Anion Conductance Behavior of the Glutamate Uptake Carrier in Salamander Retinal Glial Cells

Glutamate uptake into salamander retinal glia activates an anion conductance

Earlier experiments (Brew and Attwell, 1987) found that the glutamate-evoked current in salamander retinal glia is inward and smaller at positive potentials (see Fig. 2B,D; data for 0 mm [NO₃⁻]_o and [ClO₄⁻]_o), as expected from activation of a carrier that transports two Na⁺ ions into the cell with each glu⁻ anion while countertransporting a K⁺ and an OH⁻ ion (Bouvier et al., 1992). If, as has been shown for the cloned mammalian glutamate transporters, activation of the retinal glial carrier also activates an anion conductance, then altering the extracellular chloride concentration should influence the glutamate-evoked current. When external Cl⁻ was lowered from 126.5 to 19 mm, the inward current evoked by 200 μm glutamate was increased, consistent with an increase in Cl⁻ efflux through an anion conductance (Fig. 1A,B). In four cells at −100 mV, lowering [Cl⁻] increased the current by 15 ± 6% SEM. Interestingly, although one might expect the current increase produced by chloride removal to be larger at positive potentials (where the driving force for Cl⁻ influx would be greatest), a larger change was seen at negative voltages (Fig.1B; in 4 cells the current change at 0 mV was 34 ± 13% of that at −100 mV). This might be attributable to more activation of the anion conductance occurring at more negative potentials when the carrier is cycling more often.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

With ClO₄⁻ or NO₃⁻ present outside the cell, glutamate produces an outward current at positive potentials. A, Specimen currents evoked by 200 μm glutamate (Glu) at −50 and 0 mV with Cl⁻ as the main external anion (left; external solution as in Materials and Methods) or with 100 mm Cl⁻ replaced by ClO₄⁻ (right). B,I–V data for the glutamate-evoked current show that, with Cl⁻ outside, the current is inward at all potentials but decreases toward zero at positive potentials, whereas with ClO₄⁻ outside, the current reverses at depolarized potentials. In four cells the mean ± SEM outward current at +20 mV in 100 mm ClO₄⁻ was 60 ± 9 pA.C, Dependence on [ClO₄⁻]_o(log scale) of the reversal potential for currents studied as inB (mean ± SEM; n = 4 cells for 10 mm, 5 for 30 mm, 6 for 100 mm).D, Experiments are as in B but with NO₃⁻ replacing external Cl⁻.E, Dependence on [NO₃⁻]_oof the reversal potential for currents studied as in D(n = 5 cells for 10 mm, 7 for 30 mm, 17 for 100 mm).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Chloride dependence of the glutamate-evoked current in salamander retinal glia. A, Lowering external [Cl⁻] from 126.5 to 19 mm (replaced with gluconate⁻) increased the current evoked by 200 μm glutamate (Glu, black bar) at −60 mV. B, Voltage dependence of the glutamate-evoked current in the presence and absence of external Cl⁻. Data typical of nine cells; pipette and external solution as discussed in Materials and Methods.

Consistent with the effect of removing external Cl⁻, we found that the glutamate-evoked current was smaller in cells clamped with a pipette solution with reduced [Cl⁻]. In four and six cells clamped with a pipette solution containing 116 or 21 mm Cl⁻, respectively, the current evoked by 200 μm glutamate at −100 mV (normalized by cell capacitance) was 2.7 ± 0.2 and 2.0 ± 0.2 pA/pF. In the six cells studied with lowered [Cl⁻]_i, lowering external [Cl⁻] increased the glutamate-evoked inward current, as in Figure 1.

The anion conductance is more permeable to NO₃⁻and ClO₄⁻ than to Cl⁻

Intracellular NO₃⁻, SCN⁻, and ClO₄⁻ (replacing Cl⁻) increase the inward current generated by the uptake carrier when glutamate is applied extracellularly (Bouvier et al., 1992). With ClO₄⁻ or NO₃⁻ present extracellularly but not intracellularly, the glutamate-evoked current was outward at depolarized potentials (Fig. 2A,B,D). An outward current is not expected from external glutamate activating a carrier that transports two Na⁺ ions in with each glu⁻ and transports a K⁺ and an OH⁻ out of the cell, as proposed earlier (Bouvier et al., 1992), but it could be explained by ClO₄⁻ entering the cell through an anion conductance linked to the carrier (Eliasof and Jahr, 1996).

The glutamate-evoked currents recorded with different external [ClO₄⁻] showed I–V relations characteristic of an anion conductance with a relatively high permeability to ClO₄⁻ (Fig. 2B). Best-fitting a straight line to the dependence on log([ClO₄⁻]_o) of the reversal potential of the I–V relations gave an average shift of 55 mV per 10-fold change of [ClO₄⁻]_o (Fig.2C). Similar results were obtained with NO₃⁻ as the anion replacing Cl⁻, except that less outward current was generated at positive potentials (relative to the inward current at negative potentials) with NO₃⁻ present outside (Fig. 2D,E); on average, the reversal potential shifted by 33 mV for a 10-fold change of [NO₃⁻]_o. To test whether the anion conductance is permeable to HCO₃⁻, we changed the external solution to one buffered to pH 7.3 with 5% CO₂/26 mm HCO₃⁻ (using a highly buffered pipette solution to minimize changes of intracellular pH; see Materials and Methods). This made the glutamate-evoked current less inward at negative potentials (by 24 ± 8% SEM at −40 mV in 4 cells), consistent with some HCO₃⁻ entering through the anion conductance, but it did not result in the current becoming net outward at positive potentials (+50 mV) unlike with 30 mmNO₃⁻ or ClO₄⁻.

We interpret these results, similar to Wadiche et al. (1995), in terms of the glutamate-evoked current having two components: a current generated by the glutamate-transporting part of the molecule, which is always inward and decreases at more positive potentials, and a current generated by an anion conductance, which is highly permeable to ClO₄⁻ (giving a reversal potential that depends in an almost Nernstian manner on [ClO₄⁻]), less permeable to NO₃⁻ (producing a less-than-Nernstian dependence of reversal potential on [NO₃⁻]), and even less permeable to Cl⁻ and HCO₃⁻. With Cl⁻ as the main intra- and extracellular anion, the glutamate-evoked current at positive potentials is dominated by the transporter part of the molecule (as judged by the lack of a net outward current seen at positive potentials). With external ClO₄⁻ or NO₃⁻ present, an outward current through the anion conductance is seen at positive potentials. Because glutamate transport is greatly reduced at positive potentials, this suggests that, for the anion conductance to be activated, it may not be necessary for net glutamate transport to occur. Experiments described below will confirm this and show that ion movements through the anion conductance are not coupled to the flux of glutamate.

Activation of the anion conductance by external glutamate is dependent on external Na⁺ and internal K⁺

Replacing external sodium with choline abolished the outward current evoked by glutamate with NO₃⁻ or ClO₄⁻ present outside the cell (Fig.3A,B) (see also Eliasof and Jahr, 1996) as well as the inward current at negative potentials that may (with Cl⁻ as the main intracellular anion) be generated primarily by glutamate transport. Thus, activation of the anion conductance by external glutamate, like activation of the uptake process, requires external sodium.

Removing intracellular potassium greatly reduces the inward glutamate-evoked current at negative potentials (Barbour et al., 1988,1991) and abolishes glutamate uptake (Kanner and Sharon, 1978). It also reduces the outward current seen at positive potentials with NO₃⁻ present outside the cell (Fig. 3C,D). With K⁺ in the whole-cell pipette, at +40 mV no glutamate-evoked current is produced with Cl⁻ as the external anion, because transport of glutamate with a net positive charge into the cell is inhibited. With 50 mmNO₃⁻ outside the cell, however, an outward current of ∼0.43 pA/pF of cell capacitance is seen because of NO₃⁻ influx through the anion conductance (Fig.3C). When the same experiment was done without K⁺ in the pipette (Fig. 3D), no glutamate-evoked current was seen at any potential with Cl⁻ as the external anion (because glutamate uptake is absolutely dependent on the countertransport of K⁺), and the outward current produced at +40 mV with NO₃⁻ present was reduced by 86 ± 3% (5 cells studied with and 5 without K⁺ in the pipette). Thus, activation of the anion conductance shows a similar ionic dependence to that for activation of glutamate transport.

The anion conductance can be activated by intracellular glutamate

With sodium and glutamate present inside the cell, raising the external potassium concentration evokes an outward membrane current, which is attributed to reversed operation of the uptake carrier, transporting glutamate and net positive charge out of the cell (Szatkowski et al., 1990). Experiments described below (Fig. 6; Billups and Attwell, 1996) confirm that glutamate is released from the cell by reversed uptake in this situation.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 6.

Investigation of coupling of anion movements to glutamate transport. A, B, Test of whether increasing current flow through the anion conductance increases glutamate transport. A, Currents evoked in a Purkinje cell (clamped to −60 mV) by glutamate released from an adjacent Müller cell by reversed uptake in external solution containing Cl⁻ (left) or NO₃⁻(right) as the main anion. Reversed uptake was evoked by bathing the cells in 30 mm K⁺ solution (as in Fig. 3C with KCl replacing choline-Cl) and stepping the Müller cell voltage from −60 to +20 mV (top trace). The decrease in the Purkinje cell current response in NO₃⁻ is produced by a decrease in glutamate sensitivity of the non-NMDA receptors of the Purkinje cell and not by a decrease of glutamate release from the Müller cell (see text). Purkinje cell pipette solution as described in Materials and Methods; Müller cell pipette solution for reversed uptake as described in Materials and Methods. B, Same experiment as inA but testing the effect of external ClO₄⁻. The glutamate sensitivity of the Purkinje cell is more than doubled by ClO₄⁻ (see text), so the small decrease of response seen here implies a large decrease of glutamate release from the Müller cell. C, D, Test of whether H⁺/OH⁻ movements on the uptake carrier are through the anion conductance and are passive or are coupled to glutamate transport. C, Membrane currents evoked at −60 and 0 mV by 200 μm glutamate in a Müller cell clamped with standard internal solution, pH 7.0, but containing only 0.5 mm HEPES and also 100 μm BCECF. Standard external solution was used but with its pH adjusted to 7.7. D, Glutamate-evoked changes in intracellular pH, measured at the same time as the current records in C, are acid both below and above the reversal potential for H⁺/OH⁻, implying coupling of the movement of H⁺/OH⁻ to glutamate transport.

When the external Cl⁻ was replaced by NO₃⁻ or ClO₄⁻, the outward current evoked by a rise of [K⁺]_o was increased (Fig.4A,C), the mean increase being by a factor of 1.58 ± 0.06 (SEM; 6 cells) for NO₃⁻ and by 3.34 ± 0.41 (5 cells) for ClO₄⁻. Experiments described below (Fig. 6) show that there is no increase in the glutamate release by reversed uptake when Cl⁻ is replaced in this way. Furthermore, when sodium and glutamate were omitted from the pipette (replaced with choline-Cl), raising [K⁺]_o evoked no current in cells superfused with NO₃⁻ solution (mean current/capacitance was 0.49 ± 0.08 pA/pF in 5 cells with Na-glu inside and −0.05 ± 0.03 pA/pF in 5 cells with Na-glu omitted), as found by Szatkowski et al. (1990) with Cl⁻ outside, indicating that the extra K⁺-evoked outward current seen during Cl⁻ substitution is generated by the glutamate transporter. We therefore attribute the extra outward current to an influx of NO₃⁻ or ClO₄⁻ through the anion conductance of the carrier, with the anion conductance being activated when reversed operation of the carrier is evoked by the simultaneous presence of intracellular glu⁻ and Na⁺ and extracellular K⁺. When activated by reversed uptake, the selectivity sequence of the anion conductance for different anions is ClO₄⁻ > NO₃⁻ > Cl⁻, as was found above for activation of the conductance by transport of glutamate into the cell.

Conversely, when the [K⁺]_o was raised around cells containing 50 mm intracellular ClO₄⁻(replacing Cl⁻), instead of an outward current being evoked, an inward current shift occurred (Fig.4B,C), presumably because activation of the anion conductance leads to an efflux of ClO₄⁻, generating an inward current that is larger than the outward transport current produced by glutamate efflux.

These data show that the anion conductance can be activated by extracellular or intracellular glutamate, provided that the transport part of the molecule is allowed to cycle by provision of Na⁺ and K⁺ on appropriate sides of the membrane. A kinetic scheme consistent with these observations is presented in Discussion (Fig. 7). The following section provides evidence that the anion conductance can also be activated when the transport activity of the carrier molecule is greatly reduced.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 7.

A possible kinetic scheme for the glutamate transporter and associated anion conductance. C denotes the carrier in conformations for which the anion conductance is not activated (closed). In these conformations the carrier can bind extracellular glu⁻ and two Na⁺ ions, transport them to the inner face of the membrane, then bind K⁺ and OH⁻ at the inner membrane surface and transport them to the outside of the cell, shifting one net positive charge into the cell during the carrier cycle. (Note that the order in which K⁺and OH⁻ bind is unknown. Furthermore, although we show OH⁻ countertransport out of the cell, as discussed in the text the carrier might get energy from the transmembrane pH gradient by cotransporting H⁺ with glu⁻). Odenotes a conformation of the transporter in which the anion conductance is open. Here we postulate simply that the open state can be accessed from the closed conformation that has Na⁺ and glu⁻ bound at the inner face of the membrane. Our data would also be consistent with an O state being accessed from the closed conformation with Na⁺ and glu⁻bound at the outer membrane surface (or from both of theC.Na₂.Gluconformations).

Anion conductance activation in the absence of net glutamate transport

In the absence of intra- and extracellular K⁺, forward and reversed transport of glutamate are inhibited (Kanner and Sharon, 1978; Barbour et al., 1988; Szatkowski et al., 1990; Billups and Attwell, 1996) (homoexchange of glutamate can still occur: Kanner and Bendahan, 1982). Figure 3D shows that external glutamate does not evoke a detectable current when Cl⁻ is the main intra- and extracellular anion present. However we found that, with net glutamate transport abolished in this way, if glutamate and sodium were present inside the cell (via the whole-cell pipette), addition of extracellular glutamate did evoke a current. This current was inward at negative and outward at positive potentials and showed the pharmacology of the uptake carrier (Fig. 5A). Activation of this current was dependent on the presence of both glutamate and sodium inside the cell (Fig. 5B). Relative values of glutamate-evoked current (normalized to cell capacitance) at +20 mV with 10 mm Na-glu, 10 mm glu⁻ but no Na⁺, 10 mm Na⁺ but no glu⁻, or no Na⁺ and no glu⁻ in the pipette, were, respectively, 1.0, 0.014 ± 0.024, −0.041 ± 0.031, and 0.0 ± 0.0 (6 or 7 cells for each pipette solution).

Changing the external chloride concentration revealed that the current was produced by activation of an anion conductance (Fig.5C,D), although the absolute value of the reversal potential for the current and its less-than-Nernstian dependence on [Cl⁻]_o indicated that this conductance was not very specific for Cl⁻. Interestingly, Vandenberg and colleagues (1995) have shown the presence of a Cl⁻-dependent cation leak through a cloned human glutamate transporter, and, if present in the salamander transporter, this might explain the lack of a Nernstian dependence on [Cl⁻]_o in Figure 5D. With ClO₄⁻ instead of Cl⁻ as the major anion present inside and outside the cell (Fig. 5E), the glutamate-evoked conductance was greatly increased (in 4 cells the currents evoked at −80 and +40 mV were increased by factors of 30.1 ± 6.5 and 9.7 ± 1.8, respectively: the fact that the reversal potential was near −10 mV instead of 0 mV with 50 mm ClO₄⁻ in the pipette and outside the cell may reflect incomplete dialysis of the cell with ClO₄⁻). A similar, but smaller increase in the outward current at positive potentials was seen with NO₃⁻outside the cell (data not shown). The increase in current when Cl⁻ was replaced by NO₃⁻ or ClO₄⁻ is consistent with the selectivity sequence described above for the anion conductance evoked during forward or reversed transport of glutamate.

From these data, it seems that the anion conductance can be activated even when the carrier molecule is not producing net transport of glutamate across the membrane. We took advantage of this to investigate the external glutamate and sodium dependence of anion conductance activation without contamination from current generated by glutamate transport. With 100 mm NO₃⁻ outside the cell, the anion conductance (assessed as the outward current at +20 mV) showed a Michaelis–Menten dependence on external glutamate concentration, with a K_m (mean value 15.1 ± 0.3 μm in 5 cells) similar to that found for the glutamate transport current (Barbour et al., 1991; Eliasof and Jahr, 1996). Varying the external sodium concentration (replaced with choline; 5 cells) revealed a sigmoid dependence on [Na⁺]_o at low [Na⁺]_o (rising as [Na⁺]_o^2.5 for [Na⁺]_o ≤ 15 mm at −40 mV), similar to that for the current associated with glutamate transport (Barbour et al., 1991). These data are consistent with one glutamate anion and two Na⁺ ions having to bind to activate the anion conductance—a result that is incorporated into a kinetic scheme proposed in Discussion.

With Na⁺ and glu⁻ inside the cell, the reversed uptake current produced by raising [K⁺]_o is greatly reduced when the extracellular pH is made acid (Billups and Attwell, 1996), presumably because with an acid pH_o there is not enough OH⁻ present for countertransport into the cell (or because the carrier cannot lose H⁺ cotransported out of the cell). An acid extracellular pH had no effect, however, on the anion conductance activated by adding external glutamate with glu⁻ and Na⁺ inside the cell (and no K⁺ inside or outside; Fig. 5F), again suggesting that it is possible to dissociate the anion conductance function of the molecule from its glutamate-transporting activity. A kinetic model consistent with this observation is presented in Discussion.

Lack of coupling of anion movements to glutamate transport

To determine whether alteration of the ion flux through the anion conductance part of the transporter molecule has any effect on the rate of glutamate transport, we evoked reversed uptake in salamander retinal glial cells while monitoring glutamate release with non-NMDA receptor channels in isolated rat Purkinje cells placed just outside the glial cells (Billups and Attwell, 1996).

Glutamate release by reversed uptake was evoked with 10 mmNa-glu in the glial cell and 30 mm K⁺ in the extracellular solution by depolarizing the cell from −60 mV (at which potential glutamate release by reversed uptake is small; Szatkowski et al., 1990; Billups and Attwell, 1996) to +20 mV. This procedure produced a current in the adjacent Purkinje cell consistent with glutamate release by reversed uptake activating non-NMDA channels (Billups and Attwell, 1996). Replacing external Cl⁻ with NO₃⁻ or ClO₄⁻ increased the outward current shift evoked in the glial cell by a rise of [K⁺]_o at 0 mV (Fig. 4A), because NO₃⁻ and ClO₄⁻ can enter the cell through the anion conductance better than Cl⁻ can but had little effect on the change of membrane current evoked in the Purkinje cell by glutamate release from the glial cell (Fig.6A,B). On average, the responses with 50 mm external NO₃⁻ or ClO₄⁻ present were 0.69 ± 0.04 (3 cells) and 0.98 ± 0.10 (6 cells) of those in external Cl⁻. Control experiments, in which the sensitivity of three Purkinje cells to 3 μm glutamate was tested, showed that NO₃⁻ reduced the current evoked in the Purkinje cell to 0.76 ± 0.18 of its value in Cl⁻, whereas ClO₄⁻ increased it to 2.33 ± 0.29 (data not shown). The increased current in ClO₄⁻ is consistent with the fact that chaotropic ions like ClO₄⁻ increase the affinity of AMPA receptors (Honore and Drejer, 1988). Combining these alterations of glutamate sensitivity with the data in Figure6, A and B, suggests that in the presence of NO₃⁻ (which increases the glial cell current evoked by a rise of [K⁺]_o by a factor of 1.58; Fig. 4) glutamate release is essentially unaffected (0.91 ± 0.22 of its value in Cl⁻), whereas in ClO₄⁻ (which increases the K⁺-evoked current in the glial cell 3.34-fold) glutamate release is actually reduced to 0.42 ± 0.06 of its value in Cl⁻. Clearly, the amount of glutamate transported is not proportional to the movement of charge through the anion conductance part of the carrier molecule, as was suggested also by the experiments above in which anion conductance activation was still seen in the absence of K⁺ and at positive potentials when transport is inhibited.

Coupling of movement of pH-changing ions to glutamate transport

Changes of pH produced by glutamate uptake carriers have been attributed previously to a cotransport of H⁺ ions with glutamate or to a countertransport of OH⁻ ions (Erecinska et al., 1983; Bouvier et al., 1992)—interpretations that imply that glutamate accumulation is powered partly by the transmembrane pH gradient. However, if glutamate transporters contain an anion conductance, an obvious possibility is that the pH changes are generated by passive movement of OH⁻ ions through the anion conductance, not coupled to the transport of glutamate. To investigate this possibility, we measured changes of intracellular pH evoked by external glutamate in Müller cells clamped to different potentials. For this experiment the intra- and extracellular solutions had pH values of 7.0 and 7.7, respectively, giving a reversal potential for OH⁻ of −41 mV. Thus, at potentials more positive than −41 mV, if OH⁻ were moving passively through the glutamate-evoked anion conductance, it would move into the cell, making the cell more alkaline, whereas if movement of OH⁻/H⁺ were coupled thermodynamically to glutamate entry, then glutamate should make the cell go acid at all potentials. Experimentally, the latter was found to be the case (Fig.6C,D). Indeed, at 0 mV the ratio of the rate of acidification to the glutamate-evoked current was similar to that at −60 mV (0.27 ± 0.04 and 0.23 ± 0.05 pH units/sec per nA at 0 mV and −60 mV, respectively, in 6 cells: the slightly, although not significantly, smaller value at −60 mV might be expected because the inward glutamate-evoked current, but not the pH change, is increased by chloride efflux through the anion conductance). Thus, the movement of pH-changing ions is coupled to glutamate transport, rather than occurring through the anion conductance.

The salamander glial cell glutamate transporter has an anion conductance

Data presented here show that the glutamate transporter in salamander retinal glial cells activates an anion conductance (see alsoEliasof and Jahr, 1996). Removing external chloride increases, and lowering internal chloride decreases, the glutamate-evoked inward current, consistent with the results of Wadiche et al. (1995) on cloned mammalian transporters. With Cl⁻ as the main anion inside and outside the cell, the glutamate-evoked current remains inward at positive potentials (Fig. 1), presumably because it is dominated by the current associated with glutamate transport rather than that generated by the anion conductance. The contribution of the anion conductance can be greatly enhanced by replacing Cl⁻ with more permeant anions, resulting in the glutamate-evoked current becoming outward at positive potentials (Fig. 2). Inspection of the data in Figures 2,C and E, and 4 of this paper and in Bouvier et al. (1992), suggests a selectivity sequence SCN⁻ > ClO₄⁻ > NO₃⁻ > Cl⁻ ≃ Br⁻ ≃ I⁻ for the anion conductance. This is similar to the theoretical sequence 1 of Wright and Diamond (1977) but differs in that, for sequence 1, ClO₄⁻ > SCN⁻ and I⁻ > Br⁻ > Cl⁻. The apparent position of ClO₄⁻ in our selectivity sequence could, however, be altered by the fact that ClO₄⁻, in addition to permeating the anion conductance, seems to slow carrier cycling (see Lack of Coupling of Anion Movements to Glutamate Transport) and so may reduce opening of the anion conductance. Conceivably, the selectivity sequence of the anion conductance would be identical to sequence 1 of Wright and Diamond (1977) if currents through the open anion conductance could be investigated independently of changes in conductance activation. In earlier work, Barbour and colleagues (1991) observed a small (12%, but statistically insignificant) decrease of glutamate-evoked current on removing internal chloride, as we report here, but did not see the effect of removing external chloride shown in Figure 1. This may be attributable to the use of a nonsaturating glutamate dose or to the presence of acetate in the internal solution in the experiments of Barbour et al. (1991); we are performing experiments to examine these possibilities .

Different modes of gating of the anion conductance

The glutamate carrier anion conductance can be activated by the simultaneous presence of extracellular glutamate and sodium and intracellular potassium when the carrier operates in forward uptake mode (Figs. 1, 2, 3). However, it is also activated by the simultaneous presence of intracellular glutamate and sodium and extracellular potassium when the carrier transports glutamate out of the cell (Fig.4). These data suggest that activation of the anion conductance may occur when a particular state of the carrier cycle is reached, independent of whether that state is reached by the carrier cycling in the forward or the reversed direction. A possible example of such a scheme is shown in Figure 7.

If the anion conductance only opens once each carrier cycle (during forward uptake), its open probability could be larger at negative potentials when the carrier cycles more rapidly. This might explain why the change in glutamate-evoked current produced by removing external Cl⁻ is larger at more negative potentials (Fig. 1) rather than at positive potentials when the driving force for Cl⁻entry is greatest; similarly, it would explain why the outward shift of glutamate-evoked current produced by external ClO₄⁻ or NO₃⁻ is only slightly larger at positive potentials (Fig. 2).

One constraint on which state of the carrier cycle allows activation of anion conductance is provided by the observation that the anion conductance can be activated (Fig. 5) when net glutamate transport is inhibited by the absence of intra- and extracellular K⁺. This implies that the anion conductance is activated by a state of the carrier cycle at which the carrier does not have K⁺ bound. If, as suggested by Kanner and Bendahan (1982) and as shown in Figure7, the K⁺ translocating part of the carrier cycle is distinct from the Na⁺ and glu⁻ translocating part of the cycle, then activation of the anion conductance must occur from one of the states of the glu⁻/Na⁺transporting limb of the carrier cycle. The observation (see Anion Conductance Activation in the Absence of Net Glutamate Transport) that anion conductance activation shows a first-order dependence on external glutamate concentration and a sigmoid dependence on [Na⁺]_o implies it occurs from a state with one glu⁻ and two Na⁺ ions bound (Fig. 7). When intra- and extracellular K⁺ are absent, the anion conductance is activated by external glutamate only if glutamate and sodium are present intracellularly (Fig. 5B); this could be explained by binding of the internal glutamate and sodium resulting in the carrier spending more time in the state with glu⁻ and two Na⁺ ions bound that is suggested in Figure 7 to lead to anion conductance activation. (Similarly, external glutamate is needed to prevent the carrier accumulating in the state at the external surface with Na⁺ bound but with no glutamate bound.) Figure7 proposes that the OH⁻/H⁺ transporting part of the carrier cycle is associated with the K⁺transporting limb of the cycle rather than with the glu⁻/Na⁺ transporting limb. This would explain the fact that an acid pH (equivalent to lack of transported substrate OH⁻ in Fig. 7) does not affect the activation of the anion conductance seen in the absence of intra- and extracellular K⁺ (Fig. 5F), although it does block reversed uptake of glutamate (Billups and Attwell, 1996).

The data in Figure 6, A and B, suggest that there is no energetic coupling between the anion flux through the conformation of the carrier denoted O in Figure 7 and the cycle of reactions that transport glutamate.

Reinterpretation of the effects of intracellular ClO₄⁻ and NO₃⁻

Bouvier and colleagues (1992) found that intracellular ClO₄⁻ and NO₃⁻ increased the inward current evoked by external glutamate, that ClO₄⁻ came out of the cell when glutamate was applied, and that the presence of these ions intracellularly reduced the ratio of the pH change generated by the carrier to the current that it generated. Those results were interpreted as showing that the glutamate-evoked pH changes were generated by the transport of OH⁻ ions out of the cell and that ClO₄⁻ and NO₃⁻ could compete for transport at the OH⁻ site. It is now clear that the effects of ClO₄⁻ and NO₃⁻ were produced by these ions leaving the cell (at a much higher rate than Cl⁻) through the anion conductance associated with the uptake carrier, generating an extra inward current. This invalidates the earlier conclusion that the pH changes generated by the carrier are produced by the transport of OH⁻ out of the cell rather than the (thermodynamically equivalent) transport of H⁺into the cell: our data reopen the possibility that H⁺ is cotransported with glutamate.

Figure 6D shows that, irrespective of whether OH⁻ or H⁺ is transported, movement of the pH-changing ion is coupled to glutamate transport. Thus, glutamate uptake does derive energy from the transmembrane pH gradient, consistent with the observation that, in the kidney, a pH gradient alone can drive uptake (Nelson et al., 1983).

Therapeutic possibilities offered by the existence of the anion conductance

Our demonstration that the anion conductance part of the transporter molecule can be activated even when glutamate transport is inhibited (at positive potentials in Fig. 2 and in the absence of K⁺ in Fig. 5) suggests some independence between these two functions of the molecule and, hence, that they may be capable of being modulated separately by pharmacological agents. This suggests a possible strategy for developing drugs to treat conditions in which excessive glutamate is released, such as epilepsy. If, for glutamate transporters in presynaptic terminals, the anion conductance activation could be greatly enhanced, then whenever glutamate was released, activation of the anion conductance during glutamate re-uptake would tend to clamp the presynaptic terminal at a negative potential, reducing further exocytotic release (by making it harder for action potentials to invade the synaptic terminal) and potentiating the (voltage-dependent) re-uptake. Interestingly, in the retina at least, the glutamate transporter in cone synaptic terminals expresses a particularly large anion conductance (Sarantis et al., 1988, Eliasof and Werblin, 1993), like the human EAAT4 carrier (Fairman et al., 1995), suggesting that evolution already might have arrived at this strategy for controlling glutamate release. Recently Rothstein and colleagues (1996) have shown that preventing the expression of neuronal EAAC-1 carriers leads to epileptic behavior of neurons (whereas preventing expression of glial uptake carriers leads to a rise of extracellular glutamate concentration but no epilepsy). It is not yet known whether the antiepileptic properties of EAAC-1 transporters derive solely from their ability to take up glutamate or whether their contribution to the anion conductance of neurons is also involved.

During ischemia the glutamate concentration in glial cells rises (Storm-Mathisen et al., 1992). Because activation of the anion conductance in glial uptake carriers can be potentiated by intracellular glutamate (Fig. 5B), it is possible that the uptake carrier might contribute to the glial cell chloride conductance, which, by allowing Cl⁻ influx, could facilitate glial cell swelling in ischemia (Walz et al., 1993).