Evidence for Distinct Sodium-, Dopamine-, and Cocaine-dependent Conformational Changes in Transmembrane Segments 7 and 8 of the Dopamine Transporter

Previously we obtained evidence based on engineering of Zn2+ binding sites that the extracellular parts of transmembrane segment 7 (TM7) and TM8 in the human dopamine transporter are important for transporter function. To further evaluate the role of this domain, we have employed the substituted cysteine accessibility method and performed 10 single cysteine substitutions at the extracellular ends of TM7 and TM8. The mutants were made in background mutants of the human dopamine transporter with either two (E2C) or five endogenous cysteines substituted (X5C) that render the transporter largely insensitive to cysteine modification. In two mutants (M371C and A399C), treatment with the sulfhydryl-reactive reagent [2-(trimethylammonium)-ethyl]methanethiosulfonate (MTSET) led to a substantial inhibition of [3H]dopamine uptake. In M371C this inactivation was enhanced by Na+ and blocked by dopamine. Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A399C. The present findings add new functionality to the TM7/8 region by providing evidence for the occurrence of distinct Na+-, substrate-, and perhaps inhibitor-induced conformational changes critical for the proper function of the transporter. Previously we obtained evidence based on engineering of Zn2+ binding sites that the extracellular parts of transmembrane segment 7 (TM7) and TM8 in the human dopamine transporter are important for transporter function. To further evaluate the role of this domain, we have employed the substituted cysteine accessibility method and performed 10 single cysteine substitutions at the extracellular ends of TM7 and TM8. The mutants were made in background mutants of the human dopamine transporter with either two (E2C) or five endogenous cysteines substituted (X5C) that render the transporter largely insensitive to cysteine modification. In two mutants (M371C and A399C), treatment with the sulfhydryl-reactive reagent [2-(trimethylammonium)-ethyl]methanethiosulfonate (MTSET) led to a substantial inhibition of [3H]dopamine uptake. In M371C this inactivation was enhanced by Na+ and blocked by dopamine. Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A399C. The present findings add new functionality to the TM7/8 region by providing evidence for the occurrence of distinct Na+-, substrate-, and perhaps inhibitor-induced conformational changes critical for the proper function of the transporter. The dopamine transporter (DAT) 1The abbreviations used are: DAT, dopamine transporter; hDAT, human dopamine transporter; NET, norepinephrine transporter; SERT, serotonin transporter; CFT, 2β-carbomethoxy-3β-(4-fluorophenyl)tropane; MPP+, 1-methyl-4-phenylpyridinium; MTS, methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; SCAM, substituted cysteine accessibility method; TM, transmembrane domain; WT, wild type; ANOVA, analysis of variance; NMDG, N-methyl-D-glucamide. is primarily situated in the presynaptic membrane of dopaminergic nerve terminals and mediates rapid removal of dopamine released into the synaptic cleft upon neuronal stimulation (1Amara S.G. Kuhar M.J. Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (1001) Google Scholar, 2Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601PubMed Google Scholar). Accordingly, the transporter plays a critical role in regulating the availability of dopamine in the synaptic cleft and thus in modulating the physiological functions of dopamine (1Amara S.G. Kuhar M.J. Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (1001) Google Scholar, 2Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar). Furthermore, the DAT is the principle target for the action of widely abused psychostimulants such as cocaine and amphetamine (1Amara S.G. Kuhar M.J. Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (1001) Google Scholar, 2Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601PubMed Google Scholar). The transporter belongs to the family of Na+/Cl–-dependent transporters, along with transporters for several other neurotransmitters including the norepinephrine (NET), serotonin (SERT), γ-aminobutyric acid (GAT), and glycine transporters (3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601PubMed Google Scholar, 4Chen N. Reith M.E. Eur. J. Pharmacol. 2000; 405: 329-339Crossref PubMed Scopus (144) Google Scholar). This family of transporters have been predicted to contain 12 putative transmembrane segments (TMs) connected by alternating extracellular and intracellular loops with an intracellular location of the N and C termini (Fig. 1) (1Amara S.G. Kuhar M.J. Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (1001) Google Scholar, 2Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 5Nelson N. J. Neurochem. 1998; 71: 1785-1803Crossref PubMed Scopus (324) Google Scholar). No high resolution is for DAT or and into the of the 12 (3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601PubMed Google Scholar, 4Chen N. Reith M.E. Eur. J. Pharmacol. 2000; 405: 329-339Crossref PubMed Scopus (144) Google Scholar). the of have been in the of the human DAT L. Gether U. J. 1998; PubMed Scopus Google Scholar, L. Gether U. J. Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Gether U. 2000; PubMed Scopus Google Scholar). is the of the transporters, evidence suggests that and function as Sci. U. 2001; PubMed Scopus Google Scholar, Sci. U. 2000; PubMed Scopus Google Scholar, J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Gether U. PubMed Scopus Google Scholar). is a that Na+/Cl–-dependent transporters by an in the transporter an in the binding is to the extracellular and an in the binding is to the intracellular Reith and Google Scholar). The is to the transmembrane Na+ binding of Na+ with and of the transporter to the by of and Na+ to the intracellular is that this conformational changes in the transporter. we have into the parts of the that are in conformational changes and the parts of the that the predicted and of to the binding the extracellular and intracellular is to that cysteine accessibility have led to the of such as two in of the and Sci. U. 2000; PubMed Scopus Google a cysteine in the extracellular in J. Sci. U. 1998; PubMed Scopus Google Scholar, B. J. 2001; Full Text Full Text PDF PubMed Scopus Google and cysteines in the intracellular loops and in DAT, in J. Sci. U. 1998; PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar). In a of we have obtained evidence that a region the extracellular parts of TM7 and TM8 plays a critical role in the L. Gether U. J. 1998; PubMed Scopus Google Scholar, L. Gether U. J. Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Gether U. 2000; PubMed Scopus Google Scholar). we an endogenous high Zn2+ binding in the with two of the Zn2+ situated in this of the transporter at the extracellular of TM7 and at the extracellular of (Fig. 1) L. Gether U. J. 1998; PubMed Scopus Google Scholar, L. Gether U. J. Full Text Full Text PDF PubMed Scopus Google Scholar). we were to a of Zn2+ binding sites in the the extracellular parts of TM7 and TM8 L. J. Gether U. 2000; PubMed Scopus Google Scholar). the for binding of the the TM7 and TM8 in the of and the the of a of the TM7/8 L. J. Gether U. 2000; PubMed Scopus Google Scholar). the a role of this for transporter function Zn2+ binding to sites in a L. J. Gether U. 2000; PubMed Scopus Google Scholar). for this effect is that Zn2+ critical of TM7 to TM8 by a the two the effect of Zn2+ be by a change in the to an inhibition of the In this we have this and further the of the TM7/8 region for transporter function by of the substituted cysteine accessibility method of single cysteine substitutions was at the extracellular ends of TM7 and TM8 in background mutants of with either five (X5C) or two (E2C) endogenous cysteines substituted the transporter largely insensitive to with J. Sci. U. 1998; PubMed Scopus Google Scholar). mutants (M371C and are by the sulfhydryl-reactive reagent the inactivation of M371C on the of Na+ and is blocked by dopamine with no effect of the In inactivation of A399C is by and dopamine blocked by the that the TM7/8 region is and critical conformational changes in not to to as as to such as the wild in was by Caron The the the and J. Sci. U. 1998; PubMed Scopus Google in was by The in J. Sci. U. 1998; PubMed Scopus Google the and was by further was the and was and into the B. Caron M.G. J. Full Text PDF PubMed Google Scholar, B. Caron M.G. J. Neurosci. 1993; PubMed Google Scholar). were in this background by to the the The were with the by and into were by and an to the the in were at in in with and and transporters were into by the method as PubMed Scopus Google Scholar, U. PubMed Scopus Google Scholar). were performed B. N. Caron M.G. Pharmacol. Google were in or to an of of The were to the the were in of at were to the in and was by of [3H]dopamine in a of of of at the were with of in of and for at were to by of of The were in a was in the of dopamine were performed in were on as were in either or to a binding of of binding were performed in a of of and the of and was by the in of to in of for at were to by of of and of the in a binding was in the of were made in with MTSET in or were with of The were with the of MTSET at for 10 or as The MTSET in was and into a of of the were in of of [3H]dopamine performed as To the effect of on the were with MTSET for 10 at as the were in of and with 12 at in a of of for of the [3H]dopamine was The of and on MTSET reactivity were by with 10 or other or at for 10 in a of of by treatment with MTSET for in the of or the were in of of [3H]dopamine the of Na+ were with MTSET for 10 in either of the Na+ or by of [3H]dopamine as with MTSET in or were with of The of and on MTSET reactivity were by with or of dopamine or cocaine for 10 in a of of by treatment with MTSET for in the of or the were in of of the binding performed as the of Na+ were with MTSET for 10 in Na+ or by binding as were by analysis The used in the of were of and the the The were the the of or analysis of by either the or was used for as in to MTSET of and are situated at the extracellular of TM7 and and two of the in the endogenous high Zn2+ binding of the (Fig. 1) L. Gether U. J. 1998; PubMed Scopus Google Scholar, L. Gether U. J. Full Text Full Text PDF PubMed Scopus Google Scholar). two of the in a of sites in the of the was L. J. Gether U. 2000; PubMed Scopus Google Scholar). Zn2+ binding to inhibition of dopamine the for dopamine to that Zn2+ [3H]dopamine by the L. J. Gether U. 2000; PubMed Scopus Google Scholar). To further the this we at a and with The substitutions were in the in and Zn2+ inhibition of [3H]dopamine is by a change in the as a of the of a of and with a such as MTSET J. Sci. U. 1998; PubMed Scopus Google be to in decreased [3H]dopamine uptake. as in MTSET treatment did not to a substantial inhibition of transporter function. of with MTSET to the [3H]dopamine stimulation of [3H]dopamine in and a in [3H]dopamine in (Fig. that changes were by an in of and a decreased in of with no changes in To that reaction with MTSET had we the effect of Zn2+ on [3H]dopamine in the two In the of MTSET, Zn2+ [3H]dopamine in and with of and in (Fig. This suggests that a cysteine can for and in the endogenous Zn2+ binding treatment with MTSET, high Zn2+ inhibition of [3H]dopamine by the two mutants was (Fig. C and This that and are and with MTSET that the of a is not to a inhibition of the can either a inhibition or a for and mutants with and MTSET in a new of at the of TM7 and TM8 we performed single cysteine substitutions at at the extracellular of TM7 and at at the extracellular of TM8 in the background (Fig. The cysteine mutants were in and for to MTSET treatment in [3H]dopamine the mutants were and not the and were to a substantial by MTSET (Fig. In of treatment with MTSET [3H]dopamine by whereas in the [3H]dopamine was by (Fig. In the other [3H]dopamine was by upon MTSET treatment (Fig. and of MTSET MTSET inactivation of and was further by the of inactivation and the of The inhibition of by MTSET inactivation was that for (Fig. In the that the rate of inactivation was in in (Fig. with MTSET 10 of inhibition of at a of MTSET, whereas MTSET was required in (Fig. is with MTSET This accessibility in the of the substituted The for the inhibition on in and was by with [3H]dopamine with and MTSET that treatment with MTSET for 10 decreases in the with a in the for [3H]dopamine and Ala-399 to to the that the MTSET inactivation was by reaction with an endogenous cysteine that was present in the and that had in and mutants and were not were insensitive to MTSET treatment (Fig. accessibility of as with was further by in we the of to the the cysteine and the of MTSET, and and with the MTSET reaction rate of the this was to a by was (Fig. of MTSET of Na+ is of the in the and is believed to be by conformational we the accessibility of MTSET to and was on the of Na+ by the effect of MTSET Na+ with or no inactivation of by MTSET in the of Na+ with by or (Fig. that the extracellular of TM7 conformational changes that to to MTSET. In MTSET inactivation of was in and (Fig. of and Inhibitors on MTSET in and of and to MTSET inactivation of and was or were with or for 10 to and MTSET treatment for the of in dopamine blocked MTSET inactivation of whereas 10 cocaine had no effect. protection was at a dopamine not In contrast to in cocaine MTSET inactivation of whereas dopamine was effect (Fig. The cocaine protection was at a as not was for other and (Fig. In the DAT blocked MTSET inactivation to a as whereas the cocaine had no effect (Fig. In had no whereas blocked MTSET inactivation cocaine (Fig. in to other such as CFT, and of the high of we were to the an of Furthermore, we the and protection MTSET inactivation not that this was not the not MTSET and we that at this dopamine and cocaine were of inactivation of and not MTSET of M371C and A399C in the the the for [3H]dopamine was as with the WT, for in the This is by a of and In of we the that of the obtained in are for this The of the five cysteines for conformational changes that not in the transporter of and to M371C and A399C in a background the two extracellular cysteines and were this (E2C) contain intracellular cysteines such as and J. Sci. U. 1998; PubMed Scopus Google is largely insensitive to such as MTSET the treatment is on In with we did not inhibition of [3H]dopamine for 10 with to MTSET (Fig. MTSET treatment a inhibition of [3H]dopamine in and with that for the mutants in the background (Fig. of Na+ to a (Fig. treatment with MTSET inactivation in in Na+ inhibition was in in (Fig. of MTSET, a inactivation of was in and Na+ (Fig. the inactivation was not on the of in with in (Fig. of M371C and A399C in the in a new of of dopamine and cocaine to inactivation of and was in the dopamine not cocaine inactivation of whereas cocaine not dopamine not Because we inactivation of by MTSET in the of Na+ we were to dopamine protection of this was is evidence suggests that dopamine and other can to and transporters with in the of Na+ Reith Pharmacol. PubMed Scopus Google Scholar, N. L. Reith M.E. J. Neurochem. PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar). Accordingly, the Na+ of dopamine protection to protection and a conformational change that decreases the accessibility of M371C to MTSET. in protection by dopamine was on the of in dopamine did not alter whereas protection was in the of Na+ (Fig. the of inactivation in as with further the accessibility of MTSET to M371C (Fig. on the binding of dopamine for the transporter upon to cysteine and upon with MTSET, we performed binding the high cocaine with high to and to binding with dopamine for dopamine of in and in of decreased the of dopamine for the transporter. we were to the of dopamine for the transporter MTSET treatment of binding not This inhibition was the of a in for to of that MTSET This suggests that the not with high and binding with dopamine not reflect the of dopamine for the of M371C and A399C in the in a new on we were to binding of the of this was by binding be in The for was for and the was N. J. Neurochem. 1998; 71: 1785-1803Crossref PubMed Scopus (324) Google Scholar, L. Gether U. J. 1998; PubMed Scopus Google Scholar, L. Gether U. J. Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Gether U. 2000; PubMed Scopus Google Scholar, Sci. U. 2001; PubMed Scopus Google Scholar, Sci. U. 2000; PubMed Scopus Google Scholar, J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Gether U. PubMed Scopus Google Scholar, Reith and Google Scholar, Sci. U. 2000; PubMed Scopus Google for L. Gether U. J. Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Gether U. 2000; PubMed Scopus Google Scholar, Sci. U. 2001; PubMed Scopus Google Scholar, Sci. U. 2000; PubMed Scopus Google Scholar, J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Gether U. PubMed Scopus Google Scholar, Reith and Google for Because that cocaine A399C MTSET we to the effect of MTSET treatment on binding in in MTSET a and inhibition of binding in whereas a inhibition was not in MTSET of A399C as as binding of the cocaine we cocaine or dopamine were of inhibition of In with [3H]dopamine cocaine inhibition of binding whereas dopamine had no effect (Fig. of 1998; PubMed Scopus Google we have in the present obtained new into the role of the TM7/8 region in the human dopamine transporter. we and two in the endogenous high Zn2+ binding of the transporter (Fig. 1) L. Gether U. J. 1998; PubMed Scopus Google Scholar, L. Gether U. J. Full Text Full Text PDF PubMed Scopus Google at a with mutants were in the background that is largely insensitive to with J. Sci. U. 1998; PubMed Scopus Google Scholar). In of the Zn2+ was to the cysteines for the and in to Zn2+ binding (Fig. Zn2+ MTSET of the two cysteines did not to a inhibition of uptake. In was in a inhibition of was (Fig. the that Zn2+ inhibition of is the of the of Zn2+ to of TM7 and TM8 to the of a change in by the of a L. J. Gether U. 2000; PubMed Scopus Google Scholar). This is with findings in the γ-aminobutyric acid in a a cysteine at the extracellular of TM7 at in and at the extracellular of TM8 at in was by of the of a the two N. Gether U. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). not be with DAT or the as a to such as not The by cysteine of at the extracellular ends of TM7 and TM8 two and MTSET in contrast to was for and led to decreases in with the MTSET reagent to be to with DAT function at situated to the membrane (Fig. M371C and A399C were not the reaction and the of the inactivation by MTSET that M371C is to MTSET A399C. Because the reaction a in the that A399C is to the M371C, several other the reactivity PubMed Scopus Google Scholar). a accessibility of A399C was by the in of extracellular (Fig. To the that for M371C and A399C were for the background in five endogenous cysteines were we M371C and A399C in the background in and were to is largely insensitive to treatment with and is largely insensitive to such as MTSET used in (Fig. we were to the findings obtained in the background in the background with the and were by MTSET, the inactivation by MTSET in was in and The mutants the distinct protection with dopamine and cocaine not Furthermore, inactivation of and Na+ whereas MTSET did not inactivation in the of Na+ in we did inactivation of (Fig. we to that functions to and to the transporter. The Na+ of M371C inactivation by MTSET is with a conformational change that to be to the in to Na+ the occurrence of conformational changes the protection by of M371C inactivation by MTSET is with a conformational change that decreases the of to the The for the protection of M371C by be that the of MTSET and thus that a of the binding this for several The protection by dopamine is on the of Na+ (Fig. Because dopamine and other have been to to the and transporters with in the of Na+ Reith Pharmacol. PubMed Scopus Google Scholar, N. L. Reith M.E. J. Neurochem. PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google this the that the protection of M371C in the of Na+ is the of a transport-associated conformational a of the binding have that a into can a Zn2+ binding with the endogenous and L. J. Gether U. 2000; PubMed Scopus Google Scholar). binding to this Zn2+ as a of dopamine Zn2+ a in for [3H]dopamine L. J. 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J. Full Text Full Text PDF PubMed Scopus Google whereas Zn2+ binding at this no effect on binding of was in to J. and U. This the cocaine protection of A399C is the of the other we that the binding crevice with In this cocaine reaction with MTSET and the of A399C a in the binding crevice that The that protection of A399C with and protection of M371C with the that and conformational of the transporter that in the of conformational of the as the that for cocaine and the reactivity of and M.E. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we that and cocaine A399C reaction with MTSET, the protection by was (Fig. this cocaine and the conformational that not be the conformational by the that are not by the is an important that to be of to the of the protection of M371C MTSET inactivation and the protection of the protection were performed at a at of the protection was not was at not a of the protection for a conformational effect a is that a protection is is no to that conformational changes with the The conformational changes at the extracellular ends of TM7 and TM8 at and Sci. U. 2000; PubMed Scopus Google have that dopamine is of MTSET inactivation of a cysteine in of the norepinephrine transporter in to in and that this in contrast to the present is that perhaps conformational changes in the membrane are at and in to the extracellular of the transporter. is of to the present in the of the for the function of In the of the transporter is predicted to in an with the putative that M371C and A399C are in this In the of and the is is to in a the and with a of transporters in the the accessibility of M371C to MTSET the in the in the In we not an accessibility of that this is in the reaction with A399C an important not role of this for the transporters, such as the transporter have been predicted in the of Na+ to an as with the L. J. Google Scholar, PubMed Scopus Google Scholar). this with that accessibility of M371C is on the of Na+ the of the transporter the to the the MTSET accessibility is not on the of with this to the in the and The TM7/8 region is not the in the transporter. the extracellular of the the cysteine been to be in hDAT, SERT, and the glycine J. Sci. U. 1998; PubMed Scopus Google Scholar, B. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In SERT, was that a of cysteine substitutions in TM7 the accessibility of to in the extracellular with an the two J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the intracellular the intracellular we that of in this a of the Zn2+ of the transporter that be as a in the and in the L. Gether U. Sci. U. PubMed Scopus Google Scholar). in the been to be N. J. 2000; Full Text Full Text PDF PubMed Scopus Google and have been obtained for the in the J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In we have in this obtained evidence for distinct Na+-, substrate-, and perhaps conformational changes in the TM7/8 region of The evidence for changes the analysis of a of Zn2+ binding sites the two transmembrane The for the of and changes in this new and functionality to this of the transporter. The present in been employed with the of can substantial and into the and the function of of the is that the conformational changes are by of changes in to transporter function the of such as the of conformational changes in Gether U. PubMed Scopus Google Scholar, S.G. Gether U. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). are to for providing and and for on the and for critical of the and and for