Defining Proximity Relationships in the Tertiary Structure of the Dopamine Transporter

Recently, we have described a distance constraint in the unknown tertiary structure of the human dopamine transporter (hDAT) by identification of two histidines, His193 in the second extracellular loop and His375 at the top of transmembrane (TM) 7, that form two coordinates in an endogenous, high affinity Zn2+-binding site. To achieve further insight into the tertiary organization of hDAT, we set out to identify additional residues involved in Zn2+ binding and subsequently to engineer artificial Zn2+-binding sites. Ten aspartic acids and glutamic acids, predicted to be on the extracellular side, were mutated to asparagine and glutamine, respectively. Mutation of Glu396 (E396Q) at the top of TM 8 increased the IC50 value for Zn2+ inhibition of [3H]dopamine uptake from 1.1 to 530 μm and eliminated Zn2+-induced potentiation of [3H]WIN 35,428 binding. These data suggest that Glu396 is involved in Zn2+ binding to hDAT. Importantly, Zn2+ sensitivity was preserved following substitution of Glu396 with histidine, indicating that the effect of mutating Glu396 is not an indirect effect because of the removal of a negatively charged residue. The common participation of Glu396, His193, and His375 in binding the small Zn2+ ion implies their proximity in the unknown tertiary structure of hDAT. The close association between TM 7 and 8 was further established by engineering of a Zn2+-binding site between His375 and a cysteine inserted in position 400 in TM 8. Summarized, our data define an important set of proximity relationships in hDAT that should prove an important template for further exploring the molecular architecture of Na+/Cl−-dependent neurotransmitter transporters.

The dopamine transporter (DAT) 1 tightly controls the amount of available dopamine in the synaptic cleft by mediating rapid reuptake of released dopamine into the presynaptic nerve terminal (1,2). In this way, DAT plays a critical role in modulating the physiological effects of dopamine, including regulation of locomotor activity, cognitive functions, and neuroendocrine systems (1,2). The DAT, together with the closely related norepinephrine (NET) and serotonin transporters, forms a subfamily within a larger family of neurotransmitter and amino acid transporters that are characterized functionally by their dependence on the presence of both Na ϩ and Cl Ϫ in the extracellular fluid (3,4). Of particular interest, DAT, NET, and serotonin are transporters targets for both antidepressants drugs and several commonly abused psychostimulatory drugs, such as cocaine and amphetamine (1,2,4). Although cocaine and amphetamine inhibit all three transporters with similar potencies, it is generally assumed that inhibition of DAT is primarily responsible for the reinforcing and locomotor stimulatory effects of these drugs (5)(6)(7).
Na ϩ /Cl Ϫ -dependent transporters are believed to share a common topology characterized by 12 transmembrane segments and an intracellular location of the amino and carboxyl termini (2)(3)(4). Many studies have been carried out to characterize functional domains both in DAT and other Na ϩ /Cl Ϫ -dependent transporters. Generation of chimeric transporter molecules has provided insight into the domains determining the subtype-specific pharmacological properties of the different transporters (8 -12). Moreover, point mutagenesis (13)(14)(15)(16)(17)(18) and a variety of other mutagenesis-based approaches, such as the substituted cysteine accessibility method (19,20) and random mutagenesis techniques (21), have been applied and lead to identification of some residues thought to be involved in the transport process or binding of substrate and/or blockers. However, little is yet known about the tertiary structure of this important class of transporters, and the nature of the molecular processes responsible for the translocation mechanism remains unknown.
Recently, we have reported an important distance constraint in the tertiary structure of the hDAT based on the discovery of an endogenous high affinity Zn 2ϩ -binding site (22). Initially, we observed that Zn 2ϩ , in micromolar physiological concentrations, acts as a potent noncompetitive blocker of dopamine uptake. Furthermore, it was found that micromolar concentrations of Zn 2ϩ markedly potentiate binding of the cocaine-like blocker, WIN 35,428 (22). Systematic mutation of histidines, predicted to be on the extracellular face of the transporter, identified two histidines, His 193 in the large second extracellular loop 2 outside transmembrane segment (TM) 3 and His 375 at the top of TM 7, as two coordinates in this endogenous Zn 2ϩ -binding site ( Fig. 1) (22). The two residues are separate in the primary structure, but their common participation in Zn 2ϩ binding designated their spatial proximity in the tertiary structure (22).
The structures of many Zn 2ϩ -binding sites are known from x-ray crystallography of Zn 2ϩ -binding proteins, and therefore, the geometry of the interaction between Zn 2ϩ and different coordinating side chains is well characterized (23)(24)(25). The coordination of Zn 2ϩ is most often found to be tetrahedral involving the side chains of four residues or the side chains of three residues plus a water molecule (23)(24)(25). It is thus conceivable that His 193 and His 375 are not the only residues involved in coordinating Zn 2ϩ binding to the hDAT. In addition to the imidazole side chain of histidines, the side chains found to coordinate Zn 2ϩ are the sulfhydryl side chain of cysteines and the acidic side chains of glutamates and aspartates (25). Because mutation of several cysteines predicted to be accessible from the extracellular side did not indicate participation of these residues in Zn 2ϩ binding, 2 we decided to investigate the possible involvement of aspartates and glutamates. Previously, we demonstrated that full Zn 2ϩ susceptibility can be conveyed to the human NET (hNET) by mutational transfer of only His 193 , which is nonconserved between hDAT and hNET (22). Additional coordinating residues can therefore be expected to be conserved between the two transporters, and we could restrict our search to 10 conserved aspartates and glutamates predicted to be on the extracellular face ( Fig. 1). By mutational analyses we find that Glu 396 at the extracellular end of TM 8 is a putative third coordinate in the hDAT Zn 2ϩ -binding site, indicating proximity between extracellular loop 2, TM 7 and TM 8 in hDAT. Moreover, we show that an artificial Zn 2ϩbinding site can be engineered between TM 7 and TM 8. This binding site both verify the proximity between the two domains as wells as it supports an ␣-helical configuration at the top of TM 8.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis-The cDNA encoding the human dopamine transporter (hDAT) and norepinephrine transporter (hNET) were kindly provided by Dr. Marc G. Caron (Duke University, Durham, NC). Mutant transporters were constructed by polymerase chain reactionderived mutagenesis using Pfu polymerase according to the manufacturer's instructions (Stratagene, La Jolla, CA). The generated polymerase chain reaction fragments were digested with the appropriate enzymes, purified by agarose gel electrophoresis, and cloned into the eukaryotic expression vector, pRc/CMV (Invitrogen, Carlsbad, CA), containing either hDAT or hNET (9,26). All mutations were confirmed by restriction enzyme mapping and DNA sequencing using an ABI 310 automated sequencer.
Cell Culture and Transfection-COS-7 cells were grown in Dulbecco's modified Eagle's medium 041 01885 supplemented with 10% fetal calf serum, 2 mM L-glutamine and 0.01 mg/ml gentamicin. Wild type and mutant constructs in pRc/CMV were transiently transfected into COS-7 cells by the calcium phosphate precipitation method as described previously (27,28).
[ 3 H]Dopamine Uptake Experiments-Uptake assays were performed modified from Giros et al. (29) using 2,5,6-[ 3 H]dopamine (7-21 Ci/mmol) (Amersham Pharmacia Biotech). Transfected COS-7 cells were plated in either 24-well dishes (10 5 cells/well) or 12-well dishes (2-3 ϫ 10 5 cells/well) to achieve an uptake level of 5-10% of total added [ 3 H]dopamine. The uptake assays were carried out 2 days after transfection. Prior to the experiment, the cells were washed once in 500 l of uptake buffer (5 mM Tris base, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl 2 , 1.2 mM MgSO 4 , 1 mM L-ascorbic acid, 5 mM D-glucose, pH 7.1). The compound to be tested was added to the cells, and uptake was initiated by addition of 10 nM [ 3 H]dopamine in a final volume of 500 l. After 10 min of incubation at 37°C, the cells were washed twice with 500 l of uptake buffer, lysed in 500 l 1% SDS and left 1 h at 37°C. All samples were transferred to 24-well counting plates (Wallac, Turku, Finland), 500 l of Opti-phase Hi Safe 3 scintillation fluid (Wallac) was added followed by counting of the plates in a Wallac Tri-Lux ␤-scintillation counter (Wallac). Nonspecific uptake was determined in the presence of 1 mM nonlabeled dopamine (Research Biochemicals International, Natick, MA). All determinations were performed in triplicate.
Ligand Binding-Binding assays were carried out on whole cells using [ 3 H]WIN 35,428 (83.5 Ci/mmol) (NEN Life Science Products) as radioligand. One day after transfection, cells were seeded in either 24-well dishes (10 5 cells/well) or 12-well dishes (2-3 ϫ 10 5 cells/well) to achieve a binding level of 5-10% of total added [ 3 H]WIN 35,428. Two days after transfection, competition binding assays were performed in a final volume of 500 l of uptake buffer containing 2-4 nM [ 3 H]WIN 35,428 and indicated concentrations of compound to be tested. Binding was terminated after 2 h at 4°C by washing the cells twice in 500 l of uptake buffer prior to lysis in 500 l 1% SDS for 1 h at 37°C. All samples were transferred to 24-well counting plates (Wallac, Turku, Finland), and 500 l of Opti-phase Hi Safe 3 scintillation fluid (Wallac) was added followed by counting of the plates in a Wallac Tri-Lux ␤scintillation counter (Wallac). Nonspecific binding was determined in the presence of 10 M WIN 35,428 (RBI). Determinations were made in triplicate.
Calculations -Uptake and binding data were analyzed by nonlinear regression analysis using Prism 2.0 from GraphPad Software (San Diego, CA).  and Table I and Ref. 22). Previously, we have shown evidence that the high affinity component is due to the interaction of Zn 2ϩ with at least two residues, His 193 and His 375 , on the predicted extracellular face of the transporter ( Fig. 1 and Ref. 22). His 193 is nonconserved between hDAT and hNET, whereas His 375 is conserved between the two transporters. Full Zn 2ϩ susceptibility can be conveyed to the hNET by mutational transfer of only His 193 (22); therefore, Zn 2ϩ -coordinating residues in addition to His 193 and His 375 can be expected to be conserved between hDAT and hNET. To examine the possible participation of aspartates and glutamates in binding of Zn 2ϩ , we mutated the 10 conserved aspartates and glutamates predicted to face the extracellular environment to asparagine and glutamine, respectively ( Fig. 1 (Table I). In contrast, mutation of Glu 396 to glutamine caused an almost 500-fold increase in the IC 50 value for Zn 2ϩ inhibition of [ 3 H]dopamine uptake ( Fig. 2A and Table I). This suggests that Glu 396 could be a third coordinate in the hDAT Zn 2ϩ -binding site. Notably, the effect on Zn 2ϩ inhibition by mutating Glu 396 is similar to the effect observed by mutating the previously identified Zn 2ϩ coordi-nates, His 193 and His 375 (Ref. 22 and shown in Fig. 2A for comparison).

Glu 396 Is a Putative Third Coordinate in the hDAT
Mutation of Glu 396 in the Homologous hNET-We have shown earlier that introducing a histidine in position 189 of the hNET (hNET-K189H), corresponding to His 193 in hDAT, converts Zn 2ϩ into a potent inhibitor of [ 3 H]dopamine uptake mediated by the hNET (Ref. 22 and shown in Fig. 2B for comparison). We demonstrated also that mutation of His 372 in hNET-K189H, corresponding to His 375 in hDAT, essentially eliminated the high affinity component in hNET-K189H, suggesting that Zn 2ϩ is interacting with the same site in the hNET-K189H mutant as in WT hDAT (22). This was further supported by mutating Glu 393 to glutamine in hNET-K189H, corresponding to Glu 396 in the hDAT. As shown in Fig. 2B, Zn 2ϩ was only a rather poor inhibitor of [ 3 H]dopamine uptake in this mutant (hNET-K189H-E393Q), displaying a 30-fold increase in IC 50 value as compared with hNET-K189H ( Fig. 2B and Table I). These data indicate that Glu 393 is involved in Zn 2ϩ binding in hNET-K189H. The effect of mutating Glu 393 was, however, not as dramatic as the effect of mutating Glu 396 in the hDAT, where a 500-fold increase in IC 50 value was observed for Zn 2ϩ (Table I and Fig. 2A). Most likely, this difference is due to some subtle structural differences between hDAT and hNET. Similar to what was observed following mutation of Glu 396 in hDAT, mutation of Glu 393 in hNET-K189H did not cause changes in the K m for [ 3 (Fig. 3). This provides additional support that Glu 396 is a third coordinate in the hDAT Zn 2ϩbinding site. Following mutation of the other nine acidic residues (Asp 191 , Glu 215 , Glu 218 , Asp 232 , Asp 301 , Glu 307 , Asp 381 , Asp 385 , and Glu 476 ), Zn 2ϩ -induced potentiation was at least partially preserved (Table I).
A Histidine Can Substitute for Glutamate in Position 396 -It is possible that the loss of Zn 2ϩ sensitivity upon mutating Glu 396 to glutamine in hDAT is due to an indirect structural effect caused by the removal of a negative charge rather than the disruption of a direct interaction with Zn 2ϩ . To exclude this possibility we substituted the glutamate with another residue capable of coordinating Zn 2ϩ . As shown in Fig. 4 To ensure that Zn 2ϩ binding to hDAT-E396H involved His 193 and His 375 , we mutated His 193 and His 375 by themselves (hDAT-H193K-E396H and hDAT-H375A-E396) and combined (hDAT-H193K-H375A-E396H) ( Table II) binding of Zn 2ϩ , which confirmed a crucial role of His 375 (Table  II and Fig. 4). However, mutation of His 193 (hDAT-H193K-E396H) did only increase the IC 50 value for Zn 2ϩ inhibition 2-fold, suggesting that His 193 only has a minor role in Zn 2ϩ inhibition of [ 3 H]dopamine uptake in the hDAT-E396H mutant (Table II and Fig. 4). Possibly, insertion of a histidine in position 396 causes a structural change of the Zn 2ϩ -binding site that could lead to involvement of as yet unidentified residues in addition to His 375 and the histidine in position 396. Nonetheless, the data still support the close proximity between His 375 and Glu 396 and are consistent with Glu 396 being a third coordinate in the endogenous Zn 2ϩ -binding site.
An Engineered Zn 2ϩ -binding Site between the TM 7 and 8 -Our data suggest that His 375 at the top of TM 7 is facing Glu 396 at the top of TM 8. To further elaborate this spatial proximity between TM 7 and 8, we next wished to engineer artificial metal ion-binding sites between the two domains. Assuming that Glu 396 is situated in an ␣-helical environment, it could be expected that His 375 also is close to the iϩ4 or iϩ3 position from Glu 396 in TM 8 and thus that a bidentate Zn 2ϩ -binding site could be generated between His 375 and either the iϩ4 or iϩ3 position, whereas the iϩ2 position would be expected to be located on the opposite side of the helix (Fig. 5A). Accordingly, three mutant transporters were generated in which His 193 and Glu 396 were removed, His 375 was preserved, and cysteines residues were inserted in positions iϩ2 (hDAT-H193K-E396Q-I398C), iϩ3 (hDAT-H193K-E396Q-A399C) and iϩ4 (hDAT-H193K-E396Q-T400C). Cysteines were chosen because their smaller side chain would be expected to be better tolerated in the transmembrane regions as compared with histidines. As illustrated in Fig. 5B, Zn 2ϩ was a potent inhibitor of [ 3 H]dopamine uptake in hDAT-H193K-E396Q-T400C (iϩ4), displaying an IC 50 value of 24 M in contrast to 660 M for the background mutant, hDAT-H193K-E396Q (Table III and Fig.  5B). This almost 30-fold increase in apparent Zn 2ϩ affinity was not only dependent on the presence of a cysteine in position 400 but was also dependent on the presence of His 375 . Mutation of His 375 to alanine in the hDAT-H193K-E396Q-T400C construct (resulting in hDAT-H193K-H375A-E396Q-T400C) eliminated   Table III). This indicates that an inhibitory bidentate Zn 2ϩ -binding site had been generated between His 375 and T400C. For hDAT-H193K-E396Q-A399C (iϩ3) we observed a small 2-3-fold decrease in the IC 50 value for Zn 2ϩ , whereas a 5-fold decrease was found for hDAT-H193K-E396Q-I398C (iϩ2) (Fig. 5C and Table III). Importantly, this 5-fold decrease in the IC 50 value for Zn 2ϩ in hDAT-H193K-E396Q-I398C (IC 50 ϭ 122 M) as compared with control, hDAT-H193K-E396Q (IC 50 ϭ 664 M; Table III) was found to be independent of the presence of His 375 because a similar IC 50 value was observed in the control mutant hDAT-H193K-H375A-E396Q-I398C (IC 50 ϭ 137 M; Table III). DISCUSSION In our previous study we demonstrated that the hDAT contains an endogenous high affinity Zn 2ϩ -binding site and that Zn 2ϩ by binding to this site acts as a potent noncompetitive blocker of dopamine uptake (22). Moreover, we identified two residues on the extracellular face of the transporter, His 193 and His 375 (Fig. 1) forming two coordinates in this binding site (22). In this study, we show evidence that a highly conserved glu-tamic acid, Glu 396 , is a third coordinate in the hDAT Zn 2ϩbinding site. The presence of a third coordinating residue in the hDAT Zn 2ϩ -binding site is supported by the estimated affinities of Zn 2ϩ for different Zn 2ϩ -binding sites. The affinity of Zn 2ϩ for binding sites involving three residues has generally been observed to range from 10 Ϫ8 to 10 Ϫ6 M, whereas sites involving four coordinates ranges from 10 Ϫ9 to 10 Ϫ8 M and sites with two coordinates from 10 Ϫ5 to 10 Ϫ4 M (30 -32). The wide affinity ranges reflect the fact that the Zn 2ϩ affinity depends on a broad variety of factors in addition to the ability of the coordinating side chain to chelate Zn 2ϩ (32); however, the observed apparent affinity of Zn 2ϩ for the hDAT (IC 50 ϭ 1 M; Table I) is mostly consistent with a binding site containing three coordinating residues. It is therefore also our estimate that no additional residues are involved, in agreement with the fact that we at present have mutated the majority of candidate Zn 2ϩ -binding residues in the hDAT (this study and Ref. 22). 2 Importantly, several Zn 2ϩ -binding sites in soluble proteins have been described involving two histidines and a glutamate (25). In these sites, the geometry is most often found to be tetrahedral with the fourth coordinate being a water molecule   (25). In some cases, both oxygens of the carboxylate side chain of the glutamate coordinates to the Zn 2ϩ ion resulting in a square-based pyramidal or trigonal pyramidal geometry (25). Of additional interest, the interaction between Zn 2ϩ and histidines is, independent of the geometry, very often stabilized by hydrogen bonds between the noncoordinating nitrogen of the imidazole side chain and residues that are not part of the primary coordination shell (25). For example, a hydrogen bond between the carboxylate of a nearby aspartate or glutamate and the imidazole is believed to increase the basicity of the imidazole and orient it correctly for interacting with the Zn 2ϩ ion in several Zn 2ϩ -binding proteins (25). Asp 191 in hDAT, located only one residue apart from His 193 , is a likely candidate for such an interaction. Possibly, this could explain the 3-fold decrease in apparent Zn 2ϩ affinity by mutating Asp 191 to asparagine, consistent with a stabilizing but not direct role in binding Zn 2ϩ to the hDAT (Table I).
According to known structures of Zn 2ϩ -binding proteins, the distance between the Zn 2ϩ ion and the coordinating nitrogen of the imidazole ring or the coordinating oxygen of the carboxylate side chain vary from 2.0 Å -2.3 Å and is largely independent of the coordination geometry (25,33). Hence, the identification of three residues involved in Zn 2ϩ binding to the endogenous hDAT Zn 2ϩ -binding site and the engineering of a Zn 2ϩ -binding site between TM 7 and 8 impose an important set of distance constraints in the tertiary structure of hDAT (Fig.  6). Glu 396 is predicted to be situated right at the extracellular end of TM 8, which is separated from TM 7 by a large loop of approximately 20 residues, allowing TM 7 and 8 to be rather far apart in the tertiary structure. However, the present data outline the close association between the two transmembrane segments, which is further documented by the engineered Zn 2ϩ -binding site between His 375 and the cysteines inserted in position 400. Moreover, the participation of His 193 in binding of Zn 2ϩ to the hDAT outline the association of the outer portion of TM 7 and 8 with the large second extracellular loop connecting TM 3 and 4. In summary, the data from the present study together with our previous data (22) provide the first crude insight into the tertiary organization of a Na ϩ /Cl Ϫ -dependent transporter. It is important to emphasize that the data, moreover, are consistent with the originally proposed topology of hDAT (shown in Fig. 1) and thus support recent studies based on site-directed chemical labeling of cysteines or lysines (20,34).
Engineering of artificial Zn 2ϩ -binding sites has previously been used as a powerful tool to probe the structure of both G protein-coupled receptors and the Lac permease of Escherichia coli (30,31,35,36). In this study, we have engineered a Zn 2ϩbinding site between His 375 and a cysteine inserted in position 400 (Fig. 5). The possibility of Zn 2ϩ to coordinate not only between His 375 and Glu 396 but also between His 375 and a cysteine inserted in position 400 further defines the orientation of His 375 , and thus TM 7, in relation to TM 8. To accommodate the data, His 375 must be facing TM 8, positioned between His 396 and Thr 400 (Fig. 6). Our data are moreover consistent with Glu 396 and Thr 400 being located in an ␣-helical environment. The apparent affinity, as indicated from the IC 50 value for Zn 2ϩ inhibition of [ 3 H]dopamine uptake, increased more than 30-fold by inserting the cysteine in position 400 (iϩ4 from Glu 393 ), whereas only smaller increases were observed for positions 399 and 398 (Table III). The apparent increase in affinity for Cys 398 was also observed in the absence of His 375 (mutant hDAT-H193K-H375A-E396Q-I398C) and therefore cannot be attributed to coordination of Zn 2ϩ between these two residues.
Earlier, we have shown evidence that Zn 2ϩ by binding to the endogenous high affinity binding site of the hDAT noncompeti-  tively prevents dopamine translocation without interfering with dopamine binding to the transporter (22). It is conceivable that Zn 2ϩ , by keeping His 193 , His 375 , and Glu 396 in close spatial proximity, inhibits a conformational change critical for the translocation process. Therefore, our data provide indirect evidence for an important role of extracellular loop 2 and TM 7/TM 8 in the conformational changes accompanying translocation of dopamine. It was interesting to observe that substitution of Glu 396 with His allows Zn 2ϩ to fully inhibit the translocation process (Fig. 4) in contrast to our observation in the WT hDAT where saturation of the high affinity Zn 2ϩ -binding site (in presence of around 100 M of Zn 2ϩ ) only resulted in approximately 60 -70% inhibition of uptake ( Fig. 2A and 4) in contrast to mutation of His 375 (hDAT-H375A-E396H), which eliminated apparent high affinity binding of Zn 2ϩ (Table III). It could therefore be hypothesized that substitution of glutamate with histidine in position 396 may lead to interaction between Zn 2ϩ and so far unidentified residues in addition to His 375 and His 396 . The steep inhibition curve with a Hill slope of Ϫ2.2 in hDAT-E396H is another indication of a more complex pattern of interaction between Zn 2ϩ and the transporter (Fig. 4). Nevertheless, it is also possible that fixation of TM 7 and 8 is the key element in Zn 2ϩ -induced inhibition of dopamine uptake whereas the interaction with His 193 in extracellular loop 2 is of less importance. Thus, a histidine in position 396 could be envisioned to be more optimal for Zn 2ϩ coordination than a glutamic acid and accordingly facilitate the ability of Zn 2ϩ to constrain motions of TM 7 relative to TM 8. An increasing number of receptors and transporters in the brain have been shown to be regulated by Zn 2ϩ in low micromolar concentrations. In addition to the DAT, these include ionotropic glutamate receptors, some ␥-aminobutyric acid receptor subtypes, the strychnine-sensitive glycine receptor, dopamine D2 receptors, and the EAAT1 glutamate transporter (37)(38)(39)(40)(41)(42)(43). This is consistent with the increasing amount of evidence pointing to Zn 2ϩ as an important neuromodulator in the brain (44). It is highly likely that free Zn 2ϩ is present in the synaptic clefts in concentrations required to modulate the function of the proteins. The basal concentration of Zn 2ϩ in the brain is approximately 10 nM (45,46). However, upon neuronal stimulation Zn 2ϩ has been shown to be released in concentrations of approximately 300 M (47). Although substantial information is available about Zn 2ϩ -binding sites in soluble proteins, including transcription factors and a variety of enzymes, little is known about Zn 2ϩ -binding sites in membrane proteins. High resolution structural information is not available for any of these proteins, and at present, residues involved in Zn 2ϩ binding have been identified only in the ␥-aminobutyric acid 1 subunit (48), in the ␥-aminobutyric acid ␤1 subunit (49) and in the glutamate transporter EAAT1 (42). In the EAAT1, two histidines were identified in the predicted large second extracellular loop (42). Evidently, additional insight into the specific structural and regulatory role of Zn 2ϩ at neurotransmitter transporters and receptors is a prerequisite for further clarifying the role of Zn 2ϩ as a neuromodulator in the human brain.