These results indicate that different inhibitors of DAT, such as cocaine and benztropine, produce different conformational changes in the transporter. There are differences in the psychomotor stimulant-like effects of these compounds, and it is possible that the different behavioral effects of these DAT inhibitors stem from their different molecular actions on DAT. dopamine DA transporter 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenyl-2-propenyl) piperazine methanethiosulfonate ethyltrimethylammonium 428, 2β-carbomethoxy-3β-(4-fluorophenyl) tropane wild-type methanethiosulfonate The dopamine (DA)1transporter (DAT) in neuronal cells clears DA from the extracellular space (1Povlock S. Amara S.G. Reith M.E.A. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press Inc., Totowa, NJ1997: 1-28Crossref Google Scholar, 2Kuhar M.J. Ritz M.C. Boja J.W. Trends Neurosci. 1991; 14: 299-302Abstract Full Text PDF PubMed Scopus (915) Google Scholar), thereby playing a pivotal role in dopaminergic neurotransmission (3Iversen L.L. Br. J. Pharmacol. 1971; 41: 571-591Crossref PubMed Scopus (651) Google Scholar). DAT is also thought to mediate the uptake of potentially neurotoxic agents into DA cells (4Uhl G.R. Kitayama S. Adv. Neurol. 1993; 60: 321-324PubMed Google Scholar) and to be a principal target for psychostimulant drugs such as cocaine (5Madras B.K. Spealman R.D. Fahey M.A. Neumeyer J.L. Saha J.K. Milius R.A. Mol. Pharmacol. 1989; 36: 518-524PubMed Google Scholar, 6Ritz M.C. Lamb R.J. Goldberg S.R. Kuhar M.J. Science. 1987; 237: 1219-1223Crossref PubMed Scopus (2017) Google Scholar, 7Javitch J.A. Blaustein R.O. Snyder S.H. Mol. Pharmacol. 1984; 26: 35-44PubMed Google Scholar, 8Calligaro D.O. Eldefrawi M.E. Membr. Biochem. 1987; 7: 87-106Crossref PubMed Scopus (133) Google Scholar, 9Reith M.E.A. Meisler B.E. Sershen H. Lajtha A. Biochem. Pharmacol. 1986; 35: 1123-1129Crossref PubMed Scopus (317) Google Scholar, 10Giros B. Wang Y.M. Suter S. McLeskey S.B. Pifl C. Caron M.G. J. Biol. Chem. 1994; 269: 15985-15988Abstract Full Text PDF PubMed Google Scholar). A number of studies have demonstrated that sulfhydryl reagents inhibit DA uptake by and ligand binding to DAT (11Dwoskin L.P. Moore S.E. Shaw W. Butterfield D.A. Soc. Neurosci. Abstr. 1996; 22: 1576Google Scholar, 12Schweri M.M. Thurkauf A. Mattson M.V. Rice K.C. J. Pharmacol. Exp. Ther. 1992; 261: 936-942PubMed Google Scholar, 13Johnson K.M. Bergmann J.S. Kozikowski A.P. Eur. J. Pharmacol. 1992; 227: 411-415Crossref PubMed Scopus (55) Google Scholar, 14Reith M.E.A. Xu C. Coffey L.L. Biochem. Pharmacol. 1996; 52: 1435-1446Crossref PubMed Scopus (26) Google Scholar, 15Saadouni S. Refahi-Lyamani F. Costentin J. Bonnet J.J. Eur. J. Pharmacol. 1994; 268: 187-197Crossref PubMed Scopus (40) Google Scholar, 16Schweri M.M. Neuropharmacology. 1990; 29: 901-908Crossref PubMed Scopus (27) Google Scholar, 17Xu C. Coffey L.L. Reith M.E. Naunyn-Schmiedebergs Arch. Pharmacol. 1997; 355: 64-73Crossref PubMed Scopus (25) Google Scholar). Protection against modification of DAT by sulfhydryl reagents has been predominantly interpreted as an indication of whether the reactive cysteine residues lie inside or outside the binding domain for the protecting ligand (13Johnson K.M. Bergmann J.S. Kozikowski A.P. Eur. J. Pharmacol. 1992; 227: 411-415Crossref PubMed Scopus (55) Google Scholar, 14Reith M.E.A. Xu C. Coffey L.L. Biochem. Pharmacol. 1996; 52: 1435-1446Crossref PubMed Scopus (26) Google Scholar, 15Saadouni S. Refahi-Lyamani F. Costentin J. Bonnet J.J. Eur. J. Pharmacol. 1994; 268: 187-197Crossref PubMed Scopus (40) Google Scholar, 16Schweri M.M. Neuropharmacology. 1990; 29: 901-908Crossref PubMed Scopus (27) Google Scholar, 17Xu C. Coffey L.L. Reith M.E. Naunyn-Schmiedebergs Arch. Pharmacol. 1997; 355: 64-73Crossref PubMed Scopus (25) Google Scholar). Differences in the protective abilities of various compounds have therefore been interpreted as evidence that the binding sites are not completely overlapping. Other experimental evidence, however, also implicates conformational changes in drug action at DAT, thereby complicating the interpretation of these protection assays. Bonnet et al. (18Bonnet J.J. Benmansour S. Costentin J. Parker E.M. Cubeddu L.X. J. Pharmacol. Exp. Ther. 1990; 253: 1206-1214PubMed Google Scholar), based on a thorough thermodynamic analysis, suggested that conformational changes occur upon binding to DAT of blockers but not substrates. Héronet al. (19Heron C. Costentin J. Bonnet J.J. Eur. J. Pharmacol. 1994; 264: 391-398Crossref PubMed Scopus (37) Google Scholar) reported that the interaction between DAT and blockers requires more time than that for substrates, implying that the binding of blockers involves a significant conformational change. Similarly, Do-Rego et al. (20Do-Rego J.C. Hue H. Costentin J. Bonnet J.J. J. Neurochem. 1999; 72: 396-404Crossref PubMed Scopus (9) Google Scholar) observed a two-step reaction of 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenyl-2-propenyl) piperazine (GBR 12783) with DAT. More recently, Ferrer and Javitch (21Ferrer J.V. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Crossref PubMed Scopus (110) Google Scholar) provided evidence for conformational changes of DAT upon exposure to cocaine. Specifically, cocaine increased the reaction of Cys-90 with charged methanethiosulfonate (MTS) reagents, thereby augmenting the stimulatory effect of this reaction on the binding of the cocaine analog, [3H]2β-carbomethoxy-3β-(4-fluorophenyl) tropane (WIN 35,428). In contrast, cocaine decreased the reaction of Cys-135 and Cys-342 with MTS reagents, a reaction that inhibited [3H]WIN 35,428 binding. This divergence in the effects of cocaine suggested that differences in the protective abilities of various compounds against the modification of DAT by sulfhydryl reagents relate to differences in the ability of these compounds to alter the conformation of DAT and hence the reactivities of the various endogenous cysteine residues. The present work focuses on the ability of a number of drugs to alter the reaction of endogenous cysteines in DAT with MTS ethyltrimethylammonium (MTSET), a positively charged sulfhydryl reagent (22Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (541) Google Scholar). To focus on the role of the various reactive cysteine residues, we used DAT constructs in which Cys-90, Cys-135, or Cys-342 were replaced, one at a time, into an MTSET-insensitive mutant DAT construct (X) in which five endogenous cysteine residues had been substituted (C90A, C135A, C306A, C319F, and C342A) (see Fig.1) (21Ferrer J.V. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Crossref PubMed Scopus (110) Google Scholar). The results showed intriguing differences between the potencies with which particular drugs inhibited ligand binding to DAT and the potencies with which they modulated the effects of MTSET on ligand binding. The DAT inhibitors benztropine and cocaine appear to affect differently the reaction of Cys-90 and of Cys-135, suggesting that inhibitors do not all stabilize the same conformational state. [3H]WIN 35,428 (84.5–86.0 Ci/mmol) and [3H]mazindol (23.5 Ci/mmol) were from PerkinElmer Life Sciences. Unlabeled WIN 35,428 was from the NIDA (Research Triangle Institute, Research Triangle Park, NC), andN-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine was obtained from Research Biochemicals Inc. (Natick, MA). Mazindol was a gift from Sandoz (Basel, Switzerland), and d-amphetamine was from Smith Kline & French Laboratories. MTSET was from Toronto Research Chemicals (Toronto, Ontario, Canada). Bovine serum was from HyClone (Logan, UT). All other chemicals were from Sigma or Fisher. Glass fiber filter mats and Betaplate Scint scintillation mixture for the binding assays were from Wallac Inc. (Gaithersburg, MD). The experiments were conducted with human wild-type (WT) and mutant DAT stably expressed in HEK-293 cells as described previously (21Ferrer J.V. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Crossref PubMed Scopus (110) Google Scholar). Cell membranes were prepared as described previously (14Reith M.E.A. Xu C. Coffey L.L. Biochem. Pharmacol. 1996; 52: 1435-1446Crossref PubMed Scopus (26) Google Scholar). The general procedures for treatment with sulfhydryl reagent and wash-out were also as described in that study, with the following modifications. Membranes were pretreated in assay buffer (see below) with varying concentrations of the protecting compound at 21 °C for 5 min. MTSET or vehicle was added, and after 15 min all reagents were removed in three centrifugation steps. For compounds that were more difficult to wash out (benztropine and mazindol) five centrifugation steps were applied. The wash buffer used in these steps was the assay buffer (see below) minus tropolone. This wash in itself did not alter the pharmacological profile of [3H]WIN 35,428 binding to DAT, with 0 and 3 washes resulting in the same affinities for cocaine, WIN 35,428, benztropine, and DA in WT DAT (23Eshleman A.J. Stewart E. Evenson A.K. Mason J.N. Blakely R.D. Janowsky A. Neve K.A. J. Neurochem. 1997; 69: 1459-1466Crossref PubMed Scopus (43) Google Scholar) (data not shown).The membrane protein concentration in the MTSET treatment phase was set at ∼0.6 mg/ml for the experiments with 0.3 and 1.0 mm MTSET and at 2 mg/ml for those with 10 mm MTSET. The washed pellets obtained above were homogenized with a Brinkmann Instruments Polytron (setting 6, 15 s) in 0.4 ml of assay buffer: 30 mm sodium phosphate buffer resulting from mixing primary and half-strength secondary sodium phosphate buffer to pH 7.4 at room temperature, containing also (in mm): 122 NaCl, 5 KCl, 1.2 MgSO4, 10 glucose, 1 CaCl2, and 0.1 tropolone. The tropolone was added to inhibit catechol-O-methyltransferase. Aliquots of 20 µl of the membrane suspension (25–50 µg of protein) were assayed in triplicate for [3H]WIN 35,428 binding in a final volume of 200 µl of assay buffer in 96-well plates using a cell harvester and liquid scintillation counter as described previously (24Berfield J.L. Wang L.C. Reith M.E.A. J. Biol. Chem. 1999; 274: 4876-4882Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). For [3H]mazindol binding, 30 µl of suspension (40–75 µg of protein) was assayed in a final volume of 300 µl. In addition to the binding assays in the protection design described above, inhibitory potencies of compounds were assessed in separate experiments under conditions identical to those used for protection. Inhibition curves consisted of six concentrations evenly spaced around the IC50 value of the test compound. For routine protection and inhibition experiments, binding assays were conducted at 21 °C with 4 nm [3H]WIN 35,428 for 15 min or with 0.5 nm [3H]mazindol for 10 min. Nonspecific binding was defined with 100 µmcocaine. For saturation analysis, [3H]WIN 35,428 was present at 2 nm, along with varying concentrations of nonradioactive WIN 35,428 (0, 3, 10, 30, 100, 300, 1,000, 3,000, and 10,000 nm) (“varying cold” design). In saturation experiments with [3H]mazindol, the radioligand was added at 0.03, 0.09, 0.22, 0.75, 2.2, 7, 22, and 75 nm with or without 100 µm cocaine for nonspecific binding (“varying hot” design). Because reaction of Cys-90 and of Cys-306 stimulates binding, whereas reaction of Cys-135 and of Cys-342 inhibits binding (21Ferrer J.V. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Crossref PubMed Scopus (110) Google Scholar), the overall ability of a drug to reduce MTSET-induced inhibition of binding will also depend on the effect of the drug on the MTSET-induced stimulation of binding (see below). These effects will obviously differ in the different mutants. Thus, we defined EC50 empirically as the concentration of a compound that reduces the MTSET-induced inhibition of [3H]WIN 35,428 binding by 50%. The effect of a compound on MTSET-induced inhibition of [3H]WIN 35,428 binding was calculated as ((sample − mt)/(total − mt)) × 100, where sample is the binding after MTSET treatment in the presence of compound, total is the binding in the absence of MTSET/compound, and mt is the binding after MTSET treatment in the absence of compound. In a small number of cases, correction for residual, incompletely washed out compound was applied at the highest concentration point as described previously (26DeLean A. Munson P.J. Rodbard D. Am. J. Physiol. 1978; 235: E97-E102Crossref PubMed Google Scholar). Compounds that together with MTSET increased binding (sample) over that observed in the control without MTSET or compound (total) give values greater than 100%. In DAT constructs in which Cys-90 and Cys-306 were mutated and stimulation of binding by MTSET does not occur, we quantified the potency of a compound to protect against MTSET-induced inhibition of binding relative to its potency to inhibit [3H]WIN 35,428 binding by EC50/IC50, which is inversely related to the protective activity (see Refs. 14Reith M.E.A. Xu C. Coffey L.L. Biochem. Pharmacol. 1996; 52: 1435-1446Crossref PubMed Scopus (26) Google Scholar, 17Xu C. Coffey L.L. Reith M.E. Naunyn-Schmiedebergs Arch. Pharmacol. 1997; 355: 64-73Crossref PubMed Scopus (25) Google Scholar, and 19Heron C. Costentin J. Bonnet J.J. Eur. J. Pharmacol. 1994; 264: 391-398Crossref PubMed Scopus (37) Google Scholar). The ALLFIT equation (25Zimanyi I. Jacobson A.E. Rice K.C. Lajtha A. Reith M.E.A. Synapse. 1989; 3: 239-245Crossref PubMed Scopus (14) Google Scholar) was used for calculation of IC50 and EC50 values. K D and B max were determined by the nonlinear regression program LIGAND (27Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7759) Google Scholar) as described previously (28Xu C. Reith M.E.A. J. Pharmacol. Exp. Ther. 1997; 282: 920-927PubMed Google Scholar); for the “varying hot only” experiments, the specific binding at each radioligand concentration was entered into the program. In WT upon MTSET treatment, and in C90A regardless of treatment, a low affinity binding component was observed with highly variableK D values between 1 µm and 1 mm. The present study focuses on the high affinity component obtained by LIGAND in two-site fits, or in one-site fits with floating nonspecific binding, with similar results observed previously (29Reith M.E. Xu C. Zhang L. Coffey L.L. Naunyn-Schmiedebergs Arch. Pharmacol. 1996; 354: 295-304Crossref PubMed Scopus (22) Google Scholar). All results are expressed as mean ± S.E. Comparisons were made by one-way analysis of variance followed by Tukey-Kramer Multiple Comparisons Test and by one-sample Student's t test for testing significance of protection. For comparison of EC50/IC50 ratios, the EC50 value for each experiment was divided by the average IC50 value observed for the compound, grouped, and tested. Data were log-transformed for homogeneity of variance when required. The accepted level of significance was 0.05. In WT DAT, 10 mm MTSET caused approximately a 50% reduction in [3H]WIN 35,428 binding (Fig.2). A number of DAT inhibitors and substrates were tested for their ability to reduce this effect of 10 mm MTSET. The concentrations needed for 50% reduction of MTSET-induced inhibition of binding (EC50) ranged from nanomolar (WIN 35,428) to sub-millimolar (DA) (TableI). For each compound, the affinity with which it inhibited [3H]WIN 35,428 binding to DAT (IC50) was measured under the same conditions, and in comparison with the IC50, the EC50 was somewhat higher affinity (cocaine and WIN 35,428) or lower affinity (mazindol, benztropine, and DA).Table IPotency of compounds in reducing MTSET-induced inhibition of [3H]WIN 35,428-binding sites (EC50) and in inhibiting [3H]WIN 35,428 (4 nm) binding (IC50) in WT and C90A DATCell/compoundEC50IC50WT DATCocaine142 ± 82 × 10−9380 ± 128 × 10−9WIN 35,42816 ± 5 × 10−950 ± 5 × 10−9Mazindol244 ± 110 × 10−916 ± 2 × 10−9Benztropine28 ± 6 × 10−678 ± 33 × 10−9Dopamine113 ± 50 × 10−68.3 ± 0.4 × 10−6C90A DATCocaine23.8 ± 10.7 × 10−6348 ± 64 × 10−9WIN 35,4288.06 ± 2.96 × 10−669 ± 9 × 10−9Mazindol5.68 ± 1.49 × 10−628 ± 1 × 10−9Benztropine>300 × 10−6287 ± 34 × 10−9Dopamine13.2 ± 4.2 × 10−310.5 ± 1.5 × 10−6For EC50 measurements, DAT was exposed to varying concentrations of DAT inhibitor or substrate and 10 mmMTSET at ∼2 mg of membrane protein/ml. After wash-out, residual [3H]WIN 35,428 (4 nm) binding was measured. For IC50 measurements under the same conditions, DAT was incubated with varying drug concentrations and 4 nm [3H]WIN 35,428. The results are the average ± S.E. for 3–5 independent experiments, each assayed in sextuplicate (EC50) or triplicate (IC50). Open table in a new tab For EC50 measurements, DAT was exposed to varying concentrations of DAT inhibitor or substrate and 10 mmMTSET at ∼2 mg of membrane protein/ml. After wash-out, residual [3H]WIN 35,428 (4 nm) binding was measured. For IC50 measurements under the same conditions, DAT was incubated with varying drug concentrations and 4 nm [3H]WIN 35,428. The results are the average ± S.E. for 3–5 independent experiments, each assayed in sextuplicate (EC50) or triplicate (IC50). Incubation with various DAT inhibitors (WIN 35,428, cocaine, and mazindol) or a substrate (DA) prior to and during MTSET treatment potentiated the binding of [3H]WIN 35,428 (4 nm) above the control level observed without drug or MTSET (Fig. 3 A). In contrast, although the substrate d-amphetamine and the inhibitor benztropine restored binding to control values, even higher concentrations of these compounds did not potentiate binding beyond that of control (Fig. 3 A). Potentiation of binding above control values requires that part of the effect of reaction with MTSET be a stimulation of binding and that exposure to DAT compounds uncovers this stimulatory effect by protecting against the inhibitory effects of reaction elsewhere and/or enhances the stimulatory effect. Indeed, saturation analysis with WIN 35,428 showed that treatment of WT with MTSET greatly reduced the B max (∼6-fold), which is reflected as a decrease in overall binding, but raised the affinity of the residual binding (∼5-fold increase in affinity) (Table II). A similar 6-fold effect on B max and 5-fold effect onK D was observed when binding of [3H]mazindol to DAT was assessed after reaction with MTSET (data not shown).Table IIEffect of MTSET with or without pretreatment with cocaine (Coc) or DA on high affinity [3H]WIN 35,428 binding in WT and C90A DATTreatmentWTC90AK DB maxK DB maxnmpmol/mg proteinnmpmol/mg proteinControl46.5 ± 4.6a27.8 ± 1.4a64.8 ± 8.3a15.2 ± 5.4aMTSET8.5 ± 0.2b4.6 ± 0.7b21.4 ± 1.8b2.7 ± 0.2bCoc/MTSET5.8 ± 0.2c8.2 ± 1.1c26.7 ± 4.3b6.8 ± 1.2cDA/MTSET6.3 ± 0.7c8.4 ± 1.5c24.8 ± 1.4b4.0 ± 0.8bWT or C90A DAT was exposed to cocaine (1 µm for WT and 200 µm for C90A), DA (3 mm), or vehicle, and 10 mm MTSET at ∼2 mg of membrane protein/ml. After wash-out, residual [3H]WIN 35,428 (2 nm) binding was measured in the presence of varying [WIN 35,428] for saturation analysis. The results are the average ± S.E. for 3 independent experiments, each assayed in triplicate. p < 0.05 is used for comparison within each column of individual groups that do not share the same superscript letters (Tukey-Kramer Multiple Comparisons Test following one-way analysis of variance). Open table in a new tab WT or C90A DAT was exposed to cocaine (1 µm for WT and 200 µm for C90A), DA (3 mm), or vehicle, and 10 mm MTSET at ∼2 mg of membrane protein/ml. After wash-out, residual [3H]WIN 35,428 (2 nm) binding was measured in the presence of varying [WIN 35,428] for saturation analysis. The results are the average ± S.E. for 3 independent experiments, each assayed in triplicate. p < 0.05 is used for comparison within each column of individual groups that do not share the same superscript letters (Tukey-Kramer Multiple Comparisons Test following one-way analysis of variance). Thus, 1 µm cocaine or 3 mm DA, in conjunction with MTSET, increased [3H]WIN 35,428 binding ∼2-fold (Fig. 3 A), through a dual mechanism. Both the binding affinity and the number of binding sites were increased (K D values were reduced by 29%, andB max values were enhanced by 80% in the case of cocaine and MTSET), compared with MTSET treatment alone (Table II). The analogous effects on [3H]mazindol binding for cocaine were an 18% reduction in K D and 62% enhancement inB max values (data not shown). Because Cys-90 has been implicated in the stimulatory effects of MTSET on binding (21Ferrer J.V. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Crossref PubMed Scopus (110) Google Scholar), the C90A mutant was examined for a loss of stimulation of binding upon MTSET treatment. MTSET at 10 mm caused approximately a 70% reduction in [3H]WIN 35,428 binding, greater than the inhibition seen in WT (Fig. 2). The potentiation seen in WT with various compounds upon co-incubation with 10 mm MTSET was not observed in C90A, even at concentrations at least 10 times the C90A EC50values (Fig. 3 B and Table I). The various compounds still reversed the MTSET-induced inhibition of binding (Fig. 3 B) but less potently than in WT, due to the absence of the Cys-90-induced stimulation, which affects the entire curve in WT. Even so, the rank order of potency in reducing the MTSET-induced binding inhibition among compounds was similar to that observed in WT: WIN 35,428 > cocaine > benztropine, DA (Table I). Consistent with these observations with [3H]WIN 35,428 as the radioligand, cocaine also increased the binding of [3H]mazindol above control in WT but not in C90A, and cocaine displayed a greatly increased EC50 in C90A (data not shown). C90A still contains Cys-306, which also reacts with MTSET to stimulate binding. Indeed, MTSET reduced the K D value of [3H]WIN 35,428 binding in C90A, but the magnitude of the effect was smaller (∼3-fold) than in WT (∼5-fold) (Table II). Similar to WT, the B max value of C90A was reduced ∼5-fold by MTSET. The analogous effects on [3H]mazindol binding in C90A were a ∼2-fold reduction in K D and a 7-fold reduction inB max values (data not shown). Cocaine (200 µm) and dopamine (3 mm) before and during reaction with MTSET did not further decrease the K Dvalue of [3H]WIN 35,428 binding, compared with MTSET alone, consonant with the lack of enhancement of reaction of Cys-306, but did increase the B max value by ∼100% (Table II), reflecting protection against inhibition. A comparable lack of effect on K D and a 262% increase inB max values, respectively, were observed for cocaine on [3H]mazindol binding (data not shown). In the following experiments, we used a background DAT construct with five cysteines removed (X), which in itself is not reactive with the concentrations of MTSET used here (21Ferrer J.V. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Crossref PubMed Scopus (110) Google Scholar) (see Introduction). X-A342C, in which Cys-342 has been substituted back into its original position, reacted with 0.3 mm MTSET to inhibit [3H]WIN 35,428 binding by 60% (Fig. 3 A). In contrast to WT, no potentiation of binding was observed in the presence of any of the inhibitors or substrates (Fig.4 A), consistent with the lack of Cys-90 and Cys-306 in this DAT construct (21Ferrer J.V. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Crossref PubMed Scopus (110) Google Scholar). Thus, the effect of the compounds was simply to protect Cys-342 from reaction. The protective ratios (EC50/IC50) ranged from 7.4 for cocaine to 70 for DA (Table III). Most significantly, benztropine had a protective ratio of 32, only about 4-fold less than that for cocaine (Table III).Table IIIPotency of compounds in reducing MTSET-induced inhibition of [3H]WIN 35,428 binding sites (EC50) and in inhibiting [3H]WIN 35,428 (4 nm) binding (IC50) in X-A342C and X-A135C DATCell/compoundEC50IC50EC50/IC50X-A342C DATCocaine2.43 ± 0.75 × 10−6328 ± 41 × 10−97.4 ± 2.5aWIN 35,4280.43 ± 0.19 × 10−652 ± 1 × 10−98.3 ± 3.6aMazindol1.59 ± 0.74 × 10−639 ± 3 × 10−941 ± 19a,bBenztropine15.7 ± 6.0 × 10−6478 ± 57 × 10−932 ± 13a,bDopamine0.83 ± 0.28 × 10−311.8 ± 1.6 × 10−670 ± 26bX-A135C DATCocaine3.33 ± 0.93 × 10−6322 ± 69 × 10−910.3 ± 3.6a,cWIN 35,4280.38 ± 0.09 × 10−660 ± 19 × 10−96.3 ± 2.5a,cMazindol0.42 ± 0.06 × 10−655 ± 11 × 10−97.6 ± 1.9a,cBenztropine>300 × 10−6298 ± 77 × 10−9>1.007bDopamine0.38 ± 0.22 × 10−314.0 ± 2.4 × 10−627 ± 16cFor EC50 measurements, DAT was exposed to varying concentrations of DAT inhibitor or substrate and 0.3 (X-A342C) or 1.0 mm (X-A135C) MTSET at ∼0.6 mg of membrane protein/ml. After wash-out, residual [3H]WIN 35,428 (4 nm) binding was measured. For IC50 measurements under the same conditions, DAT was incubated with varying drug concentrations and 4 nm [3H]WIN 35,428. The results are the average ± S.E. for 3–5 independent experiments, each assayed in sextuplicate (EC50) or triplicate (IC50). The S.E. in the EC50/IC50 ratio was computed from the individual S.E. values by standard precision calculus for mixed observations.p < 0.05 is used for comparison of individual groups that do not share the same symbol superscript letters (Tukey-Kramer Multiple Comparisons Test). Open table in a new tab For EC50 measurements, DAT was exposed to varying concentrations of DAT inhibitor or substrate and 0.3 (X-A342C) or 1.0 mm (X-A135C) MTSET at ∼0.6 mg of membrane protein/ml. After wash-out, residual [3H]WIN 35,428 (4 nm) binding was measured. For IC50 measurements under the same conditions, DAT was incubated with varying drug concentrations and 4 nm [3H]WIN 35,428. The results are the average ± S.E. for 3–5 independent experiments, each assayed in sextuplicate (EC50) or triplicate (IC50). The S.E. in the EC50/IC50 ratio was computed from the individual S.E. values by standard precision calculus for mixed observations.p < 0.05 is used for comparison of individual groups that do not share the same symbol superscript letters (Tukey-Kramer Multiple Comparisons Test). In X-342C K D values for WIN 35,428 with or without MTSET were not significantly different (TableIV). Cocaine (200 µm) or DA (3 mm) also had no effect on the K D but restored the B max toward control values (TableIV).Table IVEffect of MTSET with or without pretreatment with cocaine (Coc) or DA on high affinity [3H]WIN 35,428 binding in X-A342C DATTreatmentX-A34" @default.
• W2047143254 created "2016-06-24" @default.
• W2047143254 creator A5020951431 @default.
• W2047143254 creator A5056752754 @default.
• W2047143254 creator A5076865147 @default.
• W2047143254 creator A5080307047 @default.
• W2047143254 creator A5084648810 @default.
• W2047143254 date "2001-08-01" @default.
• W2047143254 modified "2023-10-16" @default.
• W2047143254 title "The Uptake Inhibitors Cocaine and Benztropine Differentially Alter the Conformation of the Human Dopamine Transporter" @default.
• W2047143254 cites W1514724839 @default.
• W2047143254 cites W1592442520 @default.
• W2047143254 cites W1877347825 @default.
• W2047143254 cites W1904488889 @default.
• W2047143254 cites W1966183574 @default.
• W2047143254 cites W1977182351 @default.
• W2047143254 cites W1980606076 @default.
• W2047143254 cites W1985442468 @default.
• W2047143254 cites W1994024775 @default.
• W2047143254 cites W1996813192 @default.
• W2047143254 cites W2007604257 @default.
• W2047143254 cites W2008280189 @default.
• W2047143254 cites W2009547115 @default.
• W2047143254 cites W2021032401 @default.
• W2047143254 cites W2025611728 @default.
• W2047143254 cites W2027622624 @default.
• W2047143254 cites W2033247304 @default.
• W2047143254 cites W2042757964 @default.
• W2047143254 cites W2043813981 @default.
• W2047143254 cites W2045064531 @default.
• W2047143254 cites W2067753855 @default.
• W2047143254 cites W2080728281 @default.
• W2047143254 cites W2093380711 @default.
• W2047143254 cites W2095217951 @default.
• W2047143254 cites W2107441299 @default.
• W2047143254 cites W2223822401 @default.
• W2047143254 cites W2951803564 @default.
• W2047143254 cites W2052563055 @default.
• W2047143254 doi "https://doi.org/10.1074/jbc.m011785200" @default.
• W2047143254 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11395483" @default.
• W2047143254 hasPublicationYear "2001" @default.
• W2047143254 type Work @default.
• W2047143254 sameAs 2047143254 @default.
• W2047143254 citedByCount "98" @default.
• W2047143254 countsByYear W20471432542012 @default.
• W2047143254 countsByYear W20471432542013 @default.
• W2047143254 countsByYear W20471432542014 @default.
• W2047143254 countsByYear W20471432542015 @default.
• W2047143254 countsByYear W20471432542016 @default.
• W2047143254 countsByYear W20471432542017 @default.
• W2047143254 countsByYear W20471432542018 @default.
• W2047143254 countsByYear W20471432542019 @default.
• W2047143254 countsByYear W20471432542020 @default.
• W2047143254 countsByYear W20471432542021 @default.
• W2047143254 countsByYear W20471432542023 @default.
• W2047143254 crossrefType "journal-article" @default.
• W2047143254 hasAuthorship W2047143254A5020951431 @default.
• W2047143254 hasAuthorship W2047143254A5056752754 @default.
• W2047143254 hasAuthorship W2047143254A5076865147 @default.
• W2047143254 hasAuthorship W2047143254A5080307047 @default.
• W2047143254 hasAuthorship W2047143254A5084648810 @default.
• W2047143254 hasBestOaLocation W20471432541 @default.
• W2047143254 hasConcept C104317684 @default.
• W2047143254 hasConcept C149011108 @default.
• W2047143254 hasConcept C169760540 @default.
• W2047143254 hasConcept C185592680 @default.
• W2047143254 hasConcept C2776755682 @default.
• W2047143254 hasConcept C2909816965 @default.
• W2047143254 hasConcept C2910393698 @default.
• W2047143254 hasConcept C513476851 @default.
• W2047143254 hasConcept C55493867 @default.
• W2047143254 hasConcept C71924100 @default.
• W2047143254 hasConcept C86803240 @default.
• W2047143254 hasConcept C98274493 @default.
• W2047143254 hasConceptScore W2047143254C104317684 @default.
• W2047143254 hasConceptScore W2047143254C149011108 @default.
• W2047143254 hasConceptScore W2047143254C169760540 @default.
• W2047143254 hasConceptScore W2047143254C185592680 @default.
• W2047143254 hasConceptScore W2047143254C2776755682 @default.
• W2047143254 hasConceptScore W2047143254C2909816965 @default.
• W2047143254 hasConceptScore W2047143254C2910393698 @default.
• W2047143254 hasConceptScore W2047143254C513476851 @default.
• W2047143254 hasConceptScore W2047143254C55493867 @default.
• W2047143254 hasConceptScore W2047143254C71924100 @default.
• W2047143254 hasConceptScore W2047143254C86803240 @default.
• W2047143254 hasConceptScore W2047143254C98274493 @default.
• W2047143254 hasIssue "31" @default.
• W2047143254 hasLocation W20471432541 @default.
• W2047143254 hasOpenAccess W2047143254 @default.
• W2047143254 hasPrimaryLocation W20471432541 @default.
• W2047143254 hasRelatedWork W1963610972 @default.
• W2047143254 hasRelatedWork W1997837647 @default.
• W2047143254 hasRelatedWork W1997975262 @default.
• W2047143254 hasRelatedWork W2019647316 @default.
• W2047143254 hasRelatedWork W2047568835 @default.
• W2047143254 hasRelatedWork W2056476032 @default.
• W2047143254 hasRelatedWork W2090481633 @default.
• W2047143254 hasRelatedWork W2157295802 @default.

• next