Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2β-carbomethoxy-3β-(4-[125I]iodophenyl)tropane (β-CIT) in intact cells expressing these mutants, but it inactivated β-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an α-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity. Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2β-carbomethoxy-3β-(4-[125I]iodophenyl)tropane (β-CIT) in intact cells expressing these mutants, but it inactivated β-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an α-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity. The serotonin transporter (SERT, 1The abbreviations used are: SERT, serotonin transporter; β-CIT, 2β-carbomethoxy-3β-(4-[125I]iodophenyl)tropane; DAT, dopamine transporter; 5-HT, 5-hydroxytrypatamine (serotonin); IL, intracellular loop; MTS, methanethiosulfonate; MTSEA, 2-(aminoethyl)methanethiosulfonate hydrobromide; MTSES, (2-sulfonatoethyl)methanethiosulfonate; NET, norepinephrine; NHS-SS-biotin, sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate; NSS, neurotransmitter:sodium symporter; PBS, phosphate-buffered saline; TM, transmembrane. SLC6A4) is a presynaptic plasma membrane protein responsible for the reuptake of serotonin (5-hydroxytrypatamine, abbreviated 5-HT) after its release by neurons. It is a member of a large family of amine and amino acid transporters (SLC6, NSS) distributed throughout the prokaryotic and animal kingdoms. Together with norepinephrine and dopamine transporters (NET and DAT), SERT is part of a subgroup of transporters that mediate the transport of biogenic amine neurotransmitters by symport with Na+ and Cl– ions (1Rudnick G. Reith M.E.A. Neurotransmitter Transporters, Structure, Function, and Regulation. 2nd. Humana Press, Totowa, NJ2002: 25-52Google Scholar). SERT is of particular interest in neurobiology because it is the molecular target of several drugs of abuse and many therapeutic agents used to treat psychiatric disorders. Along with NET and DAT, SERT is inhibited by cocaine, and cocaine analogues bind to SERT with high affinity (2Rudnick G. Wall S.C. Mol. Pharmacol. 1991; 40: 421-426PubMed Google Scholar, 3Wall S.C. Innis R.B. Rudnick G. Mol. Pharmacol. 1993; 43: 264-270PubMed Google Scholar, 4Humphreys C.J. Wall S.C. Rudnick G. Biochemistry. 1994; 33: 9118-9125Crossref PubMed Scopus (55) Google Scholar). Amphetamine and its derivatives, including 3,4-methylenedioxymethamphetamine (MDMA, also known as ecstasy), interact with SERT, DAT, and NET as substrates (5Rudnick G. Wall S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1817-1821Crossref PubMed Scopus (406) Google Scholar, 6Wall S.C. Gu H. Rudnick G. Mol. Pharmacol. 1995; 47: 544-550PubMed Google Scholar). Inhibitors that prevent 5-HT reuptake into serotonergic neurons have been used to treat a variety of neuropsychiatric disorders, including affective disorder, anxiety disorder, obsessive-compulsive disorder, and autism (7Murphy D.L. Andrews A.M. Wichems C.H. Li Q. Tohda M. Greenberg B. J. Clin. Psychiatry. 1998; 59: 4-12PubMed Google Scholar, 8Stahl S.M. J. Affect. Disord. 1998; 51: 215-235Crossref PubMed Scopus (401) Google Scholar, 9Jones B.J. Blackburn T.P. Pharmacol. Biochem. Behav. 2002; 71: 555-568Crossref PubMed Scopus (203) Google Scholar). Furthermore, the incidence of some psychiatric disorders and the effectiveness of antidepressant drugs has been linked to polymorphisms in the promoter of the gene encoding SERT (10Lesch K.P. Bengel D. Heils A. Sabol S.Z. Greenberg B.D. Petri S. Benjamin J. Muller C.R. Hamer D.H. Murphy D.L. Science. 1996; 274: 1527-1531Crossref PubMed Scopus (4408) Google Scholar, 11Sher L. Goldman D. Ozaki N. Rosenthal N.E. J. Affect. Disord. 1999; 53: 203-210Crossref PubMed Scopus (54) Google Scholar, 12Weizman A. Weizman R. Pharmacogenomics. 2000; 1: 335-341Crossref PubMed Scopus (35) Google Scholar, 13Smits K.M. Smits L.J. Schouten J.S. Stelma F.F. Nelemans P. Prins M.H. Mol. Psychiatry. 2004; 9: 433-441Crossref PubMed Scopus (161) Google Scholar). A naturally occurring mutation in human SERT, I425V, was found to be associated with obsessive-compulsive disorder and several other 5-HT-related disorders (14Ozaki N. Goldman D. Kaye W.H. Plotnicov K. Greenberg B.D. Lappalainen J. Rudnick G. Murphy D.L. Mol. Psychiatry. 2003; 8: 933-936Crossref PubMed Scopus (183) Google Scholar). Further investigation revealed that this mutation caused SERT to be in an activated state that is reached in the wild type through the action of cGMP-dependent protein kinase (15Kilic F. Murphy D.L. Rudnick G. Mol. Pharmacol. 2003; 64: 440-446Crossref PubMed Scopus (126) Google Scholar). Hydropathy analysis of SERT predicted 12 transmembrane (TM) domains connected by intracellular and extracellular loops (16Blakely R. Berson H. Fremeau R. Caron M. Peek M. Prince H. Bradely C. Nature. 1991; 354: 66-70Crossref PubMed Scopus (686) Google Scholar, 17Hoffman B.J. Mezey E. Brownstein M.J. Science. 1991; 254: 579-580Crossref PubMed Scopus (507) Google Scholar). This analysis predicted that the second intracellular loop (IL2) was a short cytoplasmic loop containing nine residues connecting TM domains 4 and 5. Further analysis suggested that this loop contained consensus phosphorylation sites for cGMP-dependent protein kinase and protein kinase C (18Miller K.J. Hoffman B.J. J. Biol. Chem. 1994; 269: 27351-27356Abstract Full Text PDF PubMed Google Scholar). From the location of glycosylation sites in the second extracellular loop, the NH2 and COOH termini were predicted to be cytoplasmic (19Tate C. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar). Subsequent experiments using mutagenesis and chemical modification in intact cells and disrupted membranes have verified the overall topology (20Chen J.G. Liu-Chen S. Rudnick G. J. Biol. Chem. 1998; 273: 12675-12681Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 21Androutsellis-Theotokis A. Rudnick G. J. Neurosci. 2002; 22: 8370-8378Crossref PubMed Google Scholar). However, the extent of most of the loops remains unknown, with the exception of extracellular loops 4 (22Mitchell S.M. Lee E. Garcia M.L. Stephan M.M. J. Biol. Chem. 2004; 279: 24089-24099Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and 5 (23Keller II, P.C. Stephan M. Glomska H. Rudnick G. Biochemistry. 2004; 43: 8510-8516Crossref PubMed Scopus (26) Google Scholar). Previous studies of intracellular and extracellular loop residues by cysteine substitution have revealed changes in conformation of these loops in response to substrate and inhibitor binding (21Androutsellis-Theotokis A. Rudnick G. J. Neurosci. 2002; 22: 8370-8378Crossref PubMed Google Scholar, 22Mitchell S.M. Lee E. Garcia M.L. Stephan M.M. J. Biol. Chem. 2004; 279: 24089-24099Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Androutsellis-Theotokis A. Ghassemi F. Rudnick G. J. Biol. Chem. 2001; 276: 45933-45938Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 25Sato Y. Zhang Y.-W. Androutsellis-Theotokis A. Rudnick G. J. Biol. Chem. 2004; 279: 22926-22933Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To further examine the structure of SERT IL2, we have studied IL2 by cysteine-scanning mutagenesis. The results reported here suggest that it is longer than predicted and contains an α-helical structure. Mutagenesis—Mutant transporters were generated by site-directed mutagenesis using the QuikChange™ kit (Stratagene). The mutated region was excised by digestion with appropriate restriction enzymes and subcloned into the X5C mutant of rat SERT, which contains sequences encoding a c-Myc epitope tag at the NH2 terminus and a FLAG epitope tag at the COOH terminus (19Tate C. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar). X5C (C15A/C21A/C109A/C357I/C622A) is lacking all of the endogenous cysteine residues known to react with MTS reagents (25Sato Y. Zhang Y.-W. Androutsellis-Theotokis A. Rudnick G. J. Biol. Chem. 2004; 279: 22926-22933Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). All mutations were screened by restriction mapping and confirmed by DNA sequencing. Expression—The expression system used has been described in detail elsewhere (26Blakely R.D. Clark J.A. Rudnick G. Amara S.G. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (151) Google Scholar, 27Stephan M.M. Chen M.A. Penado K.M. Rudnick G. Biochemistry. 1997; 36: 1322-1328Crossref PubMed Scopus (66) Google Scholar). Briefly, HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO2 incubator. Cells were plated in 96-well culture plates and allowed to grow overnight. The confluent cells were infected with recombinant VTF7-3 virus and transfected with a plasmid containing rat SERT cDNA under the control of the T7 promoter. Transfected cells were incubated for 20–22 h at 37 °C with 5% CO2 and then assayed for transport. Protein concentration was determined with the Micro BCA protein assay reagent kit (Pierce). Cell Surface Biotinylation—Surface expression of SERT mutants was determined using the membrane-impermeant biotinylation reagent sulfo-NHS-SS-biotin (Pierce) as described previously (20Chen J.G. Liu-Chen S. Rudnick G. J. Biol. Chem. 1998; 273: 12675-12681Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). HeLa cells expressing SERT mutants were treated twice with NHS-SS-biotin for 20 min on ice. After labeling, the cells were rinsed with 100 mm glycine in phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, and 1.4 mm KH2PO4, pH 7.3) containing 0.1 mm CaCl2 and 1 mm MgCl2 (PBS/CM) for 20 min on ice to quench excess sulfo-NHS-SS-biotin. The cells were then lysed, and the biotinylated proteins were recovered using streptavidin-agarose beads (Pierce) in an overnight incubation at 4 °C with gentle agitation. The beads were washed, and the biotinylated proteins were eluted with 100 μl of SDS-PAGE sample buffer. Portions of each sample were applied to a 10% SDS-polyacrylamide gel and visualized by Western blotting. The transporters were detected using anti-FLAG polyclonal antibody (Affinity Bioreagents, Inc.) (1:1000) against the FLAG epitope tag at the COOH terminus of rat SERT (19Tate C. Blakely R. J. Biol. Chem. 1994; 269: 26303-26310Abstract Full Text PDF PubMed Google Scholar). A horseradish peroxidase-conjugated anti-rabbit IgG (1:10,000) was used to visualize the signal by Super Signal West Femto (Pierce). The amount of surface expression was determined by quantitative luminescence imaging using a UVP Biochemi imaging system. Transport Assay—[3H]5-HT influx was assayed in monolayer cultures at room temperature. Transfected HeLa cells in 96-well plates were washed with 100 μl of Transport of 5-HT was by 100 μl of containing 20 to each and for min at room temperature. The were by with phosphate-buffered The cells were then in μl of 0.1 for The extent of was determined by a were for to extracellular 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in intact Cells were with 1 mm MTSEA for min at room in and washed with 100 μl of to quench MTSEA, and the transport assay was as described Cell expressing each of the IL2 mutants were with 1 mm MTSEA for min at room and washed with 100 μl of (β-CIT) binding was by the of 100 μl of containing 0.1 β-CIT was allowed to for h at room with gentle and the assay was by the cells with 100 μl of The cells were then in μl of 0.1 for The amount of β-CIT was by a and of the high affinity cocaine β-CIT was in membrane preparations. HeLa cells in culture were transfected with rat SERT cDNA as described After overnight the cells were rinsed with room mm mm acid to pH with and into of mm pH containing of a inhibitor and 100 The cells were by of and and the membrane was by at for 20 min at 4 The membranes were in 1 of and at °C in membrane binding of membranes from cells expressing rat SERT mutants were on ice and with 1 of binding mm to pH with mm NaCl, 0.1 mm and 1 mm was in 96-well plates which were by the of μl of to each and incubated overnight at 4 The was rinsed with 100 μl of room binding and then 100 μl of the membrane was The membranes were washed twice by with μl of binding and binding was then by the of 100 μl of binding containing 0.1 β-CIT was allowed to for h at room with gentle The was by all with 100 μl of binding buffer. The were from the and after in μl of of MTSEA or sensitivity, the membranes were incubated with MTSEA or at for min at room the of the and then the MTS reagent was by the membranes with binding buffer. The β-CIT binding after MTS was by the of β-CIT as described of and were with which the The analysis was from multiple with the for analysis was using Transport of IL2 on hydropathy suggested that IL2 is a loop and Trp-271 through (16Blakely R. Berson H. Fremeau R. Caron M. Peek M. Prince H. Bradely C. Nature. 1991; 354: 66-70Crossref PubMed Scopus (686) Google Scholar, 17Hoffman B.J. Mezey E. Brownstein M.J. Science. 1991; 254: 579-580Crossref PubMed Scopus (507) Google Scholar, S. A. K. H. A. Blakely R. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). acid the residues in IL2 the neurotransmitter transporter family and the biogenic amine transporters To the and of this we the at each from Ile-270 to Ser-293, at a with The cysteine mutants were subcloned into a X5C which is to cysteine MTS reagents (25Sato Y. Zhang Y.-W. Androutsellis-Theotokis A. Rudnick G. J. Biol. Chem. 2004; 279: 22926-22933Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). X5C has of wild type transport activity. 2 the transport activity of each cysteine substitution mutant to the X5C the mutants, most transport activity with or than that of X5C with the exception of and which of X5C activity. mutants and had of 5-HT to that of and in the mutants transport activity was from to that of Full analysis was on each the for and in surface expression in The of the cysteine mutants were at than X5C on the of the mutants, was However, the surface expression of of the mutants was significantly from that of X5C A and From the and the of surface we a for each mutant of the mutants had of transport than the X5C and the wild type transporter transport activity of HeLa cells transfected with SERT IL2 cysteine HeLa cells expressing each of the IL2 cysteine mutants were assayed for 5-HT for min as described under The activity is to the The results from significantly from that of of X5C and IL2 mutants from in a surface expression of SERT IL2 cysteine substitution cells expressing the mutants were treated with sulfo-NHS-SS-biotin to surface Cells were lysed, proteins were and the biotinylated was with were detected by with a polyclonal antibody against the FLAG epitope and from the of each which the of SERT, the expression of IL2 mutants were as a of X5C expression in significantly from that of control the for these mutants from and the surface expression from we a for each mutant to the activity of SERT The activity of X5C is by the in C significantly than activity. The results from each with for each The of MTSEA on Transport and of IL2 each of the cysteine mutants we the of intact cells expressing the mutant with the reagent of these mutants was in the X5C which was to MTSEA each mutant we 5-HT influx and β-CIT binding after incubation with 1 mm MTSEA for in II, extracellular MTSEA had on transport activity or binding activity in of the cysteine mutants, all of which were to the suggesting as these residues were from the of MTSEA on 5-HT transport and β-CIT binding of IL2 cysteine mutants in intact in a MTSEA of β-CIT studies (21Androutsellis-Theotokis A. Rudnick G. J. Neurosci. 2002; 22: 8370-8378Crossref PubMed Google Scholar, 24Androutsellis-Theotokis A. Ghassemi F. Rudnick G. J. Biol. Chem. 2001; 276: 45933-45938Abstract Full Text Full Text PDF PubMed Scopus (42) Google intact cells expressing cysteine substitution mutants in intracellular domains were to MTS reagents in the However, in membrane from binding of the cocaine analog β-CIT was inactivated by the by an at the binding that the cytoplasmic of the plasma membrane to MTS reagents in the membrane preparations. this we the of MTSEA concentration on β-CIT binding to membrane from cells expressing each for of the mutants and X5C in that some mutants, for and were inactivated at of MTSEA, others, as and activity at the MTSEA of the in 4 and from the 20 other mutants were to the MTSEA concentration that to for each mutant and also the extent of MTSEA 5 the of β-CIT binding after which was from of MTSEA or inactivated all of the mutants with at and binding activity was in and with at to and to of β-CIT binding to that of the X5C after with results suggest that IL2 from Trp-271 to to MTSEA, the was also used to treat some of the failed to X5C this concentration that it was at mutants at including the mutants and The in of and to with and MTSEA were to to react with with MTSEA to 1 mm failed to these mutants from by as in this membranes were treated with of MTSEA to The concentration of MTSEA, to each was used to with the of and a of activity G. NJ2002: Google Scholar). the a of min and a of From this and the of MTSEA for the in were a as to Six cysteine mutants, and reacted with 20 which is faster than the the mutants reacted with 1 The in 5 a region of SERT from Trp-271 through Ile-290, the residues to be in with the A significantly region was initially predicted from hydropathy to the IL2 of SERT (16Blakely R. Berson H. Fremeau R. Caron M. Peek M. Prince H. Bradely C. Nature. 1991; 354: 66-70Crossref PubMed Scopus (686) Google Scholar, 17Hoffman B.J. Mezey E. Brownstein M.J. Science. 1991; 254: 579-580Crossref PubMed Scopus (507) Google Scholar, S. A. K. H. A. Blakely R. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). The region is because mutants with cysteine at these were inactivated by MTSEA in membrane and but react with extracellular MTSEA in intact cells It is possible that IL2 this because residues to Trp-271 or to react with MTSEA with for transport or binding activity. However, it is to in the of a in activity. were mutations the reactive and substitution with cysteine transport or binding activity and which a of activity at high MTSEA the was to each of the mutants mutants at were inactivated by the the that the modified cysteine a with was in structure to the endogenous residues at these that the modification in which an cysteine a was described for the dopamine J.A. D. Chen J. Mol. Pharmacol. 1996; Google Scholar). was that MTSEA or because of from with this with MTSEA failed to these mutants against by MTSES, as be MTSEA modified these it is most that MTSEA react with and cysteine substitution mutants were inactivated with than of the to be in a as an the more reactive to a the of the This was because structure this region as the the E. L. J. Mol. Biol. 1998; PubMed Scopus Google the B. G. J. 2004; PubMed Scopus Google Scholar) predicted structure in this The pattern or that at part of this region is in an α-helical conformation. To this is the of structure in the cytoplasmic of a neurotransmitter transporter in the that the of and results from of the cysteine to that the of these to of the cysteine or to a concentration of the of these because at pH we of the cysteine to be and the from a in be The more reactive mutants to more reactive than the reactive It is also possible that the of cysteine at the reactive the of in of an MTSEA we that is the most of in this of for were on a of MTSEA or concentration than at a a all these were on the amount of in a of the MTS reagents this with the of the However, by using the for each we the in the of reagent mutants that reacted at the studied were in disrupted plasma membrane that residues the we have used β-CIT binding than transport as an assay to modification of these However, the with MTSEA and were in the of β-CIT and the of The in from of the to the other that other of the protein or associated proteins to some of the residues but to The region be an of of the transmembrane domains as in the of C. M. H. H. Nature. 2000; PubMed Scopus Google Scholar) and a transporter D. Y. E. Nature. 2004; PubMed Scopus Google Scholar) and for SERT extracellular loop 4 (22Mitchell S.M. Lee E. Garcia M.L. Stephan M.M. J. Biol. Chem. 2004; 279: 24089-24099Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). the IL2 be of the transmembrane domains as in the transporter E. C. R. G. Nature. 2003; PubMed Scopus Google Scholar). also the that a part of the region part of and that the of this region that part of the for substrates and the of the from the reactive residues residues the animal of the family the prokaryotic of the family at residues by suggesting that this the IL2 is an or of these was with cysteine in or MTSEA was suggesting that MTSEA was by the of the Cells expressing many of the cysteine mutants in this region 5-HT more than the X5C results and suggest that the activity of these mutants was to of surface several of these mutants had transport activity for surface expression than wild type The pattern of cysteine substitution elevated that of wild type was a α-helical pattern but a of at an to the the of this region is a was in the at this then the residues cysteine activity a The of mutations in this region to transport activity the that the region and a in the of of the an in this region and a the N. S. J. Neurosci. 2004; PubMed Scopus Google Scholar) suggested that as a of transport by a the substrate IL2 an by SERT activity through then mutations in SERT IL2 its by the and the activity of the this that mutations or other in this region activity by the structure and
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