Analysis of transmembrane domain 2 of rat serotonin transporter by cysteine scanning mutagenesis.

The second transmembrane domain (TM2) of neurotransmitter transporters has been invoked to control oligomerization and surface expression. This transmembrane domain lies between TM1 and TM3, which have both been proposed to contain residues that contribute to the substrate binding site. Rat serotonin transporter (SERT) TM2 was investigated by cysteine scanning mutagenesis. Six mutants in which cysteine replaced an endogenous TM2 residue had low transport activity, and two were inactive. Most of the reduction in transport activity was due to decreased surface expression. In contrast, M124C and G128C showed increased activity and surface expression. Random mutagenesis at positions 124 and 128 revealed that hydrophobic residues at these positions also increased activity. When modeled as an alpha-helix, positions where mutation to cysteine strongly affects expression levels clustered on the face of TM2 surrounding the leucine heptad repeat conserved within this transporter family. 2-(Aminoethyl)-methanethiosulfonate hydrobromide (MTSEA)-biotin labeled A116C and Y136C but not F117C, M135C, or Y134C, suggesting that these residues may delimit the transmembrane domain. None of the cysteine substitution mutants from 117 through 135 were sensitive to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) or MTSEA. However, treatment with MTSEA increased 5-hydroxytryptamine transport by A116C. Activation of A116C by MTSEA was observed only in mutants containing Cys to Ile mutation at position 357, suggesting that modification of Cys-116 activated transport by compensating for a disruption in transport in response to Cys-357 replacement. The reactivity of A116C toward MTSEA was substantially increased in the presence of substrates but not inhibitors. This increase required Na+ and Cl-, and was likely to result from conformational changes during the transport process.

The second transmembrane domain (TM2) of neurotransmitter transporters has been invoked to control oligomerization and surface expression. This transmembrane domain lies between TM1 and TM3, which have both been proposed to contain residues that contribute to the substrate binding site. Rat serotonin transporter (SERT) TM2 was investigated by cysteine scanning mutagenesis. Six mutants in which cysteine replaced an endogenous TM2 residue had low transport activity, and two were inactive. Most of the reduction in transport activity was due to decreased surface expression. In contrast, M124C and G128C showed increased activity and surface expression. Random mutagenesis at positions 124 and 128 revealed that hydrophobic residues at these positions also increased activity. When modeled as an ␣-helix, positions where mutation to cysteine strongly affects expression levels clustered on the face of TM2 surrounding the leucine heptad repeat conserved within this transporter family. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA)-biotin labeled A116C and Y136C but not F117C, M135C, or Y134C, suggesting that these residues may delimit the transmembrane domain. None of the cysteine substitution mutants from 117 through 135 were sensitive to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) or MTSEA. However, treatment with MTSEA increased 5-hydroxytryptamine transport by A116C. Activation of A116C by MTSEA was observed only in mutants containing Cys to Ile mutation at position 357, suggesting that modification of Cys-116 activated transport by compensating for a disruption in transport in response to Cys-357 replacement. The reactivity of A116C toward MTSEA was substantially increased in the presence of substrates but not inhibitors. This increase required Na ؉ and Cl ؊ , and was likely to result from conformational changes during the transport process.
Serotonin transporter (SERT) 1 functions to terminate the action of serotonin (5-hydroxytryptamine, 5-HT) released from central and peripheral neurons (1). It is a principal target for antidepressant drugs like imipramine and fluoxetine (Prozac TM ) as well as psychostimulants like cocaine and 3,4methylenedioxymethamphetamine (MDMA, also known as ecstasy) (2)(3)(4)(5)(6). It belongs to a large family of neurotransmitter and amino acid transporters called neurotransmitter sodium symporters (NSS) (7). Other members of the family include transporters for dopamine (DAT), norepinephrine, and ␥-aminobutyric acid (GAT). SERT is thought to transport 5-HT, Na ϩ , and Cl Ϫ into cells and to transport K ϩ out in the same reaction cycle (8).
The transmembrane topology of SERT predicted from its sequence (9,10) consists of 12 transmembrane domains (TM) connected by six external and five internal loops (EL and IL), with the NH 2 and COOH termini cytoplasmic. Studies using site-directed chemical labeling of SERT have confirmed this topology (11,12). However, the boundaries between TM and loop domains remain largely unknown. Residues in TM1 and TM3 have been proposed to determine substrate and inhibitor selectivity and to contribute to the substrate binding site and permeation pathway (13)(14)(15)(16)(17). Mutations in TM2 have been shown to affect surface expression of GAT-1 and DAT (18,19), but the functional contribution of TM2 to the mechanism of 5-HT transport is unclear.
Multiple lines of evidence suggest that SERT exists as a multimer in the membrane (20 -22). These findings extend to the related transporters GAT-1, DAT, and norepinephrine (19,(22)(23)(24). Many transporters in the NSS family contain, in TM2, a leucine repeat motif similar to that found in coiled-coil proteins (25,26). Experiments with DAT and GAT-1 have shown that mutations in the leucine repeat of TM2 prevent oligomerization and interfere with the cell surface expression of these transporters (18,19). These results suggest the possibility that residues in TM2 control oligomerization, cell surface expression, or both. However, the role of the leucine repeat in this TM is far from certain.
In the present work, we have used cysteine scanning mutagenesis to examine the role of TM2 in transport by replacing each residue, one at a time, with cysteine. Subsequent reaction of the individual cysteine mutants with methanethiosulfonate (MTS) reagents revealed information about the reactivity and importance of those positions (27). Our results provide information about the boundaries of the transmembrane region and provide additional evidence that TM2 is involved in cell surface expression. Furthermore, they suggest a functional interaction between the region of EL1 adjacent to TM2 and Cys-357 in IL3. * This work was supported by grants from the National Institute on Drug Abuse (to G. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡

EXPERIMENTAL PROCEDURES
Mutagenesis-Mutant transporters were generated by site-directed mutagenesis using the QuikChange TM kit (Stratagene, La Jolla, CA). The mutated region was excised by digestion with appropriate restriction enzymes and subcloned back into the original plasmid. All mutations were confirmed by DNA sequencing.
Expression-Confluent HeLa cells were infected with recombinant vTF-7 vaccinia virus and transfected with plasmid bearing SERT cDNA under control of T7 promoter as described previously (28). Transfected cells were incubated for 16 -20 h at 37°C and then used for the determination of transport and binding activities.
Transport Assay-Transfected HeLa cells in 48-well culture plates were washed twice with 250 l of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , and 1.4 mM KH 2 PO 4 , pH 7.3) containing 0.1 mM CaCl 2 and 1 mM MgCl 2 (PBS/CM). Transport of 5-HT was measured by adding 100 l of PBS/CM containing 20.5 nM [ 3 H] 5-HT (PerkinElmer) to each well and incubating for 10 min at room temperature. The substrates were then removed by washing rapidly three times with ice-cold PBS. The cells were lysed in 250 l of 1% SDS for 20 min, transferred into scintillation vials, and counted in 3 ml of Optifluor scintillant (Packard Instrument Co.).
Treatment with MTS Reagents-Mutants were tested for their sensitivity to the MTS reagents (2-(trimethylammonium)ethyl)methanethiosulfonate bromide (MTSET), 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and (2-sulfonatoethyl)methanethiosulfonate (MTSES); MTS reagents were from Toronto Research Chemicals (Ontario, Canada). Cells were preincubated with these reagents for 10 min at room temperature in PBS/CM and then washed three times with 250 l of PBS/CM. The third wash was left in the wells until the transport assay was performed as described above. For the study of the effect of substrates or cocaine on the action of MTSEA, cells were coincubated with MTSEA and varying concentration of each substrate or cocaine for 10 min. The cells were washed twice with indicated buffer and incubated for 10 min. The cells were washed twice again with PBS/CM to quench residual substrates and unreacted MTSEA, then the transport assay was performed as described above.
Membrane Preparation and Binding Assay-HeLa cells grown in 75-cm 2 cell culture flasks were transfected with SERT cDNA as described above. The cells were rinsed once with 10 mM HEPES buffer (adjusted to pH 8.0 with NaOH) and scraped into 15 ml of homogenation buffer (10 mM HEPES, pH 8.0, containing protease inhibitor mixture (1:100) (Sigma, P8340) and 100 M phenylmethylsulfonyl fluoride (1: 500)). The cells were disrupted by sonication, and the membranes were collected by centrifugation at 20,000 ϫ g for 20 min at 4°C. The membranes were resuspended in 1 ml of homogenation buffer and stored at Ϫ80°C until used.
Cell Surface Expression-Cell surface expression of SERT mutants was determined using the membrane-impermeant biotinylation reagent NHS-SS-biotin (Pierce) as described previously (11). Cells expressing transporters in a 12-well plate were treated twice with 500 l of 1.5 mg/ml NHS-SS-biotin in 20 mM HEPES, pH 8.6, 2 mM CaCl 2 , and 150 mM NaCl for 20 min on ice. After labeling, the cells were rinsed with 500 l of 100 mM glycine in PBS/CM for 20 min on ice to quench excess NHS-SS-biotin. The cells were then lysed with 120 l of SDS-lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% SDS, 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5% Sigma protease inhibitor mixture) for 30 min on ice with gentle shaking until the cells were completely lysed. The cell lysate was diluted to 1.2 ml with cold lysis buffer (same as SDS-lysis buffer but without SDS and protease inhibitors). The biotinylated proteins were recovered by adding 100 l of streptavidin-agarose beads (Pierce) and incubating overnight at 4°C with gentle agitation. The beads were washed once with 1.2 ml of lysis buffer and high salt lysis buffer (lysis buffer containing 500 mM NaCl and only 0.1% Triton X-100) and twice with 50 mM Tris-HCl (pH 7.5). The biotinylated proteins were eluted with 100 l of SDS-PAGE sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 1% mercaptoethanol, 0.003% bromphenol blue) for 10 min at 85°C. Portions of each sample (15 l) were applied to a 10% SDS-PAGE and visualized by Western blotting. The transporters were detected using anti-FLAG polyclonal antibody (Affinity Bioreagents, Inc.) (1:400) against the FLAG epitope tag at the C terminus of SERT (30). A horseradish peroxidase conjugated anti-rabbit IgG (1:10,000) was used to visualize the signal by Super Signal West Femto (Pierce).
External Accessibility-The accessibility of introduced cysteine residues in mutant transporters was tested by determining their reactivity to the biotinylating reagent MTSEA-biotin (Toronto Research Chemicals). Cells expressing transporters in a 12-well plate were washed twice with PBS/CM. The cells were then incubated with 500 l of 1 mM MTSEA-biotin for 10 min at room temperature. After washing the reagent away three times with PBS/CM, the cells were lysed and MTSEA-biotin-labeled proteins were recovered from the cell lysate with streptavidin-agarose as described above. Portions of each sample (15 l) were subjected to SDS-PAGE and analyzed by Western blotting using the COOH-terminal FLAG tag on SERT (30). SERT was detected by reacting with anti FLAG polyclonal antibody (Affinity Bioreagents, Inc., 1:400) followed by anti-rabbit IgG (1:10,000). The signals were visualized by chemiluminescence using Super Signal West Femto (Pierce).

RESULTS
Transport Activity of Cysteine Mutants-To generate a suitable background construct from rSERT, five endogenous cysteine residues, which were shown to react with MTS reagents (12,31), were replaced with other amino acids. Among the endogenous cysteines in SERT, Cys-109 in EL1 and Cys-357 in IL3 are responsible for inactivation by cysteine-specific MTS reagents. Together with these two cysteines, we replaced reactive cysteines at positions 15, 21, and 622, all of which are located in the NH 2 -and COOH-terminal regions, with alanine. This X5C construct (C15A/C21A/C109A/C357I/C622A) retained significant transport activity (ϳ50 -60% of C109A) and was used to construct 21 single cysteine mutants in TM2. The transport activity of these mutants varied widely (Fig. 1A). Two of the mutants (P131C and Y121C) were completely inactive, in six other mutants (E136C, M135C, F133C, L119C, L118C, and F117C) 5-HT uptake was measurable but severely impaired (less than 30% compared with X5C), and in three mutants (M124C, A125C, and G128C) transport activity was increased (240%, 150%, and 230% of X5C, respectively). The remaining mutants had levels of transport similar to that of X5C.
Binding Activity of Cysteine Mutants-Membrane preparations from HeLa cells expressing cysteine mutants were used to determine binding of the cocaine analog ␤-CIT. For this analysis, we chose the cysteine mutants with activity levels significantly different from that of X5C. As shown in Table I, transport and binding activity are well correlated in most mutants. Mutant Y121C, however, was inactive for transport but retained some ␤-CIT binding affinity. 5-HT affinity was measured by its ability to displace ␤-CIT. From nonlinear regression analysis, the K D for 5-HT was found to be 1.0 Ϯ 0.5 M and 1.3 Ϯ 0.1 M for C109A and X5C, respectively. Relative to X5C, apparent K D values for mutants M124C and Y134C were slightly increased (2.5 Ϯ 0.3 M and 2.2 Ϯ 0.8 M, respectively). Other mutants showed less significant increases in K D values (1.5 Ϯ 0.15 M for G128C and 1.85 Ϯ 0.26 M for Y121C). These results suggest that a change in 5-HT affinity is unlikely to account for the change in transport activity observed for M124C, G128C, and Y121C.
Surface Expression of TM2 Mutants-Like transport activity, cell surface expression levels varied widely for the TM2 cysteine mutants, and transport activity generally reflected surface expression. To measure cell surface expression, we labeled cell surface proteins by treating cells expressing each cysteine substitution mutant with NHS-SS-biotin. We then used Western blotting against a FLAG epitope tag to identify SERT in the mixture of solubilized biotinylated proteins recovered using streptavidin beads. The diffuse band at ϳ97 kDa was previously identified as the mature glycosylated form of SERT (11,30). C109A and X5C were expressed on the surface at similar levels (data not shown), but surface levels of TM2 mutants varied markedly from those of the parental X5C (Fig.  1B). Surface expression of two inactive mutants, Y121C and P131C, was dramatically decreased (17.3% and 5.5%, respectively), and two other mutants, F117C and L118C, were also expressed at low levels (11.4% and 32.9%, respectively), consistent with their poor transport ability. In contrast, expression of G128C and M124C was significantly increased when compared with X5C (129 and 108%, respectively). Other functional mutants were expressed at levels similar to or slightly less than that of X5C.
These results described above suggest that the difference in transport activity observed for TM2 mutants may be attributed, at least in part, to the change in their surface expression levels. However, the transport data were from a single assay point at 20 nM 5-HT. To determine the nature of the increased activity for mutants M124C, A125C, and G128C, we measured the 5-HT concentration dependence for each of these mutants and compared them with C109A and X5C. The results presented in Table II indicate that the increased activity in these mutants is primarily in their V max , with very little change in K m , consistent with an increase in surface expression.
Mutations of Met-124 and Gly-128 -To further examine the increased activity of M124C and G128C, we substituted those positions with other amino acid residues by mutating position 124 and 128 using degenerate primers that encoded all possible amino acid substitutions. Clonal plasmids obtained from the degenerate product were used to transfect HeLa cells that were subsequently screened for 5-HT transport activity. We obtained several clones with transport activity similar to that of the corresponding cysteine mutant. Table III shows that the replacement of the endogenous methionine at 124 with valine, leucine, or isoleucine enhanced transport activity as well as cysteine. Similarly, the replacement of glycine at position 128 with isoleucine and threonine also increased activity. However, replacing both Met-124 and Gly-128 with isoleucine failed to increase activity in the X5C background and actually decreased activity in the C109A background.
The X5C mutant, although it is resistant to modification with MTS reagents, has only about half the activity of wild type SERT or the C109A mutant. Most of the activity decrease results from the replacement of Cys-357 (32). To test the possibility that the M124C or G128C mutations might stimulate merely by reversing the effect of the C357I mutation, we also

TABLE II Kinetic characteristics of mutants with increased activity
Mutants with greater transport activity than the parent X5C construct were tested to measure transport rate over a range (0.02-5 M) of 5-HT concentrations. K m and V max were calculated by fitting the rate versus concentration data using Origin software (Originlab, Northhampton, MA). Differences in K m were not sufficiently great to be statistically significant. The results are from two experiments, which differed by less than 15% for all values measured. Differences in V max were significant according to the following criteria: a , p Ͻ 0.006; b , p Ͻ 0.04; c , p Ͻ 0.004.   Fig. 2A shows that replacing either Met-124 or Gly-128 with cysteine increased transport activity in both backgrounds, although the stimulation (50 -100%) was somewhat less than in X5C (150%, Fig. 1A). As in the X5C background, both M124C and G128C also increased surface expression (Fig. 2B). Accessibility of TM2 Cysteine Residues to MTSEA-biotin-We have previously used labeling with MTSEA-biotin to define the accessibility of internal and external loop residues (11,12). Residues Cys-109 and Gln-111 in EL1 and Leu-137 and Ile-157 in IL1 were shown to be accessible to biotin labeling in intact and permeabilized cells, respectively. However, the residues forming the boundaries between TM2 and these loops had not been identified. We used intact cells expressing the TM2 cysteine mutants A116C through L118C, and digitoninpermeabilized cells expressing TM2 cysteine mutants Y134C through L137C to address this question.
Cells expressing mutant transporters were labeled with MTSEA-biotin and lysed, and the biotinylated proteins precip-itated with streptavidin-agarose beads. After elution from the beads with buffer containing reducing agents, SERT was specifically detected by SDS-PAGE followed by Western-blotting using anti-FLAG antibody. Fig. 3A shows results for those residues near the extracellular end of TM2. E493C contains a highly reactive cysteine in EL5, which reacted strongly with MTSEA-biotin 2 and was used here as a positive control. X5C contains no reactive cysteines and was used as a negative control. Mutants A116C reacted slightly more than X5C and F117C reacted about the same as X5C, whereas L118C and L119C (not shown) did not react at all, suggesting that the transition between EL1 and TM2 may occur in the vicinity of 2 P. C. Keller II, M. Stephan, H. Glomska, and G. Rudnick, in press.

FIG. 2. 5-HT influx and surface expression of M124C and
G128C mutants in either C109A or C357I/C109A background. A, 5-HT influx was expressed as a percentage of C109A as in Fig. 1A. B, surface expression levels of biotinylated mutants precipitated with streptavidin beads and visualized on Western blots with antibodies against the FLAG epitope tag as in Fig. 1B. The results represent combined data from two experiments. Asterisks indicate values significantly different (p Ͻ 0.05) from control (either C109A or C109A/C357I).
FIG. 3. Accessibility of TM2 cysteines. Cells expressing wild type and mutant transporters were labeled with 1 mM MTSEA-biotin. The cells were solubilized, biotinylated proteins were recovered by streptavidin-agarose beads, and biotinylated SERT was visualized using SDS-PAGE and Western blotting. Quantitation of band density was based on the diffuse 97-kDa band with the "No DNA" control subtracted as a background. A, predicted external residues, labeled in intact cells. B, predicted intracellular residues. Where indicated, the cells were treated with digitonin (0.0025%) for 4 min to permeabilize the plasma membrane prior to labeling with MTSEA-biotin. Lines adjacent to the immunoblot show the positions of 66-and 96-kDa markers. The data represent combined data from two experiments. The data in the bar graph represent combined results from two experiments, one of which is shown in the immunoblot representations. E493C, A116C, E136C, and L137C were significantly different from X5C (p Ͻ 0.05). Ala-116. Results for the intracellular end of TM2 are shown in Fig. 3B. Cells were labeled both with and without permeabilization with digitonin. Fig. 3B also shows that E493C but not X5C reacted in the absence of digitonin. In digitonin-treated cells, L137C reacted much more strongly than in intact cells, demonstrating its intracellular location (12). Even with permeabilization, Y134C and M135C barely reacted and E136C reacted weakly. These results suggest that the transition between TM2 and IL1 may occur in the region between Tyr-134 and Leu-137.
Sensitivity of Cysteine Mutants to MTS Derivatives-Cysteine scanning of TM1 and TM3 suggested that some of the residues in these transmembrane domains were accessible to aqueous reagents added from the cell exterior, supporting the existence of an aqueous transmembrane permeation pathway (13,14,17). To determine whether residues in TM2 also were exposed to external reagents through a permeation pathway, we treated cells expressing mutant transporters with the positively charged and membrane-impermeant reagent MTSET or with the smaller and more permeant reagent MTSEA and then assayed for transport activity. Fig. 4 demonstrates that none of mutants were sensitive to a 10-min treatment with 1 mM MTSET, and most were also insensitive to 1 mM MTSEA. However, when cells expressing the A116C mutant were treated with MTSEA, transport activity was enhanced by ϳ100%. The stimulation was maximal in a 10-min treatment with 0.6 -1.0 mM MTSEA, and was incomplete at lower MTSEA concentrations, whereas the same treatment of X5C led to no stimulation of transport (Fig. 5A). As shown in Fig. 5B, MTSEA modification of A116C significantly decreased the K m for 5-HT transport from 110 Ϯ 7 to 34 Ϯ 10 nM. The bulkier and more highly charged reagents MTSET and MTSES were unable to stimulate transport by A116C at concentrations up to 10 mM (Table IV). The failure of MTSES and MTSET to affect transport in A116C could have resulted from an inability to react with Cys-116 or a lack of effect once reacted. When cells expressing A116C were treated first with either MTSET or MT-SES, the treatment was unable to block the stimulation of A116C transport activity by MTSEA (Table IV)

TABLE IV Inability of MTSET or MTSES to react with A116C
Cells expressing SERT A116C were treated for 10 min with 10 mM MTSES or MTSET or with 1 mM MTSEA, and the excess reagent was removed by washing twice with PBS/CM. In the first three cases, the cells were assayed directly after washing and in the last two cases, a second 10-min incubation was performed with 1 mM MTSEA after washing off MTSES and MTSET. Transport rates are expressed relative to that of cells expressing A116C, which was 0.10 Ϯ 0.001 pmol/ mg/min. The results represent data from a single experiment with duplicate determinations, reproduced at least twice with similar results. Transport was not significantly different (p Ͼ 0.05) after MTSET or MTSES. Stimulation by MTSEA was not significantly different after pretreatment with MTSET or MTSES. and MTSEA were washed away from the cells prior to measuring transport. At the optimal concentration of 5-HT, the stimulation by 0.1 mM MTSEA was equivalent to that observed at 1 mM MTSEA in the absence of 5-HT, suggesting that the effect of 5-HT was to make Cys-116 10-fold more reactive toward MTSEA. The stimulatory effect of 5-HT required the presence of Na ϩ in the incubation with MTSEA. However, in contrast to the effect of 5-HT on the reactivity of other positions (12-14, 32, 34), there was no corresponding stimulation by cocaine, either in the presence or absence of Na ϩ (Fig. 6D).
Other transported substrates such as amphetamine and MDMA were, like 5-HT, effective in potentiating the MTSEA effect, and both these alternative substrates required Na ϩ for their action (Fig. 6, B and C). The inhibition at high concentrations of MDMA (Fig. 6C) is similar to the inhibition of efflux at high concentrations of amphetamine derivatives previously observed in biogenic amine transporters (5). Although data are not shown, the replacement of chloride ion with isethionate inhibited the potentiation of MTSEA reactivity toward A116C, suggesting both Na ϩ and Cl Ϫ are simultaneously required for substrates to increase the reactivity of Cys-116.
To test whether MTSEA activation of the A116C mutant represented a reversal of the decreased activity in X5C or an absolute increase in transport activity, we constructed mutants containing A116C in the C109A and C109A/C357I backgrounds. Fig. 7 shows that the stimulation of activity was observed only in backgrounds containing the C357I mutation.
There was no stimulation in C109A/A116C, either in the presence or absence of 5-HT. However, addition of the C357I mutation to generate C109A/A116C/C357I rendered the transporter sensitive to activation by MTSEA and that reaction was stimulated by 5-HT (Fig. 7). DISCUSSION Cysteine scanning of the region of SERT including TM2 has revealed two insights into the role of this domain. The first is that the extracellular end of TM2 becomes more reactive toward external reagents as SERT proceeds through the transport cycle. The second insight is that SERT expression levels are both enhanced and reduced by mutations on one face of TM2. These results provide a picture of TM2 function that is distinctly different from those of its immediate neighbors in the primary sequence, TM1 and TM3. Those domains were both proposed to contribute residues to the substrate binding site (13,14,17). Both TM1 and TM3 contained several central positions where replacement with cysteine rendered the transporter sensitive to inactivation with MTS reagents (13,17). However, TM2 does not appear to contain residues that directly affect substrate binding or are accessible to external reagents.
A striking finding observed for the TM2 cysteine mutants was the pattern of two groups of residues for which replacement with cysteine caused a loss of activity and expression: Group I includes Phe-117, Leu-118, Leu-119, and Tyr-121 near the extracellular end of TM2, and Group III includes Pro-131, Phe-133, Met-135, and Glu-136 near the cytoplasmic end. Between these two groups, we found Group II, consisting of three residues, Met-124, Ala-125, and Gly-128, where cysteine substitution increased transporter expression and activity. When modeled as an ␣-helix, these groups form three distinct patches along one face of the helix, shown as a helical net diagram in Fig. 8. Of these, Phe-117, Tyr-121, and Pro-131 are completely conserved among mammalian NSS transporters. Replacing Phe-98 of DAT, which corresponds to SERT Phe-117, also caused a dramatic decrease in transport and expression (34). Each of the three patches overlaps a line connecting Leu-118, Ala-125, and Leu-132, which are in the leucine heptad repeat proposed to be involved in oligomerization of SERT and other members of the NSS family.
Almost all of the inhibitory substitutions by cysteine were accompanied by a decrease in cell surface expression, although the extent of inhibition was not always equivalent to the decrease in expression. The one exception was F133C, which had normal surface expression but only 22% of the transport activity of X5C. It is apparent that replacement of Phe-133 has a deleterious effect on substrate translocation or binding, and future experiments will address the nature of this defect. Of the replacements that increased transport activity, M124C and G128C were expressed at higher levels on the cell surface, although the increased surface expression did not match the increase in transport rates. The lack of strict correspondence between changes in transport and expression raises the possibility that mutations along this face of TM2 affect not only the efficiency of SERT delivery to the cell surface but also the functional competence of the transporters that are inserted into the plasma membrane. However, it is also possible that our measurements of surface expression were limited in sensitivity and that the actual surface expression of M124C and G128C may be higher than we estimated and proportionally closer to the increased levels of transport activity in those mutants.
The leucine heptad repeat that runs through the helical face sensitive to mutagenesis has been invoked in SERT and other members of the NSS family as an oligomerization domain. Torres et al. (19) showed that inactive DAT mutants decreased surface delivery of co-expressed wild type DAT, but not if the leucine repeat was disrupted. Sitte and coworkers showed homo-oligomerization of SERT and GAT-1 by fluorescence techniques (22) and found that disruption of the leucine heptad repeat of GAT-1 prevented oligomerization and cell surface localization but not transport activity (18). Although the current results do not address the issue of oligomerization, it may be significant that the regions of TM2 that affected surface expression either positively or negatively lay on the same helical face as the leucine repeat.
Mutation of either Met-124 or Gly-128 to cysteine dramatically increased transport activity. Part of the increase could be accounted for by an increase in surface expression. We considered the possibility that the presence of a cysteine residue in this region of TM2 compensated for the loss of Cys-357 in the X5C mutant. However, we found that valine, isoleucine, and leucine had the same effect as cysteine as a replacement for Met-124 and that isoleucine and threonine replaced cysteine at Gly-128. Moreover, this increase was not an artifact of the X5C background, from which reactive endogenous cysteine residues were removed, but was observed also in SERT C109A.
In contrast to M124C and G128C, the stimulation of transport activity in A116C, when treated with MTSEA, depends on the X5C background, because MTSEA did not stimulate C109A/A116C but did stimulate C109A/A116C/C357I. The connection between Ala-116 and Cys-357 is somewhat surprising. These two residues are not only far apart in the primary sequence but also topologically separated by the bilayer, because Cys-357 was shown to be intracellular (12,32) and A116C reacts with external MTSEA. It is possible that the C357I mutation creates a conformational distortion in the transmembrane region of SERT and that this distortion is overcome by modifying a cysteine at 116. However, the low reactivity of cysteine at both 116 and 357, when compared with other residues in internal or external loops, suggests another possibility. This relatively slow rate of reaction with MTSEA suggests that positions 116 and 357 both have limited access to the aqueous medium, as if they were not exposed on the surface of SERT but were both partially buried in the protein interior. If so, the physical distance between positions 116 and 357 might be smaller than predicted.
The limited reactivity of A116C toward MTSEA (Fig. 5A) and MTSEA-biotin (Fig. 3A) suggests that, although Cys-116 reacted with extracellular reagents, its reactivity is at least partially limited, probably for reasons of accessibility. This is apparent in Table IV where MTSET and MTSES were unable to modify Cys-116, and in Fig. 3A where labeling of Cys-493 in EL5 is much more complete than labeling of Cys-116. Another residue in EL1, Cys-109, reacted much more rapidly in the presence of Li ϩ (31,33). The residues predicted to constitute EL1 may be associated with other loops or TM domains and therefore not as reactive toward extracellular reagents. Although this may provide interesting insight into the structure of EL1, it makes assignment of the extracellular border of TM2 more difficult. The lack of effect by MTSET and MTSEA on L119C through E136C, however, supports their location within the transmembrane region. In contrast with residues at the extracellular end of TM2, cysteine replacement mutants at the intracellular end showed a dramatic rise in reactivity from M135C through L137C (Fig. 3B). The conclusion is somewhat tempered by the low activity of M135C and E136C, but expression levels of M135C, E136C, and L137C were comparable (Fig. 1B and Ref. 12).
The conditions leading to activation of A116C by MTSEA suggest that its reactivity changes during transport of 5-HT or other substrates. Activation by 5-HT occurred only if both Na ϩ and Cl Ϫ were present. Previous data suggest that 5-HT binds in the absence of Na ϩ , so it is clear that the activation requires additional Na ϩ -and Cl Ϫ -dependent steps. Accessibility of other positions, such as Cys-357, was affected by 5-HT and cocaine only in the presence of Na ϩ , but the activation of A116C differs in that cocaine, an inhibitor, is not effective. Moreover, other substrates, such as amphetamine and MDMA, act similarly to 5-HT. These properties suggest that the cysteine at position 116 is more reactive when SERT is in an intermediate form (populated only when the transporter is progressing through its catalytic cycle) than it is in the form that predominates when one of the required components (substrate, Na ϩ , or Cl Ϫ ) is absent. From the increase in reactivity of Cys-116 in the presence of 5-HT (ϳ10-fold judging by the increased potency of MTSEA), we can predict that the intermediate form of SERT has at least 10-fold increased reactivity toward MTSEA, presumably because of increased exposure to the external medium. However, this prediction assumes that the transporter spends essentially all of its time in that more reactive interme- FIG. 8. Helical net projection of TM2 results. Substitution of residues in white on black (groups I and III) with cysteine led to lower activity and those in group II had higher activity. Position numbers in boxes represent the residues with decreased cell surface expression, and underlined numbers represent the residues with significantly increased surface expression. The dotted line shows the putative leucine zipper motif. diate form when transporting 5-HT. Therefore, we can regard the 10-fold increase in reactivity as a minimum. If, during transport, SERT spends significantly less time in this form, its reactivity would need to be even more than 10-fold increased over the resting state in the absence of substrate. This analysis also assumes that Ala-116 in the wild type transporter has similar exposure through the transport cycle as does Cys-116 in the mutant.
Although it is premature to attempt a prediction of the relative positions of TM1-3, it is clear that TM2 differs functionally in that it lacks potential binding site residues. However, some evidence from the closely related DAT suggests that mutations in TM2 can affect inhibitor binding (35). From our results, the transmembrane portion of TM2 would seem to extend at most from Phe-117 through Glu-136. However, such a conclusion is subject to some uncertainty, because the low activity of mutants F117C, L118C, M135C, and E136C precludes a firm conclusion regarding accessibility of cysteine residues at those positions. Our data do not address the positions in TM2 that might possibly contact TM1 or TM3, although it may be significant that Phe-105 in DAT, which, when mutated, affected cocaine binding (35) corresponds to Met-124 in SERT, where mutation increased expression. Thus, there might be an indirect interaction between TM2 and the binding site residues of TM1 and TM3 through helix-helix contacts, but further structural studies will be required to evaluate this possibility.