(Received for publication, January 5, 1996; and in revised form, February 28, 1996)
From the
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 (
pH). With the use of these
agents, we show that the transmembrane potential regulates the apparent
affinity for glutamate, whereas the
pH antagonizes the effect of
high chloride concentrations and is important for retaining glutamate
inside the vesicles. Selective dissipation of
pH 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
pH
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
pH 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
pH 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-dependent carrier
located in the plasma membrane, and the other is a low affinity,
Na
-independent transport system located in the
synaptic vesicles (Kanner, 1983; Maycox et al., 1990).
Glutamate uptake into synaptic vesicles is driven by a
µH+ (
)formed across the vesicle membrane by a
bafilomycin A
-sensitive vacuolar H
-ATPase
(Disbrow et al., 1982; Naito and Ueda, 1983, 1985; Maycox et al., 1988; Cidon and Sihra, 1989; Floor et al.,
1990). As H
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
(Maycox et al., 1988; Cidon and Sihra, 1989; Hartinger and
Jahn, 1993; Moriyama and Yamamoto, 1995) or by both the
and
pH components of the
µH+ (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
concentrations (>20 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
would be related to the
dissipation of
(Maycox et al., 1988). 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
µH+ 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.
Figure 1:
Time course of glutamate uptake into
synaptic vesicles. The reaction was carried out at 35 °C in medium
containing 10 mM Mops-Tris (pH 7.0), 4 mM MgATP, 1 mg
synaptic vesicle protein/ml, with 50 µM (A) or 4
mM (B) L-[H]glutamate,
and 140 mM potassium gluconate (
), 136 mM potassium gluconate plus 4 mM KCl (
), or 60 mM potassium gluconate plus 80 mM KCl (
). The reaction
was started by the addition of synaptic vesicles and stopped after the
indicated times. The values are the means of three independent
experiments with three vesicle
preparations.
Figure 2:
Effect of glutamate and chloride
concentration on initial rate (A) and steady state (B) glutamate uptake. The glutamate uptake was measured in the
presence of various glutamate concentrations and 140 mM potassium gluconate (), 136 mM potassium gluconate
plus 4 mM KCl (
), or 60 mM potassium gluconate
plus 80 mM KCl (
). The reaction was started by the
addition of synaptic vesicles and stopped after 1 (A) or 20 (B) min at 35 °C. The values are the means ± S.E.
of four independent experiments with three vesicle preparations. Other
conditions are the same as described in the legend to Fig. 1.
Figure 3:
(NH)
SO
and A23187 alter the balance between
(A and B) and
pH (C and D).
was
measured in a medium containing 10 mM Mops-Tris (pH 7.0), 140
mM potassium gluconate and 1.5 µM oxonol V.
pH was measured in a medium containing 10 mM Mops-Tris
(pH 7.0), 140 mM KCl, and 2 µM acridine orange.
The reaction was started by the addition of MgATP at 2 mM final concentration. After 4-5 min., 10 mM (NH
)
SO
(A and C) or 10 M A23187 (B and D) was
added. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone at
10 µM final concentration was added to dissipate
in A and B. The traces are representative
of ten experiments with four different vesicle
preparations.
Figure 4:
Effect of increasing chloride
concentrations on (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
was varied by using a mixture of potassium
gluconate and KCl, keeping total K
concentration at
140 mM. A,
in the presence of 10 mM K
SO
(
) or 10 mM (NH
)
SO
(
). B,
in A23187-free medium (
) or at 10 µM A23187 (
). C,
pH in
K
SO
-free medium (
) or at 10 mM K
SO
(
), 10 µM A23187
(
), or 10 mM (NH
)
SO
(
). The inset details the
pH found in the
range of 0-4 mM Cl
when neither A23187
or (NH
)
SO
is present. The values
represent a typical experiment of four independent experiments with two
vesicle preparations.
Figure 5:
Dual effect of chloride at various
glutamate concentrations and in the presence of A23187. The reaction
was carried out at 35 °C in medium containing 10 mM Mops-Tris (pH 7.0), 4 mM MgATP, 1 mg of synaptic vesicle
protein/ml, a mixture of potassium gluconate and KCl to achieve the
desired chloride concentrations, maintaining total K at 140 mM, either in the absence (
) or in the
presence of 10 µM A23187 (
). Glutamate
concentrations were 10 µM (A), 50 µM (B), or 1 mM (C). The reaction was
stopped after 20 min of incubation at 35 °C. The values represent
the average of three different experiments with two different vesicle
preparations. glu, glutamate.
Figure 6:
Dual effect of chloride at various
glutamate concentrations and in the presence of
NH. The conditions were the same as
described in the legend to Fig. 5. Glutamate concentrations were
50 µM (A) or 1 mM (B) in the
presence of either 10 mM K
SO
(
)
or 10 mM (NH
)
SO
(
).
The reaction was stopped after 10 min of incubation at 35 °C. The
values represent the average of three different experiments with three
different vesicle preparations. glu,
glutamate.
Figure 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 µM (
,
) or 4
mML-[
H]glutamate (
,
), 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
(
,
) to a final concentration of 10 µM, or no
A23187 was added (
,
). 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 µM A23187 with 50 M (
,
) or 4 mML-[
H]glutamate (
,
).
After steady state was reached (10 min, arrow) KCl was added
(
,
) to a final concentration of 80 mM, or no KCl
was added (
,
). The values are means of three experiments
with three different vesicle preparations.
Figure 8:
Effect of dissipation of pH on
steady state uptake and influx of glutamate (A) and
H
-ATPase activity (B). A, the assay
medium contained 10 mM Mops-Tris (pH 7.0), 4 mM MgATP, 4 mML-[
H]glutamate, 120 mM potassium gluconate, 20 mM KCl, and 1 mg of synaptic
vesicles/ml. After steady state was reached (10 min, arrow)
A23187 was added (
,
) to a final concentration of 10
µM. Glutamate influx (
) was measured by loading the
vesicles with nonradioactive glutamate and after 10 min (arrow) a tracer amount of L-[
H]glutamate plus A23187 (final
concentration, 10 µM) was added. B, bafilomycin
A
-sensitive ATPase activity was measured in the presence of
10 mM Mops-Tris (pH 7.0), 4 mM MgATP, 4 mML-glutamate, 120 mM potassium gluconate, 20
mM KCl, and 0.1 mg of synaptic vesicles/ml. After 10 min (arrow), A23187 was added (
) to a final concentration of
10 µM, or no A23187 was added (
). The reaction was
stopped by the addition of trichloroacetic acid to 10% final
concentration. The values are the means of three to five experiments
with three different vesicle preparations.
Figure 9:
Chloride dependence of glutamate efflux
in the absence of pH. The assay medium contained 10 mM Mops-Tris (pH 7.0), 4 mM MgATP, 60 mM potassium
gluconate, 4 mML-[
H]
glutamate, 10 µM A23187, and 1 mg of synaptic vesicles/ml.
After 10 min of reaction, potassium gluconate (
) or KCl (
)
was added to final concentrations ranging from 4 to 80 mM. The
initial rate of glutamate efflux was measured 40 s after Cl
addition. The inset shows a double-reciprocal plot. The
values are the means of three independent experiments with three
different vesicle preparations.
Figure 10:
Effect of dissipation of pH on
steady state glutamate uptake in the presence of 4 mM
chloride. Glutamate uptake was measured in the presence of 10 mM Mops-Tris (pH 7.0), 4 mM KCl, 4 mM MgATP, 136
mM potassium gluconate, 1 mg of synaptic vesicles/ml with 50
µM (A) or 4 mML-[
H]glutamate (B). After
steady state was reached (10 min, arrow) A23187 was added
(
,
) to a final concentration of 10 µM, or no
A23187 was added (
,
). The values are means of three
experiments with three different vesicle preparations. glu,
glutamate.
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
µH+ 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 1and Fig. 5Fig. 6Fig. 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
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
flux is provided by the vacuolar
H
-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 abolished with either A23187 or
(NH
)
SO
(Fig. 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
for glutamate and
altered the V
even in the presence of A23187 (Table 1); (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 (Maycox et al., 1988).
Figure 11:
Proposed mechanisms for glutamate uptake
into synaptic vesicles. The sequence includes two distinct functional
states of the glutamate transporter, T and T
.
When in the T
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
faces the vesicles lumen. The
drives the conversion
of T
into T
, which accounts for the
reorientation of the glutamate and Cl
binding sites.
In the T
form the glutamate binding site faces the vesicles
lumen and the Cl
site, the outer surface. The
conversion of T
into T
is associated with a
large change of the carrier affinity for the species transported.
T
has a higher affinity to both glutamate and
Cl
than the form T
. In the absence of
Cl
(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
(B and C, reactions 1, 2, 3, and 4). glu or Glu,
glutamate.