Regulation of glutamate transport into synaptic vesicles by chloride and proton gradient.

Glutamate uptake into synaptic vesicles is driven by an electrochemical proton gradient formed across the membrane by a vacuolar H-ATPase. Chloride has a biphasic effect on glutamate transport, which it activates at low concentrations (2-8 mM) and inhibits at high concentrations (>20 mM). Stimulation with 4 mM chloride was due to an increase in the V of transport, whereas inhibition by high chloride concentrations was related to an increase in K to glutamate. Both stimulation and inhibition by Cl were observed in the presence of A23187 or (NH)SO, two substances that dissipate the proton gradient (ΔpH). With the use of these agents, we show that the transmembrane potential regulates the apparent affinity for glutamate, whereas the ΔpH antagonizes the effect of high chloride concentrations and is important for retaining glutamate inside the vesicles. Selective dissipation of ΔpH in the presence of chloride led to a significant glutamate efflux from the vesicles and promoted a decrease in the velocity of glutamate uptake. The H-ATPase activity was stimulated when the ΔpH component was dissipated. Glutamate efflux induced by chloride was saturable, and half-maximal effect was attained in the presence of 30 mM Cl. The results indicate that: (i) both transmembrane potential and ΔpH modulate the glutamate uptake at different levels and (ii) chloride affects glutamate transport by two different mechanisms. One is related to a change of the proportions between the transmembrane potential and the ΔpH components of the electrochemical proton gradient, and the other involves a direct interaction of the anion with the glutamate transporter.

Glutamate is the major excitatory neurotransmitter found in the mammalian central nervous system and is released into the synaptic cleft by synaptic vesicles exocytosis (Jahn and Sudhof, 1994). Re-uptake of the released glutamate is mediated by two transport systems. One is a high affinity, Na ϩ -dependent carrier located in the plasma membrane, and the other is a low affinity, Na ϩ -independent transport system located in the synaptic vesicles (Kanner, 1983;Maycox et al., 1990). Glutamate uptake into synaptic vesicles is driven by a ⌬Hϩ 1 formed across the vesicle membrane by a bafilomycin A 1 -sensitive vacuolar H ϩ -ATPase (Disbrow et al., 1982;Ueda, 1983, 1985;Maycox et al., 1988;Cidon and Sihra, 1989;Floor et al., 1990). As H ϩ is pumped into the vesicle lumen, a ⌬pH, acidic inside, and a ⌬⌿, positive inside, are built across the membrane. The relative proportions of ⌬pH and ⌬⌿ vary greatly. In the absence of a permeating anion, the proton charge is not counterbalanced, and thus ⌬⌿ predominates over ⌬pH. When the concentration of the physiological permeating anion chloride is increased there is a progressive fall of the ⌬⌿, and a significant ⌬pH is formed across the membrane (Van Dyke, 1988). There is no consensus in the literature on whether glutamate uptake into synaptic vesicles is driven solely by ⌬⌿ Cidon and Sihra, 1989;Hartinger and Jahn, 1993;Moriyama and Yamamoto, 1995) or by both the ⌬⌿ and ⌬pH components of the ⌬Hϩ (Naito and Ueda, 1985;Shioi and Ueda, 1990;Tabb et al., 1992). Low concentrations (2-8 mM) of chloride stimulate glutamate uptake, and high Cl Ϫ concentrations (Ͼ20 mM) inhibit it. Tabb et al. (1992) proposed that low chloride stimulates glutamate uptake because it increases the vesicle ⌬pH, and the inhibition by high Cl Ϫ would be related to the dissipation of ⌬⌿ . Recently, Hartinger and Jahn (1993) found that high concentrations of chloride prevented inhibition of glutamate transport promoted by DIDS, an anion transporter blocker. These authors proposed that the vesicular glutamate transporter possesses a DIDS-sensitive chloride binding site. In the present study we examined the effects of chloride on steady state glutamate uptake. Different ⌬pH dissipating agents were used to alter the balance between the ⌬pH and ⌬⌿ contribution to the ⌬Hϩ formed across the synaptic vesicles membrane. It was found that ⌬⌿ controls the apparent affinity for glutamate, whereas ⌬pH is important for antagonizing the effect of high chloride concentrations.

EXPERIMENTAL PROCEDURES
Vesicle Preparation-Synaptic vesicles were isolated from rat brains as described in detail by Hell et al. (1988). The vesicles were used omitting the chromatography on controlled pore glass beads, as described by Hell et al. (1990). The glutamate uptake and purity of this preparation have been previously determined Hartinger and Jahn, 1993). The vesicles were stored under liquid nitrogen until use. Protein concentration was determined by the method of Lowry et al. (1951). All the experiments were performed at least three times with different vesicle preparations.
Neurotransmitter Uptake-The vesicles were incubated with L-[ 3 H]glutamate at 35°C in different media as described in the figure legends. The Cl Ϫ concentration was varied using different proportions of potassium gluconate and KCl and maintaining K ϩ concentration fixed at 140 mM. Glutamate uptake was started by the addition of 4 mM MgATP, and the reaction was stopped by filtration of the assay medium through 0.45-m Millipore filters (100 g of protein/filter). The filters were quickly flushed with 20 ml of Mops-Tris (pH 7.0) solution. Glutamate uptake was corrected for the nonspecific binding measured in the absence of ATP. Radioactivity was counted in a liquid scintillation counter.
Electrochemical Proton Gradient-Fluorescence measurements were made at 25°C in a F-3010 Hitachi fluorescence spectrophotometer using ⌬pH and potential-sensitive dyes as described previously Cidon and Sihra, 1989). The ⌬pH was determined by measuring the fluorescence quenching of acridine orange (2 M) at 492 (excitation) and 537 (emission) nm. The ⌬⌿ was determined by measuring the fluorescence quenching of oxonol V (1.5 M) at 617 (excitation) and 643 (emission) nm. The reaction was started by the addition of 2 mM ATP in a 2-ml stirred cuvette containing 0.1 mg of synaptic vesicle protein/ml, 10 mM Mops-Tris (pH 7.0), and different amounts of KCl and potassium gluconate. The reaction was stopped after 5-10 min by the addition of the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone to a final concentration of 10 M.
ATPase Activity-Bafilomycin A 1 -sensitive ATPase activity was determined by measuring the rates of P i production in the absence and in the presence of 10 nM bafilomycin A 1 . P i released was determined colorimetrically (Fiske and Subbarow, 1925).
Materials-L-[ 3 H]glutamic acid was purchased from Amersham Corp. L-Glutamate was obtained from Merck. A23187, bafilomycin A 1 , acridine orange, and MgATP were obtained from Sigma. Oxonol V was purchased from Molecular Probes. Other reagents were of analytical grade.

RESULTS
The Dual Effect of Cl Ϫ on the Kinetics of Glutamate Transport-Previous reports have shown that the transport of glutamate is stimulated by 2-8 mM Cl Ϫ . This effect is not observed with gluconate, a nonpermeating anion (Naito and Ueda, 1985;Maycox et al., 1988;Cidon and Sihra, 1989). The present study shows that the effect of Cl Ϫ varies depending on the glutamate concentration in the medium and on whether the initial velocity or the steady state level of glutamate uptake is measured (Fig. 1). The steady state is achieved when the rate of glutamate efflux equals the rate of glutamate influx. The initial rate of glutamate uptake was measured after 1 min of incubation, and the steady state level of glutamate uptake was measured after 10 -20 min of incubation (Fig. 1). Cl Ϫ (4 mM) stimulated the initial rate of uptake regardless of the glutamate concentration used (Fig. 1). Measurement of the initial rate of uptake in the presence of different glutamate concentrations indicated that the activation promoted by Cl Ϫ is related to an increase in the V max of glutamate transport ( Fig. 2A and Table I). The activation was abolished, and inhibition of glutamate uptake was observed when the Cl Ϫ concentration was raised from 4 to 80 mM ( Figs. 1 and 2). The effect of high Cl Ϫ concentrations seems to be related to an increase of the apparent K m of the transport system for glutamate; the inhibition by Cl Ϫ was more pronounced in the presence of low glutamate concentrations and decreased progressively as the concentration of glutamate was raised to 4 mM (Figs. 1B and 2A and Table I). Therefore, although Cl Ϫ modified the initial rate of glutamate uptake in the presence of high glutamate concentrations ( Fig. 2A), it did not alter the maximal filling capacity of the vesicles observed after the steady state was reached (Figs. 1B and 2B). The values shown in Table I for the K m and V max of glutamate uptake in the absence of A23187 are in agreement with those previously reported by Naito and Ueda (1985). Different mechanisms have been proposed in the literature for the activation of glutamate transport promoted by low Cl Ϫ . Some authors have suggested that the effect is essentially due to an increase of ⌬pH promoted by Cl Ϫ (Naito and Ueda, 1985;Shioi and Ueda, 1990;Tabb et al., 1992). Other research groups favor a direct effect of the anion on the glutamate carrier, a mechanism that does not involve changes of the proton gradient (Maycox

TABLE I
Modulation of V max and K m of glutamate transport by chloride and ⌬Hϩ components The initial rate of glutamate uptake was measured either in the absence or in the presence of 10 M A23187. Other conditions were as described in the legend to Fig. 1A. V max and K m were determined by double-reciprocal plots. The effect of A23187 at each Cl Ϫ concentration was analyzed by a paired t test, and the results are shown in parentheses under each pair of values. The effects of Cl Ϫ within each column were compared by one-way ANOVA, followed by Duncan's multiple range test. The values are means Ϯ S.E. of 4 -8 experiments with four different preparations. N.S., not a significant difference.
a Value that is statistically different from that obtained without Cl Ϫ in the same column at p Ͻ 0.01. b Value that is statistically different from that obtained with 4 mM Cl Ϫ in the same column at p Ͻ 0.01. c Value that is statistically different from that obtained without Cl Ϫ in the same column at p Ͻ 0. 05. et al., 1988;Hartinger and Jahn, 1993;Moriyama and Yamamoto, 1995). In the following experiments, different combinations of ⌬pH dissipating agents and Cl Ϫ and glutamate concentrations were used. The aim was to estimate the relative contributions of ⌬⌿ and ⌬pH to the kinetics of glutamate transport.
Alteration of ⌬Hϩ Components by (NH 4 ) 2 SO 4 and A23187-The electrochemical proton gradient formed across the vesicle membrane varies with the permeating anion concentration used (Van Dyke, 1988). Without added Cl Ϫ , a large ⌬⌿ and a small ⌬pH are formed across the membrane. Increasing Cl Ϫ concentration promotes a progressive decrease of the ⌬⌿, and this is associated with an increase of ⌬pH (Figs. 3 and 4). The effect of chloride can be modified with the use of ammonium sulfate and A23187 (Figs. 3 and 4). These two substances dissipate ⌬pH and increase the ⌬⌿ (Johnson and Scarpa, 1976). Note that these effects were observed over a wide range of Cl Ϫ concentrations (Fig. 4). During H ϩ pumping, the uncharged species NH 3 derived from (NH 4 ) 2 SO 4 diffuse into the lumen of the vesicles and associate with H ϩ forming NH 4 ϩ . A23187 is a dicarboxylic ionophore capable of transporting 2H ϩ in exchange for either one Mg 2ϩ or one Ca 2ϩ ion (Johnson and Scarpa, 1976;Pressman, 1976;Romani and Scarpa, 1992). Under the conditions of the present study, the collapse of the ⌬pH by A23187 is promoted by the exchange between H ϩ and Mg 2ϩ . Removal of contaminating Ca 2ϩ from the medium with EGTA did not modify the effect of A23187 on the ⌬Hϩ (data not shown). In the subsequent experiments, we show that the effect of A23187 and (NH 4 ) 2 SO 4 on the steady state level of glutamate uptake varies with the Cl Ϫ concentrations used.
The Effect of A23187 and (NH 4 ) 2 SO 4 in the Presence of Low Cl Ϫ Concentrations-Both A23187 (Fig. 5) and (NH 4 ) 2 SO 4 ( Fig.  6) stimulated 2-fold the uptake when the latter was measured with glutamate concentrations far below the K m value and either in the absence or in the presence of 4 mM Cl Ϫ (Figs. 5, A and B, and 6A). Under these conditions, these agents not only increased the rate of uptake but also enhanced the amount of glutamate stored by the vesicles after the steady state was reached. This could be best seen if A23187 was added to the medium after the steady state uptake was reached (Fig. 7A). The enhancement of the filling capacity of the vesicles was only observed with the use of low glutamate concentrations. A23187 had no effect on steady state glutamate uptake when saturating glutamate concentrations were used (Fig. 7B). The different effects observed with the use of low and high glutamate concentrations can be ascribed to the 2-fold decrease of the K m for glutamate promoted by A23187 (Table I). The experiments illustrated in Figs. 3 and 4 show that A23187 dissipates the preexisting ⌬pH and increases the ⌬⌿ component of the gradient. This indicates that ⌬⌿ alone can drive glutamate uptake and that the apparent affinity of the vesicles for glutamate is determined by the magnitude of ⌬⌿.
The Combined Effects of High Cl Ϫ and ⌬pH on Glutamate Efflux-High Cl Ϫ concentrations seem to activate the efflux of glutamate by interacting directly with the glutamate transporter of the membrane, but this is only observed after the ⌬pH is collapsed with A23187. Thus, the ⌬pH seems to be important to antagonize the effect of high Cl Ϫ and for the retention of glutamate inside the vesicles. The data that led to these conclusions are the following: (i) In the absence of Cl Ϫ , A23187 had no effect on the V max of glutamate transport. In the presence of 4 mM Cl Ϫ , A23187 promoted a small decrease of V max , but this decrease become more pronounced when the Cl Ϫ concentration  ⌬ (A and B) and ⌬pH (C). Oxonol V or acridine orange fluorescence was measured as described in the legend to Fig. 3, except that the concentration of Cl Ϫ was varied by using a mixture of potassium gluconate and KCl, keeping total K ϩ concentration at 140 mM. A, ⌬⌿ in the presence of 10 mM K 2 SO 4 (E) or 10 mM (NH 4 ) 2 SO 4 (q). B, ⌬⌿ in A23187-free medium (Ç) or at 10 M A23187 (å). C, ⌬pH in K 2 SO 4 -free medium (Ç) or at 10 mM K 2 SO 4 (E), 10 M A23187 (å), or 10 mM (NH 4 ) 2 SO 4 (q). The inset details the ⌬pH found in the range of 0 -4 mM Cl Ϫ when neither A23187 or (NH 4 ) 2 SO 4 is present. The values represent a typical experiment of four independent experiments with two vesicle preparations. was raised to 80 mM (Table I). (ii) The amount of glutamate retained by the vesicles depends on both the velocity of uptake and the rate of glutamate efflux from the vesicles. The effect of Cl Ϫ and A23187 can be analyzed better after steady state is reached, a condition in which the rate of glutamate uptake equals the rate of glutamate efflux. Using saturating concentrations of glutamate and in the absence of Cl Ϫ (Fig. 7B), there was no change in the level of glutamate retained by the vesicles when A23187 was added to the medium, indicating that dissipation of the pre-existing ⌬pH did not lead to a change in either the rate of glutamate uptake or the rate of glutamate efflux. However, when 20 mM Cl Ϫ was present in the medium, the addition of A23187 led to an increase in the efflux rate (Fig. 7,  C and D), and although the H ϩ -ATPase was still pumping protons and the driving the transport of glutamate (Fig. 8), the rate of efflux was faster than the rate of uptake. As a result, there was a net decrease in the amount of glutamate retained by the vesicles until a new steady state was reached (Fig. 8A). A comparison of Fig. 7 (D and F) indicates that glutamate efflux rate increased with Cl Ϫ concentration in the medium. This was observed in the presence of both a low and a high glutamate concentration (Fig. 7, E and F) regardless of the order of A23187 and Cl Ϫ addition to the medium. (iii) In the presence of Cl Ϫ , dissipation of ⌬pH by A23187 promoted a significant stimulation of the bafilomycin A 1 -sensitive ATPase activity (Fig. 8B). This suggests that similar to other H ϩtransport ATPases (Dufour et al., 1982), the vesicular H ϩ -ATPase is also back-inhibited by the accumulation of protons inside the vesicles (⌬pH) and that the dissipation of the ⌬pH by A23187 uncouples the glutamate uptake from the ATPase ac-tivity (Fig. 8). (iv) The effect of Cl Ϫ on glutamate efflux revealed a saturation kinetics, with the half-maximal effect being attained with 30 mM Cl Ϫ (Fig. 9). Notice in Fig. 9 that gluconate did not induce glutamate efflux from the vesicles, thus indicating that the effect is specific for the chloride anion and is not related to a possible osmotic imbalance of the system.
The Critical Cl Ϫ and Glutamate Concentration Ranges-The  7. Effect of dissipation of ⌬pH (A, B, C, and D) and chloride addition (E and F) at steady state glutamate uptake. A, B, C, and D, glutamate uptake was measured in the presence of 10 mM Mops-Tris (pH 7.0), 4 mM MgATP, 120 mM potassium gluconate, 1 mg of synaptic vesicles/ml, with 50 M (E, q) or 4 mM L-[ 3 H]glutamate (Ç, å), either in the absence (A and B) or in the presence of 20 mM KCl (C and D). After steady state was reached (10 min, arrow) A23187 was added (q, å) to a final concentration of 10 M, or no A23187 was added (E, Ç). E and F, glutamate uptake was measured in the presence of 10 mM Mops-Tris (pH 7.0), 4 mM MgATP, 60 mM potassium gluconate, 1 mg of synaptic vesicles/ml, 10 M A23187 with 50 M (E, q) or 4 mM L-[ 3 H]glutamate (Ç, å). After steady state was reached (10 min, arrow) KCl was added (q, å) to a final concentration of 80 mM, or no KCl was added (E, Ç). The values are means of three experiments with three different vesicle preparations. two effects of Cl Ϫ can be detected when the concentration of this anion in the medium varies between 4 and 10 mM. In this range, Cl Ϫ increases both the rates of glutamate uptake (Figs. 1 and 5) and of glutamate efflux (Fig. 10). These two effects become more apparent when the ⌬pH is abolished with A23187. Under these conditions, the effect varied depending on the concentrations of glutamate in the two sides of the vesicle membrane. The amount of glutamate retained by the vesicles depends on the rates of both uptake and efflux. The rate of uptake depends on the concentration of substrate in the medium and on the affinity of the transport system for glutamate. The rate of efflux depends on both the membrane permeability coefficient and on the glutamate concentration inside the vesicles. When added to a medium containing subsaturating concentrations of glutamate, A23187 promotes an increase of the transport system affinity for glutamate, which in turn leads to an increase of the rate of uptake (Fig. 10A). Under this condition, the total amount of glutamate inside the vesicles is about 15 times smaller than that found inside the vesicles in the situation illustrated in Fig. 10B. Therefore, the change of the membrane permeability coefficient promoted by Cl Ϫ and A23187 does not lead to a large increase of efflux. Thus, the overall balance between the increment of uptake and efflux favors the net accumulation of glutamate (Fig. 10A). On the other hand, a net efflux of glutamate is measured when a saturating concentration of glutamate is present in the medium (Fig. 10B). Under this condition, the decrease of the apparent K m for glutamate does not lead to an increase of uptake, and a small increase in the membrane permeability promoted by Cl Ϫ leads to a substantial increase in the efflux due to the large amount of glutamate found inside the vesicles at steady state. The net effect is release of glutamate (Fig. 10B).

DISCUSSION
One mechanism of modulation of glutamate uptake by chloride is the requirement of this anion for the formation of a ⌬pH across the membrane. The present data indicate that ⌬⌿ and ⌬pH components play distinct roles in glutamate uptake and that both components of ⌬Hϩ are important for optimal glutamate uptake. The ⌬⌿ seems to modulate the apparent affinity for glutamate and is essential for glutamate accumulation into the vesicles (Table I and Figs. 5-7). The ⌬pH antagonizes the efflux of glutamate promoted by chloride and is important for retaining glutamate inside the vesicles (Fig. 7). The inhibition of glutamate uptake observed after dissipation of ⌬pH occurs under physiologically relevant glutamate and chloride concentrations (Fig. 9). The intraneuronal glutamate concentration is in the range of 1-10 mM (McMahon and Nicholls, 1991), whereas the intracellular chloride concentration is within 2-15 mM (Shepherd, 1988;Albers et al., 1989). After exocytosis, the entire recycling of synaptic vesicles takes approximately 1 min (Sudhof, 1995). Several drugs such as neuron blockers are accumulated in synaptic vesicles, leading to selective dissipation of the ⌬pH component (Moriyama et al., 1993). As suggested by the present results under these conditions, small changes of intraneuronal chloride concentration may lead to the release of glutamate accumulated by the vesicles. This mechanism may contribute to the inhibitory action of neuron blockers in neurotransmission.
Under optimal conditions, the ⌬pH across synaptic vesicles was found to be of one unit (Tabb et al., 1992). The pK a of the ␥-carboxylic group of glutamate is 4.25 and is far from the ⌬pH range of this study. After decreasing the ⌬pH from 7.0 to 6.0, the concentration of the negative forms of glutamate decreases from 99.8 to 98%, whereas a neutral species concentration increases from 0.2 to 2% of the total. Under the conditions of our study, only 1-5% of the glutamate was taken up by the vesicles. Thus, if the neutral species of glutamate will be less permeable than the negative forms, the possibility exists that glutamate will be progressively trapped inside the vesicles, because a continuous H ϩ flux is provided by the vacuolar H ϩ -ATPase. Another possibility is a direct effect of internal ⌬pH on the glutamate transporter protein as suggested by Tabb et al. (1992). Hartinger and Jahn (1993) found that high chloride concentration prevents inhibition of glutamate uptake by DIDS, indicating that the glutamate carrier has a chloride binding site on the cytoplasmic side. In this view, a second mechanism for the action of chloride implies a direct interaction with the glutamate transporter protein, and in the present study this is supported by the following data: (i) Both activation and inhibition of glutamate uptake by chloride can be observed at subsaturating glutamate concentrations after the ⌬pH is abol-ished with either A23187 or (NH 4 ) 2 SO 4 (Figs. 5, A and B, and  6A). Thus, stimulation with 4 mM chloride is not essentially due to the formation of a ⌬pH as previously suggested by Tabb et al. (1992); (ii) Chloride significantly increased the K m for glutamate and altered the V max even in the presence of A23187 (Table I); (iii) In the absence of a ⌬pH, the addition of chloride led to glutamate efflux, an effect that exhibited saturation kinetics (Fig. 9). A possible explanation for the two effects of chloride is that chloride may bind to the glutamate carrier and act as a counter anion to glutamate (Fig. 11, B and C, reactions  1-4). Glutamate influx during glutamate accumulation may be coupled to chloride movement in the opposite direction (Fig.  11B). For the influx of glutamate, Maycox et al. (1990) have already proposed that chloride may act as a counter anion. These authors, however, did not observe an effect of high chloride on efflux. We now raise the possibility that release of glutamate will also be coupled to chloride influx. The occurrence of this reaction is inversely related to the magnitude of the ⌬pH component. In this view, the affinity of the glutamate carrier for chloride would vary depending on the side of the membrane to which glutamate binds. When glutamate binds to the external surface of the membrane, the carrier would bind chloride with high affinity to the part of the carrier that faces the vesicle lumen (Fig. 11B). Conversely, during efflux, the binding of glutamate in the vesicles lumen would be coupled with the binding of chloride to a low affinity site located in the part of the carrier facing the external surface of the membrane. In the first situation, the binding of chloride will facilitate the uptake and in the second the release of glutamate. It is not clear whether binding of chloride and glutamate to the carrier will be simultaneous (Fig. 11B) or follow a sequential pattern (Fig. 11C, reactions 1-4). Although a chloride-glutamate counter transport has still to be directly demonstrated, it would provide charge balance and thus explain the previous finding that the membrane potential remains largely intact during glutamate accumulation . FIG. 11. Proposed mechanisms for glutamate uptake into synaptic vesicles. The sequence includes two distinct functional states of the glutamate transporter, T 1 and T 2 . When in the T 1 conformation, the site that translocates glutamate across the membrane faces the outer surface of the vesicles, and the region of the protein that translocates Cl Ϫ faces the vesicles lumen. The ⌬⌿ drives the conversion of T 1 into T 2 , which accounts for the reorientation of the glutamate and Cl Ϫ binding sites. In the T 2 form the glutamate binding site faces the vesicles lumen and the Cl Ϫ site, the outer surface. The conversion of T 1 into T 2 is associated with a large change of the carrier affinity for the species transported. T 1 has a higher affinity to both glutamate and Cl Ϫ than the form T 2 . In the absence of Cl Ϫ (A and C, reactions 2, 5, 6, and 7), the rate of glutamate transport will be slower than that measured in the presence of Cl Ϫ (B and C, reactions 1, 2, 3, and 4). glu or Glu, glutamate.