Functional Role of Critical Stripe Residues in Transmembrane Span 7 of the Serotonin Transporter EFFECTS

Mutations at critical residue positions in transmembrane span 7 (TM7) of the serotonin transporter affect the Na dependence of transport. It was possible that these residues, which form a stripe along one side of the predicted a-helix, formed part of a water-filled pore for Na. We tested whether cysteine substitutions in TM7 were accessible to hydrophilic, membrane-impermeant methanethiosulfonate (MTS) reagents. Although all five cysteine-containing mutants tested were sensitive to these reagents, noncysteine control mutants at the same positions were in most cases equally sensitive. In all cases, MTS sensitivity could be traced to changes in accessibility of a native cysteine residue in extracellular loop 1, Cys-109. Moreover, none of the TM7 cysteines reacted with the biotinylating reagent MTSEA-biotin when tested in the C109A background. It is thus unlikely that the critical stripe forms part of a water-filled pore. Instead, studies of the ion dependence of the reaction between Cys-109 and MTS reagents lead to the conclusion that TM7 is involved in propagating conformational changes caused by ion binding, perhaps as part of the translocation mechanism. The critical stripe residues on TM7 probably represent a close contact region between TM7 and one or more other TMs in the transporter’s three-dimensional structure.


INTRODUCTION
The serotonin transporter (SERT) is responsible for re-uptake of the neurotransmitter serotonin from the synaptic cleft following neurotransmission. SERT cDNA's have been cloned and sequenced from a number of species, as well as from several different human tissues (for reviews, see (1)(2)(3). The DNA and predicted amino acid sequences of these SERT isoforms are highly homologous, not only to each other but also to the transporters for the other biogenic amines, dopamine and norepinephrine. The biogenic amine transporters in turn belong to a larger superfamily of neurotransmitter and bioactive amine transporters, each of which couples uptake to the transmembrane Na + and Clgradients. Hydropathy analysis predicts that these transporters contain 12 membrane-spanning α-helices, connected by hydrophilic loops lying inside and outside the cell (Figure 1).
Our group and others are using site-directed mutagenesis to test these structural predictions about SERT and its relatives, and to correlate structural information with the physical mechanisms by which the Na + and Clgradients are coupled to the inward movement of serotonin. Functionally important residues have been identified in several regions, including transmembrane span 1 (TM1) (4-6), TM3 (6-10), TM5 (11), TM7 (6,(12)(13)(14)(15), TM11 (11), and TM12 (16). 4 filled pore or channel, through which ions and/or serotonin enter the cell. To examine this possibility, we decided to test the accessibility of cysteine substitutions in TM7 to the aqueous environment, using membrane-impermeant methanethiosulfonate (MTS) reagents. MTS reagents have previously been used to investigate the external accessibility of cysteines in both the serotonin (8,17,18) and dopamine transporters (19,20). The reaction of these reagents with cysteine residues introduces a positively or negatively charged group into the protein's structure (21). If the reacting cysteine lies in a functionally important position, transporter activity will be sensitive to the modification.
In the course of the random mutagenesis study, we isolated five mutants containing cysteine substitutions in TM7: N368C-C369G, F373C, F377C, F380C, and Y385C-I379V (two mutants contain multiple substitutions due to the random mutagenesis protocol) (14). The predicted positions of these cysteine substitutions with respect to the membrane ranged from Asn-368, which is predicted to lie well within the membrane, to Tyr-385, which is predicted to lie near the end of the helix and thus might be exposed to the external environment. Sensitivity to hydrophilic reagents at Tyr-385 might allow us to define the end of the membrane-spanning helix. Sensitivity at other positions might indicate the presence of a water-filled pore, or alternatively, that this region of the protein does not lie buried in the membrane as predicted by hydropathy analysis. Here we report the results of these studies, which have lead to the conclusion that the critical stripe residues of TM7 do not form part of a water-filled pore, since they do not appear to be accessible to hydrophilic MTS reagents. Our results suggest instead that TM7 is involved in the propagation of conformational changes occurring after ion binding to the transporter, perhaps as part of the translocation mechanism. The critical stripe region is likely to represent a close contact region between TM7 and one or more other TM's. Determination of external accessibility using MTSEA-biotin. TM7 mutants containing novel cysteines were also tested for their ability to react with the biotinylating reagent, MTSEA-8 biotin (Toronto Research Chemicals). HeLa cells were grown and transfected as described above except in 12 well plates, with appropriate scaling up of DNA and other reagents. Eighteen to twenty-four hours post-transfection, each well was washed twice with 1.2 mL room temperature PBSCM. After removing the second wash, 450 uL of PBSCM was added, and 50 uL of MTSEAbiotin (10 mM, dissolved in water) was added to each well. Cells were allowed to incubate with MTSEA-biotin for 10 min at room temperature before washing the reagent away three times with 600 uL room temperature PBSCM.
Wild type SERT and mutant transporters were detected in the mixture of biotinylated proteins using the M2 anti-FLAG antibody (Sigma Chemical Co.) directed against the FLAG by guest on  http://www.jbc.org/ Downloaded from epitope tag located at the C-terminus. Blots were incubated with 10 µg/mL anti-FLAG in blocking buffer (5% nonfat dry milk, 40 mM Tris (pH 7.5), 300 mM NaCl, 0.1% Tween 20) overnight with gentle rocking, washed, and then further incubated with goat anti-mouse IgGhorseradish peroxidase conjugate (Pierce). After further washing, bands were detected by chemiluminesence using Super Signal West Pico (Pierce). MTSEA-biotinylated samples were compared with two controls: NHS-SS-biotinylated samples and total cell expression. NHS-SSbiotin (sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate; Pierce) was used to detect the total surface expression of each transporter, as described in (26). Total cell expression levels were determined without biotinylation by lysing cells directly in SDS-PAGE sample buffer, heating to 85° C for 10 min., and running them on the gel without further sample preparation.

Accessibility of TM7 cysteine substitutions to membrane-impermeant MTS reagents.
As described in the Introduction, we had previously isolated by random mutagenesis five mutants containing cysteine substitutions in TM7: Y385C-I379V, F380C, F377C, F373C and the MTS reagents. However, it was also possible that cysteine mutations in TM7 might make the transporter sensitive by changing the structure of SERT so that a previously inaccessible native cysteine (or cysteines) became exposed. Due to the large number of mutants isolated in the random mutagenesis project, we also had at least one non-cysteine substitution at each of the five positions to use as controls for this possibility. These controls were: Y385N, F380Y, F377Y, F373Y, and N368I (the single mutations C369G and I379V were also tested separately and found to have no effect on SERT sensitivity to MTS reagents, data not shown).
Cells expressing TM7 cysteine mutants, non-cysteine control mutants, or the wild type transporter were pretreated with MTSET or MTSES in the presence of 150 mM NaCl, 150 mM NMDG-Cl, or 150 mM LiCl as described in Experimental Procedures. After pretreatment, unreacted reagents and the salts were washed away, and [ 3 H]-serotonin uptake measured under normal, high Na + conditions. Figure 2 shows the results of these experiments, using 1 mM MTSET (solid bars) or 2 mM MTSES (hatched bars). As had been shown previously (17), wild type SERT was highly sensitive to MTSET or MTSES in the presence of Li + but not in Na + (panels C and A). Wild type SERT was also resistant to both reagents in NMDG (panel B), indicating that it is the presence of Li + rather than the absence of Na + that results in MTS sensitivity. Panel A shows that, in Na + , all of the TM7 cysteine mutants were more sensitive than wild type to one or both reagents, albeit to varying degrees. Mutants Y385C-I379V and N368C-C369G were most sensitive to MTSET, while mutant F380C was most sensitive to MTSES. A similar pattern was found for these mutants in the presence of NMDG (panel B). In Li + (panel C), all of the mutants were highly sensitive to MTSET, in all cases much more than wild type.
Two mutants, F380C and F373C, also showed a much higher than wild-type sensitivity to MTSES.

11
The enhanced sensitivity of the TM7 cysteine mutants, especially in Na + and NMDG, would seem to suggest that the novel cysteines in TM7 were accessible to reaction with the MTS reagents. However, Fig. 2, panels D -F, shows that in many cases the non-cysteine control mutants were also more sensitive than wild type to the MTS reagents. In Na + , mutant F380Y was more sensitive than both wild type and its cysteine-containing counterpart F380C, to both reagents (panels A and D). In NMDG, F380Y and F377Y were more sensitive to MTSET than both wild type and their counterparts F380C and F377C (panels B and E). In Li + , all of the noncysteine TM7 mutants except Y385N show the same enhanced sensitivities as the TM7 cysteine mutants. These results suggest that it is not the presence of novel cysteines in the TM7 mutants that confers enhanced MTS sensitivity, but rather that mutations in TM7 disturb the structure of SERT to expose a previously unexposed native cysteine or cysteines to these reagents.

Effect of incorporating the C109A mutation into TM7 mutants. The observation that
Li + even further enhanced the sensitivity of the TM7 mutants suggested that the newly exposed native cysteine could be Cys-109, previously identified in wild type SERT as the source of its sensitivity in Li + . We tested this possibility by incorporating the C109A mutation into all of the TM7 cysteine mutants. The C109A mutation was added by replacement of a restriction fragment in the SERT cDNA (described in Experimental Procedures). For the most part, the Cys-109-less mutants retained a significant portion of the transport activity found in the parent mutants (Table   I). The results of MTSET and MTSES treatment of these mutants are shown in Figure 3 (panels B and D), compared to wild type, the C109A construct alone, and the parent mutants (A and C).
In all TM7 cysteine mutants, removal of Cys-109 resulted in nearly complete loss of sensitivity to the MTS reagents. This was true for both reagents and under all three ionic conditions. The 12 C109A mutation was also incorporated into the non-cysteine TM7 control mutants, resulting in the same complete loss of MTS sensitivity in these mutants (data not shown). These results indicate that C109 was the major source of enhanced MTS sensitivity in the TM7 mutants, whether they contained novel cysteines or not. 13

Ion dependence of the reaction between MTSET and Cys-109.
We further studied the dependence of the reaction between MTSET and Cys-109 on Na + and Li + concentrations, using NMDG to maintain the same ionic strength in all samples (see Experimental Procedures). Figure   5 shows the effects of changing the ionic conditions on the rate of MTSET inactivation of the wild type (panels A and D), F380Y (panels B and E), and Y385C-I379V (panels C and F). After MTSET preincubation in the indicated salt concentrations, the unreacted reagents and salts were washed away and transport measured under high Na + conditions. MTSET-treated wells were compared to control wells that were subjected only to the varying salt conditions without the addition of MTSET. We chose two MTSET concentrations for each mutant and the wild type: a low concentration at which inhibition was limited, and a high concentration at which inhibition was complete (both as observed in 150 mM LiCl, see Fig. 4A). These concentrations allowed us to observe whether the ions could accelerate or reduce the rate of MTSET inhibition.
The results for wild type SERT are shown in panels A and D. Wild type SERT was equally insensitive to MTSET in all concentrations of Na + , at all MTSET concentrations tried (up to 5 mM). As expected, Li + increased the sensitivity of the wild type transporter. However, this effect was apparent only at higher concentrations of MTSET (panel D, results for 0.5 and 1 mM MTSET were similar to the curve shown here for 5 mM MTSET.) MTSET at 0.05 mM had little or no effect on wild type SERT regardless of the Li + concentration (panel A). At 5 mM MTSET, Li + increased the rate of inactivation in a saturable, concentration-dependent fashion, with a K ½ of 252 ± 48 mM.
The F380Y mutant, shown in panels B and E, revealed a very different phenotype. F380Y is highly sensitive to MTSET when treated in NMDG, unlike the wild type (Fig. 2E). At a low concentration of MTSET (0.05 mM), the rate of inactivation in NMDG was low but measurable The Y385C-I379V mutant presented yet another phenotype. Like F380Y, Y385C-I379V is sensitive to 1 mM MTSET in the presence of NMDG (Fig. 2). However, this reaction was neither stimulated by increasing Li + concentrations, nor prevented by increasing Na + concentrations, whether tested at low or high MTSET concentrations (Fig. 5, panels C and F).

Accessibility of TM7 cysteines to MTSEA-biotin.
The results shown thus far indicate that neither MTSET nor MTSES had a discernable effect on the activity of the TM7 cysteine mutants, beyond that which can be traced to effects on the accessibility of Cys-109. However, the possibility remained that one or both reagents might react with one of the introduced cysteines without having any effect on transport activity. This possibility might particularly apply to the cysteines at positions 373 and 377, since these positions are not part of the "critical stripe" where mutations were shown to be highly likely to have deleterious effects on activity. For the other positions, which do lie in the critical stripe region, the possibility was less likely but did still exist. For example, Asn-368 can be mutated to aspartate without loss of activity (14), suggesting that the introduction of a negative charge at this position by MTSES might also be harmless.
However, a lysine mutation here abolishes activity, suggesting that reaction at this position with MTSET was likely to be deleterious (14). In opposite fashion, mutation of Tyr-385 to aspartate abolishes activity, suggesting potential sensitivity to MTSES (14). The effect of a positively charged substitution at this position is unknown, as is the effect of introducing either a positive or a negative charge at position 380.
To address these possibilities, we used a third MTS reagent, MTSEA-biotin. This reagent reacts with exposed cysteine residues to attach a covalent biotin "tag." The presence of the tag can then be detected by the ability of the transporter to be captured on streptavidin-coupled beads. This measurement is thus independent of effects on activity. Figure 6A Fig 6B, lanes 1 and 2; the mutants look essentially the same). This interpretation is further supported by the observation that these forms also predominate when biotinylation is performed in the presence of 50 µg/ml digitonin to permeabilize the cells (Fig. 6B, lanes 3 and 4). and supporting the conclusion that the novel cysteines they contain are not accessible to MTS reagents from outside the cell. Figure 6C shows a further control for this experiment. Although most of the mutants show fairly robust activity, indicating good cell surface expression, a few have low activity (Table   I). It was thus possible that these mutants might not be recovered simply because they were not well expressed on the cell surface. In Figure 6B, a different biotinylating reagent was used, NHS-SS-biotin. This reagent reacts with lysines instead of cysteines, and has been used previously to measure the cell surface expression of other mutant SERTs (26,29). All of the mutants have the same number of lysine residues on their external surfaces. Figure 6C shows that, although there were differences in cell surface expression, all of the mutants were expressed in sufficient amounts to be detectable in the MTSEA-biotinylation experiment. In particular, the two mutants with the lowest transport activities, F380C-C109A and N368C-C369G-C109A, appear to be at least as well expressed on the cell surface as the other mutants (Fig. 6C, lanes 3 and 6).

DISCUSSION
We had previously identified a pattern of functionally important residues in TM7 of SERT: a "critical stripe" that runs down one side of the putative α-helix, ranging from its predicted extracellular end nearly to the intracellular side of the membrane. The residues in this stripe lie on a predominantly polar face of the predicted helix. Mutant transporters with substitutions at these critical stripe positions were found to have impaired Na + dependence for transport, such that serotonin transport was decreased compared to wild type at low Na + concentrations (14). These observations led us to hypothesize that this stripe might form one 18 wall of a water-filled pore through which Na + , as well as possibly the other substrates serotonin and Cl -, might enter the cell. However, the results presented in this paper do not support this hypothesis. We tested the accessibility of cysteine substitutions in TM7 to hydrophilic MTS reagents added from the outside of intact cells. The cysteine substitutions lay at positions ranging from close to the predicted extracellular end of TM7 (position 385) to near its predicted intracellular end (position 368). We found no evidence that cysteine residues at any of these positions were accessible to hydrophilic reagents from outside the cell, as would be expected if these positions lined a water-filled pore (Figures 2, 3, 4 and 6). The evidence included measurements of the effects of MTS reagents on transport activity (Figs. 2-4), as well as direct physical detection of the MTS reaction using the biotinylating reagent MTSEA-biotin (Fig. 6).
In the course of these studies, however, we observed an unexpected and interesting property of the TM7 mutants. Many of the TM7 mutations we studied affected the accessibility of a native cysteine residue, Cys-109, to MTS reagents (Figs. 2-4). This residue lies on extracellular loop 1 between TM's 1 and 2, some distance away from TM7 in the primary structure of the transporter. This result suggests that structural changes in TM7 affect the conformation of EL1. Such an effect could occur relatively directly, for example, if TM7 lies in direct contact with TM1 or TM2. Alternatively, this effect could occur more indirectly, via global changes in SERT structure occurring when TM7 is mutated. In either case, these considerations lead us to consider an alternative hypothesis for the functional role of the critical stripe residues: as an important region of close contact with one or more other TM's. For reasons described below, we think this contact region may be involved in the propagation of conformational changes brought about when ions, specifically Na + and Li + , bind to the transporter. It is possible that these conformational changes are the same changes that link ion binding to the translocation of serotonin through the membrane as part of the transport cycle.
In earlier studies, it had been observed that Cys-109, the only free cysteine on the extracellular face of SERT, becomes accessible to MTS reagents in the presence of Li + but not in Na + (17). This result suggested that Li + binding changes SERT structure so that Cys-109 is exposed to the aqueous environment. In this paper we show that mutations in TM7 have a similar effect of exposing Cys-109. In many of the TM7 mutants, Cys-109 reacts with MTS reagents in Na + and/or NMDG, unlike in wild type. This result suggests that Cys-109 is permanently more exposed in the conformation adopted by these mutants. Two such mutants were examined in more detail: F380Y and Y385C-I379V. In F380Y, Cys-109 is highly exposed in the presence of NMDG, as evidenced by a very high rate of reaction. Li + further stimulates this reaction. Na + , on the other hand, promotes a conformation in which Cys-109 is less exposed and reduces the rate of reaction (Fig. 5, B and E). By contrast, in Y385C-I379V, Cys-109 is also more exposed than wild type in NMDG, but neither Li + nor Na + has any further effect on the degree of this exposure (Fig. 5, C and F).
As mentioned above, most mutants in the critical stripe region of TM7 show impaired Na + dependence (14). Such a result might indicate that TM7 is directly involved in Na + binding, or alternatively, that TM7 participates in a later step of the translocation process occuring after Na + is bound. Our results with these two mutants would seem to favor the latter hypothesis. In the case of Y385C-I379V, the mutant does not appear to respond to Na + or Li + , as detected by the ability of these ions to drive changes in the accessibility of Cys-109. However, these results are unlikely to reflect a serious defect in ion binding, at least for Na + , since this mutant is able to 20 transport serotonin at a nearly wild type level. Its K M for Na + is 39 ± 13 mM, compared to 14 ± 3 mM for the wild type. Mutant F380Y, on the other hand, has a K M for Na + of 101 ± 16 mM, seven times higher than wild type, suggesting a potential defect in Na + binding. However, this mutant shows a K 1/2 for the protective effect of Na + on Cys-109 exposure of 22 ± 6 mM. This value is comparable to the wild type K M for transport, suggesting that this mutant interacts with Na + almost as well as wild type, but that the subsequent coupling to transport is inefficient. Both of these results support the idea that residues in TM7 act to propagate conformational changes that occur after Na + has bound. There is no evidence for their direct involvement as part of the Na + binding site. Our inability to detect a water-filled pore involving these residues also argues against a direct role for TM7 in Na + binding.
If the TM7 residues were part of the lining of the translocation pathway, it might be expected that mutating them would affect serotonin binding affinity. Several other groups have studied the effects of TM7 mutations on substrate binding, in the closely related dopamine (6,(11)(12)(13)30) and norepinephrine transporters (15). These studies got their first impetus from studies of the β-adrenergic receptors showing that three residues, an aspartate in TM1 and two serines in TM5, were principally responsible for substrate binding in this family (31). By analogy, an aspartate in TM1 and two serines in TM7 of DAT were hypothesized to play a role in dopamine binding. It was thought that the negatively charged aspartyl side chain might bind to the amine moiety of dopamine, while the serines might hydrogen bond to the hydroxyl groups on the catechol ring. Mutant DATs in which these serines were changed to alanine showed reduced dopamine uptake, characterized by a decrease in affinity for dopamine as well as a decrease in V max . These mutants also showed increased uptake of MPP + , supporting a potential role in 21 substrate recognition (12,13). However, later studies of the analogous serines in NET argue strongly against the hypothesis of a receptor-like binding site (15). Although mutants in one serine residue showed a loss of affinity for both dopamine and the inhibitor nisoxetine, mutants in the other serine residue had nearly wild type affinities for both. In addition, studies with substrates containing either one or the other hydroxyl on the catechol ring showed no evidence of interaction between a particular hydroxyl and a particular serine, as would be expected if these serines interacted directly with the substrate. Interestingly, mutations at both serines affected the rate of dopamine efflux but not influx, suggesting a potential role in the translocation mechanism rather than direct interaction with substrate (15). Other mutagenesis studies have focused on the roles of aromatic, proline and polar residues in TM7 of DAT (6,11,30). Although many of the mutants showed large defects in transport ability, there was little evidence for direct involvement in dopamine binding. These results support our conclusion that TM7 is not part of the translocation pathway, since residues lying in such a pathway would be expected to be involved in direct substrate interaction.
Other groups besides ours have suggested a role for TM7 in the conformational changes accompanying substrate translocation. Recent studies of the dopamine transporter (DAT) have shown that the divalent cation Zn 2+ inhibits dopamine transport but does not prevent dopamine binding by DAT (32,33). Zn 2+ inhibits transport in DAT by interacting with an endogenous binding site, made up of three residues: His-193 in EL2, His-375 in EL4 near the predicted top of TM7, and Glu-396, also lying in EL4 but nearer to the top of TM8. The authors of these studies have suggested that Zn 2+ binding inhibits transport by holding these residues in a fixed conformation relative to one another, preventing the conformational changes necessary for substrate translocation (32).
In summary, the results described here argue strongly against a direct role for TM7 critical stripe residues in lining a pore or as part of the translocation pathway. Instead, it seems likely that this group of residues forms an important contact region with one or more other TM's. TM7 appears to mediate at least some of the conformational changes that occur when Na + or Li + bind to the transporter, since at least one mutation in TM7 (Y385C-I379V) disables the connection between ion binding and changes in Cys-109 accessibility. We will continue to investigate the functional role of these residues by studying how TM7 mutations affect serotonin and Clbinding. We will also examine whether these substrates can also drive conformational changes that expose Cys-109, similar to those driven by Na + and Li + in mutant F380Y. These studies will allow us to further elucidate the role of TM7 in the conformational changes that bring about the translocation of ions and serotonin across the membrane.   separate experiments for each mutant. As in Fig. 2 (see legend), names of mutants have been shortened due to space considerations. "CA" stands for the C109A mutation.  NMDG-Cl was added at each concentration to maintain the same ionic strength in all wells. Cells were then washed and assayed for transport activity as described in Experimental Procedures.
Wild type SERT is represented by the circles, mutant F380Y by the squares, and mutant Y385C-I379V by the triangles. Results are shown for representative experiments. Error bars smaller than the height of the symbols have been omitted.