Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an (cid:1) -Helical Conformation*

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is reg-ulated 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 2 (cid:2) -carbome-thoxy-3 (cid:2) -(4-[ 125 I]iodophenyl)tropane ( (cid:2) -CIT) in intact cells expressing these mutants, but it inactivated (cid:2) -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 (cid:1) -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, 1 SLC6A4) is a presynaptic plasma membrane protein responsible for the reuptake of serotonin (5-hydroxytrypatamine, abbreviated 5-HT) after at 37 °C in a humidified 5% CO 2 incubator. Cells were plated in 96-well culture plates and allowed to grow overnight. confluent cells were

The serotonin transporter (SERT, 1 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 ϩ and Cl Ϫ ions (1).
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 (2)(3)(4). Amphetamine and its derivatives, including 3,4-methylenedioxymethamphetamine (MDMA, also known as ecstasy), interact with SERT, DAT, and NET as substrates (5,6). 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 (7)(8)(9). 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 (10 -13).
A naturally occurring mutation in human SERT, I425V, was found to be associated with obsessive-compulsive disorder and several other 5-HT-related disorders (14). 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 (15).
Hydropathy analysis of SERT predicted 12 transmembrane (TM) domains connected by intracellular and extracellular loops (16,17). 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 (18). From the location of glycosylation sites in the second extracellular loop, the NH 2 and COOH termini were predicted to be cytoplasmic (19). Subsequent experiments using mutagenesis and chemical modification in intact cells and disrupted membranes have verified the overall topology (20,21). However, the extent of most of the loops remains unknown, with the exception of extracellular loops 4 (22) and 5 (23).
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 (21,22,24,25). 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 ␣-helical structure.

EXPERIMENTAL PROCEDURES
Mutagenesis-Mutant transporters were generated by site-directed mutagenesis using the QuikChange TM 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 NH 2 terminus and a FLAG epitope tag at the COOH terminus (19). X5C (C15A/C21A/C109A/ C357I/C622A) is lacking all of the endogenous cysteine residues known to react with MTS reagents (25). All mutations were screened by restriction mapping and confirmed by DNA sequencing.
Expression-The expression system used has been described in detail elsewhere (26,27). 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 g/ml streptomycin at 37°C in a humidified 5% CO 2 incubator. Cells were plated in 96-well culture plates and allowed to grow overnight. The confluent cells were * This work was supported by grants from the National Institute on Drug Abuse. 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  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% CO 2 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 (20). 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 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) for 20 min on ice to quench excess sulfo-NHS-SSbiotin. 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 l 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 (19). 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. Mutants were tested for their accessibility to extracellular 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) (Toronto Research Chemicals, Ontario Canada) in intact cells. Cells were preincubated with 1 mM MTSEA for 10 min at room temperature in PBS/CM and washed three times with 100 l of PBS/CM to quench unreacted MTSEA, and the transport assay was performed as described above.
Whole Cell Binding Assay-Cells expressing each of the IL2 mutants were preincubated with 1 mM MTSEA for 10 min at room temperature and washed three times with 100 l of PBS/CM. 2␤-Carbomethoxy-3␤-(4-[ 125 I]iodophenyl)tropane (␤-CIT) binding was initiated by the addition of 100 l of PBS/CM containing 0.1 nM ␤-CIT (RTI-55; PerkinElmer Life Sciences). Binding was allowed to proceed for 1.5 h at room temperature with gentle rocking, and the assay was terminated by washing the cells three times with 100 l of ice-cold PBS. The cells were then solubilized in 30 l of 0.1 M NaOH for 30 min. The amount of ␤-CIT bound was measured by a Wallac MicroBeta plate counter.
Membrane Preparation and Binding Assay-Binding of the high affinity cocaine analogue ␤-CIT was measured in crude membrane preparations. HeLa cells grown in 75-cm 2 cell culture flasks were transfected with rat SERT cDNA as described above. After overnight transfection, the cells were rinsed once with room temperature 10 mM lithium-HEPES buffer (10 mM HEPES-free acid brought to pH 8.0 with LiOH) and scraped into 10 ml of homogenization buffer (10 mM HEPES, pH 8.0, containing 0.5% of a protease inhibitor mixture (Sigma) and 100 M phenylmethylsulfonyl fluoride). The cells were lysed by two cycles of freeze-thawing and sonication, and the resulting crude membrane fraction was collected by centrifugation at 15,000 ϫ g for 20 min at 4°C. The membranes were resuspended in 1 ml of homogenization buffer and stored at Ϫ80°C in 0.1-ml aliquots until used.
For membrane binding assays, aliquots of membranes from cells expressing rat SERT mutants were thawed on ice and diluted with 1 ml of binding buffer (10 mM HEPES, adjusted to pH 8.0 with NaOH, 150 mM NaCl, 0.1 mM CaCl 2 , and 1 mM MgCl 2 ). Binding was measured in Multiscreen-FB 96-well filtration plates (Millipore, Bedford, MA), which were pretreated by the addition of 200 l of 0.1% polyethyleneimine to each well and incubated overnight at 4°C. The polyethyleneimine was rinsed away with 3ϫ 100 l of room temperature binding buffer, and then 100 l of the diluted membrane solution was added per well. The membranes were washed twice by filtration with 200 l of binding buffer, and binding was then initiated by the addition of 100 l of binding buffer containing 0.1 nM ␤-CIT (RTI-55; PerkinElmer Life Sciences). Binding was allowed to proceed for 1.5 h at room temperature with gentle rocking. The reaction was stopped by washing all wells three times with 100 l of ice-cold binding buffer. The filters were removed from the plate and counted after soaking in 150 l of Optifluor.
For determination of MTSEA or (2-sulfonatoethyl)methanethiosulfonate (MTSES) sensitivity, the membranes were incubated with MT-SEA or MTSES at varying concentrations for 15 min at room temperature following the initial washing of the membranes, and then the MTS reagent was removed by washing the membranes three times with binding buffer. The ␤-CIT binding after MTS treatment was measured by the addition of ␤-CIT as described above.
Data Analysis-Nonlinear regression fits of experimental and calculated data were performed with Origin (Microcal Software, Northampton, MA), which uses the Marquardt-Levenberg nonlinear least squares curve-fitting algorithm. The statistical analysis given was from multiple experiments. Data with error bars represent the mean S.D. for triplicate measurements. Statistical analysis was performed using Student's paired t tests.

RESULTS
Transport Activity of IL2 Mutants-Initial predictions based on hydropathy profiles suggested that IL2 is a nine-residue loop between TM4 and TM5 (from Trp-271 through Lys-279) (16,17,28). Amino acid alignment indicates the residues in IL2 are highly conserved within the neurotransmitter transporter family and especially so within the biogenic amine transporters (Fig. 1). To investigate the structural and functional properties of this region, we substituted the native residue at each position from Ile-270 to Ser-293, one at a time, with cysteine. The cysteine mutants were individually subcloned into a background construct designated X5C (C15A/C21A/C109A/C357I/ C622A), which is relatively insensitive to cysteine MTS reagents (25). X5C has ϳ25-30% of wild type transport activity. Fig. 2 shows the transport activity of each cysteine substitution mutant relative to the parental X5C construct. Of the twentyfour mutants, most retained transport activity comparable with or greater than that of X5C with the exception of V291C and L292C, which retained Ͻ50% of X5C activity. Seven mutants (W271C, K272C, K275C, T276C, A285C, F287C, and I290C) had levels of 5-HT uptake similar to that of X5C, and in the remaining mutants transport activity was increased from 150 to 400% that of X5C. Full kinetic analysis was performed on each mutant, yielding the data for K m and V max shown in Table I. Actual surface expression levels are shown in Fig. 3. The majority of the cysteine mutants were expressed at lower levels than X5C on the cell surface. One of the mutants, L292C, was almost undetectable. However, the surface expression of about one-third of the mutants was not significantly different from that of X5C (Fig. 3, A and B). From the V max values and the levels of surface expression, we calculated a normalized V max for each mutant (Fig. 3C). Several of the mutants had maximal rates of transport higher than both the parent X5C construct (Fig. 3C, white dashes) and the wild type transporter (black dashes).
The Effect of Extracellular MTSEA on Transport and Binding Activity of IL2 Cysteine Mutants-For each of the cysteine replacement mutants we tested the effect of incubating intact cells expressing the mutant with the cysteine-modifying reagent MTSEA. Each of these mutants was constructed in the X5C background, which was relatively insensitive to MTSEA (Table II). For each mutant we measured 5-HT influx and ␤-CIT binding after incubation with 1 mM MTSEA for 10 min. As shown in Table II, extracellular MTSEA had little effect on transport activity or binding activity in any of the cysteine mutants, all of which were similar to the background X5C, suggesting that, as expected, these residues were not accessible from the external medium.
MTSEA Inactivation of ␤-CIT Binding-In previous studies (21,24), intact cells expressing cysteine substitution mutants in putative intracellular domains were insensitive to MTS reagents in the external medium. However, in membrane preparations from those cells, binding of the cocaine analog ␤-CIT was inactivated by the same reagents, presumably by inducing an indirect conformational change at the binding site. We infer that the cytoplasmic face of the plasma membrane became accessible to MTS reagents in the membrane preparations. For this reason, we tested the effect of MTSEA concentration on ␤-CIT binding to membrane preparations from cells expressing each mutant.
Representative data for four of the mutants and X5C are shown in Fig. 4. These inactivation profiles show that some mutants, for example, G273C and V274C, were completely inactivated at low concentrations of MTSEA, whereas others, such as K272C and K275C, retained partial activity even at the highest MTSEA concentrations used. Each of the curves in Fig.  4 and similar curves from the 20 other mutants were fit to estimate the MTSEA concentration that led to half-maximal inhibition for each mutant and also the maximal extent of MTSEA inhibition.

FIG. 3. Cell surface expression of SERT IL2 cysteine substitution mutants.
A, cells expressing the indicated mutants were treated with sulfo-NHS-SS-biotin to label cell surface proteins. Cells were lysed, proteins were solubilized, and the biotinylated fraction was extracted with streptavidin beads. Transporters were detected by immunoblotting with a polyclonal antibody against the FLAG epitope tag. B and C, from the relative integrated density of each 96-kDa band, which represents the mature, fully glycosylated form of SERT, the expression levels of IL2 mutants were estimated as a percentage of X5C expression (B). Asterisks in panel B indicate values significantly different (p Ͻ 0.05) from that of control (X5C). Using the V max values for these mutants from Table I and the surface expression levels from panel B, we calculated a normalized V max for each mutant (C). These normalized values are expressed relative to the activity of SERT C109A (black dashes). The activity of X5C is shown by the white dashes. Asterisks in panel C indicate values significantly higher (p Ͻ 0.05) than C109A activity. The results represent data combined from three experiments, each with triplicate measurements for each mutant.
Y289C, and I290C. Mutants with cysteines at positions proximal to 270 and distal to 290 retained levels of ␤-CIT binding similar to that of the parental construct X5C after treatment with MTSEA. These results suggest that IL2 functionally extends from Trp-271 to Ile-290.
In addition to MTSEA, the negatively charged MTSES was also used to treat some of the mutants. Although MTSES failed to inactivate X5C over this concentration range, Fig. 6 shows that it was quite effective at inactivating mutants at positions 272-279 including the three lysine-to-cysteine mutants K272C, K275C, and K279C.
The differences in sensitivity of K272C, K275C, and K279C to treatment with MTSES and MTSEA were apparently due to their inability to react with MTSEA. Treatment with MTSEA up to 1 mM failed to protect these mutants from subsequent inactivation by MTSES as shown in Fig. 7. In this experiment, membranes were treated with various concentrations of MT-SEA prior to MTSES.
The EC 50 concentration of MTSEA, sufficient to half-maximally inactivate each mutant, was used to calculate rate constants with the assumption of bimolecular kinetics and a firstorder time course of activity loss (29). In the 15-min inactivation reaction, half-maximal inactivation gives a t1 ⁄2 of 15 min and a pseudo first-order rate constant of 0.046 min Ϫ1 . From this value and the concentrations of MTSEA required for half-maximal inactivation, the rate constants shown in Fig. 8   were calculated. We interpret a higher inactivation rate as greater accessibility to MTSEA. Six cysteine mutants, G273C, V274C, S277C, V280C, V281C, and T284C, reacted with rate constants over 20 s Ϫ1 M Ϫ1 , which is Ͼ75 times faster than the background X5C, whereas the remaining mutants reacted with rate constants below 1 s Ϫ1 M Ϫ1 .

DISCUSSION
The data presented in Fig. 5 clearly define a region of SERT from approximately Trp-271 through Ile-290, where the residues are likely to be in contact with the cytoplasm. A significantly shorter region (271-279) was initially predicted from hydropathy profiles to constitute the IL2 of SERT (16, 17, 28). The region is likely intracellular, because mutants with cys-teine at these positions were inactivated by MTSEA in membrane preparations (Figs. 4, 5, and 8) but did not react with extracellular MTSEA in intact cells (Table II). It is possible that IL2 extends even beyond this range, because residues proximal to Trp-271 or distal to Ile-290 might react with MT-SEA with no consequence for transport or binding activity. However, it is problematic to measure reactivity in the absence of a change in activity.
There were two mutations within the reactive region, K275C and K279C, where substitution with cysteine did not adversely affect transport or binding activity and which retained a considerable fraction of activity even at high MTSEA concentrations (Fig. 5), although the negatively charged MTSES was able to totally inactivate each of the lysine-to-cysteine mutants (Fig. 6). Cysteine mutants at neighboring positions were mostly inactivated by the same treatment. We considered the possibility that the modified cysteine residue, a disulfide with 2-thioethylamine, was sufficiently similar in structure to the endogenous lysine residues at these positions so that the modification did not disrupt function. An example in which an MTSEA-modified cysteine mimicked a lysine residue was described for the dopamine D2 receptor (30). An alternative possibility was that MTSEA did not fully modify K275C or K279C, possibly because of electrostatic repulsion from neighboring residues. Consistent with this latter hypothesis, pre-treatment with MTSEA failed to protect these mutants against subsequent inactivation by MTSES, as would be expected if MTSEA modified these positions (Fig. 7). Thus, it is most likely that MTSEA did not react with K275C and K279C.
At six positions, cysteine substitution mutants were inactivated with dramatically higher rate constants than those of the remaining positions tested (Fig. 8). These six positions appeared to be in a repeating pattern, every 3-4 residues, within IL2. When modeled as an ␣-helix (Fig. 9), the more reactive positions mapped to a vertical stripe along the side of the helix encompassing four helical turns. This was somewhat surprising, because structure prediction algorithms did not identify this region as ␣-helical. Neither the Garnier-Osguthrope-Robson algorithm, nor the Agadir algorithm (31), nor the Predict-Protein algorithm (32) predicted helical structure in this region. The pattern or reactivity, however, strongly suggests that at least part of this region is in an ␣-helical conformation. To our knowledge, this is the first observation of secondary structure in the cytoplasmic domain of a neurotransmitter transporter in the NSS family.
We assume that the increased reactivity of G273C, V274C, S277C, V280C, V281C, and T284C results from increased accessibility of the substituted cysteine residue to aqueous MT-SEA. Other possibilities are that the microenvironments of these positions lead either to greater ionization of the cysteine sulfhydryl group (pK a ϭ 8.33) or to a higher local concentration of MTSEA. We consider the first of these possibilities unlikely, because at pH 7.3 we expect ϳ10% of the cysteine sulfhydryl groups to be ionized, and the maximum increase from a decrease in pK a would be 10-fold. The more reactive mutants are up to 100-fold more reactive than the less reactive ones (Fig. 8).
It is also possible that limited aqueous accessibility increases the pK a of cysteine at the less reactive positions. In the absence of any evidence in favor of an increased local MTSEA concentration, we consider that accessibility is the most likely determinant of reactivity in this region.
Our estimates of first-order rate constants for inactivation were based on measurements over a range of MTSEA or MT-SES concentration rather than time courses at a single concentration. As a consequence, all these estimates were based on the amount of inactivation in a 15-min incubation. Because hydrolysis of the MTS reagents during this time competes with inactivation, the concentrations are only estimates of the true rates. However, by using the same time point for each measurement we minimize the variability in the fraction of reagent hydrolyzed between mutants that reacted at different rates.
Because the positions studied were accessible only in disrupted plasma membrane preparations that expose residues facing the cytoplasm, we have used ␤-CIT binding rather than transport as an assay to follow modification of these mutants. However, the incubations with MTSEA and MTSES were performed in the absence of ␤-CIT and presumably reflect the properties of unliganded SERT.
The dramatic change in reactivity from one side of the helix to the other suggests that other parts of the protein or associated proteins must restrict access to some of the residues but not to others. The helical region could be an extension of one of the adjacent transmembrane domains as seen in the structures of Ca 2ϩ -ATPase (33) and a glutamate transporter homologue (34) and postulated for SERT extracellular loop 4 (22). Alternatively, the IL2 helix could be independent of the transmembrane domains as in the mitochondrial adenine nucleotide transporter (35). We must also consider the possibility that a major part of the accessible region represents part of TM5 and that the accessibility of this region indicates that TM5 lines part of the permeation pathway for substrates and ions.
On the opposite side of the putative helix from the reactive residues are three lysine residues. These residues are highly conserved within the animal members of the NSS family (Fig.  1). Even among the prokaryotic members of the family there are usually at least two lysine residues separated by 2-3 residues, suggesting that this basic stripe along the IL2 helix is an important structural or functional element. When one of these lysines was replaced with cysteine (as in K275C or K279C), MTSEA (but not MTSES) reactivity was reduced dramatically (Figs. 4 -6), suggesting that MTSEA was repelled by the electrostatic influence of the remaining lysine residues.
Cells expressing many of the cysteine mutants in this region transported 5-HT more rapidly than the parental X5C construct (Fig. 2). Our results (Figs. 3 and 9) suggest that the increased activity of these mutants was not due to higher levels of surface expression. Moreover, several of these mutants had greater maximal transport activity when normalized for surface expression than wild type SERT. The pattern of positions where cysteine substitution elevated V max over that of wild type was not a typical ␣-helical pattern (Fig. 9B) but rather delineated a set of positions at an acute angle to the putative helical axis. In the middle of this region is Pro-288. If a kink was formed in the helix at this point, then the residues where cysteine replacement increased activity could potentially define a contiguous region.
The ability of mutations in this region to increase transport activity suggests the possibility that the region between TM4 and TM5 may fulfill a conformationally active role in the catalytic cycle of SERT. Disruption of the native conformation, evidently an ␣-helix, may allow greater flexibility in this region and a consequently increased turnover. In the related GABA transporter, GAT-1, Hansra et al. (36) suggested that IL4 acts similarly as a negative regulator of transport rate, presumably by forming a barrier near the substrate permeation path. If IL2 represents an inhibitory element by decreasing SERT activity through intramolecular interactions, then mutations in SERT IL2 might disrupt its inhibitory influence, possibly by destabilizing the ␣-helix, and increase the intrinsic activity of the transporter. We are currently investigating this possibility that mutations or other modifications in this region affect activity by disrupting the apparently helical structure between Gly-273 and Thr-284. FIG. 9. Helical projections of SERT IL2. A, IL2 is represented as an ␣-helix using helical net representation. Positions at which cysteine replacement led to high rates of inactivation are shown in white letters on black backgrounds. At the remaining positions, cysteine substitution mutants were 10-to 100-fold less reactive. B, shown is the same helical net with the positions highlighted where substitution with cysteine increased activity above that of wild type.