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Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy and Pharmaceutical Sciences, West Lafayette, Indiana 47907, USA

(RECEIVED June 5, 2006; FINAL REVISION July 13, 2006; ACCEPTED July 14, 2006)

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
The human serotonin transporter (hSERT) regulates neurotransmission by removing released serotonin (5-HT) from the synapse. Previous studies identified residues in SERT transmembrane helices (TMHs) I and III as interaction sites for substrates and antagonists. Despite an abundance of data supporting a 12-TMH topology, the arrangement of the TMHs in SERT and other biogenic amine transporters remains undetermined. A high-resolution structure of a bacterial leucine transporter that demonstrates homology with SERT has been reported, thus providing the basis for the development of a SERT model. Zn²⁺-binding sites have been utilized in transporters and receptors to define experimentally TMH proximity. Focusing on residues near the extracellular ends of hSERT TMHs I and III, we engineered potential Zn²⁺-binding sites between V102 or W103 (TMH I) and I179–L184 (TMH III). Residues were mutated to either histidine or cysteine. TMH I/III double mutants were constructed from functional TMH I mutants, and Zn²⁺ sensitivity was assessed. Dose-response assays suggest an approximately twofold increase in sensitivity to Zn²⁺ inhibition at the hSERT V102C/M180C and approximately fourfold at the V102C/I179C mutant compared to the hSERT V102C single mutant. We propose that the increased sensitivity to Zn²⁺ confirms the proximity and the orientation of TMHs I and III in the membrane. Homology modeling of the proposed Zn²⁺-binding sites using the coordinates of the Aquifex aeolicus leucine transporter structure provided a structural basis for interpreting the results and developing conclusions.

Keywords: serotonin transporter; cocaine; metal binding; antidepressant; psychostimulant

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
The human serotonin transporter (hSERT) belongs to a family of Na⁺/Cl^–-dependent transporters with 12 putative membrane-spanning -helices, intracellular N and C termini, and a large extracellular loop between transmembrane helices (TMHs) III and IV (extracellular loop 2). The high-resolution structure of a bacterial homolog of the Na⁺/Cl^–-dependent transporter family, as well as the available biochemical data, should aid in the determination of the hSERT structure.

Structural information for the Na⁺/Cl^–-dependent transporter family was initially obtained by Norregaard et al. (1998), who identified a histidine in extracellular loop 2 that, along with a histidine and a glutamate at the extracellular ends of TMHs VII and VIII, respectively, form an endogenous Zn²⁺-binding site in the dopamine transporter (DAT). This study was the first to establish proximity between any of the TMHs within the Na⁺/Cl^–-dependent family of transporter proteins. Subsequently, the Zn²⁺-binding site was mutated into the norepinephrine transporter (NET), dopamine transporter (DAT), the -aminobutyric acid transporter (GAT1), and hSERT, providing evidence for structural similarities within the family (Norregaard et al. 1998; MacAulay et al. 2001; Meinild et al. 2004; Mitchell et al. 2004). Furthermore, investigation into the effects of Zn²⁺ on wild-type hSERT function suggested that concentrations >1 mM were necessary to inhibit [³H]1-methyl-4-phenylpyridinium (MPP⁺) uptake >25%, as opposed to low micromolar Zn²⁺ concentrations in DAT (Scholze et al. 2002).

Amino acid side chains involved in a Zn²⁺-binding site must be within 4–5 Å of each other, allowing 2–2.3 Å between Zn²⁺ and the coordinating atoms (Alberts et al. 1998). This strict structural requirement makes an engineered Zn²⁺-binding site suitable for determining proximity relationships. These studies, along with similar experiments in receptors, validate the use of engineered Zn²⁺-binding sites to define protein structure in the absence of a high-resolution crystal structure (Elling et al. 1995; Elling and Schwartz 1996; Thirstrup et al. 1996; Holst et al. 2000).

Several studies have highlighted the importance of TMHs I and III in substrate and antagonist recognition by hSERT. TMH I residue Y95 in hSERT is an important determinant for interaction with the antagonists mazindol and citalopram as well as tryptamine derivatives (Barker et al. 1998; Adkins et al. 2001). A separate study identified D98 in TMH I of rat SERT as critical for 5-HT (5-hydroxytryptamine or serotonin) transport (Barker et al. 1999). This aspartate is conserved throughout the biogenic amine transporters. Substituted cysteine accessibility method (SCAM) analysis of hSERT showed that 5-HT could protect D98C as well as G100C and N101C from thiol inactivation (Henry et al. 2003). SCAM was also utilized to identify residues I172 and Y176 in TMH III of rat SERT that may reside in the substrate permeation pathway (Chen et al. 1997b). Despite the obvious involvement of both TMHs I and III in the 5-HT permeation pathway, until recently no studies have been able to confirm the proximity of these TMHs (Zomot et al. 2005).

In the present study, we engineered Zn²⁺-binding sites into locations near the extracellular ends of hSERT TMHs I and III. In TMH I, hSERT residues N101, V102, and W103 were selected for their location above D98. These were mutated to either histidine or cysteine, two residues commonly found in Zn²⁺-binding proteins, although histidine is thought to coordinate Zn²⁺ with higher affinity than cysteine (Alberts et al. 1998). From TMH III, we selected residues above Y176 for mutation to cysteine, including I179, M180, A181, W182, A183, and L184. These positions are known to tolerate mutation to cysteine (Chen et al. 1997b). All mutations were made in the background of the hSERT C109A single mutant (hereinafter referred to simply as C109A), which is located in the first extracellular loop and is the only endogenous cysteine known to be sensitive to modification by methanethiosulfonates (Chen et al. 1997a). In addition, 5-HT uptake activity and pharmacology of C109A is similar to wild-type hSERT (Henry et al. 2003).

TMH I/III double mutants were constructed from functional TMH I mutants. The ability of Zn²⁺ to inhibit the mutants was assessed by a change in [³H]5-HT uptake compared to the Zn²⁺-insensitive C109A. In this study, we have identified mutant transporters that become sensitive to Zn²⁺ inhibition in a dose-dependent manner when residues from TMHs I and III are mutated simultaneously. We propose that the increased potency of Zn²⁺ inhibition in these mutant transporters confirms the proximity and relative orientation of TMHs I and III to each other in the membrane. Furthermore, a homology model of hSERT derived from the recently solved structure of the Aquifex aeolicus leucine transporter (LeuT_Aa) (Yamashita et al. 2005) supports our experimental findings and suggests that the SERT model derived from the LeuT_Aa structure will be valuable in exploring the structure of SERT and other members of the Na⁺/Cl^–-dependent family of transporters.

	Results

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
Activity and expression of TMH I, III, and I/III mutants
Prior to determining the ability of TMH I/III double mutations to form a potential Zn²⁺-binding site, all hSERT C109A mutants were transiently expressed in HeLa cells, and [³H]5-HT transport was assessed. Mutation of residues in TMH I showed decreased [³H]5-HT transport compared to C109A, which had transport kinetics comparable to wild-type hSERT (hSERT C109A transport kinetics: K _m = 1.3 ± 0.3 µM, V _max = 8.3 ± 0.5 x 10^–17 mol/min/cell; data not shown) in agreement with previous studies with this mutant (Chen et al. 1997b; Henry et al. 2003). Mutation of N101 to either histidine or cysteine reduced [³H]5-HT transport to <1% of C109A, even when [³H]5-HT concentrations were increased to 50 nM (data not shown). This result contrasts with an earlier study that reported 40% activity at N101C/hSERT (Henry et al. 2003). Mutation of V102 and W103 to histidine also resulted in nearly complete loss of [³H]5-HT uptake, with V102H exhibiting <1% of C109A uptake. W103C retained 50% C109A uptake activity, compared to only 15% for the W103H mutant (Fig. 1A). Due to the lack of 5-HT transport activity at the N101H, N101C, and V102H mutants, TMH I/III double mutants were not constructed from these mutations. All TMH III mutants, except I179C (35%), exhibited 80%–100% of C109A activity, again in agreement with previous studies (Fig. 1A) (Chen et al. 1997b).

View larger version (42K): [in this window] [in a new window]   Figure 1. TMH I exhibits greater sensitivity to mutation than TMH III. [3H]5-HT uptake activity of hSERT C109A, TMH I, TMH III, and TMH I/III mutants was assessed in transiently transfected HeLa cells as previously described (Roman et al. 2003); nonspecific uptake was determined using 10 µM fluoxetine. Specific [3H]5-HT uptake of TMH I or TMH III single mutants (A) and TMH I/III double mutants (B) was normalized to total specific uptake of C109A. Bars represent mean ± SEM of at least three independent experiments performed in triplicate. (N.D.) Not detectable.

A decrease in [³H]5-HT uptake could be attributed to a change in cell surface expression of the mutant transporters rather than a change in substrate recognition or translocation. To address this issue, surface binding assays in HEK-293 cells were performed at 4°C as previously described (Rodriguez et al. 2003). Briefly, the amount of transporter expressed at the cell surface can be determined by subtracting the amount of [³H]citalopram that binds in the presence of MPP⁺ from total specific [³H]citalopram binding. Total specific binding of [³H]citalopram is determined by subtracting the amount bound in the presence of saturating concentrations of fluoxetine from binding in buffer only. MPP⁺ is not transported at 4°C, thus effectively separating surface from internal transporters (Rodriguez et al. 2003). Surface expression of C109A did not differ from wild-type hSERT (Fig. 2A). TMH I mutants N101C and V102H retained minimal [³H]5-HT transport activity, but neither total nor surface [³H]citalopram binding was reduced compared to C109A (Fig. 2A). Although total binding at hSERT mutants V102C, V102C/M180C, W103H/I179C, W103H/M180C, and W103H/W182C was decreased compared to C109A, the percentage of the total transporter pool that was expressed on the surface remained constant (Fig. 2B). For the remaining mutants tested, neither the total nor the percentage of surface binding deviated significantly from C109A. In many cases, the transporter surface expression levels did not correspond to the 5-HT transport activity observed for a given mutant. For example, the W103H/I179C mutant exhibited reductions in both uptake activity and surface expression, whereas the W103C/I179C mutant demonstrated reduced 5-HT transport activity, but its surface expression levels were comparable to wild-type hSERT (Fig. 2). These discrepancies suggest that some mutations impair actual transport function as opposed to disrupting trafficking or surface expression.

View larger version (53K): [in this window] [in a new window]   Figure 2. Changes in SERT expression do not account for mutation-induced reductions in [3H]5-HT uptake. Surface binding assays were performed in HEK-293 cells transiently transfected with mutants possessing <50% parental uptake activity, as described in Materials and Methods. (A) V102C, V102C/M180C, W103H/I179C, W103H/M180C, and W103H/W182C exhibited a decrease in total binding compared to hSERT, but (B) the percentage of surface binding compared to the total protein was approximately the same for C109A and all of the mutants. Nonspecific binding was determined using 10 µM fluoxetine. Internal binding was determined as the binding of [3H]citalopram in the presence of 200 µM MPP+. Specific binding on the surface was calculated as (total binding – nonspecific binding) – (binding in the presence of MPP+ – nonspecific binding). Bars represent the mean ± SEM for assays performed in triplicate in at least three independent experiments, except $ = n of 2.

View larger version (36K): [in this window] [in a new window]   Figure 3. hSERT TMH I/III double mutants exhibit significant Zn2+ inhibition. Inhibition of [3H]5-HT uptake by 1 mM Zn2+ in transiently transfected HeLa cells was performed as described in Materials and Methods; nonspecific uptake was determined using 10 µM fluoxetine. (A) Only the V102C mutant exhibited a significant inhibition by Zn2+ compared to C109A. (B) Mutation of residues V102C/I179C, V102C/M180C, W103H/M180C, W103H/W182C, W103H/A183C, and W103C/W182C resulted in statistically significant Zn2+ inhibition. The dashed lines represent the maximum Zn2+ inhibition by V102C alone (V102C/TMH III mutants) or C109A (W103H/TMH III and W103C/TMH III mutants). Bars represent the mean ± SEM for at least three independent experiments performed in triplicate. One-way ANOVA with Bonferroni's post-hoc test was performed for statistical analysis. () p < 0.001 vs. V102C; (**) p < 0.01 vs. C109A; (***) p < 0.001 vs. C109A; (N.D.) not determined.

Dose-response for Zn²⁺ inhibition of [³H]5-HT uptake
To verify that the inhibition of [³H]5-HT uptake in the presence of Zn²⁺ was due to the construction of a high-affinity Zn²⁺-binding site and not merely due to cytotoxic or nonspecific effects of the high Zn²⁺ concentration, the dose-dependency of Zn²⁺ inhibition at Zn²⁺-sensitive mutants was determined. IC₅₀ values for Zn²⁺ inhibition are summarized in Table 1. Despite significant inhibition by 1 mM Zn²⁺ in Krebs-Ringer-HEPES (KRH)/glucose, IC₅₀ values for [³H]5-HT uptake inhibition in Zn²⁺-binding buffer were >1 mM for W103H/A183C (Table 1) and W103C/W182C (data not shown). Single hSERT mutants W103H and M180C exhibited an IC₅₀ value shift for Zn²⁺ from >1 mM to 390 ± 60 µM when mutated together (Table 1). Similarly, the V102C/I179C and V102C/M180C mutants showed statistically significant decreases in IC₅₀ values compared to the single mutant V102C (Table 1). These results confirm our earlier findings that Zn²⁺ inhibits the TMH I/III cysteine double mutants to a greater extent than the single mutants.

To explore the possible formation of a disulfide bond, we performed the Zn²⁺ inhibition assays in the presence or absence of 12 mM dithiothreitol (DTT). In the presence of DTT, V102C/A183C gained sensitivity to Zn²⁺ similar to that of V102C in the absence of DTT (Fig. 4A), suggesting that we had successfully reduced a disulfide bond between V102C and A183C, freeing V102C to coordinate Zn²⁺. Control V102C, however, exhibited a decrease in sensitivity to Zn²⁺ when DTT was present compared to when DTT was absent. Moreover, total [³H]5-HT uptake decreased to 65 ± 5% of the total in the absence of DTT for V102C and 71 ± 4% for V102C/A183C (Fig. 4B).

View larger version (26K): [in this window] [in a new window]   Figure 4. DTT increases inhibition by 1 mM Zn2+ at the hSERT V102C/A183C mutant. Assays were performed in transiently transfected HeLa cells in the presence or absence of 12 mM DTT, as described in Materials and Methods. (A) In KRH/glucose, V102C/A183C was insensitive to inhibition by 1 mM Zn2+ compared to V102C. Zn2+ inhibition at V102C/A183C was increased in the presence of DTT. (B) Total specific uptake of [3H]5-HT was reduced in the presence of DTT. (***) p < 0.001 vs. V102C without DTT; () p < 0.001 vs. total specific [3H]5-HT uptake in KRH/glucose. One-way ANOVA with post-hoc Bonferroni's multiple-comparison test was performed for statistical analysis. Data represent mean ± SEM from three separate experiments.

View larger version (49K): [in this window] [in a new window]   Figure 5. Zn2+ significantly potentiates MTSET inhibition of [3H]5-HT uptake at the hSERT V102C and W103C mutants. HeLa cells were transiently transfected with the indicated cDNAs. At the time of assay, cells were treated with 1mM MTSET for 10 min at 22°C in the presence or absence of 1 mM Zn2+ in Zn2+-binding buffer or with 1 mM Zn2+ alone, washed twice with Zn2+-binding buffer, and [3H]5-HT uptake was then performed as described in Materials and Methods. V102C was insensitive to 1 mM MTSET in the absence of Zn2+. In the presence of 1 mM Zn2+, V102C and W103C MTSET inhibition of [3H]5-HT uptake was potentiated, but C109A remained insensitive to MTSET. Comparison of inhibition at V102C, W103C, and W103H by Zn2+ alone or Zn2+ plus MTSET suggested that the potentiation of MTSET inactivation was due to the mutated cysteine and not to the unmasking of an endogenous cysteine; W103H inhibition by Zn2+ plus MTSET was not different from inhibition by Zn2+ alone. The increased inhibition by Zn2+ compared to that previously shown (Fig. 3) was most likely due to the use of Zn2+-binding buffer as opposed to KRH/glucose. Data represent the mean ± SEM for at least three independent experiments performed in triplicate.

Zn²⁺ significantly enhances the rate and the extent of inactivation of the hSERT V102C mutant by MTSET
Because the presence of Zn²⁺ allows almost complete inactivation of V102C by MTSET, we decided to investigate the rate of inactivation as described by others (Rudnick 2002; Keller et al. 2004). This analysis involves adding increasing concentrations of MTSET to the cysteine mutant of interest and determining the concentration that results in half-maximal uptake inhibition (Rudnick 2002). MTSET inhibition of C109A, V102C, and W103C was performed in the presence or absence of 1 mM Zn²⁺. The rate of inactivation did not vary in C109A, regardless of whether or not Zn²⁺ was present (data not shown). The inactivation rate of W103C was nearly doubled in the absence of Zn²⁺ to 570 ± 90 M^–1 min^–1 from 290 ± 50 M^–1 min^–1 with Zn²⁺ present (Fig. 6A). At V102C, however, Zn²⁺ increased the rate of MTSET inactivation 30-fold over that observed in the absence of Zn²⁺ (6300 ± 600 M^–1 min^–1 vs. 210 ± 40 M^–1 min^–1, respectively) (Fig. 6B). The maximum level of inactivation observed with MTSET was also greater in the presence of Zn²⁺ for the hSERT V102C mutant (Fig. 6B).

View larger version (20K): [in this window] [in a new window]   Figure 6. Zn2+ increases the rate of inactivation by MTSET at the hSERT V102C and W103C mutants. HeLa cells transiently transfected with V102C or W103C cDNAs were treated with increasing concentrations of MTSET in the presence or absence of 1 mM Zn2+ in Zn2+-binding buffer followed by assessing [3H]5-HT uptake as described in Materials and Methods. For MTSET inhibition assays performed in the absence of Zn2+, activity remaining was normalized to total uptake. In the presence of Zn2+, activity remaining was determined by normalizing MTSET uptake inhibition in the presence of Zn2+ to the uptake inhibition by 5 mM MTSET alone (i.e., in the absence of Zn2+). Nonspecific uptake was determined using 10 µM fluoxetine. (A) W103C exhibited an increase in the reaction rate of MTSET inactivation in the presence vs. the absence of Zn2+ with rates of 570 ± 90 M–1 min–1 and 290 ± 50 M–1 min–1, respectively. (B) MTSET inactivated V102C at a rate of 6300 ± 600 M–1 min–1 vs. 210 ± 40 M–1 min–1 in the presence or absence of Zn2+, respectively. Data represent the mean ± SEM for at least three independent experiments performed in triplicate.

View larger version (32K): [in this window] [in a new window]   Figure 7. MTS-3-MTS cross-links V102C to M180C. HeLa cells transiently expressing hSERT C109A, M180C, V102C, or V102C/M180C cDNA were incubated with 1 mM MTS-3-MTS or MTSET in PBS/CM for 10 min at 22°C and washed twice before being assayed for [3H]5-HT uptake, as described in Materials and Methods. (A) MTS-3-MTS significantly inhibited [3H]5-HT uptake at V102C/M180C compared to V102C. Data were normalized to total uptake of the respective mutant. However, (B) MTSET inhibited uptake only at V102C/M180C. (#) p < 0.05 vs. V102C; (***) p < 0.001 vs. C109A. One-way ANOVA with post-hoc Bonferroni's multiple comparison test was performed for statistical analysis. Data represent mean ± SEM for three experiments performed in triplicate.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

View larger version (73K): [in this window] [in a new window]   Figure 8. Proximity of TMHs I and III in a SERT homology model. (A) A homology model of SERT was generated from the structure of LeuTAa using the hSERT sequence and energy minimization (Yamashita et al. 2005). The 12 TMHs of SERT are shown as ribbons, with each TMH assigned a unique color. TMH I is light blue, and TMH III is bright yellow. The area expanded in B is defined by the yellow box. (B) A close-up visualization of TMH I residues V102 and W103 and residues in TMH III. The C backbone of TMH I is a light blue ribbon, and the backbone of TMH III is shown as a bright yellow ribbon. Only residues examined in this study are shown. Approximate S-S distances between V102 and TMH III residues were estimated as if the residues had been mutated to cysteines in the homology model. Measured distances were V102–I179, 8.5 Å; V102–M180, 8.6 Å; V102–A181, inaccessible on opposite side of helix; V102–W182, 9.9 Å; V102–A183, 7.5 Å; V102–L184, inaccessible on opposite side of helix. These distances are in general agreement with the experimental data.

All active mutants were tested for Zn²⁺ sensitivity. Zn²⁺ had a minimal effect on 5-HT uptake in most single mutants. Uptake at V102C, however, was reduced 35% by Zn²⁺. Our hSERT homology model positions D98 of TMH I approximately one turn below V102C (data not shown). Because acidic residues can participate in Zn²⁺-binding sites (Alberts et al. 1998), Zn²⁺ may coordinate between residues D98 and V102C. Furthermore, D98 is critical for 5-HT recognition by hSERT, and mutation to any residue other than glutamate results in an inactive transporter (Barker et al. 1999).

An initial screen of TMH I/III double mutants identified seven mutants with increased Zn²⁺ sensitivity, compared to any single mutant alone. Several of these showed significantly increased dose-dependent Zn²⁺ inhibition of 5-HT uptake compared to any single mutant. Surprisingly, W103C/I179C was not among them. Recently, Kanner and colleagues (Zomot et al. 2005) reported the proximity of the homologous residues in GAT1, W68C/I143C, using Cd²⁺ and copper phenanthroline inhibition of GABA uptake. Although both GAT1 and SERT are members of the Na⁺/Cl^–-dependent transporter family, subtle differences may exist in the relative orientation of their helices during translocation due to distinct structural differences between their respective substrates.

W103H/M180C exhibited a 2.5-fold increased sensitivity to Zn²⁺ inhibition compared to W103H. The IC₅₀ value for W103H/M180C is also within the expected range of 2 x 10^–4 to 2 x 10^–6 M Zn²⁺ predicted for two coordinating residues (Regan 1995). In contrast, W103C/M180C was insensitive to Zn²⁺ in the initial screen. There could be two explanations for this difference: First, histidine has increased affinity for Zn²⁺ relative to cysteine (Alberts et al. 1998); second, histidine is slightly longer than cysteine (4.9 Å from C to the most distal N on the imidazole ring vs. 2.9 Å from C to S). Thus, two cysteines at these positions probably cannot achieve the relatively strict distance requirement for Zn²⁺ coordination. Because the IC₅₀ values for W103H/M180C and V102C are identical (390 ± 40 vs. 360 ± 20 µM Zn²⁺, respectively), another nearby amino acid, potentially D98, may be participating in a Zn²⁺-binding site with V102C as described above. Mutation of V102C with either TMH III mutation I179C or M180C resulted in a significant increase in Zn²⁺ sensitivity compared to V102C alone. These two double mutants exhibited the greatest Zn²⁺ sensitivity of all the TMH I/III mutants, strongly suggesting a Zn²⁺-binding site comprised of three amino acids that likely includes D98 participation.

None of the Zn²⁺-binding sites engineered here produced mutant transporters that were as sensitive to Zn²⁺ as the endogenous Zn²⁺-binding site in DAT (IC₅₀ = 1 µM) (Loland et al. 1999) or the equivalent site introduced into hSERT (K_1/2 = 3.2 µM) (Mitchell et al. 2004). These differences may be attributed to the location of the Zn²⁺-binding site and the subsequent disruption by Zn²⁺ of conformations necessary for substrate translocation. Additionally, the differences could be attributed to the use of a histidine and a cysteine in the Zn²⁺-binding sites rather than multiple histidines (Loland et al. 1999).

Previous studies in GAT1 have provided evidence for the proximity of hSERT V102C and I179C, as GABA uptake activity at the equivalent GAT1 mutant, V67C/I143C/GAT1, is sensitive to Cd²⁺ inhibition (Zomot et al. 2005). Further, V67C/I143C/GAT is not as sensitive to Cd²⁺ inhibition as W68C/I143C/GAT1 (hSERT W103C/I179C), suggesting differences in the orientation of TMHs I and III in GAT1 versus hSERT (Zomot et al. 2005). The hSERT double mutant N101C/I179C is sensitive to inhibition by Cd²⁺, further suggesting the proximity of these domains (Henry et al. 2006). In our studies, hSERT mutant N101C unexpectedly resulted in a nonfunctional transporter. This loss of function was unexpected because a previous study had reported that it retained 40% of the transport activity of C109A (Henry et al. 2003). We have no explanation for this discrepancy, as the only apparent difference between the studies was the expression vector that was utilized.

The hSERT V102C/A183C double mutant was the only V102C/TMH III mutant to exhibit a significant decrease in Zn²⁺ sensitivity. Homology modeling of the proposed Zn²⁺-binding sites suggests 7.5 Å separates V102C and A183C (Fig. 8). We hypothesized that the proximity of V102C and A183C allows them to form a disulfide bond, thus preventing V102C from coordinating Zn²⁺ with the endogenous residue. As predicted by this hypothesis, DTT treatment of V102C/A183C increased sensitivity to Zn²⁺ to a degree that was not significantly different from V102C alone. This result is consistent with our hypothesis, suggesting that DTT reduces a disulfide bond between V102C and A183C, freeing V102C to coordinate Zn²⁺. This conclusion is confounded, however, by the fact that V102C alone exhibited a significant decrease in Zn²⁺ sensitivity in the presence of DTT. Previous studies have suggested that the conformation of SERT is altered in the presence of DTT, as determined by changes in imipramine, but not paroxetine, binding (Tarrant and Williams 1995).

To characterize further the proximity of SERT TMHs I and III, we performed cross-linking studies. MTS-3-MTS reduced V102C transport activity to 20% but completely inactivated V102C/M180C. Although we cannot rule out the possibility that MTS-3-MTS is cross-linking V102C or M180C to other endogenous cysteines, the simplest explanation for the increased Zn²⁺ sensitivity of V102C/M180C is the proximity of V102C and M180C. MTSET inhibition of V102C/M180C but not V102C or M180C (data not shown) suggests a change in accessibility of at least one of the mutations in the double mutant. We speculate that the sensitivity of the double mutant to MTSET may result from subtle alterations in hSERT conformation that are induced by the mutations. Therefore, the increase in MTSET sensitivity could be similar to the increased sensitivity of V102C to MTSET in the presence of Zn²⁺. Despite sensitivity of V102C/M180C to MTSET treatment, MTS-3-MTS inhibition of [³H]5-HT was significantly greater than inactivation by MTSET alone, confirming the orientation of these residues toward each other.

Although participation of these TMHs in the formation of a common pore remains undefined, previous studies of SERT have established roles for TMHs I and III in the permeation pathway (Chen et al. 1997b; Barker et al. 1998, 1999). The work reported here on Zn²⁺-binding sites constructed between TMHs I and III supports that residues in these TMHs are within proximity to each other. Our homology model for SERT is consistent with proximity relationships identified for SERT TMHs I and III (Fig. 8). Furthermore, residues within these two TMHs also were found to be important in other members of the Na⁺/Cl^–-dependent family of neurotransmitter transporters (Kitayama et al. 1992; Bismuth et al. 1997; Lee et al. 1998; Barker et al. 1999), and a recent study identified an interaction between TMH I and III for antidepressant recognition (Henry et al. 2006). Our results also provide direct experimental support for the use of the LeuT_Aa as a valuable template for homology modeling of SERT and related transporters.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
Site-directed mutagenesis
hSERT C109A pBluescript KSII- (Stratagene Corp.) was subcloned into pcDNA3.1+ using NotI and AgeI. TMH I, III, and I/III mutants were generated in the parental hSERT C109A using oligonucleotides containing the desired mutation (Integrated DNA Technologies). Mutagenesis was performed using Pfu Ultra polymerase (Stratagene Corp.) per the manufacturer's protocol, utilizing the mutants C109A/V102C, C109A/W103H, or C109A/W103C as templates for the TMH I/III double mutants. All mutant cDNAs were screened using coding region silent restriction enzyme sites designed into the oligonucleotides. Mutation-positive cDNAs were verified by sequencing (University of Michigan DNA Sequencing Core, Ann Arbor) and then subcloned into the parental vector using NotI and AgeI sites. Although all mutations were generated in hSERT C109A, the mutants will be referred to only as their TMH I, III, or I/III mutation.

[³H]5-HT uptake inhibition assays
HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 1% penicillin/streptomycin (10,000 U/mL), 5% FetalClone I, 5% Bovine Calf Serum, and 2 mM glutamine in a humidified 37°C CO₂ incubator, and seeded into 24-well tissue culture plates 18 h prior to transfection. The cells (1 x 10⁵) were transiently transfected with C109A, TMH I, III, or I/III mutant cDNAs using the vaccinia virus method and Lipofectin (Life Technologies) (Fuerst et al. 1986; Blakely et al. 1991; Roman et al. 2003). Six to eight hours following transfection, [³H]5-HT uptake assays were performed in KRH/glucose buffer (1.2 mM MgSO_4, 4.7 mM KCl, 10 mM HEPES, 2.2 mM CaCl₂, 1.2 mM KH₂PO₄, 120 mM NaCl, and 1.8 g/L glucose at pH 7.4) as previously described (Roman et al. 2003). For hSERT mutants V102C and V102C/A183C assays involving DTT, DTT (12 mM final) in KRH/glucose (KRH/DTT) was made fresh for each experiment. Six to eight hours post-transfection, cells were washed once with KRH/glucose before KRH/glucose or KRH/DTT were added to the appropriate wells and incubated 10 min at 22°C. Next, 1 mM Zn²⁺, KRH/glucose, or 10 µM fluoxetine was added and incubated 10 min at 37°C, followed by an additional 10 min incubation at 37°C in the presence of [³H]5-HT (20 nM). Uptake was terminated by washing three times with cold KRH/glucose. Cells were solubilized with Microscint-20, shaken overnight, and radioactivity determined using a PerkinElmer TopCount NXT (PerkinElmer Life Sciences). Data were analyzed using GraphPad Prism v3.0 (GraphPad Software). Statistical comparison of data normalized to an untreated group was performed using one-way ANOVA followed by a post-hoc Bonferroni's multiple-comparison test comparing all test groups. Values of p < 0.05 were considered to be significant.

MTS-3-MTS and MTSET inhibition of [³H]5-HT uptake
hSERT mutants C109A, M180C, V102C, or V102C/M180C cDNAs were transfected into HeLa cells as described above. Six to eight hours following transfection, cells were washed once with phosphate-buffered saline supplemented with calcium and magnesium (PBS/CM) (137 mM NaCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, 2.7 mM KCl, 0.1 mM CaCl₂, and 1 mM MgCl₂ at pH 7.4). Cells were incubated 10 min at 22°C with 1 mM MTS-3-MTS or MTSET (made fresh in deionized water) in PBS/CM. Cells were washed twice with PBS/CM, and uptake was performed as previously described (Roman et al. 2003). Nonspecific uptake was determined with 10 µM fluoxetine.

Zn²⁺ dose-response assays
Because Zn²⁺ can form an insoluble complex with phosphate-containing buffers, Zn²⁺ dose-response assays were performed in Zn²⁺-binding buffer (120 mM NaCl, 1 mM MgCl₂, 15 mM HEPES, 5.4 mM KCl, and 0.1 mM CaCl₂ at pH 7.1) as previously described (Mitchell et al. 2004). HeLa cells were plated in the same manner as the [³H]5-HT uptake inhibition assays. Six to eight hours post-transfection, cells were washed once with Zn²⁺-binding buffer. Increasing Zn²⁺ concentrations (10^–7 to 10^–3 M) in Zn²⁺-binding buffer were added in triplicate wells and allowed to incubate at 37°C for 10 min. Next, 20 nM [³H]5-HT diluted in Zn²⁺-binding buffer supplemented with 100 µM pargyline and 100 µM ascorbic acid was added to each well and incubated for another 10 min at 37°C before being washed three times with cold Zn²⁺-binding buffer. Nonspecific uptake was determined in the presence of 10 µM fluoxetine, and total uptake was determined in the presence of Zn²⁺-binding buffer alone.

Cell surface radioligand binding assays
Human embryonic kidney (HEK-293) cells maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 1% penicillin/streptomycin (10,000 U/mL), and 10% dialyzed fetal bovine serum in a humidified 37°C CO₂ incubator were seeded into 24-well tissue culture plates coated with poly-D-lysine. Eighteen hours after plating, the cells (1 x 10⁵) were transfected using Lipofectamine 2000 per manufacturer's directions (Life Technologies). Forty-eight hours post-transfection, the surface binding experiments were performed as previously published (Rodriguez et al. 2003). Briefly, cells were maintained on ice to prevent translocation of MPP⁺. Cells were washed with 4°C KRH/glucose and incubated with 10 µM fluoxetine (nonspecific binding), 200 µM MPP⁺ (internal binding), or KRH/glucose alone (total binding) and 20 nM [³H]citalopram. The reaction was allowed to incubate 1 h at 4°C in order to reach equilibrium before being washed quickly with 4°C KRH/glucose. Cells were solubilized with Microscint-20 and overnight shaking before radioactivity was determined using a PerkinElmer TopCount NXT.

MTSET reaction rates
HeLa cells were maintained and transfected as described above. Six to eight hours post-transfection, the cells were washed once with Zn²⁺-binding buffer, and Zn²⁺ (1 mM) or Zn²⁺-binding buffer was added to the appropriate wells with increasing concentrations of MTSET (prepared and diluted fresh in deionized water). Cells were incubated for 10 min at 22°C. The cells were washed twice with Zn²⁺-binding buffer, and [³H]5-HT uptake was performed in KRH/glucose as previously described (Roman et al. 2003). Rates of inactivation were calculated as previously described after determining the concentration necessary to obtain half-maximal inactivation (Rudnick 2002).

Materials
Dulbecco's modified Eagle's medium, pargyline, MPP⁺, L-ascorbic acid, DTT, and zinc chloride were purchased from Sigma-Aldrich. FetalClone I and Bovine Calf Serum were purchased from Hyclone Laboratories. Dialyzed fetal bovine serum was obtained from Atlanta Biologicals. MTS-3-MTS was from Toronto Research Chemicals, and MTSET was from Biotium, Inc. [³H]5-HT (125 Ci/mmol) and [³H]citalopram (80 Ci/mmol) were obtained from Amersham Biosciences, Inc. Microscint 20 and 24-well tissue culture plates were purchased from Perkin Elmer Life Sciences. All other reagents were purchased from commercial sources and were of the best available grade.

	Footnotes

 
Reprint requests to: Eric L. Barker, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy and Pharmaceutical Sciences, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA; e-mail: ericb{at}pharmacy.purdue.edu; fax: (765) 494-1414.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062386106.

Abbreviations: 5-HT, 5-hydroxytryptamine or serotonin; LeuT_Aa, Aquifex aeolicus leucine transporter; DAT, dopamine transporter; DTT, dithiothreitol; EL, extracellular loop; hSERT, human serotonin transporter; GAT1, -aminobutyric acid transporter; KRH, Krebs-Ringer-HEPES buffer; MPP⁺, 1-methyl-4-phenylpyridinium; MTS-3-MTS, 1,3 propanediyl-bismethanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate; SCAM, substituted cysteine accessibility method; TMH, transmembrane helix.

	Acknowledgments

TOP Abstract Introduction Results Discussion Materials and methods Acknowledgments References

 
This work was supported by National Institute of Mental Health grant MH60221 (E.L.B.) and NIH Training in Biochemistry and Molecular Biology 5 T32 GM008737-05 (K.J.W.).

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