Introduction

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Zn²⁺ is found in many parts of the brain and has been reported to modulate glutamate receptors (Peters et al., 1987; Westbrook and Mayer, 1987), -aminobutyric acid and glycine receptors (Smart and Constanini, 1983; Laube et al., 1995), glutamate transporters (Balcar and Johnston, 1972; Gabrielsson et al., 1986; Spiridon et al., 1998), and the Na⁺K⁺ATPase (Hexum, 1974); thus, it may modulate neurotransmission in a number of different ways (reviewed by Frederickson, 1986). In particular regions of the brain, most notably in the mossy fibers of the hippocampus, Zn²⁺ is co-localized with glutamate in synaptic vesicles and is released into the synapse in a calcium-dependent manner; it may reach concentrations of up to 118 µM (Howell et al., 1984; Spiridon et al., 1998). The different glutamate receptor subtypes show differential sensitivity to Zn²⁺. The NMDA receptor subtype is potently inhibited by Zn²⁺ in a rapid and reversible manner, yet the kainate and quisqualate receptor subtypes are relatively insensitive to Zn²⁺ (Westbrook and Maher, 1987). Furthermore, Zn²⁺ has differential effects on different subtypes of NMDA receptors (Chen et al., 1997).

Balcar and Johnston (1972) and Gabrielsson et al. (1986) reported inhibition of [³H]glutamate transport in brain slices and synaptosomes, respectively, by moderate concentrations of Zn²⁺ (30-100 µM). However, in these experimental systems, Zn²⁺ may have a number of effects, including inhibition of Na⁺K⁺ATPase activity, which may indirectly reduce glutamate transport activity by disrupting the Na⁺ and K⁺ gradients required for glutamate uptake. More recently, Zn²⁺ has been demonstrated to inhibit glutamate transport in Müller cells and cone cells of the salamander retina (Spiridon et al., 1998). The effects of Zn²⁺ in this study were rapid in onset and fully reversible, which suggests a direct role in transport inhibition. Zn²⁺ does not seem to compete with glutamate, sodium, potassium or protons binding to the transporter, which implies that Zn²⁺ allosterically modulates transporter function. In addition to inhibiting glutamate transport, Zn²⁺ seems to have opposing effects on the chloride conductance associated with the glutamate transporter in the two cell types investigated in the salamander retina. In Müller cells, the chloride conductance is increased by Zn²⁺; in the cone cells, however, the chloride conductance is inhibited by Zn²⁺. The transporter in Müller cells is likely to be similar in properties to EAAT1/GLAST1, whereas the cone-cell glutamate transporter is most similar to EAAT5 (Eliasof et al., 1997).

We have investigated the effects of Zn²⁺ on two of the most abundant human glutamate transporter subtypes, EAAT1 and EAAT2 (Arriza et al., 1994) expressed in Xenopus laevis oocytes. Zn²⁺ is a noncompetitive inhibitor of glutamate transport by EAAT1, but has no effect on transport by EAAT2. Zn²⁺ seems to cause an increase in the relative chloride component of the EAAT1 transport current while inhibiting the transport component. In addition, analysis of the Zn²⁺ sensitivity of point mutated transporters has been used to characterize the molecular basis for differential Zn²⁺ inhibition of glutamate transport by EAAT1 compared with EAAT2.

	Materials and Methods

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Chemicals. L-glutamate-Na salt, D-aspartic acid, HEPES-hemi Na salt, gluconate salts and methanesulfonic acid were obtained from Sigma/Aldrich, Sydney, Australia. Zinc sulfate (analytical grade) was obtained from Standard Laboratories, Melbourne, Australia. Stock solutions of zinc sulfate in frog Ringer buffer were made up fresh on the day of use. All other buffer components were of analytical grade or acceptable for use in high performance liquid chromatography.

Expression of transporters and electrophysiological recording. cDNAs encoding the human glutamate transporters, EAAT1 and EAAT2, were subcloned into pOTV for synthesis of RNA and expression in X. laevis oocytes as described previously (Arriza et al., 1994). Site-directed mutagenesis was carried out using the Altered Sites kit (Stratagene, La Jolla, CA) as described in the manufacturer's instructions. Two to seven days after RNA injection, current recordings were made at 22° with a Geneclamp 500 interfaced with an IBM-compatible computer using a Digidata 1200 A/D controlled by pCLAMP software (version 6.0.2; Axon Instruments). The standard frog Ringer recording solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5 mM HEPES, pH 7.55. In experiments in which chloride was removed, the oocytes were dialyzed overnight in a chloride-free frog Ringer solution in which all the chloride was replaced with gluconate (96 mM Na-gluconate, 2 mM K-gluconate, 1 mM Mg (gluconate)₂, 1.8 mM Ca (gluconate)₂, and 5 mM HEPES, pH 7.55). It has been estimated that this procedure reduces the intracellular chloride concentration from 40 mM to 4 mM (Wadiche et al., 1995). The next day, recordings were made using a chloride-free Ringer solution in which the chloride was replaced with either gluconate, as described above, or methanesulfonate (96 mM methanesulfonic acid, 2 mM KOH, 1 mM MgSO₄, 1.8 mM Ca-acetate, 5 mM HEPES and the pH adjusted with NaOH to pH 7.55). When recording using the chloride-free solutions, junction potentials were minimized by the use of a 3 M KCl-agar bridge from the recording chamber to a reservoir containing 3 M KCl and Ag/AgCl electrodes connected to a bath clamp headstage. The oocytes were voltage clamped at 30 mV and the current-voltage relations were determined by subtraction of steady state current measurements in the absence of substrate, obtained during 200-msec voltage pulses to potentials between 100 mV and +50 mV, from corresponding current measurements in the presence of L-glutamate or D-aspartate. In experiments concerned with the pH dependence of Zn²⁺ inhibition of glutamate transport, the pH of the bath solution was adjusted with HCl. In some oocytes, Zn²⁺ blocked a current that was present in uninjected oocytes, water-injected oocytes, and also oocytes injected with either EAAT1 or EAAT2. Therefore, all base-line current measurements were made in the presence of the appropriate Zn²⁺ concentration so that only glutamate transport currents were measured.

Analysis of kinetic data. Zn²⁺ chelates glutamate; therefore, to estimate the free Zn²⁺ and free glutamate concentrations, we have used the correction methods described by Dawson et al. (1986) and also employed by Spiridon et al. (1998). When calculating values for IC₅₀, EC₅₀, and %Inhibition, we have used the free Zn²⁺ and free glutamate concentrations. Current (I) as a function of L-glutamate concentration ([S]) was fitted by least squares to I = I_max · [S]/(EC₅₀ + [S]), where I_max is the maximal current and EC₅₀ is the concentration of L-glutamate generating half the maximal current. I_max and EC₅₀ values are expressed as mean ± standard error and were determined by fitting data from individual oocytes. For Zn²⁺ dose responses, current (I) as a function of Zn²⁺ concentration ([Zn²⁺]) was fitted by least squares to I = I_max(Z)  [(I_max(Z) · [Zn²⁺])/(IC₅₀ + [Zn²⁺])] + I_r, where I_max(Z) is the maximal current generated by L-glutamate that is inhibited by Zn²⁺, IC₅₀ is the concentration of Zn²⁺ that reduced I_max(Z) by 50% and I_r is the residual glutamate transport current in the presence of a maximal dose of Zn²⁺. %Inhibition = I_max(Z)/(I_max(Z)+I_r). IC₅₀ and %Inhibition values are expressed as mean ± standard error.

	Results

Top Summary Introduction Materials & Methods Results Discussion References

Application of L-glutamate to X. laevis oocytes expressing the human glutamate transporters, EAAT1 or EAAT2, generates dose-dependent inward currents when voltage clamped at 60 mV (Arriza et al., 1994). Co-application of 100 µM Zn²⁺ with 30 µM glutamate reduced the EAAT1 transport current but had no effect on EAAT2 (Fig. 1A). The inhibition of transport currents in EAAT1 are rapid in onset and fully reversible, which indicates a direct action of Zn²⁺ on the transporter. EC₅₀ values for glutamate transport measured in the presence and absence of 100 µM Zn²⁺ were similar (Table 1) but the maximal transport current was decreased by approximately 50% in the presence of 100 µM Zn²⁺ compared with its absence (Fig. 1B). This suggests that Zn²⁺ is acting as a noncompetitive inhibitor of glutamate transport. Inhibition of EAAT1 transport currents by Zn²⁺ is dose dependent with an IC₅₀ value of 9.9 ± 2.3 µM and maximal inhibition of 59 ± 4% (Fig. 1D). In contrast, Zn²⁺, at concentrations up to 300 µM, had little or no effect on glutamate transport currents for oocytes expressing EAAT2 (Fig. 1, C and D). Thus, the actions of Zn²⁺ on EAAT1 seem to be similar (although less potent for EAAT1) to that observed for glutamate transporters of salamander retina Müller cells.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Zn2+ inhibits glutamate transport by EAAT1 and has no effect on EAAT2. A, Representative current traces at 60 mV for the effect of 100 µM Zn2+ (filled bar) on 30 µM glutamate (open bar) transport by oocytes expressing EAAT1 or EAAT2. For the purposes of this figure, we selected traces from cells in which Zn2+ alone had no effect on the base-line current. Zn2+ has no effect on the EC50 values for glutamate transport by either EAAT1 (B) or EAAT2 (C) but reduces the maximal glutamate transport current for EAAT1. Dose-dependent glutamate transport currents at 60 mV were measured in the presence () and absence () of 100 µM Zn2+. Data from individual oocytes were normalized to a maximal dose of glutamate in the absence of Zn2+ and fit to the equation I = Imax · [S]/(EC50 + [S]) (see Methods). The data plotted represent the mean ± standard error (n = 4). D, Glutamate (1 mM) transport currents at 60 mV for EAAT1 () and EAAT2 () were measured in the presence of increasing doses of Zn2+. Current measurements were normalized to the current in the absence of Zn2+ and the mean ± standard error from four cells plotted. The errors for EAAT2 are smaller than the size of the symbol.

Glutamate transport is coupled to 2-3 Na⁺, 1 H⁺ (or possibly counter-transport of 1 OH) and the counter-transport of 1 K⁺ (Kanner and Sharon, 1978; Barbour et al., 1988; Zerangue and Kavanaugh, 1996). The different transporter subtypes also allow a varying degree of chloride ion flux, which is thermodynamically uncoupled to the rate of glutamate transport (Fairman et al., 1995; Wadiche et al., 1995; Billups et al., 1996; Eliasof and Jahr, 1996). In salamander-retina Müller cells, Zn²⁺ seems to increase the chloride conductance activated by glutamate transport (Spiridon et al., 1998). We investigated the effects of Zn²⁺ on the chloride conductance activated by transport by EAAT1. D-aspartate transport by EAAT1 activates a significantly greater chloride conductance than L-glutamate transport (Wadiche et al., 1995), which allows more accurate measures of the chloride conductance. The D-aspartate transport current reverses direction at 3 ± 2 mV (n = 4), with the outward current caused by the uncoupled chloride flux (Wadiche et al., 1995). The membrane potential at which the current reverses direction may be used as an indicator of the chloride conductance relative to the transport component of the current. In the presence of 100 µM Zn²⁺ the D-aspartate transport current reverses direction at 14 ± 4 mV (n = 4). This shift in transport current reversal potential toward the chloride reversal potential for X. laevis oocytes (~ 20 mV; Barish, 1983; Fairman et al., 1995; Wadiche et al., 1995) suggests that in the presence of Zn²⁺, chloride ions contribute a larger proportion of the transport current compared with conditions in which Zn²⁺ is absent. At membrane potentials greater than the reversal potentials for D-aspartate, the net transport currents in the presence and absence of Zn²⁺ converge (Fig. 2). This suggests that although the chloride conductance relative to substrate flux has increased, the absolute chloride conductance has not increased significantly.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Zn2+ modulates the transport activated chloride conductance of EAAT1. Zn2+ inhibits D-aspartate transport by EAAT1 and shifts the current reversal potential toward the chloride reversal potential. The transport currents elicited by 1 mM D-aspartate in the presence () and absence () of 100 µM Zn2+ were measured for membrane potentials between 100 mV and +50 mV in 10 mV increments, using the voltage pulse protocol described in the methods section with the standard frog Ringer solution. Data from a representative cell are shown.

The voltage dependence of glutamate transport block by Zn²⁺ was also measured. For these experiments, we used oocytes that had been dialyzed in chloride-free Ringer solution (gluconate substituted for chloride; see Materials and Methods for buffer details) to avoid chloride effects on the transport currents, which would otherwise create an apparent voltage dependence, separate from any Zn²⁺ effects. Zn²⁺ inhibition of glutamate transport by EAAT1 was then measured using a chloride-free Ringer solution in which the chloride was replaced with either gluconate or methanesulfonate. After taking into account the chelation of Zn²⁺ by gluconate, as described by Dawson et al. (1986), and Spiridon et al. (1998), the IC₅₀ for Zn²⁺ inhibition of 1 mM glutamate transport currents, measured using gluconate-substituted buffer solutions, was at least an order of magnitude greater than similar measurements using the chloride-containing frog Ringer solution. In contrast, the IC₅₀ value measured using a buffer with methanesulfonate substituted for chloride was similar to IC₅₀ measurements made in the presences of chloride. Therefore, we used the methanesulfonate-substituted frog Ringer solution to measure the voltage dependence of Zn²⁺ inhibition of glutamate transport. The IC₅₀ for Zn²⁺ inhibition of glutamate transport was relatively constant over the membrane potential range of 100 mV to +50 mV, varying from 10.8 ± 2.6 µM at 100 mV to 15.7 ± 6.0 µM at +50 mV (Fig. 3). The %Inhibition was also relatively constant over this membrane potential range at around 60%. This suggests that the Zn²⁺ binding site on the transporter is not influenced by the electric field of the membrane and is likely to be near the external surface of the transporter. Again, similar voltage dependence of Zn²⁺ inhibition of glutamate transport by Müller cells was observed, consistent with the identification of the transporter in this cell type as similar to EAAT1.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Zn2+ inhibition of glutamate transport is voltage independent. A, Dose dependent Zn2+ inhibition of 1 mM glutamate transport by a representative oocyte expressing EAAT1 was measured in a chloride-free frog Ringer solution using oocytes that had been placed in chloride-free Ringer solution overnight to reduce the intracellular chloride. For the purposes of this figure, concentrations of Zn2+ shown are 300 µM (), 100 µM (), 30 µM (), 10 µM () and 3 µM (). B, Current measurements at the indicated membrane potentials were fit to the equation I = Imax(Z)  [(Imax(Z) · [Zn2+])/(IC50 + [Zn2+])] + Ir (see Methods) to obtain estimates for IC50 values for each cell. Zn2+ concentrations from 1 µM to 1 mM were used in calculations of IC50 values. Error bars, mean ± standard error from four cells.

It has been demonstrated that a reduction in pH reduces Zn²⁺ inhibition of transport in Müller cells and that this is unlikely to reflect competition between Zn²⁺ and protons for a site on the transporter (Spiridon et al., 1998). Zn²⁺ inhibition of glutamate transport currents for EAAT1 was similarly abolished at pH 6.0 (Fig. 4). This suggests that the protonation state of the Zn²⁺ binding site on EAAT1 is altered at pH 6.0 compared with pH 7.5, which indicates the role of histidine and also possibly cysteine residues in forming part of the Zn²⁺ binding site (e.g., see Wang et al., 1995). To identify amino acid residues that may form part of a Zn²⁺ binding site on glutamate transporters and also explain the differential Zn²⁺ sensitivity of EAAT1 compared with EAAT2, we targeted histidine, cysteine, and negatively charged amino acid residues for mutagenesis using the following criterion: residues located in regions likely to be present within extracellular domains and regions that are distinct from the putative pore region of the transporters (carboxyl-terminal domain; Pines et al., 1995; Vandenberg et al., 1995; Kavanaugh et al., 1996; Mitrovic et al., 1998).

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Zn2+ inhibition of glutamate transport by EAAT1 is pH dependent. The inhibition of glutamate transport by Zn2+ at 60 mV was measured at pH 7.5 () and at pH 6.0 (); see Methods for details. Error bars, mean ± standard error from four cells.

Histidine 146, of EAAT1, is conserved among all the cloned glutamate transporters, except EAAT3, and is located at the extracellular edge of the putative transmembrane domain 3 (Seal et al., 1996; Arriza et al., 1997; Wahle and Stoffel, 1997). Application of glutamate to oocytes expressing the EAAT1-H146A mutant generates dose dependent transport currents of similar magnitude and with an EC₅₀ value similar to that of oocytes expressing wild type EAAT1. However, the EAAT1-H146A mutant shows marked reduction in sensitivity to Zn²⁺, with concentrations up to 1 mM required to cause any significant inhibition of the glutamate transport currents (Figs. 5 and 6; Table 1). Thus, the H146A mutation selectively alters the Zn²⁺ sensitivity without affecting glutamate transport. Therefore, it is likely that in the wild type EAAT1 transporter, Zn²⁺ interacts with this histidine residue. Further evidence in favor of this conclusion comes from analysis of Zn²⁺ inhibition of an additional mutant, in which the histidine residue has been changed to an aspartic acid residue. Aspartic acid residues have also been identified at the Zn²⁺ binding sites of various zinc finger proteins; therefore, it was expected that this mutant might yield more detailed information about the Zn²⁺ binding site. The H146D mutant is significantly less sensitive to Zn²⁺ (IC₅₀ = 199 ± 60 µM) than the wild type (IC₅₀ = 9.9 ± 2.3 µM). These results demonstrate that subtle changes in the side chain of the amino acid residue at position 146 are sufficient to significantly alter the Zn²⁺ sensitivity of EAAT1.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Point mutations of histidine residues at positions 146 and 156 of EAAT1 and glycine 154 of EAAT2 alter Zn2+ inhibition of glutamate transport. Representative traces from cells expressing EAAT1, EAAT1 H146A, EAAT1 H156A, EAAT2, and EAAT2 G154H. Glutamate (100 µM; filled bar) transport currents at 60 mV are presented in the presence and absence of 100 µM Zn2+ (open bar). Note that the current amplitudes for EAAT1 and the EAAT1 mutants are significantly larger than the currents amplitudes for EAAT2 and the EAAT2 mutant.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Glutamate and Zn2+ dose response curves for the mutated glutamate transporters. Glutamate dose response curves (A) for EAAT1 () EAAT1 H146A (), EAAT1 H156A () and (B) EAAT2 () and EAAT2 G154H (). Current amplitudes were normalized to the maximal transport current for each cell. Data represent mean values ± standard error (n = 4-8). Zn2+ dose dependent inhibition of glutamate transport currents (C) for EAAT1 (), EAAT1 H146A (), EAAT1 H156A () and EAAT1 H146D () and (D) EAAT2 () and EAAT2 G154H (). Errors are mean ± standard error from four cells. A summary of EC50 values for glutamate transport and IC50 values for Zn2+ inhibition of glutamate transport is presented in Table 1.

The amino acid residue in EAAT2 at the position corresponding to histidine 146 of EAAT1 is also a histidine; therefore, the presence of this histidine residue alone cannot explain the differential Zn²⁺ sensitivity of EAAT1 compared with EAAT2. An additional EAAT1 mutant, H156A, showed reduced Zn²⁺ sensitivity with an IC₅₀ value of 237 ± 37 µM (Figs. 5 and 6; Table 1), which is approximately 24-fold higher than for the wild type EAAT1. The maximal glutamate transport currents and the EC₅₀ for glutamate of this mutant were not significantly different from that of wild type EAAT1 (Fig. 6; Table 1), which suggests that the mutation has specifically changed the Zn²⁺ binding site without altering the overall structure or expression of the transporter. Alignment of the amino acid sequences of the glutamate transporters shows some distinct differences between subtypes corresponding to this histidine residue (Arriza et al., 1997). The amino acid residue in EAAT2 that corresponds to histidine 156 of EAAT1 is glycine; as such, the presence or absence of a histidine residue at this position may explain the differential Zn²⁺ sensitivity of glutamate transporter subtypes. We tested this hypothesis by introducing a histidine into EAAT2 at position 154 in place of the glycine residue, expecting to cause an increase in Zn²⁺ sensitivity compared with wild type EAAT2. As predicted, Zn²⁺ inhibits glutamate transport of the EAAT2 mutant G154H (Fig. 5), with an IC₅₀ value of 44 ± 7 µM and a maximal inhibition of 59 ± 3% (Fig. 6; Table 1). Further, Zn²⁺ (at 100 µM) decreases the I_max for glutamate transport currents without affecting the EC₅₀ value for glutamate (Table 1), as observed for EAAT1. Thus, it may be concluded that the Zn²⁺ binding site on EAAT1 includes two histidine residues and the differential inhibition of glutamate transporter subtypes may be explained by the presence or absence of a histidine residue corresponding to position 156 of EAAT1. Additional mutations in EAAT1 that did not affect Zn²⁺ sensitivity include E86Q (extracellular loop 1); E184Q, C186A (extracellular loop 2); E303A (extracellular loop 3) (Table 1).

	Discussion

Top Summary Introduction Materials & Methods Results Discussion References

This study has demonstrated that Zn²⁺ is a noncompetitive, partial inhibitor of glutamate transport by EAAT1 and has no effect on glutamate transport by EAAT2. In addition, although Zn²⁺ inhibits transport by EAAT1, it seems to increase the relative chloride conductance of EAAT1. The results observed for EAAT1 are similar in many respects to the effects of Zn²⁺ on the salamander-retina Müller cell glutamate transporter (Spiridon et al., 1998). The major difference is that Zn²⁺ is less potent on EAAT1 than the Müller cell transporter. The cone cell glutamate transporter does show some differences with respect to the effects on the chloride conductance. The chloride conductance activated by glutamate transport in cone cells is inhibited whereas for EAAT1 and the Müller cell transporter the chloride conductance is increased. This discrimination by Zn²⁺ in modulation of the different transporter functions is particularly interesting. An explanation suggested by Spiridon et al. (1998) for the different effects on transport versus chloride conductance activation, is that Zn²⁺ may stabilize a conformation of the transporter that slows the passage of glutamate through the transporter. With glutamate being bound to the transporter for a longer period a more efficient activation of the chloride conductance may be achieved.

Molecular basis for Zn²⁺ modulation of EAAT1. To better understand the molecular basis for the mechanism of action of Zn²⁺ on glutamate transporters, we have begun to characterize the Zn²⁺ binding site on EAAT1. In this study, we demonstrated that two histidine residues of EAAT1 at positions 146 and 156 are likely to form part of the Zn²⁺ binding site. Although the histidine residue at position 146 is conserved between glutamate transporter subtypes (except EAAT3), a histidine residue corresponding to position 156 of EAAT1 is found in only EAAT1 and EAAT4. We postulate that the nature of the amino acid residue at this position is an important determinant of Zn²⁺ sensitivity. Consistent with this suggestion is that a glycine-to-histidine substitution at this position in EAAT2 generates a Zn²⁺ sensitive EAAT2 mutant. EAAT5 has a glutamate residue at the position corresponding to histidine 156 of EAAT1; because glutamate residues have also been identified as forming Zn²⁺ binding sites on other proteins (e.g., Vazeux et al., 1996) it may be expected that Zn²⁺ would also interact with EAAT5. It would be of interest to confirm this prediction, because the glutamate transporter found in cone cells of the salamander retina is inhibited by Zn²⁺ (Spiridon et al., 1998) and is likely to be closely related to EAAT5 (Eliasof et al., 1997). Although this study has identified two amino acid residues required for the formation of the Zn²⁺ binding site on EAAT1 and also the molecular basis for differential sensitivity between transporter subtypes, it is anticipated, based on the structures of other zinc binding sites on other proteins (see Branden and Tooze, 1991) that at least one other amino acid residue is required to bind Zn²⁺.

Physiological implications of the differential modulation of glutamate transporter subtypes by Zn²⁺. The modulation of neurotransmission by zinc ions has been most extensively studied in the CA3 region of the rat hippocampus (e.g., Assaf and Chung, 1984; Frederickson, 1986). Although both GLAST1 (the rat equivalent of EAAT1) and glutamate transporter1 (GLT-1; rat equivalent of EAAT2) are expressed in the CA3 region of hippocampus, GLT-1 is the more abundant transporter (Rothstein et al., 1994; Chaudhry et al., 1995). Thus, the differential modulation of glutamate transport by Zn²⁺ ions in the CA3 region is unlikely to play a major role in shaping normal glutamatergic neurotransmission.