Zn2+ Inhibits the Anion Conductance of the Glutamate Transporter EAAT4

Glutamate transport by the excitatory amino acid transporters (EAATs) is coupled to the co-transport of 3 Na+ ions and 1 H+ and the counter-transport of 1 K+ ion, which ensures that extracellular glutamate concentrations are maintained in the submicromolar range. In addition to the coupled ion fluxes, glutamate transport activates an uncoupled anion conductance that does not influence the rate or direction of transport but may have the capacity to influence the excitability of the cell. Free Zn2+ ions are often co-localized with glutamate in the central nervous system and have the capacity to modulate the dynamics of excitatory neurotransmission. In this study we demonstrate that Zn2+ions inhibit the uncoupled anion conductance and also reduce the affinity of l-aspartate for EAAT4. The molecular basis for this effect was investigated using site-directed mutagenesis. Two histidine residues in the extracellular loop between transmembrane domains three and four of EAAT4 appear to confer Zn2+inhibition of the anion conductance.

Glutamate is the predominant excitatory neurotransmitter in the mammalian brain, and excitatory amino acid transporters (EAATs) 1 serve the role of controlling extracellular glutamate concentrations to maintain normal neurotransmission. Five different glutamate transporters have been identified in humans, termed EAAT1-5 (1)(2)(3). The rat homologues of EAAT1-3 are termed GLAST1 (4), GLT1 (5), and EAAC1 (6), respectively. The stoichiometry of substrate ion flux-coupling has been determined for the EAAT3 and GLT1 subtypes where glutamate is co-transported with 3 Na ϩ and 1 H ϩ followed by the counter-transport of 1 K ϩ ion (7,8). Glutamate transporters also allow an uncoupled flux of anions through the transporter, which requires the presence of glutamate and Na ϩ ions but does not appear to influence the rate or direction of glutamate transport (2,9). The magnitude of the uncoupled anion flux relative to the glutamate-coupled ion fluxes varies with the different transporters, with the anion flux greatest for EAAT4 and EAAT5, followed by EAAT1 and then EAAT3 and EAAT2. Thus, electrophysiological measurements of glutamate transport have components derived from the ion-coupled transport conductance and the uncoupled chloride conductance. Zn 2ϩ is found in a number of regions of the brain, with Ͼ90% bound to proteins such as various Zn 2ϩ finger proteins. Che-latable Zn 2ϩ is enriched in a number of regions of the brain, especially in the mossy fibers of the hippocampus where it is stored in synaptic vesicles with glutamate (reviewed in Ref. 10). Stimulation of the mossy fibers leads to release of Zn 2ϩ (11), and Zn 2ϩ has been shown to modulate the activity of a number of proteins within excitatory synapses, including the N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors (12,13), Ca 2ϩ channels (14), and also glutamate transporters (15,16). Spiridon et al. (15) demonstrated that Zn 2ϩ inhibits glutamate transport currents, whereas stimulating the uncoupled chloride conductance associated with the transporters in Muller cells of the salamander retina. In contrast to the Muller cells, Zn 2ϩ inhibits the chloride conductance associated with glutamate transporters of the retinal cone cells (15). The predominant glutamate transporter in Muller cells shows most similarity to the human EAAT1, and in a previous study we demonstrated that Zn 2ϩ inhibits the transport current of EAAT1, but had little effect on the uncoupled chloride flux associated with the transport process (16).
In many proteins, the side chains of histidine, cysteine, glutamate, or aspartate residues (17) coordinate the binding of Zn 2ϩ . In EAAT1, we established that the side chains of histidine 146 and histidine 154 form part of the Zn 2ϩ binding site. Alignment of the amino acid sequences of EAAT1-5 in this region ( Fig. 1) shows that histidine 146 is conserved in EAAT1, -2, -4, and -5, and that the residue corresponding to histidine 154 of EAAT1 is divergent. In EAAT2, which is insensitive to Zn 2ϩ , a glycine residue corresponds to the second histidine residue of EAAT1, and mutation of this residue to histidine generates a transporter that is inhibited by Zn 2ϩ at comparable Zn 2ϩ concentrations to that of EAAT1 (16). From these results we hypothesized that the chemical nature of the side chains of the residues at the two positions will determine the sensitivity to Zn 2ϩ . In this study we have investigated the actions of Zn 2ϩ on the glutamate transporter EAAT4 and found that Zn 2ϩ increases the EC 50 for L-aspartate activation of the chloride conductance and also decreases the extent of activation of the chloride conductance. Both of the Zn 2ϩ binding site histidine residues identified in EAAT1 are conserved in EAAT4, and mutations of these residues reduce Zn 2ϩ modulation of the anion conductance of the transporter.

EXPERIMENTAL PROCEDURES
Chemicals-All chemicals were obtained from Sigma Chemical Co. (Sydney, Australia) unless otherwise stated. TBOA was kindly supplied by Dr. Keiko Shimamoto and was also obtained from Tocris. ZnCl 2 was diluted from a stock solution of 20 mM in frog ringer's solution. The stock solution was stored at 4°C and was made fresh each week.
Expression of Excitatory Amino Acid Transporters in Xenopus laevis Oocytes and Electrophysiological Recordings-The wild-type and mutant EAAT4 transporters subcloned in the pOTV plasmid (1) were linearized with BamHI and cRNA transcribed from the cDNA construct with T7 RNA polymerase and capped with 5Ј-7 methyl guanosine using the mMESSAGE mMACHINE kit (Ambion Inc., Austin, TX). Mutations in EAAT4 were generated using the QuickChange site-directed mu-tagenesis kit from Stratagene and used according to the manufacturer's instructions.
Oocytes were harvested from X. laevis as previously described (18) and 50 nl of cRNA was injected into defoliculated stage V X. laevis oocytes and incubated in standard frog ringers solution (ND96; 96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES, pH 7.55), supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline, 50 g/ml gentamicin. 2-8 days later, current recordings were made using the two electrode voltage clamp technique with a Geneclamp 500 (Axon Instruments, Foster City, CA) interfaced with an MacLab 2e chart recorder (ADInstruments, Sydney, Australia) using the Chart software and a Digidata 1200 (Axon Instruments) controlled by an IBM compatible computer using the pCLAMP software (version 7, Axon Instruments).
The current-voltage relationships for L-glutamate or L-aspartate transport were determined by subtraction of steady state current measurements in the absence of L-glutamate or L-aspartate, obtained during 300-msec voltage pulses to potentials between Ϫ100 and ϩ60mV in 10 mV steps, from corresponding current measurements in the presence of substrate. In recordings where the extracellular chloride concentration was altered, a 3 M KCl agar bridge was used to connect the ground electrode to the bath solution to minimize offset potentials. The chloride-free buffer used contained 96 mM sodium gluconate, 2 mM potassium gluconate, 1.8 mM calcium(gluconate) 2 , 1 mM magnesium(gluconate) 2, 5 mM HEPES, pH 7.5. Zn 2ϩ chelates L-glutamate and L-aspartate, and the free L-glutamate and free Zn 2ϩ concentrations were calculated according the method of Dawson et al. (19) and employed by Spiridon et al. (15). In all figures and calculations free Zn 2ϩ and L-glutamate or L-aspartate concentrations are used. The following protocol was used to isolate the effects of Zn 2ϩ on the transport, and the transport-activated anion conductances of the transporters. The conductance elicited by L-glutamate (or L-aspartate) was first measured and after washoff of substrate, Zn 2ϩ was applied and baseline conductance measurements made, followed by co-application of substrate with Zn 2ϩ , and the conductance measured again. After washoff of Zn 2ϩ and substrate, substrate alone was re-applied to ensure that the conductance measurements return to control values.
Analysis of Kinetic Data-Analysis of kinetic data was carried out using the Kaleidagraph Software version 3.1. Substrate (L-glutamate and L-aspartate) dose responses were fitted by least squares as a function of current (I) to I/I max ϭ [S]/(EC 50 ϩ [S]), where I max is the maximal current, EC 50 the concentration of substrate that generates a halfmaximal current, and [S] is the substrate concentration. Dose-dependent Zn 2ϩ inhibition of substrate-activated currents were fitted by least squares to Equation 1, where I (Glu/Asp) is current due to L-glutamate or L-aspartate alone, I max is the maximal current inhibited by Zn 2ϩ , [Zn 2ϩ ] is the Zn 2ϩ concentration, IC 50 is the concentration of Zn 2ϩ causing half-maximal inhibition, and R is the residual transport current at a maximal dose of Zn 2ϩ . EC 50 , IC 50 , and I max values are presented in Table I.

RESULTS
Zn 2ϩ Inhibits the Anion Conductance of EAAT4 -Application of L-aspartate or L-glutamate to oocytes expressing EAAT4 generates a conductance that is carried predominantly by chloride ions (2). Co-application of 100 M Zn 2ϩ with 100 M Laspartate to oocytes expressing EAAT4 causes a reduction in amplitude of the aspartate-evoked conductance compared with 100 M L-aspartate alone with little or no change in reversal potential ( Fig. 2A). Whereas the reduction in current amplitude in the presence of Zn 2ϩ was apparent at both positive and negative membrane potentials, the reduction in current amplitude was greater at positive potentials. A slightly different result was obtained for L-glutamate-evoked currents. In the absence of Zn 2ϩ , L-glutamate generates a conductance ϳ50% of the conductance evoked by L-aspartate (Ref. 2, see Fig. 2B). In the presence of 100 M Zn 2ϩ , the reduction in the L-glutamateevoked conductance was only apparent at positive potentials with little or no change in conductance at negative potentials (Fig. 2B). In subsequent experiments on EAAT4 and the various mutants of EAAT4, we have presented the results for the effects of Zn 2ϩ on L-aspartate-evoked conductances, because the larger conductance changes are more reliably measured than for L-glutamate and the extent of Zn 2ϩ modulation of the currents is greater. The onset of inhibition by Zn 2ϩ was rapid and reversible with washoff of Zn 2ϩ from the bath solution, which suggests a direct interaction between Zn 2ϩ and the transporter.
Inhibition of substrate-evoked currents by Zn 2ϩ could be due to inhibition of the coupled transport conductance or inhibition of the uncoupled anion conductance or both conductances. Under the conditions of this experiment and assuming that the stoichometry of ion flux coupling for EAAT4 is the same as for EAAT3 and GLT1, a net inward flux of L-glutamate or Laspartate is expected at membrane potentials up to ϩ60 mV, and therefore any outward current at positive potentials is caused by the uncoupled anion conductance. The reduction in outward current at positive membrane potentials suggests that Zn 2ϩ inhibits the anion conductance. If the extracellular chloride ions are replaced with the more permeant anions bromide, iodide, or nitrate, significantly greater L-aspartate-evoked outward currents are observed, with a relative order of magnitude (and relative current amplitude at ϩ60 mV) of NO 3 Although 100 M Zn 2ϩ reduced the anion conductance for all 4 anions, the degree of reduction varied, with the greatest reduction observed for iodide (76 Ϯ 2%, n ϭ 4), followed by chloride (66 Ϯ 3%, n ϭ 7) and bromide (66 Ϯ 7%, n ϭ 4) and with the smallest reduction observed for nitrate (48 Ϯ 4%, n ϭ 5) (Fig. 3B). In each case there was no significant change in reversal potential measured in the presence and absence of 100 M Zn 2ϩ . These results are consistent with Zn 2ϩ inhibiting the uncoupled anion conductance, and although there are variations in the extent of inhibition for different anions, Zn 2ϩ does not alter the relative anion permeability.
To measure the effects of Zn 2ϩ on the ion-coupled transport conductance, oocytes expressing EAAT4 were incubated in a buffer in which chloride ions were completely replaced with the impermeant anion gluconate for Ͼ40 h. This procedure has been reported to reduce the intracellular chloride concentration of the oocyte to Ͻ4 mM (9) and allows transport conductances to be measured in the absence of a significant uncoupled anion conductance. Application of L-aspartate to oocytes in chloridefree buffer generates inward currents at potentials up to ϩ 60 mV, as opposed to an outward current measured under standard conditions. At Ϫ60 mV, currents measured under both chloride-free and standard conditions were inward but differed in relative amplitude. The L-aspartate-evoked current measured under chloride-free conditions was 15 Ϯ 3 nA (n ϭ 7) compared with 74 Ϯ 8 nA (n ϭ 3, from the same batch of oocytes) under standard conditions, which confirms previous observations that under standard conditions a majority of the L-aspartate-evoked conductance is caused by activation of a chloride conductance. Under chloride-free conditions, co-application of 100 M Zn 2ϩ with 100 M L-aspartate generated a similar conductance compared with 100 M L-aspartate alone ( Fig. 3C), which suggests that Zn 2ϩ has no effect on the coupled transport component of the currents mediated by EAAT4. Thus, Zn 2ϩ inhibition of the substrate-gated conductance of EAAT4 is most likely to be due to inhibition of the uncoupled anion conductance.
In further experiments on EAAT4 and EAAT4 mutants, we have not distinguished between the coupled transport and the uncoupled anion components of the conductance and have assumed that Zn 2ϩ has a selective effect on the uncoupled anion conductance. The EC 50 for L-aspartate activation of the chloride conductance measured in the presence of 100 M Zn 2ϩ (5.1 Ϯ 0.5 M, n ϭ 7) was increased compared with the EC 50 measured in the absence of Zn 2ϩ (3.4 Ϯ 0.5 M, n ϭ 7, p ϭ 0.05 2-tailed t test) (Fig. 4A), which suggests that Zn 2ϩ modulates the interaction between L-aspartate and EAAT4. At Ϫ100 mV, Zn 2ϩ caused a maximal inhibition of 58 Ϯ 10% (n ϭ 4), whereas at ϩ60 mV a maximal dose of Zn 2ϩ inhibited the current by 81 Ϯ 5% (n ϭ 4) (Fig. 4B). The IC 50 values for Zn 2ϩ inhibition of the anion conductance also differed at the different membrane potentials. At Ϫ100 mV, the IC 50 was 86 Ϯ 29 M (n ϭ 4) and at ϩ60 mV the IC 50 was 38 Ϯ 10 M (n ϭ 4).
Mutations of Histidine 154 and Histidine 164 Abolish Zn 2ϩ Sensitivity of EAAT4 -We have previously identified two histidine residues in the large extracellular loop between transmembrane domains 3 and 4 of EAAT1 that form part of the Zn 2ϩ binding site. Alignment of the amino acid sequences of EAAT4 with EAAT1 shows that both histidine residues are conserved between the two transporters. In the following experiments we have used site-directed mutagenesis to investigate whether the conserved histidine residues also form the Zn 2ϩ binding site on EAAT4 that mediates inhibition of the anion conductance.
The two histidine residues were mutated to alanine to re-move the imidazole group that is thought to interact with Zn 2ϩ . In addition the second histidine residue, at position 164, was changed to glutamate because a glutamate residue is found at this position of the glutamate transporter EAAT5. Application of L-aspartate to oocytes expressing the EAAT4 mutants, H154A, H164A, and H164E generated dose-dependent conductances that reversed direction at similar membrane potentials to that of wild type EAAT4 (Table I). This suggests that the mutations have not caused significant structural changes to the pore of the transporter. In contrast to wild type EAAT4, co-application of 100 M Zn 2ϩ with 100 M L-aspartate to oocytes expressing the EAAT4 mutant, H154A, had no significant effect on the conductance compared with L-aspartate alone (Fig. 5) or the EC 50 for L-aspartate-evoked conductance. This suggests that the mutation has selectively disrupted Zn 2ϩ modulation of the substrate-activated anion conductance. Similar results were also observed for the second site mutants H164A  Fig. 2, but in a buffer in which 96 mM NaCl was replaced with 96 mM NaI. The data represent the mean currents Ϯ S.E. from four cells and in for each cell the current measurements are normalized to the current because of 100 M L-aspartate at 0 mV. B, the percent reduction in slope conductance (over the range 0 -40 mV) because of 100 M Zn 2ϩ in which the 96 mM NaCl in the extracellular buffer was changed to 96 mM NaBr, NaI, or NaNO 3 . C, L-aspartate elicited conductances measured from oocytes that had been incubated in a chloride-free buffer (gluconate substituted for chloride) for Ͼ40 h prior to recording. Recordings were then made in the same chloride-free buffer. Data represent the mean Ϯ S.E. of current measurements from five cells. and H164E (Fig. 5). Thus, histidines residues at positions 154 and 164 appear to influence Zn 2ϩ affinity for EAAT4, which is analogous to the results observed for EAAT1 (16).
Cysteine residues have also been identified in other proteins as forming Zn 2ϩ binding sites and as EAAT4 contains two cysteine residues we investigated whether either of these two residues play a role in mediating the effects of Zn 2ϩ on EAAT4. Application of 100 M L-aspartate to oocytes expressing the EAAT4 C194A and EAAT4 C400A mutants showed similar current-voltage relationships as wild type EAAT4, and co-application of 100 M Zn 2ϩ caused similar reductions in the conductance as observed for the wild type EAAT4. Thus, the C194A and C356A mutations do not appear to alter the functional properties of the transporter or the sensitivity to Zn 2ϩ and are unlikely to form part of the Zn 2ϩ binding site on EAAT4.
In most cells expressing EAAT4, but not in uninjected oocytes, application of Zn 2ϩ alone appears to block a constitutive conductance. This constitutive conductance could be an intrin-sic property of EAAT4 or could be because of the expression of an endogenous oocyte protein as a consequence of overexpression of the transporter. The following observations suggest that an endogenous oocyte ion channel mediates the constitutive conductance. First, if the constitutive conductance were an intrinsic property of the transporter it would be expected that there should be a correlation between the amplitude of the leak conductance and the amplitude of the anion conductance. The amplitude of the constitutive conductance was variable both between, and within, batches of oocytes. The amplitude of the leak conductance blocked by 100 M Zn 2ϩ varied from 250% of the substrate activated anion conductance to Ͻ5% of the anion conductance. Second, application of the glutamate transport blocker TBOA to oocytes expressing EAAT4, at concentrations that inhibit the substrate-activated anion conductance, does not block the constitutive conductance. Third, the amplitude of the Zn 2ϩ -blocked leak conductance does not appear to influence any of the transporter-mediated functional properties, such as the EC 50 for Zn 2ϩ inhibition of the anion conductance or the extent of inhibition. Fourth, the same variability in amplitude of the leak conductance observed for EAAT4 was also observed for the EAAT4 mutants, EAAT4 H154A, EAAT4 H164A, EAAT4 H164E, EAAT4 C194A, and EAAT4 C356A. Finally, other researchers have also described various Zn 2ϩ -blocked conductances in oocytes (20) that could be responsible for the leak conductance in oocytes expressing EAAT4. Although we cannot completely rule out the possibility that the constitutive conductance, or some proportion of the conductance, is an intrinsic property of the transporter that functions independently of the transport function, the above observations make this interpretation unlikely.

Zn 2ϩ Inhibition of the Anion Conductance of EAAT4 -Zn 2ϩ
is found throughout the brain and may modulate the actions of glutamate by influencing the activity of NMDA receptors (12,13), Ca 2ϩ channels (14), and also glutamate transporters (15,16). The actions of Zn 2ϩ are most clearly demonstrated in the mossy fibers of the hippocampus where Zn 2ϩ is co-released with glutamate upon stimulation (11). Whereas there are a number of physiological and pathological implications of Zn 2ϩ modulation of excitatory neurotransmission, Zn 2ϩ may also be used to study potential mechanisms for modulation of various proteins, including glutamate transporters. In this study we have used Zn 2ϩ as a molecular probe to identify the molecular basis for differential modulation of the coupled and uncoupled conductance states of the glutamate transporter EAAT4.
There are two distinct types of conductances associated with glutamate transporter function: a coupled flux of Na ϩ , H ϩ , K ϩ , and glutamate ions (7,8); and an uncoupled anion conductance that requires the presence of L-glutamate and Na ϩ ions (2,9,21,22). The relative contributions of the two components vary with the different transporter subtypes, and the effects of Zn 2ϩ on these conductances also vary between transporter subtypes. In the case of EAAT1, we have previously demonstrated that Zn 2ϩ inhibits the coupled Na ϩ , K ϩ , H ϩ , and glutamate fluxes with little, if any, effect on the uncoupled anion conductance, whereas application of Zn 2ϩ to oocytes expressing EAAT2 or EAAT3, does not appear to modulate any of the transporterassociated conductances (16). 2 In the present study we have investigated the actions of Zn 2ϩ on the uncoupled anion conductance and the coupled substrate transport conductance of the EAAT4 subtype.
We have demonstrated that Zn 2ϩ inhibits the uncoupled anion conductance of EAAT4 and also causes a small, but 2 R. J. Vandenberg, unpublished observations.  Table I. B, Zn 2ϩ causes a dose-dependent inhibition of L-aspartate elicited conductances in oocytes expressing EAAT4. 100 M L-aspartate was co-applied with increasing doses of Zn 2ϩ and the currents measured at Ϫ100 mV (squares) and ϩ60 mV (circles). Current measurements were normalized to the current measured in absence of Zn 2ϩ and fit to the modified Michaelis Menton equation for inhibition as described under "Experimental Procedures." C, IC 50 values for Zn 2ϩ inhibition of the anion conductance are plotted for membrane potentials between Ϫ100 and ϩ60 mV. significant, increase in L-aspartate EC 50 . Although Zn 2ϩ reduced the amplitude of the anion conductances of EAAT4, Zn 2ϩ did not change the reversal potentials of the anion conductances when carried by chloride, bromide, iodide or nitrate ions. The lack of changes in anion permeability suggests that Zn 2ϩ binds to a site on EAAT4 that is distinct from the pore region of the transporter. The IC 50 for Zn 2ϩ inhibition of the anion conductance decreases with an increase in membrane potential from 86 M at Ϫ100 mV to 38 M at ϩ60 mV and the extent of maximal inhibition of the anion conductance also changes from 58% at Ϫ100 mV to 81% at ϩ60 mV. These observations could be explained if the time spent in transport mode compared with anion-conducting mode is also dependent on membrane potential. At positive membrane potentials, the anion-conducting mode may predominate and as Zn 2ϩ selectively inhibits the anion conductance of the transporter, the measured effects of Zn 2ϩ may be more apparent at these membrane potentials.
Molecular Basis for Differential Zn 2ϩ Modulation of Glutamate Transporter Subtypes-We have previously demonstrated that two histidine residues within the extracellular loop between transmembrane domains 3 and 4 form part of the Zn 2ϩ binding site on EAAT1. Mutations of either of these histidine residues to alanine do not alter the glutamate transport kinetics of EAAT1 but do diminish the effects of Zn 2ϩ on EAAT1 and therefore these Zn 2ϩ binding site residues are unlikely to form part of the pore through which glutamate, Na ϩ , K ϩ , H ϩ and possibly Cl Ϫ ions pass during the transport process. We have now extended this work to include a description of the Zn 2ϩ binding sites on the EAAT4 subtype of excitatory amino acid transporters.
Alignment of the amino acid sequences of the EAATs shows that the two histidine residues of EAAT1 that bind Zn 2ϩ are conserved in EAAT4. Mutation of either of these histidine residues to alanine abolishes Zn 2ϩ inhibition of the anion conductance of EAAT4 and also Zn 2ϩ modulation of L-aspartate EC 50 , which demonstrates that Zn 2ϩ interacts with EAAT4 at a similar site to that of EAAT1. Although the Zn 2ϩ binding sites are similar on EAAT1 and EAAT4, the effects of Zn 2ϩ are different. In the case of EAAT1, Zn 2ϩ inhibits the coupled fluxes of L-glutamate, Na ϩ , K ϩ , and H ϩ with minimal effect on the anion conductance (16), whereas for EAAT4 Zn 2ϩ causes a small increase in EC 50 for L-aspartate with no change in the level of inhibition of L-aspartate transport and significant inhibition of the anion conductance. There are a number of possible explanations for these differences. Wadiche and Kavanaugh (23) have demonstrated that glutamate transporters do not simultaneously function as a coupled transporter and an anion channel, but rather the transporters are likely to switch between the two modes of function. As the chloride conductance dominates the combined coupled transport/uncoupled chloride channel conductance in the cases of EAAT4, it may be predicted that the time spent in the anion channel mode is significantly greater than the transporter mode compared with EAAT1. Thus, Zn 2ϩ modulates the dominant process, i.e. the anion conductances of EAAT4 and the coupled glutamate, Na ϩ , K ϩ , H ϩ fluxes of EAAT1.
If we compare the results of the human glutamate transporters expressed in oocytes with that observed for the actions of Zn 2ϩ on glutamate transporters in the salamander retina there are a number of similarities, but also some distinct differences. In Muller cells  of the salamander retina the predominant transporter is homologous to EAAT1, and in these cells Zn 2ϩ inhibits glutamate transport currents, but in contrast to human EAAT1 Zn 2ϩ stimulates the uncoupled chloride conductance. Furthermore, the K 0.5 for Zn 2ϩ modulation of the Muller cell transporter is 0.66 M, which is ϳ10-20-fold less than that observed for EAAT1. In other Zn 2ϩbinding proteins the number of coordinating residues roughly correlates with the affinity of Zn 2ϩ . With 2 coordinating histidine residues affinities range from 10-100 M whereas with 3 coordinating residues affinities in the range of 0.01-1 M have been observed (17). Thus, the higher affinity of Zn 2ϩ for the salamander EAAT1 may be due to the presence of an additional coordinating residue. If the amino acid sequences of the human and salamander EAAT1 s are compared in the putative Zn 2ϩ binding site region a number of subtle differences are apparent that could explain the different effects of Zn 2ϩ (Fig. 1). The salamander EAAT1 contains an extra histidine residue between the two histidine residues conserved between the human and salamander EAAT1s that could possibly influence the binding affinity or could create different conformational changes when Zn 2ϩ is bound compared with Zn 2ϩ binding to EAAT1 such that the different functional effects are created. The differences could also arise because of the expression of other glutamate transporter subtypes or accessory proteins (24)(25)(26) in the Muller cell with each subtype responding differently to Zn 2ϩ . In cone cells of the salamander retina, Zn 2ϩ inhibits the chloride conductance associated with glutamate transporters of the retinal cone cells (27). The predominant glutamate transporter in cone cells is homologous to human EAAT5, but in oocytes expressing human EAAT5, Zn 2ϩ appears to stimulate the anion conductance. 3 The quarternary structure of glutamate transporters is poorly defined, but recent characterization of electron micrographs of X. laevis oocyte membranes containing the EAAT3 transporters suggests that transporters may exist as homomultimers consisting of between 3-6 subunits, with 5 subunits the most favored option (28). Furthermore, it was suggested that the subunits may function as separate transporters, but the chloride channel function of the transporters may arise through the association of the subunits to form a central channel. The binding of substrate to each of the subunits may alter the association of the subunits to change the conformation of the central chloride channel and allow passage of chloride ions. If this functional model is correct then the actions of Zn 2ϩ offer a particularly interesting insight into the mechanisms for differentially modulating the dual roles of glutamate transporters. Thus, the functional role of Zn 2ϩ ions may be to modify the association between subunits so as to change the rate of conformational changes required for the different modes of function of the transporters.
Possible Physiological Roles of Zn 2ϩ Modulation of Glutamate Transporters-The concentrations of Zn 2ϩ required to modulate EAAT4 are within the reported concentration range of the free extracellular Zn 2ϩ found in various regions of the brain. EAAT4 is expressed predominantly in Purkinje cells of the cerebellum, which also co-express EAAT3 (29,30). Whereas Zn 2ϩ is found in the cerebellum, and numerous studies have investigated the effects of exogenously applied Zn 2ϩ on synaptic neurotransmission in this region, the levels of free Zn 2ϩ found in regions accessible to the transporters under normal or pathological conditions are not well established (10). If present in sufficiently high concentrations, Zn 2ϩ would inhibit activation of the anion conductance of EAAT4, which may alter the excitability of the Purkinje cells. An alternative suggestion for the physiological role of Zn 2ϩ binding to glutamate transporters is that the functional consequences of the action (inhibition of transport or modulation of the chloride conductance) may not be of particular relevance and that the primary role of Zn 2ϩ binding to transporters may be to provide a mechanism to limit exposure of other synaptic proteins to the deleterious effects of Zn 2ϩ during pathological insults. In a number of brain regions glutamate transporters are expressed at very high levels in close proximity of the synapse (29,31,32), with one estimate being that the number of transporters in close proximity to the glutamate release site is greater than the number of glutamate molecules released from a single synaptic vesicle (32). Thus, when Zn 2ϩ concentrations are elevated, which may occur under various pathological conditions (33), transporters may provide a sink for excessive Zn 2ϩ and thereby limit exposure of other synaptic proteins, such as ionotropic glutamate receptors, which are unlikely to be present at the same density as the transporters. The binding of Zn 2ϩ to highly abundant glutamate transporters may also serve to limit the extent of Zn 2ϩ uptake, which can be excitotoxic to neurons (33).