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Volume 16, Number 21,
Issue of November 1, 1996
pp. 6722-6731
Copyright ©1996 Society for Neuroscience
Anion Conductance Behavior of the Glutamate Uptake Carrier in
Salamander Retinal Glial Cells
Brian Billups,
David Rossi, and
David Attwell
Department of Physiology, University College London, London WC1E
6BT, United Kingdom
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Glutamate uptake is driven by the cotransport of Na+
ions, the countertransport of K+ ions, and either the
countertransport of OH or the cotransport of
H+ ions. In addition, activating glutamate uptake carriers
has been shown to lead to activation of an anion conductance present in
the carrier structure. Here we characterize the ion selectivity and
gating of this anion conductance. The conductance is small with
Cl as the permeant anion, but it is large with
NO3 or ClO4 present, undermining
the earlier use of NO3 and ClO4 to
suggest that OH countertransport rather than
H+ cotransport helps drive uptake. Activation of the anion
conductance can be evoked by extra- or intracellular glutamate and can
occur even when glutamate transport is inhibited. By running the
carrier backward and detecting glutamate release with AMPA receptors in
neurons placed near the glial cells, we show that anion flux is not
coupled thermodynamically to glutamate movement, but
OH/H+ transport is. The possibility that cell
excitability is modulated by the anion conductance associated with
glutamate uptake suggests a target for therapeutic drugs to reduce
glutamate release in conditions like epilepsy.
Key words:
glutamate;
transporter;
anion conductance;
uptake;
pH;
glial cell
INTRODUCTION
The extent to which glutamate uptake can lower the
extracellular glutamate concentration in the CNS is determined by the
ionic stoichiometry of the uptake process (Attwell et al., 1993 ). Entry
of each glutamate ion into the cell is thought to be accompanied by the
cotransport of two Na+ ions (Baetge et al., 1979; Stallcup
et al., 1979; Erecinska et al., 1983) and the countertransport of one
K+ ion (Kanner and Sharon, 1978; Barbour et al., 1988;
Amato et al., 1994). In addition, glutamate uptake carriers generate pH
changes, acid inside the cells and alkaline outside (Erecinska et al.,
1983; Bouvier et al., 1992). Bouvier and colleagues (1992) suggested
that, for the glutamate transporter in salamander retinal glia, power
is obtained from the transmembrane pH gradient by the transport of an
OH ion out of the cell rather than the (thermodynamically
equivalent) transport of an H+ ion into the cell. This was
based on the observation that, when certain anions
(NO3, ClO4, and SCN)
were inside the cell, the inward current evoked by external glutamate
was larger, but the pH change generated was unaffected or reduced, and
that anion-sensitive electrodes detected the efflux of
ClO4 from cells containing ClO4. It
was suggested that NO3, ClO4, and
SCN competed for transport on a carrier site that
normally transports OH.
The cloned mammalian glutamate transporters (Fairman et al., 1995;
Wadiche et al., 1995) and the transporters in salamander photoreceptors
and glia and in fish bipolar cells (Sarantis et al., 1988; Eliasof and
Werblin, 1993; Grant and Dowling, 1995; Picaud et al., 1995; Eliasof
and Jahr, 1996) activate an anion conductance when they bind external
glutamate and Na+. However, it is unclear how the binding
of substrate to the carrier gates the anion conductance. Can the anion
conductance component of the transporter be activated by intracellular
glutamate during reversed uptake as well as by external glutamate
during forward uptake? Is it even necessary for glutamate transport to
occur for the anion conductance to open, or is glutamate binding
sufficient? The presence of an anion conductance in the transporter
structure also raises the possibility that the effects of
NO3, ClO4, and SCN on
the salamander glial carrier were exerted not through the postulated
OH-binding site but through the anion conductance
component of the transporter molecule (Eliasof and Jahr, 1996). It
further brings into question whether the observed pH changes are a
result of substrate transport or are attributable to pH-changing anions
passing through the anion conductance.
Here we investigate the gating of the anion conductance in the
salamander glial glutamate transporter. We show that the anion
conductance can be activated by glutamate binding to either side of the
membrane and can occur independently of whether net glutamate transport
occurs, suggesting that it is activated by a conformation change, which
is allowed when the carrier is in a particular state of its uptake
cycle. Transport of OH (or H+) is shown to be
coupled to glutamate transport and shown not to occur through the anion
conductance.
MATERIALS AND METHODS
All experiments were done at room temperature, 25°C.
Salamander retinal glial cells. Glial (Müller) cells
were isolated from tiger salamander retinae by using papain, as
described previously (Barbour et al., 1991), and whole-cell-clamped
with pipettes of series resistance (in whole-cell mode) ~3 M ,
which leads to negligible series resistance voltage errors (<2 mV).
Large pipettes are essential for dialyzing the cell adequately in
experiments removing intracellular potassium from the cell (Szatkowski
et al., 1991). When currents were compared in different cells, they
were normalized by cell capacitance to compensate for variations in
cell size (Barbour et al., 1991).
Solutions. Unless otherwise stated, the extracellular
solution contained (in mM): NaCl 105, KCl 2.5, CaCl2 3, MgCl2 0.5, glucose 15, HEPES 5, and
BaCl2 6 (to block the inward rectifier potassium channels
of the cells), pH-adjusted to 7.3 with NaOH. A 1 M NaCl
agar bridge was used as the bath electrode to reduce (to <0.4 mV)
junction potential changes when changing the anion in the external
solution. Unless otherwise stated, the standard pipette solution for
uptake experiments contained (in mM): KCl 95, NaCl 5, HEPES
5, MgCl2 7, Na2ATP 5, CaCl2 1, and
K2EGTA 5, pH-adjusted to 7.0 with 14 mM KOH.
The pipette solution for studying the effect of
[K+]i on the anion conductance (see Fig.
3C,D) was as above [but pH-adjusted with
N-methyl-D-glucamine (NMDG) and with
(NMDG)2EGTA instead of K2EGTA] or with KCl
replaced by choline-Cl; for these experiments, the external solution
was as above, but with 0.1 mM ouabain added, KCl omitted
(Barbour et al., 1988), and with 105 mM NaCl replaced by 25 mM NaCl and 30 mM choline-Cl plus 50 mM of either NaCl or NaNO3 (pH was adjusted
with NMDG). The pipette solution for studying uptake with strong pH
buffering contained (in mM): KCl 50, HEPES 71, NMDG 26, NaCl 5, (NMDG)2EGTA 5, CaCl2 1, MgCl2 7, and Na2ATP 5, pH 7.0; that for
reversed uptake usually contained (in mM): Na-glutamate 10, choline-Cl 40, HEPES 71, NMDG 25, (NMDG)2EGTA 5, CaCl2 1, MgCl2 7, and Na2ATP 5, pH
set to 7.0 with NMDG. When we studied the effects of internal anions on
the anion conductance activated during reversed uptake (see Fig.
4B), the pipette solution contained (in
mM): Na-glu 10, choline-Cl 85 (for Cl) or 35 (ClO4), choline-ClO4 0 (Cl) or 50 (ClO4), NaCl 5, HEPES 5, (NMDG)2EGTA 5, Na2ATP 5, CaCl2 1, and MgCl2 7, pH set to 7.0 with NMDG. When we studied
activation of the anion conductance with net glutamate transport
inhibited (see Fig. 5A), the pipette solution contained (in
mM): Na-glu 100, MgATP 5, HEPES 5, CaCl2 1, (NMDG)2-EGTA 5, and MgCl2 2, pH set to 7.0 with
NMDG; the external solution contained (in mM): NaCl 100, choline-Cl 10, MgCl2 0.5, CaCl2 3, HEPES 5, glucose 15, BaCl2 6, and ouabain 0.1. When we studied the
effect of intracellular [Na+] and [glu]
on the anion conductance with net glutamate transport inhibited (see
Fig. 5B), the pipette solution contained blockers of the
Krebs' cycle, glutamate transaminase, and glutamine synthetase to
allow better control of [glu]i and
comprised (in mM): NaCl 0 or 10, NMDG-glutamate 0 or 10, choline-Cl 100 (for 0 Na+/0 glu), 90 (for 10 Na+/0 glu or 0 Na+/10
glu), or 80 (for 10 Na+/10
glu), CaCl2 1, (NMDG)2EGTA 5, MgATP 5, HEPES 5, MgCl2 2, malonic acid 0.2, amino-oxyacetic acid 5, and L-methionine sulfoximine 2, pH
set to 7.0 with NMDG; the external solution was as just described, but
with choline-Cl replaced by NaCl. Electrode junction potentials were
compensated (Fenwick et al., 1982).
Fig. 3.
[Na+]o and
[K+]i dependence of anion conductance
activation evoked by 200 µM glutamate. A,
Glutamate-evoked currents at 0 mV with ClO4 as the
external anion (solutions as in Fig. 2) and with external sodium
present (left) or absent (right; replaced
by choline, conducted on 5 cells). B, Average
I-V data from experiments as in A on
three cells. Sodium removal abolishes both the outward current at
positive potentials (produced by ClO4 entry through
the anion conductance of the carriers) and the inward current at
negative potentials (primarily current produced by the cotransport into
the cell of a net positive charge with each glutamate anion). Similar
results were obtained in four cells when NO3 was the
external anion. C, D, Mean I-V relations
(normalized by cell capacitance) for currents evoked by 200 µM glutamate, with K+ either present
(C, 5 cells) or omitted (D, 5 cells) from
the pipette and with 50 mM NO3 either
present in (curves labeled
NO3) or absent from (curves
labeled Cl) the external solution. Pipette and
external solutions are described in Materials and Methods.
[View Larger Version of this Image (27K GIF file)]
Fig. 4.
Activation of the anion conductance during
reversed uptake evoked by raising [K+]o from
0 to 30 mM (black bars) around cells
whole-cell-clamped with solutions containing 10 mM Na-glu.
A, K+-evoked currents at 0 mV in a
Müller cell bathed sequentially in external solutions containing
Cl as the main anion, including 50 mM
NO3 or ClO4, and then with just
Cl again. External solution as in Figure
3C and with 30 mM choline-Cl replaced by KCl
when [K+] was raised. Pipette solution for reversed
uptake as in Materials and Methods. B,
K+-evoked currents in two different cells (normalized by
cell capacitance) clamped with pipette solutions (see Materials and
Methods) containing either Cl or ClO4
as the main anion. External solution as in A for
Cl. C, Mean (±SEM) K+-evoked
currents measured as in A and B for six
cells with external NO3, for five with external
ClO4, and for five cells clamped with internal
ClO4 (normalized to 3 cells clamped with internal
Cl).
[View Larger Version of this Image (19K GIF file)]
Fig. 5.
Activation of the anion conductance with glutamate
transport inhibited by the absence of extra- and intracellular
K+ and with Na+ and glu added
intracellularly. A, Currents evoked at  5 and 65 mV
by 100 µM glutamate and analogs. Pipette and external
solution are described in Materials and Methods. Analogs that activate
non-NMDA, NMDA, and metabotropic receptors produced no current change.
Glutamate and D-aspartate (also transported on uptake
carriers) evoked a conductance increase. B, Specimen
glutamate-evoked currents (normalized by cell capacitance) as in
A but with varying [Na+] and
[glu] in the pipette. C, Specimen
I-V data from one cell as in A with
varying external [Cl]. External solution as in
A, but with no ouabain, with choline-Cl replaced by 7 mM NaCl, and Cl replaced by gluconate as
needed. Internal solution as in Figure 4A.
D, The reversal potential of data obtained as in
C (data from 5-8 cells/point) changes by 28 mV per
10-fold change of [Cl]o. E,
I-V data (normalized by cell capacitance) obtained as
in A from five cells with Cl and five
cells with ClO4 as the main internal and external
anion. With ClO4 the mean ± SEM outward current
at +20 mV was 122 ± 14 pA. External solutions as Figure
3C, but with ClO4 replacing
NO3; pipette solution as Figure
4B. F, Dependence on external pH
(pHo) of the anion conductance current for five cells
studied as in A (current evoked by 1 mM
glutamate at 40 mV, squares). Pipette solution as
Figure 4A; external solution as for
Cl in Figure 3C. For comparison, we show
the pHo dependence of the reversed uptake current (at 0 mV,
circles) produced by raising [K+] from 0 to 30 mM with 10 mM glu and 20 mM Na+ in the pipette (Billups and Attwell,
1996).
[View Larger Version of this Image (26K GIF file)]
I-V plots. These were derived from the steady-state current
measured at the end of 150 msec voltage steps from a holding potential
of 50 mV. Glutamate-evoked currents were obtained from the
I-V data in glutamate by subtracting the average of control
I-V data obtained before and after glutamate.
Sensing glutamate release with isolated Purkinje cells. This
was performed as described by Billups and Attwell (1996). Cerebellar
Purkinje cells were isolated from 200-µm-thick slices of cerebellum
from 12-d-old rats by incubation in papain, as for salamander glia
(Barbour et al., 1991), except that the tonicity of the incubation and
washing solution was increased to that for rat cells by raising the
NaCl concentration by 20 mM. After isolation and plating of
the cells into the recording chamber, the solution outside the cells
was altered to standard solution of the tonicity for salamander cells:
this did not seem to damage the cells. Purkinje cells were recognized
by their large cell bodies and stumps of dendrites and axon. They were
whole-cell-clamped with a pipette solution containing (in
mM): CsCl 110, HEPES 10, MgCl2 2, CaCl2 0.5, (NMDG)2EGTA 5, and
Na2ATP 5, pH set to 7.0 with NMDG. Desensitization of
Purkinje cell non-NMDA receptors was reduced with 1 mM
trichlormethiazide, and the Purkinje cell then generated a non-NMDA
current related to the glutamate concentration by a Hill equation with
an EC50 of 23 µM and a Hill coefficient of
1.2 (Billups and Attwell, 1996).
Measurement of intracellular pH. This was done as described
previously with the pH-sensitive fluorescent dye
2 ,7-bis(carboxyethyl)carboxy-fluorescein (BCECF; 100 µM) loaded into the cell in the standard whole-cell
pipette solution but buffered with only 0.5 mM HEPES
(Bouvier et al., 1992). Calibration of the pH was obtained from the
response to a weak acid and two concentrations of a weak base (Bouvier
et al., 1992).
RESULTS
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.
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 in
B (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]o
of the reversal potential for currents studied as in D
(n = 5 cells for 10 mM, 7 for 30 mM, 17 for 100 mM).
[View Larger Version of this Image (18K GIF file)]
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.
[View Larger Version of this Image (14K GIF file)]
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 mM
NO3 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 mM
NO3 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.
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 in
A 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.
[View Larger Version of this Image (15K GIF file)]
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.
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). O
denotes 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 the
C.Na2.Glu
conformations).
[View Larger Version of this Image (20K GIF file)]
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 Km (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+]o2.5 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 pHo 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 mM
Na-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 Figure
6, 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.
DISCUSSION
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 also
Eliasof 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 Figure
7, 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.) Figure
7 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).
FOOTNOTES
Received May 23, 1996; revised Aug. 13, 1996; accepted Aug. 16, 1996.
This work was supported by the Wellcome Trust, Medical Research
Council, and European Community (contract CT95-0571). We thank Alasdair
Gibb, Akiko Momiyama, and Angus Silver for comments on this
manuscript.
Correspondence should be addressed to Dr. David Attwell at the above
address.
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