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Twenty residues in the third transmembrane domain of the serotonin transporter (SERT) were mutated, one at a time, to cysteine. Almost all of these mutants were fully active for serotonin (5-HT) transport and insensitive to inactivation by the positively charged cysteine reagent 2-(trimethylammonium)ethylmethanethiosul-fonate (MTSET). Two active mutants, I172C and I179C, were sensitive to rapid inactivation by MTSET but were relatively insensitive to the negatively charged reagent (2-sulfonatoethyl)methanethiosulfonate (MTSES). Inactivation of I172C was blocked by 5-HT and cocaine, but I179C was not similarly protected. Replacement of Tyr-175 with cysteine resulted in a mutant with low transport activity, and, at the neighboring Tyr-176, cysteine replacement completely blocked transport. The Y175C and Y176C mutants were expressed on the cell surface at levels 84% and 69%, respectively, that of wild type (C109A) SERT. Mutants Y175C and Y176C had lower cocaine affinity than C109A, as measured by displacement of the high affinity cocaine analog 2β-carbomethoxy-3β-(4-125Iiodophenyl)tropane (β-CIT). For Y176C, 5-HT affinity also was decreased. MTSET inactivated β-CIT binding to I172C and Y176C, but only slightly inhibited binding to I179C and C109A. The MTSET sensitivity of cysteine replacements at positions 172, 176, and 179 was not observed when these positions were replaced with alanine, serine, or methionine. The results suggest that Ile-172, Tyr-176 and Ile-179 are on one face of an α-helical transmembrane element, and that Ile-172 and Tyr-176 are in proximity to the binding site for 5-HT and cocaine. Twenty residues in the third transmembrane domain of the serotonin transporter (SERT) were mutated, one at a time, to cysteine. Almost all of these mutants were fully active for serotonin (5-HT) transport and insensitive to inactivation by the positively charged cysteine reagent 2-(trimethylammonium)ethylmethanethiosul-fonate (MTSET). Two active mutants, I172C and I179C, were sensitive to rapid inactivation by MTSET but were relatively insensitive to the negatively charged reagent (2-sulfonatoethyl)methanethiosulfonate (MTSES). Inactivation of I172C was blocked by 5-HT and cocaine, but I179C was not similarly protected. Replacement of Tyr-175 with cysteine resulted in a mutant with low transport activity, and, at the neighboring Tyr-176, cysteine replacement completely blocked transport. The Y175C and Y176C mutants were expressed on the cell surface at levels 84% and 69%, respectively, that of wild type (C109A) SERT. Mutants Y175C and Y176C had lower cocaine affinity than C109A, as measured by displacement of the high affinity cocaine analog 2β-carbomethoxy-3β-(4-125Iiodophenyl)tropane (β-CIT). For Y176C, 5-HT affinity also was decreased. MTSET inactivated β-CIT binding to I172C and Y176C, but only slightly inhibited binding to I179C and C109A. The MTSET sensitivity of cysteine replacements at positions 172, 176, and 179 was not observed when these positions were replaced with alanine, serine, or methionine. The results suggest that Ile-172, Tyr-176 and Ile-179 are on one face of an α-helical transmembrane element, and that Ile-172 and Tyr-176 are in proximity to the binding site for 5-HT and cocaine. The serotonin transporter (SERT) 1The abbreviations used are: SERT, serotonin transporter; 5-HT, serotonin; DAT, dopamine transporter; TM, transmembrane domain; EL, external loop; MTS, methanethiosulfonate; MTSET, 2-(trimethylammonium)ethylmethanethiosulfonate; MTSES, (2-sulfonatoethyl)methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate; PBS, phosphate-buffered saline; β-CIT, 2β-carbomethoxy-3β- (4-125Iiodophenyl)tropane. 1The abbreviations used are: SERT, serotonin transporter; 5-HT, serotonin; DAT, dopamine transporter; TM, transmembrane domain; EL, external loop; MTS, methanethiosulfonate; MTSET, 2-(trimethylammonium)ethylmethanethiosulfonate; MTSES, (2-sulfonatoethyl)methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate; PBS, phosphate-buffered saline; β-CIT, 2β-carbomethoxy-3β- (4-125Iiodophenyl)tropane. is responsible for reuptake of 5-HT released during neurotransmission (1Rudnick G. Clark J. Biochim. Biophys. Acta. 1993; 1144: 249-263Crossref PubMed Scopus (366) Google Scholar). Inhibitors of SERT are clinically effective as antidepressants (2Boyer W. Int. Clin. Psychopharm. 1992; 6: 5-12Crossref PubMed Scopus (51) Google Scholar). Psychostimulants such as cocaine and amphetamine derivatives also interact with SERT, either as inhibitors or alternative substrates (3Wall S.C. Gu H.H. Rudnick G. Mol. Pharmacol. 1995; 47: 544-550PubMed Google Scholar). The site at which nontransported inhibitors, such as cocaine, bind to the transporter has been a matter of speculation. They could bind at the same site as 5-HT but not be transported, or they could bind at another, allosteric site. However, the individual amino acid residues that contribute to this binding site have not been identified. Competitive inhibition by cocaine, like most other inhibitors of biogenic amine transport, suggests that it binds at the same site as substrate, or at a mutually exclusive site. The possibility that cocaine binds at a separate site would justify searching for drugs that antagonize cocaine binding without inhibiting transport as therapeutic agents for cocaine abuse. Serotonin is cotransported with Na+ and Cl−and countertransported with K+ (4Rudnick G. Reith M. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press, Totowa, NJ1997: 73-100Crossref Google Scholar). Previous work suggests that external Na+ and Cl− are bound together with 5-HT and translocated together (5Humphreys C.J. Wall S.C. Rudnick G. Biochemistry. 1994; 33: 9118-9125Crossref PubMed Scopus (55) Google Scholar). After dissociation of 5-HT, Na+, and Cl− to the cytoplasm, internal K+ is thought to bind and be translocated to the external medium (6Nelson P.J. Rudnick G. J. Biol. Chem. 1979; 254: 10084-10089Abstract Full Text PDF PubMed Google Scholar). The translocation steps for 5-HT and K+ are envisaged as conformational changes that expose a substrate binding site (formed by α-helical transmembrane domains) alternately to one side of the plasma membrane or the other. These conformational changes could be triggered by binding of external substrates (5-HT, Na+, and Cl−) or internal K+ at this site. SERT is a member of a large gene family containing transporters for many other neurotransmitters and other substances (7Amara S. Kuhar M. Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (991) Google Scholar, 8Uhl G. Johnson P. J. Exp. Biol. 1994; 196: 229-236PubMed Google Scholar, 9Nelson N. Lill H. J. Exp. Biol. 1994; 196: 213-228PubMed Google Scholar, 10Borowsky B. Hoffman B.J. Int. Rev. Neurobiol. 1995; 38: 139-199Crossref PubMed Scopus (97) Google Scholar, 11Lester H.A. Cao Y.W. Mager S. Neuron. 1996; 17: 807-810Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Each of these transporters is believed to consist of 12 transmembrane domains connected by hydrophilic loops on the external and cytoplasmic surfaces of the plasma membrane, with the N and C termini in the cytoplasm. Within this family are the closely related transporters for the biogenic amines 5-HT, norepinephrine, and dopamine (1Rudnick G. Clark J. Biochim. Biophys. Acta. 1993; 1144: 249-263Crossref PubMed Scopus (366) Google Scholar, 4Rudnick G. Reith M. Neurotransmitter Transporters: Structure, Function, and Regulation. Humana Press, Totowa, NJ1997: 73-100Crossref Google Scholar). Mutagenesis of biogenic amine transporters has identified some amino acid residues as contributing to transport or binding of substrates and inhibitors (12Kitayama S. Shimada S. Xu H. Markham L. Donovan D. Uhl G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar, 13Kitayama S. Wang J. Uhl G. Synapse. 1993; 15: 58-62Crossref PubMed Scopus (91) Google Scholar, 14Tate C. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar, 15Wang J. Moriwaki A. Uhl G. J. Neurochem. 1995; 64: 1416-1419Crossref PubMed Scopus (65) Google Scholar, 16Barker E.L. Blakely R.D. Mol. Pharmacol. 1996; 50: 957-965PubMed Google Scholar, 17Lin F. Lester H.A. Mager S. Biophys. J. 1996; 71: 3126-3135Abstract Full Text PDF PubMed Scopus (73) Google Scholar). In the dopamine transporter (DAT), an aspartate residue in the first transmembrane domain (TM 1) was found to be critical for transport and two serine residues in TM 7 were found to affect substrate and inhibitor binding (12Kitayama S. Shimada S. Xu H. Markham L. Donovan D. Uhl G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar). Serine residues in TM 7 and TM 11 of DAT were found to affect the K m andV max for 1-methyl-4-phenylpyridinium but not dopamine (13Kitayama S. Wang J. Uhl G. Synapse. 1993; 15: 58-62Crossref PubMed Scopus (91) Google Scholar). Differences in imipramine affinity between rat and human SERT has been traced to a phenylalanine in TM 12 (16Barker E.L. Blakely R.D. Mol. Pharmacol. 1996; 50: 957-965PubMed Google Scholar). However, the removal of consensus glycosylation sites in the second external loop of SERT (EL 2), although they resulted in a lower expression level, did not affect the K m for transport (14Tate C. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar). Wang et al. (15Wang J. Moriwaki A. Uhl G. J. Neurochem. 1995; 64: 1416-1419Crossref PubMed Scopus (65) Google Scholar) mutated three cysteines in EL 1 and EL 2 of DAT. Replacement of the single cysteine in EL 1 (equivalent to Cys-109 in SERT) with alanine had no effect on DAT function, but replacement of either cysteine in EL 2 (corresponding to Cys-200 and Cys-209 in SERT) blocked the appearance of DAT immunofluorescence on the plasma membrane and no dopamine transport was observed. We obtained similar results with SERT using serine replacement mutants and further concluded, from a variety of approaches, that Cys-200 and Cys-209 in SERT were likely to form a disulfide in the native protein (18Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; PubMed Scopus Google Scholar). Cys-200 Cys-209 with external cysteine but Cys-109 in EL 1 was sensitive to with mutant in which Cys-109 was replaced with alanine was fully and insensitive to inactivation by (18Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; PubMed Scopus Google Scholar). have been in residues to the external medium in and M. D. Xu M. A. 1992; PubMed Scopus Google Scholar, M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J. J. A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, C. P. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, A. B. Neuron. 1996; 17: Full Text Full Text PDF PubMed Scopus Google Scholar). Two and (2-sulfonatoethyl)methanethiosulfonate are but is to M. Xu G. 1996; PubMed Scopus Google Scholar). In a transmembrane cysteine residues that are on the external or are in with a or binding with external mutants in which native amino acid residues are replaced with cysteine be inactivated by the the cysteine replacement is and is in a for The mutant of SERT a for cysteine transport in was insensitive to and MTSET (18Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; PubMed Scopus Google Scholar). et al. F. Lester H.A. Mager S. Biophys. J. 1996; 71: 3126-3135Abstract Full Text PDF PubMed Scopus (73) Google Scholar) found that at in TM of SERT the by the protein in the of These results are with the which the transporter as a at affect the these and to the was related to binding sites for 5-HT and cocaine, mutated of the residues in TM one at a time, to cysteine and of the mutants transporters were using the The mutated was by with and and a SERT that and at the N and C (14Tate C. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar). and a they were using the sites and in were by were with and with mutant SERT of the as R.D. Clark Rudnick G. PubMed Scopus Google Scholar). were for at and used to transport activity, β-CIT or surface surface expression of the transporters was using the by a of the of et al. C. L. M. J. 1995; Google Scholar) as (18Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; PubMed Scopus Google Scholar). were with the reagent was and the were surface were from the cell with and transporter was in the of surface by and using an were using an of 5-HT was measured by of phosphate-buffered containing and 1 containing to and for at were by of the substrate and rapid three with The were with of and the to for were used to protein J. Biol. Chem. Full Text PDF PubMed Google Scholar). were by the measured in the of cocaine. of the high affinity cocaine 2β-carbomethoxy-3β-(4-125Iiodophenyl)tropane was measured as (18Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; PubMed Scopus Google Scholar). in were with and with MTSET or as MTSET or in were and of in to the After the were with of The of MTSET and on binding were measured a with in the of 1 reagent at with MTSET for the of β-CIT similar results to the of MTSET and of and were with which the The was with from single the were a of two to and in all the same with the for the SERT which is not sensitive to external as a the residues from to were one at a time, with cysteine. was not is to form the third transmembrane domain (TM of SERT. Almost all of the cysteine replacement mutants had transport when expressed in using the expression R.D. Clark Rudnick G. PubMed Scopus Google Scholar). the mutants, only Y175C and Y176C were for transport In transport Y176C was from but Y175C 5-HT at of the wild type the other replacement mutants, only I179C was active than C109A, from to in the for the of Y176C, and I179C, measured the of transporter expression on the cell surface mutant were with an and surface were using The cell and the surface were to and using the to the SERT C (14Tate C. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar). were in the expression of the cysteine replacement mutants and only in surface expression were to C109A, the levels of surface expression for Y176C, and I179C were 69%, and surface expression for the in transport in I179C, but the transport for Y175C and Y176C was lower than from expression with 1 MTSET for had no effect on transport by and most of the cysteine However, I172C and I179C were sensitive to these and I172C was with a for inactivation of and I179C was inactivated with a of I172C was inactivated also at lower MTSET lower In to the rapid inactivation by MTSET, the negatively charged was effective at transport by I172C and I179C 1 and is as as MTSET A. Biochemistry. 1994; 33: PubMed Scopus Google used a of in these The of effect of could be to a lower or but to a transporter that which of these was the I172C and I179C with for and 1 MTSET for a second The on of that with did not either mutant from inactivation by MTSET is than the I172C and I179C of MTSET and to I172C and with I172C or I179C were with 1 MTSET or for or 1 MTSET for by for The were and for transport. are as a of the observed in a which did not MTSET or in a with I172C or I179C were with 1 MTSET or for or 1 MTSET for by for The were and for transport. are as a of the observed in a which did not MTSET or The of I172C inactivation by MTSET was sensitive to 5-HT and cocaine. for the transporter the of inactivation by MTSET was at 5-HT and cocaine. These are in the same as the of these for SERT G. Wall S.C. Mol. Pharmacol. Google Scholar) and are likely to 5-HT and cocaine binding to the active site of the In 5-HT cocaine inhibited MTSET inactivation of the I179C mutant lower the of MTSET with a protein a cysteine was to I172C and I179C that had been inactivated with MTSET to the of the cysteine I179C was by with 12 cysteine for but I172C the same was to that to Ile-172 is than to Ile-179 not The two mutants with transport activity, Y175C and Y176C, affinity for the high affinity cocaine β-CIT these mutants also wild type affinity for 5-HT and cocaine, measured the of these to β-CIT from binding sites on from these mutant to C109A, Y176C affinity for 5-HT was although Y175C affinity was to The affinity for cocaine was in Y175C and Y176C Y175C affinity was and Y176C affinity was I172C and I179C mutants 5-HT and cocaine from of the dissociation for 5-HT and cocaine and were similar to G. Wall S.C. Mol. Pharmacol. Google Scholar) and in the that MTSET inactivation to I172C containing the mutant the of MTSET to β-CIT In MTSET was effective at than in with 1 MTSET to of of the binding of although transport in was completely to such (18Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; PubMed Scopus Google Scholar). that 1 MTSET completely inactivated β-CIT binding to I172C and Y176C 2 and although I179C was to this of β-CIT by I172C and Y176C was also completely inactivated by the reagent not In one other cysteine replacement was as sensitive to than was C109A, that this reagent further TM of β-CIT binding to cysteine mutants by from the SERT mutants C109A, Y176C, and I179C were with MTSET, and β-CIT binding was measured as For the of binding is to a with no MTSET in a from the SERT mutants C109A, Y176C, and I179C were with MTSET, and β-CIT binding was measured as For the of binding is to a with no MTSET The inactivation of β-CIT binding by MTSET was not by by and that when from SERT mutants I172C or Y176C were with MTSET and inactivation was observed. The of this inactivation was in the of either 5-HT or cocaine. a of the binding was when 5-HT or cocaine was during the inactivation of with low MTSET resulted in inactivation of I172C when with MTSET of inactivation at for in with inactivation at for in For this lower of MTSET were used in with by 5-HT and cocaine MTSET inactivation of β-CIT of from the SERT mutants I172C and Y176C were in at a of of this for I172C and for was to in the same and with MTSET for I172C and for in the or of 5-HT or cocaine as for at were with with or without cocaine. The was by at in a for 2 at and The were for β-CIT binding as The results are expressed as the of the binding to the for I172C and for Y176C at The results are from a which was for I172C and for in a from the SERT mutants I172C and Y176C were in at a of of this for I172C and for was to in the same and with MTSET for I172C and for in the or of 5-HT or cocaine as for at were with with or without cocaine. The was by at in a for 2 at and The were for β-CIT binding as The results are expressed as the of the binding to the for I172C and for Y176C at The results are from a which was for I172C and for The mutant used as a for cysteine replacement cysteine residues but was not inactivated by external MTSET (18Chen J.G. Liu-Chen S. Rudnick G. Biochemistry. 1997; PubMed Scopus Google Scholar). was that replacement of residues with cysteine the of the protein as to other cysteine residues to with MTSET in the mutant this replaced Ile-172, Tyr-176, and Ile-179 with alanine, serine, and methionine. that all of these replacements at Ile-172 were although the from than of for to for the of all these mutants were insensitive to with 1 MTSET for results were observed with replacements of was for transport, a of activity, and was similar in to I179C was sensitive to MTSET when Ile-172 or Ile-179 was replaced with cysteine did MTSET the mutant and MTSET sensitivity of replacements at Ile-172 and transport of Ile-172 and Ile-179 replacement mutants to was measured and is in the first and third The same mutants were with 1 MTSET for transport. The results in the second and the transport as a of the a similar in the of in a The transport of Ile-172 and Ile-179 replacement mutants to was measured and is in the first and third The same mutants were with 1 MTSET for transport. The results in the second and the transport as a of the a similar in the of results were obtained with replacements of are not and were for transport, like of these mutants affinity for that β-CIT binding to and 2 and was inactivated by 1 MTSET to the same as in In of cysteine at this the binding sensitive to the same MTSET and MTSET sensitivity of replacements at from C109A, and Y176C were with in the or of 1 MTSET for at The of β-CIT bound is expressed as a of the bound a similar in the of in a from C109A, and Y176C were with in the or of 1 MTSET for at The of β-CIT bound is expressed as a of the bound a similar in the of The results suggest that residues in the third transmembrane domain of SERT are in proximity and the binding sites for 5-HT and cocaine. other residues in the transporter also are to contribute to this binding these results the first of residues in SERT with of these Tyr-176, is the transporter family and is likely to form of the binding in of these Replacement of residues with cysteine SERT sensitive to MTSET, a reagent 1 and For a mutant to be sensitive to MTSET, the cysteine be in with the external and of that cysteine residue to a in The results in 1 and that Ile-172, Tyr-176, and Ile-179 these The of Ile-172, Tyr-176, and Ile-179 suggests that they form a on an α-helical a of TM The of three sensitive positions the sensitive at the that TM is an and that one side of the the binding from these cysteine replacement at other positions between and did not the MTSET sensitivity of the These are not to the external or are not in a critical for transporter is of that was not sensitive to MTSET, the that is in the of the by SERT F. Lester H.A. Mager S. Biophys. J. 1996; 71: 3126-3135Abstract Full Text PDF PubMed Scopus (73) Google Scholar). of β-CIT to of I172C or Y176C was completely inactivated by MTSET and this inactivation was in the of 5-HT or cocaine These results suggest that Ile-172 and Tyr-176 are to the β-CIT binding site that with binding and that binding with MTSET of these For Tyr-176, further suggests that this residue contribute to the binding site for 5-HT and cocaine. mutated to cysteine in Y176C, the affinity for 5-HT and cocaine was The that this residue is the gene family further that it is for an function, such as substrate In with this for Tyr-176, et al. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) that of the in the acid binding of acid and the inhibitor residues in this Ile-172, and The of residues in this transmembrane domain the that it is critical for transporter For Ile-172, of a similar in binding 5-HT and cocaine. The of these to the I172C mutant from inactivation by MTSET that bound MTSET from the sensitive cysteine residue in and and of the cysteine at with MTSET the cocaine analog β-CIT from with binding site these two suggest that Ile-172 is to the binding site. The only that this is the that replacement of Ile-172 with or alanine did not transport function, in to Tyr-176 the same completely blocked transport. Replacement of Ile-172 with serine, resulted in a mutant with than of wild type Ile-172 and Tyr-176 in and and alanine are likely to be replacements for than for the not the possibility that Ile-172 and Tyr-176 in an allosteric site that is not when the binding site is the the that these two residues are in proximity to the 5-HT and cocaine binding site. 5-HT and cocaine completely I172C from inactivation in high of these only MTSET inactivation of I172C or Y176C in with the MTSET sensitivity of I172C in this suggests that inactivation in to is for that in but not in MTSET to positions and from the cytoplasmic face of the transporter and that this is not completely blocked by bound The that replacement of Tyr-176 with cysteine affinity for 5-HT and cocaine, and that 5-HT and cocaine I172C from MTSET suggest that the binding sites for substrate and cocaine are to other in the biogenic amine and The of Ile-172 and Tyr-176 in the of the third transmembrane domain further suggests that the binding sites for substrate and cocaine are by transmembrane domains and are not on the external surface of the is with a for transport in which the substrate and cotransported bind to a site the protein that is from the cell transport would be by a conformational that blocked to that site from the external medium and a for dissociation from that site to the cytoplasm. In to the of Ile-172 and Tyr-176 with the 5-HT and cocaine binding these did not mutant I179C from MTSET inactivation did of I179C with MTSET β-CIT binding these results that Ile-179 is not with the binding MTSET inactivated transport by I179C 1 and Ile-179 be in some transport or conformational that substrate possibility is that Ile-179 is of the that to bound substrates from to the external of this in I179C with MTSET a positively charged that or with the conformational with the The positively charged reagent MTSET inactivated I172C and I179C than did the negatively charged of the between the of these two agents is likely to from A. Biochemistry. 1994; 33: PubMed Scopus Google Scholar). However, this MTSET than with these is that a negatively charged residue from of the protein the the cysteine In SERT, only two negatively charged residues are found in transmembrane They are in TM 1 and in TM and in the dopamine transporter are to be for of these transporters (12Kitayama S. Shimada S. Xu H. Markham L. Donovan D. Uhl G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar, R. 1993; Google Scholar). has been that the of this residue in transport is to the amino of 5-HT or dopamine (12Kitayama S. Shimada S. Xu H. Markham L. Donovan D. Uhl G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7782-7785Crossref PubMed Scopus (349) Google Scholar). positively charged amino is a to all substrates by of the transporter although the aspartate in TM 1 is found only in biogenic amine is that to the amino of biogenic amine substrates in the binding site by the also the of in I172C and in the proximity of to the TM residues to form of the binding site. We for and for critical of the
Chen et al. (Sat,) studied this question.
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