The Cytoplasmic Substrate Permeation Pathway of Serotonin Transporter

Serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin (5-HT) and is a target for antidepressant drugs and psychostimulants. It is a member of a large family of neurotransmitter and amino acid transporters. A recent study using site-directed cysteine modification identified a helical region of the transporter with high accessibility to the cytoplasm. Subsequently, the high resolution structure of LeuT, a prokaryotic homologue, showed that the residues corresponding to this helical region are part of the fifth transmembrane domain. The accessibility of these positions is now shown to depend on conformational changes corresponding to interconversion of SERT between two forms that face the extracellular medium and the cytoplasm, respectively. Binding of the extracellular inhibitor cocaine decreased accessibility at these positions, whereas 5-HT, the transported substrate, increased it. The effect of 5-HT required the simultaneous presence of Na+ and Cl-, which are transported into the cell together (symported) with 5-HT. In light of the LeuT structure, these results begin to define the pathway through which 5-HT diffuses between its binding site and the cytoplasm. They also confirm a prediction of the alternating access model for transport, namely, that all symported substrates must bind together before translocation. Serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin (5-HT) and is a target for antidepressant drugs and psychostimulants. It is a member of a large family of neurotransmitter and amino acid transporters. A recent study using site-directed cysteine modification identified a helical region of the transporter with high accessibility to the cytoplasm. Subsequently, the high resolution structure of LeuT, a prokaryotic homologue, showed that the residues corresponding to this helical region are part of the fifth transmembrane domain. The accessibility of these positions is now shown to depend on conformational changes corresponding to interconversion of SERT between two forms that face the extracellular medium and the cytoplasm, respectively. Binding of the extracellular inhibitor cocaine decreased accessibility at these positions, whereas 5-HT, the transported substrate, increased it. The effect of 5-HT required the simultaneous presence of Na+ and Cl-, which are transported into the cell together (symported) with 5-HT. In light of the LeuT structure, these results begin to define the pathway through which 5-HT diffuses between its binding site and the cytoplasm. They also confirm a prediction of the alternating access model for transport, namely, that all symported substrates must bind together before translocation. Serotonin transporter (SERT) 2The abbreviations used are: SERT, serotonin transporter; 5-HT, 5-hydroxytryptamine; symport, transport together in the same direction; antiport, transport together in opposite directions; β-CIT, 2β-carbomethoxy-3β-(4-iodophenyl)tropane; MTS, methanethiosulfonate; MTSEA, 2-(aminoethyl)methanethiosulfonate hydrobromide; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate; X5C, SERT C15A/C21A/C109A/C357I/C622A; TM, transmembrane domain; EL, extracellular loop; NMDG, N-methyl-d-glucamine. is a polytopic membrane protein responsible for reuptake of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) following its release (1Rudnick G. Reith M.E.A. Neurotransmitter Transporters, Structure, Function, and Regulation. Humana Press, Totowa, NJ2002: 25-52Google Scholar). It is a member of the NSS or SLC6 family of sodium-coupled neurotransmitter and amino acid transporters, which also includes transporters for dopamine, norepinephrine, γ-aminobutyric acid, and glycine (2Chen N.H. Reith M.E. Quick M.W. Pfluegers Arch. 2004; 447: 519-531Crossref PubMed Scopus (344) Google Scholar). SERT is a target for antidepressant drugs such as fluoxetine (Prozac) and for psychostimulants such as cocaine and 3,4-methylenedioxymethamphetamine (also known as ecstasy) (3Stahl S.M. J. Affect. Disord. 1998; 51: 215-235Crossref PubMed Scopus (416) Google Scholar, 4Rudnick G. Wall S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1817-1821Crossref PubMed Scopus (415) Google Scholar). Antidepressants, cocaine and the high affinity cocaine analog 2β-carbomethoxy-3β-(4-iodophenyl)tropane (β-CIT, or RTI-55) are competitive inhibitors of SERT-mediated 5-HT uptake and are likely to bind from the extracellular medium close to the 5-HT binding site on SERT (5Humphreys C.J. Wall S.C. Rudnick G. Biochemistry. 1994; 33: 9118-9125Crossref PubMed Scopus (55) Google Scholar, 6Rudnick G. Wall S.C. Mol. Pharmacol. 1991; 40: 421-426PubMed Google Scholar, 7Wall S.C. Innis R.B. Rudnick G. Mol. Pharmacol. 1993; 43: 264-270PubMed Google Scholar). SERT couples the transport of one 5-HT molecule into the cell together with one Na+ and one Cl- ion and in exchange for one K+ ion in each catalytic cycle (8Nelson P.J. Rudnick G. J. Biol. Chem. 1979; 254: 10084-10089Abstract Full Text PDF PubMed Google Scholar, 9Talvenheimo J. Fishkes H. Nelson P.J. Rudnick G. J. Biol. Chem. 1983; 258: 6115-6119Abstract Full Text PDF PubMed Google Scholar, 10Rudnick G. J. Bioenerg. Biomemb. 1998; 30: 173-185Crossref PubMed Scopus (71) Google Scholar). According to the alternating access model, a transporter contains a central binding site for substrates that is exposed alternately to either side of the membrane through conformational changes (11Jardetzky O. Nature. 1966; 211: 969-970Crossref PubMed Scopus (911) Google Scholar, 12Tanford C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3701-3705Crossref PubMed Scopus (39) Google Scholar). As a corollary to the alternating access model, when a transporter couples the transmembrane movement of more than one substrate, the process requires, for maximum efficiency, that the conformational change occurs only when all substrates that are transported in the same direction (symport) are bound (13Stein W.D. Transport and Diffusion across Cell Membranes. Academic Press, Orlando1986: 397-404Google Scholar). When substrates are transported in opposite directions (antiport) efficient coupling requires that the conformational change occurs only when substrate is bound and not when the binding site is empty (14Jencks W.P. Adv. Enzymol. Relat. Areas Mol. Biol. 1980; 51: 75-106PubMed Google Scholar, 15Stein W.D. book and Diffusion across Cell Membranes. Academic Press, Orlando1986: 306-315Google Scholar). Applied to SERT, these rules for efficient coupling suggest that extracellular 5-HT, Na+, and Cl- must all be bound before the transporter will undergo the conformational change that opens the permeation pathway connecting the binding site to the cytoplasm and that cytoplasmic K+ must bind before the cytoplasmic pathway closes and the extracellular pathway opens. It is unlikely that the substrate-permeation pathways are both open simultaneously, because that would create a channel that would rapidly dissipate any accumulated substrate. Thus, we expect that the cytoplasmic permeation pathway should close before the extracellular pathway opens, and vice versa. In 2003 we demonstrated that TnaT from Symbiobacter thermophilum, one of the many previously uncharacterized prokaryotic proteins homologous to SERT, was a functional amino acid transporter (16Androutsellis-Theotokis A. Goldberg N.R. Ueda K. Beppu T. Beckman M.L. Das S. Javitch J.A. Rudnick G. J. Biol. Chem. 2003; 278: 12703-12709Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Subsequently, another prokaryotic transporter from the NSS family, LeuT from Aquifex aeolicus, was crystallized (17Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1374) Google Scholar). The high resolution structure obtained from these crystals represents a major development in the understanding of this family of transporters. It allows us to interpret results from site-directed chemical modification studies within a structural context. Nevertheless, many aspects of transporter function, including conformational changes accompanying transport, are not apparent from the LeuT structure. A similar situation exists for other transport proteins, many of which have recently been crystallized (18Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1636) Google Scholar, 19Dutzler R. Campbell E.B. 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Only for those proteins, such as Ca2+ ATPase and lac permease, for which there is an abundance of biochemical data available has the structure been very informative as to transport mechanism. To address the conformational changes that SERT undergoes as a result of substrate and inhibitor binding, we have employed a strategy of site-directed chemical modification (26Frillingos S. Sahin-Toth M. Wu J.H. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (322) Google Scholar, 27Rudnick G. Quick M.W. book Transporters. Wiley-Liss, Inc., Hoboken, NJ2002: 125-141Google Scholar), replacing endogenous SERT residues with cysteine and testing their reactivity toward methanethiosulfonate (MTS) reagents either in intact cells, where these reagents have access to the extracellular face of SERT, or in unsealed membrane preparations. In both cases, we have used, as a background construct, a SERT with endogenous cysteine residues or Y. A. Rudnick G. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). this we and have identified many positions in SERT where the reactivity of an cysteine is substrate or inhibitor positions, this is to (17Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1374) Google Scholar, A. Rudnick G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), for of the positions where binding the is Y. A. Rudnick G. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Rudnick G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Rudnick G. J. 2002; PubMed Google Scholar, S.M. E. M.L. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In only substrates not inhibitors the reactivity of the and the effect required the simultaneous presence of Na+ and of the for Na+ and Cl- in transport, we that the conformational change substrate, Na+, and Cl- to of SERT to a in the transport we with which to that to a The in this allows us to a to the previously the cytoplasmic accessibility of SERT in the region to an between transmembrane and Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). that cysteine residues for through to 2-(aminoethyl)methanethiosulfonate in membrane from cells, not in those intact cells, that those positions from the cytoplasm. In this positions, and to be more to with an Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In the high resolution structure of LeuT, the region corresponding to SERT the cytoplasmic of (17Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1374) Google Scholar, T. Javitch J.A. H. Mol. Pharmacol. PubMed Scopus Google Scholar). The more residues are in which in the of the structure of SERT as from the LeuT structure. the structure of LeuT the cytoplasmic permeation we the that the region identified in SERT to this The previously identified cysteine to in reactivity in to substrates and inhibitors in a with and of this permeation of of the cysteine used was previously Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). was into the background which is all of the endogenous cysteine residues known to with reagents Y. A. Rudnick G. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The used has been in J.A. Rudnick G. 1991; PubMed Scopus Google Scholar). Binding of the high affinity cocaine was in membrane from as previously Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). membrane binding from SERT on to and with of binding or of other as To the effect of the of 5-HT or cocaine to the and for in binding of or as was to the the was for at and the to and binding was the of of the binding and Binding was to for at with The was with of binding The from the and with a in of of and data with which the The was from In the data with the from at the in the results Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google a of positions on one face of SERT as a of residues on this face and as a of the each of these we the of and to the of with In these we from a with the of and and at a to in the of this the of and into binding and or was only the with and not the binding The of was as previously shown Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), from the in binding to the In that we showed that binding to with a cysteine at positions be reagents Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). and not to and the was shown to be with than in the protein Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). studies showed that with than Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). using of MTSEA, we to of the binding in each and to the of on that In this and the is In the of MTSEA, of a to more than with this or decreased and a in increased reactivity toward The results for substrate and inhibitor on are shown in 5-HT and cocaine decreased the reactivity of from MTSEA, at the was in the presence of and when either Na+ or Cl- was or the of 5-HT and cocaine was when both Na+ and Cl- to when either Na+ or Cl- was The in was to be to for 5-HT and for the results similar with cocaine with 5-HT. decreased reactivity toward all and the for this effect was to in the presence of both Na+ and Cl- 5-HT, reactivity not any In 5-HT more toward only when both Na+ and Cl- To confirm that the of in the presence of cocaine and 5-HT in to of and not a effect of modification MTSEA, we of binding to as a of As shown in the presence of 5-HT or cocaine decreased or the required to of the binding of cocaine increased the required for and 5-HT decreased that changes in the of The was for in the presence of 5-HT, and in the presence of results are with changes in the reactivity of in to 5-HT and cocaine and for that this in the presence of cocaine of 5-HT and cocaine on the and of of for in a previously showed that was to binding to the cysteine only in membrane and not in intact Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). To confirm that 5-HT and cocaine not the of we the of extracellular to 5-HT transport and binding in intact MTSEA, which in membrane within we of either transport or binding either in the presence or of 5-HT or cocaine of SERT to in intact in a The of 5-HT and cocaine on reactivity not to these two a of with cysteine at positions on the of of these such as and very to MTSEA, for of the was in the of 5-HT or cocaine When the with MTSEA, both 5-HT and at decreased the reactivity of and for as shown in and also both where Na+ or Cl- was 5-HT to and in the presence of and was to in The results that 5-HT not the reactivity of any residues on the face of In 5-HT increased the reactivity the of of many positions on the face of this required the presence of both Na+ and that and to a when 5-HT, Na+, and Cl- the 5-HT effect on these residues in the of Na+ or Cl- In for all these and for all cocaine decreased their reactivity toward MTSEA, from of the residues to the substrate binding was the residues in because was 5-HT in the of Na+ or Cl- was 5-HT in In to Na+, Cl-, and 5-HT, SERT also K+ as part of the cycle (8Nelson P.J. Rudnick G. J. Biol. Chem. 1979; 254: 10084-10089Abstract Full Text PDF PubMed Google Scholar, G. Nelson P.J. Biochemistry. PubMed Scopus (71) Google Scholar). To the of K+ on the accessibility of we the of to cysteine in the presence of In to Na+ and we also the of which was previously shown to conformational changes and binding in SERT A. C.J. E. Rudnick G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the effect of of Na+, and on of binding SERT was to As shown in Na+ effect on the both K+ and increased the reactivity toward in a The effect of K+ on reactivity was for residues to the cytoplasmic of that cysteine through all showed increased reactivity in the presence of K+ through not increased the reactivity of all cysteine both there was apparent between positions on the more or face of The results the that the cytoplasmic permeation pathway of SERT, through which 5-HT, Na+, Cl-, and K+ in between their binding and the cytoplasm, is in According to this and with this pathway is open to the cytoplasm in one of two of SERT, and in a which is to bind extracellular inhibitors such as results also one of the alternating access model for transport, which is that interconversion between these two should when the binding for 5-HT, Na+, and Cl- all are from residues from through to the because of their accessibility to in membrane not in intact Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The corresponding residues in the high resolution structure of LeuT that this region the cytoplasmic of (17Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1374) Google Scholar). In that structure, many positions not residues corresponding to those we in The results suggest that was because to the cytoplasmic substrate-permeation which is in the LeuT structure open in other of LeuT and The region in SERT The corresponding in LeuT is which is within of one of the Na+ bound in the LeuT structure. The region of is to the from the cytoplasm to the binding The of results is that 5-HT and cocaine opposite on accessibility in the permeation The reactivity of cysteine residues on the face of positions and was decreased the presence of cocaine with 5-HT, increased reactivity toward the same residues and interpret this as to the of 5-HT and not cocaine to as a substrate for As a competitive inhibitor that from the cocaine is to bind and the of SERT in which the substrate binding site is the extracellular the cytoplasmic pathway from and with modification of a cysteine in the cytoplasmic pathway is to SERT in the cytoplasmic in which the extracellular where cocaine and is an for the that MTSEA, from the cytoplasmic face of SERT, binding to an extracellular In to 5-HT is to interconversion of SERT to the cytoplasmic from which substrates are this release of 5-HT requires of the cytoplasmic permeation is as 5-HT is transported to the cytoplasm, residues that pathway will be more to reagents from the cytoplasmic face of the are on an that the of 5-HT and cocaine are and that these not SERT a with the site of 5-HT, the effect on reactivity requires both Na+ and Cl-, was shown that ion is required for 5-HT binding (5Humphreys C.J. Wall S.C. Rudnick G. Biochemistry. 1994; 33: 9118-9125Crossref PubMed Scopus (55) Google Scholar). 5-HT bind in the of Na+ or Cl- the reactivity of cysteine because 5-HT than the reactivity of that we are in the permeation is unlikely that the 5-HT binding site the positions that with It is also likely that the effect of cocaine is from the of the cell to 5-HT whereas extracellular not with these cysteine in intact only in membrane Rudnick G. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google these two the reactivity of at positions from to and is unlikely that both the 5-HT and cocaine binding this all these is likely that 5-HT and cocaine to or reactivity with these cysteine results for other conformational changes in to the interconversion of SERT between extracellular and cytoplasmic and that cocaine and 5-HT decreased when both Na+ and Cl- the to the and change in affinity conformational changes that 5-HT binding and interconversion from extracellular to cytoplasmic forms of another conformational change be responsible for 5-HT the reactivity of that face from the permeation pathway and It should be that these residues are in the of and any conformational changes that the accessibility of this region are likely to a large in In the alternating access model of transport, substrates that are transported together as 5-HT, Na+, and Cl- in the of are to bind together before the protein between extracellular to cytoplasmic To this should not one or more of the substrates are To the that all substrates be bound before conformational of the transporter has before been and that both Na+ and Cl- must be for the in reactivity of residues be the the conformational to the for binding of all symported It is to that Na+ and Cl- are not required for 5-HT binding, because 5-HT was shown to bind in the of Na+ or Cl- Y. A. Rudnick G. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the of cocaine and 5-HT in the of Na+ or Cl- and In studies of the reactivity of cysteine residues into extracellular and we have of conformational change in SERT that on the presence of 5-HT, Na+, and Cl- Y. A. Rudnick G. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. E. M.L. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), in the of the and the LeuT structure, the of the conformational change was not It is likely that the previously reactivity changes that required 5-HT, Na+, and Cl- all are to the same conformational change of the The of K+ and and are to conformational increased the reactivity of cysteine the effect of K+ was to the cytoplasmic of the was previously shown to reactivity in extracellular A. C.J. E. Rudnick G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and both of result from its to as a The effect of at as as a more on SERT which is from that of 5-HT or K+ is part of the SERT and not K+ in this (8Nelson P.J. Rudnick G. J. Biol. Chem. 1979; 254: 10084-10089Abstract Full Text PDF PubMed Google Scholar). It is that a the of SERT to the extracellular the increased reactivity at positions we that the cytoplasmic of increased its conformational in the presence of The structure of LeuT an structural in which to with an (17Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1374) Google Scholar). Thus, the extracellular of to the cytoplasmic of previously that at positions from through with the 2-(trimethylammonium)ethyl methanethiosulfonate M. H. Rudnick G. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). residues to through of LeuT, which the and of the (17Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1374) Google Scholar). The LeuT structure has been as a where the in the to the bound substrate molecule (17Yamashita A. Singh S.K. Kawate T. Jin Y. Gouaux E. Nature. 2005; 437: 215-223Crossref PubMed Scopus (1374) Google Scholar). the of the are and In the region of corresponding to the exposed residues in SERT the of the and within of the bound substrate. Thus, the exposed region of that of is to the from the binding site to the of the the extracellular of a similar to that of the cytoplasmic of the of the cytoplasmic of LeuT in the structure similar to the extracellular be a model for the extracellular of SERT in the cytoplasmic this from studies with and of at positions from and in toward in the presence of 5-HT, and this required both Na+ and Cl- as the 5-HT effect in S.M. E. M.L. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). A cysteine at in was more toward in the presence of cocaine and in the presence of 5-HT J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). is opposite to we with and is with the that the permeation pathway opens when the pathway the open of the extracellular face of LeuT as a model for the cytoplasmic face of SERT in the cytoplasmic The residues in face and and is in to the cytoplasmic permeation pathway is these In this is that residues in the cytoplasmic of and recently shown to more in the presence of substrate in the transporter from M. H. Goldberg N.R. T. H. Javitch J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and of and the of for the structural data used in the and model shown in