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The Journal of Neuroscience, October 1, 1998, 18(19):7650-7661
Macroscopic and Microscopic Properties of a Cloned Glutamate
Transporter/Chloride Channel
Jacques I.
Wadiche and
Michael P.
Kavanaugh
Vollum Institute, Oregon Health Sciences University, Portland,
Oregon 97201
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ABSTRACT |
The behavior of a Cl
channel associated with a
glutamate transporter was studied using intracellular and patch
recording techniques in Xenopus oocytes injected with
human EAAT1 cRNA. Channels could be activated by application of
glutamate to either face of excised membrane patches. The channel
exhibited strong selectivity for amphipathic anions and had a minimum
pore diameter of ~5Å. Glutamate flux exhibited a much greater
temperature dependence than Cl flux. Stationary
and nonstationary noise analysis was consistent with a sub-femtosiemen
Cl conductance and a maximum channel
Po 
1. The glutamate binding rate was
similar to estimates for receptor binding. After glutamate binding,
channels activated rapidly followed by a relaxation phase. Differences
in the macroscopic kinetics of channels activated by concentration
jumps of L-glutamate or D-aspartate were
correlated with differences in uptake kinetics, indicating a close
correspondence of channel gating to state transitions in the
transporter cycle.
Key words:
glutamate transporter; uptake; kinetics; astrocyte; postsynaptic; chloride channel
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INTRODUCTION |
Glutamate is the primary excitatory
neurotransmitter at central synapses, and its effects on receptors are
terminated by diffusion and by the actions of glutamate transporters.
These molecules are members of a large amino acid transporter gene
family (Malandro and Kilberg, 1996
), and they exhibit discrete
anatomical localizations. GLAST (EAAT1) and GLT-1 (EAAT2) are
found primarily in glial cells, whereas EAAC1 (EAAT3), EAAT4,
and EAAT5 are primarily expressed in neuronal cells (Rothstein et al.,
1994; Lehre et al., 1995; Yamada et al., 1996; Eliasof et al., 1998).
Glutamate transport is electrogenic and coupled to sodium and proton
influx and potassium efflux (Kanner and Sharon, 1978; Stallcup et al.,
1979; Nelson et al., 1983; Barbour et al., 1988; Zerangue and
Kavanaugh, 1996a). In addition to the coupled transport current, a
substrate-activated chloride current has been observed in oocytes
expressing cloned glutamate transporters (Fairman et al., 1995; Wadiche
et al., 1995b; Arriza et al., 1997; Eliasof et al., 1998). The ratio of the glutamate flux current to anion current varies among known glutamate transporters (EAAT2 > EAAT3 > EAAT1 > EAAT4 
EAAT5). A similar glutamate-dependent anion current is
also observed in neurons and glia (Sarantis et al., 1988; Grant and
Dowling, 1995; Picaud et al., 1995b; Billups et al., 1996; Eliasof and
Jahr, 1996; Larsson et al., 1996; Bergles and Jahr, 1997; Bergles et al., 1997; Otis et al., 1997). Although the physiological role of the
chloride flux is unclear, in retinal neurons this current may play a
role in visual processing (Grant and Dowling, 1995; Picaud et al.,
1995a). In brain slice preparations, transporter-associated anion
currents have been used to monitor the dynamics of synaptically released glutamate (Bergles and Jahr, 1997; Bergles et al., 1997; Otis
et al., 1997).
The molecular and biophysical basis of the chloride conductance is
unclear, but it appears to be associated with all eukaryotic glutamate
transporters as well as with a neutral amino acid transporter belonging
to the same gene family (Zerangue and Kavanaugh, 1996b). This study was
designed to compare and contrast the properties of the channel and
transport functions of EAAT1, a human glutamate transporter in which
these functions can be readily resolved (Wadiche et al., 1995b). The
results suggest that chloride and
glutamate/Na+/K+/H+
permeate by two distinct mechanisms, although the chloride channel gating is intrinsically linked to state transitions in the transporter cycle.
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MATERIALS AND METHODS |
Transporter expression and intracellular recording.
Capped mRNA transcribed from the cDNA encoding the human brain
glutamate transporter EAAT1 (Arriza et al., 1993) was injected into
stage V-VI Xenopus oocytes (~50-150 ng/oocyte). Membrane
currents were recorded 2-5 d later. Recording solution (frog Ringer's
solution) contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM
CaCl2, and 5 mM HEPES, pH 7.4, unless
stated otherwise. Two electrode voltage-clamp recordings were performed
at 22°C (unless stated otherwise) with a Geneclamp 500 interfaced to
an IBM-compatible PC using a Digidata 1200 A/D controlled with the
pCLAMP 6.0 program suite (Axon Instruments, Foster City, CA). The
currents were low-pass-filtered at 1 kHz and digitized at 5 kHz.
Microelectrodes were filled with 3 M KCl and had tip
resistances of <1 M
. The bath was connected to ground by a 3 M KCl-agar bridge from the recording chamber to a 3 M KCl reservoir containing a Ag/AgCl electrode. The
voltage-dependence of currents induced by glutamate was determined by
subtraction of control currents from currents recorded in the presence
of glutamate during 200 msec pulses to different test potentials. The
equilibrium potential for chloride was calculated assuming [Cl]in = 41 mM (Wadiche
et al., 1995b).
Radiotracer flux measurement. Membrane currents were
recorded in voltage-clamped oocytes during bath perfusion of 100 µM [3H]D-aspartate (0.42 Ci/mmol) (Amersham, Arlington Heights, IL) at indicated membrane
potentials. After washout of the radiotracer (<20 sec), oocytes were
rapidly transferred into a scintillation tube and lysed, and
radioactivity was measured. Currents were recorded using Chart software
(ADInstruments, New Castle, NSW, Australia), and integrated currents
were compared with radiolabel flux in the same oocytes. Control
measurements of radioactivity incorporated into uninjected oocytes
represented <8% of uptake into oocytes expressing EAAT1. The
nonspecific uptake was subtracted from the total uptake measured in
EAAT1-expressing oocytes. All data are expressed as mean ± SE.
Patch recordings. After manual removal of vitelline
membrane, inside-out or outside-out patch recordings were obtained
using pipettes (3-4 M) that were fire-polished and coated with
silicone plastic (Sylgard 184, Dow Corning, Midland, MI). Unless
stated otherwise, intracellular solution contained 100 mM
KCl or KSCN, 10 mM KCl, 3 mM
MgCl2, 5 mM Na-HEPES, and 10 mM EGTA adjusted to pH 7.5 with Tris-base. Extracellular
solutions contained 110 mM NaCl or NaSCN, 3 mM
MgCl2, and 5 mM Na-HEPES adjusted to pH 7.5. Membrane currents were recorded with an Axopatch 200A voltage clamp (Axon Instruments). Solution exchanges were made using a piezoelectric translator (Burleigh Instruments, Fishers, NY) mounted with a drawn glass theta tube (Warner Instruments, Hamden, CT) through
which control and experimental solutions flowed continuously. Solution
exchange times were measured after each experiment by rupturing the
patch and recording junction currents across the open pipette tip. Only
patches with membrane seal resistances of 
10 G were used for noise
analysis. The ensemble variance for consecutive sweeps was calculated
in bins of three sweeps to minimize any contribution of rundown of the
mean current. Steady-state subregions of individual sweeps were also
analyzed to verify the magnitude of substrate-dependent changes in
variance. The variance induced by injection of a 10 pA current through
a 10 G resistor was >100-fold lower than the
D-aspartate-induced variance. Records for spectral analysis
were low-pass Bessel filtered at 2-5 kHz and digitized at 10 kHz.
Spectra were calculated on data blocks containing 2048 points. To
produce a final spectrum, 50-500 spectra were averaged.
Estimate of unitary conductance-open probability product.
The number of transporters per oocyte was estimated from least squares fitting dihydrokainate (DHK)-sensitive charge movement to a Boltzmann function as described in Wadiche et al. (1995a). The
glutamate-activated chord anion conductances in the presence of
external Cl or SCN were
measured at various potentials after subtraction of the coupled
transport current. At 0 mV, the ratio of the chord anion conductance to
number of transporters was 1.37 × 1017
S/transporter (Cl) and 2.65 × 1016 S/transporter (SCN). At
+80 mV, the respective values were 9.08 × 1018 S/transporter and 2.86 × 1016 S/transporter. This value was used to
calculate a corrected chord conductance of 6.69 × 1016 S/transporter (+80 mV) for patches in
symmetrical SCN solutions from the
Goldman-Hodgkin-Katz (GHK) current equation (Hille, 1992).
Kinetic modeling. A kinetic model was developed using SCoP
software (Simulation Resources, Berrien Springs, MI) based on
modifications of a cyclical alternating access scheme (Kavanaugh, 1993)
with state transition rates fitted or assigned as described in the text.
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RESULTS |
Selectivity of the transporter-associated anion conductance
The EAAT1-dependent current activated by bath application of the
transporter substrate D-aspartate has been proposed to be composed of an inward current resulting from movement of
thermodynamically coupled ions with glutamate together with an
uncoupled chloride conductance that is increased during glutamate
transport (Wadiche et al., 1995b). In accord with this, the reversal
potential of the net D-aspartate current varied with
[Cl]out, but was 15-20 mV
more positive than ECl (Fig.
1A,B) [also see
Wadiche et al. (1995b); Eliasof and Jahr (1996)]. The chloride current
was resolved from the coupled transport current based on the assumption
that at ECl the transporter-mediated chloride current is zero (Wadiche et al., 1995b). The voltage-dependence of the
coupled transport current was then determined from measurement of
D-aspartate-induced inward currents at different
ECl values by varying
[Cl]out between 10 and 200 mM (Fig. 1A). The mean inward currents at
five different values of ECl were fitted to an
exponential function (e-fold 
89.7 ± 16.4 mV; n = 5) (Fig. 1A, dashed line). This function
is similar to the voltage dependence of
[3H]D-aspartate uptake (e-fold 75
mV) (Wadiche et al., 1995a), consistent with the current at
ECl reflecting the
Na+/H+/K+
coupled transport current.
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Figure 1.
Two currents are mediated by EAAT1.
A, Average currents induced by 100 µM
D-aspartate application on oocytes expressing EAAT1 with
recording solutions containing various Cl
concentrations ( , 10 mM;  , 30 mM;  , 60 mM;  , 100 mM;  , 200 mM). The
dashed line corresponds to the predicted coupled uptake
current for this group of cells. It represents the mean of exponential
fits (e-fold, 89.7 ± 16.4 mV; n = 5) through current values at the respective chloride equilibrium
potentials. Recording solutions were standard Ringer's solutions with
gluconate substitution for Cl to obtain the
indicated chloride concentration. Tris-Cl (100 mM) was
added to the solutions in experiments with Cl = 200 mM. The equilibrium potential for chloride is +35, +8,
10, 22, and 39 mV in 10, 30, 60, 100, and 200 mM
external chloride, respectively. B, The reversal
potential of the net current (,
Itotal) and the chloride-dependent
current (, Ichloride) induced by 100 µM D-aspartate are dependent on
[Cl]out. The reversal potentials of
the chloride-dependent current (IChloride)
were obtained from current-voltage relationships after the calculated
uptake current was subtracted from the total D-aspartate
currents (Itotal). The dashed
line represents the predicted Nernst equilibrium potential for
chloride assuming [Cl]in = 41 mM (see Materials and Methods). C,
Normalized anion-specific currents activated by 100 µM
D-aspartate in oocytes expressing EAAT1. Recording
solutions contained 10 mM Na salts of various test anions
(×, SCN;  ,
ClO4; ,
NO3; , I; ,
Br; , Cl) plus gluconate
substitution to obtain 90 mM Na+, 1.8 mM Ca2+, 1 mM
Mg2+, and 2 mM K+.
The average steady-state currents were obtained by subtraction of
control currents from the corresponding D-aspartate (100 µM) currents and normalized to a test dose of
D-aspartate in Ringer's solution. Current records with
glutamate as a test anion () represent records in 100 mM
Na gluconate subtracted from 100 mM Na
L-glutamate; this current did not reverse up to +80 mV.
D, Radiolabeled D-aspartate uptake from
oocytes expressing EAAT1 measured under voltage clamp
(Vm = 50 mV) with the indicated anion
substitution (0.47 ± 0.04, 0.46 ± 0.15, and 0.44 ± 0.10 pmol/sec for Cl,
NO3, and
gluconate, respectively; n = 4-7).
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Glutamate transporter currents are increased in the presence of certain
anions, including SCN,
ClO4, and
NO3, and I
(Wadiche et al., 1995b; Billups et al., 1996; Eliasof and Jahr, 1996;
Kavanaugh et al., 1997; Otis et al., 1997). With these more permeant
anions in the extracellular solution, larger
D-aspartate-induced outward currents are observed (Fig.
1C). In contrast, with gluconate as the sole extracellular
anion, outward currents were not observed, consistent with the
conclusion that it is impermeant (Wadiche et al., 1995b). The relative
permeabilities of a number of anions were quantified using the
Goldman-Hodgkin-Katz voltage equation after isolating the anion current
by subtraction of the coupled transport current as described above.
This approach relies on the assumption that the coupled transport
current is not altered by the permeant anion, which was verified by
comparing uptake of [3H]D-aspartate in
the presence of anions more (NO3) and
less (gluconate) permeant than
Cl (Fig. 1D). The ion
permeabilities (relative to Cl) ranged from <0.08
to 67 (Table 1). The data show that the
minimum pore diameter of the anion channel is ~5 Å, corresponding to
the diameter of the largest permeant ion measured,
ClO4 (Halm and Frizzell, 1992).
Significantly, the uncoupled anion conductance was not measurably
permeable to L-glutamate, because no outward current was
observed on switching from a 96 mM gluconate extracellular
solution to a 96 mM glutamate one (Fig. 1C).
This result indicates that glutamate flux occurs solely by a
cation-coupled mechanism without "short-circuit" permeation
occurring via the anion conductance that would diminish the
theoretically achievable glutamate gradient.
The transporter anion conductance displays channel-like
permeation properties
The transporter-mediated glutamate flux and the associated anion
currents indicate that different anions can permeate at different rates
(and directions) without affecting glutamate flux. The anion permeation
was investigated further to determine whether its properties were more
consistent with channel-like or carrier-like transport. Changes in
temperature are predicted to have less effect on uncoupled ion flux
through a channel than on carrier-mediated transport as a consequence
of the difference in thermal dependence of ion diffusion compared with
the protein conformational changes predicted to accompany carrier
gating. Steady-state currents induced by 100 µM
D-aspartate were examined at temperatures between 5 and 25°C. The current magnitude decreased with decreasing temperatures at
negative potentials, but was much less affected at positive potentials
where anion flux is a greater component of the net current (Fig.
2A). An Arrehnius plot
of the normalized currents at two potentials is shown in Figure
2B. At ECl, where all
of the charge is carried by the coupled uptake current, the currents exhibited a steep dependence on temperature. In contrast, at +80 mV,
where the majority of the current is caused by flux of chloride ions,
the current was much less dependent on temperature. Uptake of
radiolabeled [3H] D-aspartate was also
compared at 25 and 15°C and found to be reduced to the same extent as
the coupled uptake current (Fig. 2B, filled
circles). The temperature coefficient (Q10
between 10 and 20°C) for the D-aspartate currents was
3.2 ± 0.2 and 1.0 ± 0.1 at 30 mV and +80 mV,
respectively. These data suggest that the coupled uptake current
reflects kinetic processes that involve large energy barriers, whereas
the mechanism of chloride flux is more consistent with ionic diffusion
through an aqueous medium.
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Figure 2.
EAAT1 anion conductance properties.
A, D-Aspartate (1 mM)-dependent
current-voltage relationship for a representative oocyte
expressing EAAT1 at several bath temperatures (recording solution is
Ringer's solution). B, Arrehnius plot of normalized
currents mediated by EAAT1. The temperature coefficients
(Q10) between 10 and 20°C are
0.96 ± 0.1 and 3.2 ± 0.2 at +80 mV () and 30 mV (,
ECl), respectively. The
Q10 for the normalized radiolabeled uptake
performed under voltage clamp (60 mV) was 2.9 ().
C, Concentration dependence of the anion-specific chord
conductance (+60 mV) activated by application of
D-aspartate (100 µM). Conductances were
normalized to the maximum Cl chord conductance.
The apparent EC50 values are 54 ± 5.4 and 5.5 ± 1.6 mM for NO3 and
Cl, respectively (n = 4).
D, Lack of anomalous mole fraction behavior
[(NO3) + (Cl) = 3 mM] for the anion chord conductance (+60 mV) in cells
expressing EAAT1 (n = 3-4).
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The conductance of an ion-selective channel will generally exhibit a
saturable dependence on the permeant ion concentration as a consequence
of the interaction between the channel and the ion (Hille, 1992). The
conductance-concentration relationship was compared for
Cl and NO3, two
anions with different permeabilities. After subtraction of the coupled
transport current, the chord conductance at +60 mV activated by
application of 1 mM D-aspartate was measured as a function of the extracellular anion concentration (Fig.
2C). The conductance for both Cl and
NO3 revealed saturable kinetics with
K0.5 values of 5.7 ± 0.9 mM and 54.4 ± 15.8 mM for Cl and
NO3, respectively (n = 4). The saturation of the anion conductance is consistent with the
permeant anion interacting with a site or sites in the transporter pore
in contrast to simple diffusion-mediated flux. Multi-ion occupancy and
ion-ion interactions are common in many ion channel pores and may be
manifested as conductance minimums as the mole fraction of two
distinct permeant ions is varied. When the anion conductance was
measured in recording solutions containing varying mole fractions of
Cl and NO3, it
was found to change monotonically, yielding no evidence of multiple
anion occupancy of the channel pore (Fig. 2D).
Intracellular glutamate activates anion currents in
inside-out patches
The high permeability of anions like SCN
suggested the possibility of measuring an anion current activated by
transport in excised inside-out membrane patches containing glutamate
transporters. With a KCl-containing pipette (extracellular) solution,
inside-out patches were excised into a NaSCN-containing bath
(intracellular) solution. Application of L-glutamate or
D-aspartate to the internal membrane face induced a
voltage-dependent current (Fig. 3).
D-Aspartate did not induce any currents in patches excised
from uninjected oocytes (n = 4). The current-voltage
relationship was strongly rectifying in asymmetric anion solutions,
consistent with activation of the same anion conductance by forward or
reverse transport (Billups et al., 1996; Kavanaugh et al., 1997).
Currents in patches were activated by amino acids (300 µM) with the order of efficacy D-asp > L-glu > THA > tPDC > D-glu
(Fig. 3B and data not shown). The apparent affinity for
D-aspartate at the intracellular face was 203 ± 85 µM (80 mV; n = 5 patches), ~10-fold
lower than at the extracellular face determined in intact cells
(20.6 ± 3.0 µM; 80 mV; n = 4).
Furthermore, consistent with previous work demonstrating a requirement
for trans-K+ for transport (Kanner and
Sharon, 1978; Barbour et al., 1988; Szatkowski et al., 1990), the
D-aspartate current recorded from inside-out patches was
found to be dependent on the extracellular cation (Fig. 3C).
With K+ as the trans cation, large inward
currents were activated by application of 3 mM
D-aspartate. Substitution of K+ by
choline in the pipette failed to support D-aspartate
currents from patches excised from the same oocytes (Fig.
3C). However, substitution of K+ by
Na+ supported a D-aspartate-dependent
current that was 15-20% of the magnitude of the
trans-K+ currents in the same group of
oocytes (n = 4-7 patches), indicating that
Na+ is able to partially substitute for
K+ as a trans cation.
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Figure 3.
Reverse transport currents in inside-out patches.
A, Currents in a representative inside-out patch from an
EAAT1 oocyte. The currents were obtained by subtraction of control
currents from corresponding currents in the presence of 3 mM D-aspartate (Vm = +70 and 80 mV). Pipette solution contained 100 mM KCl, 3 mM MgCl2, 5 mM HEPES, pH
7.45, and bath solution contained 100 mM NaSCN, 3 mM MgCl2, 10 mM EGTA, and 5 mM HEPES, pH 7.45. B, Voltage dependence of
EAAT1-mediated currents (n = 3 patches) induced by
application of D-glutamate (, 3 mM),
L-glutamate (, 3 mM), and
D-aspartate (, 3 mM). Recording solutions
were the same as in A. C, Effect of the
external (trans) ion on the steady-state
D-aspartate (3 mM)-induced currents. Excised
inside-out patch currents from EAAT1-expressing oocytes were recorded
with pipettes containing 110 mM choline chloride
(; n = 6), NaCl (; n = 4), or KCl (n = 7) plus 3 mM
MgCl2 and 5 mM HEPES, pH 7.4. Bath solutions
contained 100 mM NaSCN, 10 mM NaCl, 3 mM MgCl2, 10 mM EGTA, and 5 mM HEPES, pH 7.4. D, Relative permeability
of SCN/Cl in EAAT1 inside-out patches. Pipettes contained 50 mM KSCN/56 mM KCl. The mean reversal potentials
for patches (n = 3-9) are plotted as a function of
the internal SCN concentration. The drawn curve
corresponds to nonlinear least squares fit to the function
Erev = RT/zF
ln((PSCN[SCN]o + PCl[Cl]o)/(PSCN[SCN]i + PCl[Cl]i)) and
results in a
PSCN/PCl
of 62.7. Representative D-aspartate currents in response to
voltage jumps in different [SCN]in (30 and 3 mM) are shown to the right (dotted
line represents zero current).
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Anion substitution experiments were also performed in inside-out
patches to compare the permeability of the anion channels activated by
internally and externally applied transporter substrates. Steady-state
difference currents were measured before and after application of 3 mM D-aspartate with pipette solutions
containing a mixture of 50 mM KSCN/56 mM KCl,
whereas the bath composition was changed to vary the ratio of NaCl and
NaSCN (SCN + Cl = 100 mM). A plot of the reversal potentials as a function of the
bath (internal) SCN concentration was fitted by
least squares to the GHK voltage equation (Fig. 3D).
Disregarding the coupled transport current, which is not expected to
contribute significantly in these conditions, the relative permeability
ratio for SCN/Cl was 62.7, close to the value obtained from whole-cell experiments (66.9) (Fig.
1C, Table 1).
Channel kinetics depend on transported substrates
To obtain information about the activation and deactivation
kinetics of the anion channel, outside-out patches excised from oocytes
expressing EAAT1 were held at 80 mV and exposed to
D-aspartate or L-glutamate using a
piezo-activated solution exchange system (Maconochie and Knight, 1989).
With KSCN in the pipette (intracellular) and NaCl in the bath
(extracellular), inward currents activated by pulses of saturating (10 mM) L-glutamate or D-aspartate
exhibited distinct kinetic differences. After the rise to peak,
currents evoked by a pulse of L-glutamate decayed
significantly more than currents evoked by D-aspartate
(Fig. 4A). The decay
time constants were 14.1 ± 2.4 msec (n = 18) and
85.4 ± 15.7 msec (n = 9), and the ratios of the
peak current to the steady-state current were 1.56 ± 0.11 (n = 18) and 1.05 ± 0.02 (n = 17)
for L-glutamate and D-aspartate, respectively.
This ratio was voltage-independent for both amino acids (Fig.
4B,C). At 80 mV, the steady-state response to a
saturating pulse of D-aspartate was 1.97 ± 0.36 times
larger than the L-glutamate response in the same patch
(n = 5). The activation and deactivation kinetics of
the anion current also differed for L-glutamate and
D-aspartate, with significantly faster kinetics seen in
response to L-glutamate pulses. Rise time constants
(determined from exponential fits) (Fig.
5A) were 0.96 ± 0.07 msec (n = 19) and 2.66 ± 0.22 msec
(n = 19) for 10 mM pulses of
L-glutamate and D-aspartate, respectively. The
deactivation time constant after removal of the amino acid was
approximately three times faster for L-glutamate than for
D-aspartate (80 mV; 22.8 ± 3.9 msec,
n = 9 vs 75.1 ± 10.5 msec, n = 9)
(Fig. 4A,D).
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Figure 4.
Macroscopic outside-out patch kinetics.
A, Rapid application of 10 mM
D-aspartate and L-glutamate to a representative
outside-out patch from an oocyte expressing EAAT1
(Vm = 80 mV). The pipette solution
contained 100 mM KSCN, 10 mM KCl, 3 mM MgCl2, 5 mM HEPES, and 10 mM EGTA, pH 7.5, whereas the external recording solutions
contained 110 mM NaCl, 3 mM
MgCl2, 5 mM HEPES, and 100 µM LaCl3. The application of excitatory amino
acids was delivered via flow pipes attached to a piezo-electric device.
After the patch was ruptured, the solution exchange time was tested by
switching between solutions of different osmolarities. The open tip
controls ordinarily had 10-90% rise and decay times of 350 µsec
(shown above current traces). B, Rapid
application of L-glutamate (10 mM) to a
representative outside-out patch expressing EAAT1 at the indicated
holding potentials. Open tip control is shown above current traces.
C, Voltage dependence of the peak to steady-state
current for L-glutamate (, 10 mM) or
D-aspartate (, 10 mM). D,
Voltage dependence of current deactivation time constant (single
exponential) for L-glutamate (, 10 mM) or
D-aspartate (, 10 mM). Only patches with
open tip controls of <500 µsec were used for analysis (10-90% rise
and decay times).
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Figure 5.
[L-Glutamate] dependence of
currents. A, Rapid exchange of various
L-glutamate concentrations to a representative EAAT1
expressing outside-out patch. Inset, Normalized currents
emphasize the concentration dependence of the current activation rate.
Vm = 80 mV. Open tip solution exchange
control is shown above current traces (10-90% rise = 250 µsec). B, Concentration dependence of the time
constant (single exponential) for activation and deactivation of
L-glutamate currents (80 mV; n = 8).
The limiting slope for the activation time constants equals 6.8 × 106 M1
sec1.
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The activation rates of the currents were dependent on amino acid
concentration. Figure 5A shows representative patch currents induced by application of varying concentrations of
L-glutamate between 10 µM and 1 mM. An expanded time scale shows the rising phase of the
currents (Fig. 5A). At low concentrations of glutamate, the
activation rate was proportional to concentration, whereas at higher
concentrations, the rate reached a plateau of ~1000 sec1 (Fig. 5B). The limiting slope of
the activation rate was 6.8 × 106
M1 sec1, estimated by
linear regression of the activation rates recorded in response to the
three lowest concentrations of glutamate (Fig. 5B). This
value represents a minimum for the glutamate binding rate constant.
After the rise to peak, currents decayed in the continued presence of
L-glutamate, with more decay observed at higher
concentrations (Fig. 5A). After removal of
L-glutamate, the current deactivated in a
concentration-independent manner (44 ± 7 sec1; n = 4) (Fig.
5B).
Predicted unitary properties of EAAT1 currents
No glutamate-dependent unitary events were seen in patches
containing EAAT1 transporters, precluding a direct analysis of the
properties of single anion channels. Indirect information about the
unitary anion current (i) was therefore obtained from transporter density estimates (Wadiche et al., 1995a) as well as
stationary and nonstationary noise analysis (see below) (Anderson and
Stevens, 1973; Sigworth, 1980). From measurement of the macroscopic current (I) in a patch or cell containing a number of
transporters (N), the product of the open probability
and the unitary current amplitude
(Poi) can be determined because
Poi = I/N.
The number of transporters was estimated by fitting capacitive charge
movements blocked by the nontransported amino acid analog DHK to a
Boltzmann function (Wadiche et al., 1995a). In oocytes expressing
EAAT1, voltage pulses revealed an analogous transient current blocked by high concentrations of DHK (10 mM) (Fig.
6A, inset).
This current was Na-dependent and not seen in uninjected oocytes, and
the DHK-sensitive current-time integrals during the voltage pulse were
equal to the current-time integral after the return to the holding
potential (data not shown) (r = 0.92 ± 0.2;
n = 7). The DHK-sensitive charge movement had an
EC50 of 1.43 ± 0.24 mM, close to the
affinity estimated from Schild analysis of steady-state
L-glutamate currents (data not shown). Finally, the
DHK-sensitive transient current-time integrals were saturable and
obeyed a Boltzmann function with a V0.5 = 12.1 ± 3 mV and slope factor 74.3 ± 2 mV (Fig.
6A). The number of transporters was calculated from
the charge movement measurement using the equation N = Qtotal/eoz
,
where Qtotal represents the total charge
movement blocked by a saturating DHK concentration, eo is the elementary charge (1.6 × 1019 C), and z is the effective
valence of the DHK-sensitive charge movement
[RT/(F * 74.3 mV)]. The average number of
transporters in seven oocytes was 4.9 ± 0.2 × 1011. Based on an oocyte surface area of 2.85 × 107 µm2 (Wadiche et al.,
1995a; Zampighi et al., 1995), this corresponds to an average
transporter density of ~17,000 µm2. This
density is similar to levels of other transport proteins expressed in
Xenopus oocytes (Mager et al., 1993; Wadiche et al., 1995a;
Zampighi et al., 1995; Klamo et al., 1996). Turnover rates for
saturating concentrations (1 mM) of both
D-aspartate and L-glutamate were calculated
using current measurements and transporter density estimates in
individual oocytes assuming the movement of two charges per transport
cycle at ECl, which has been determined
for the EAAT3 (Zerangue and Kavanaugh, 1996a,b) and EAAT1
transporters (A. Zable and M. Kavanaugh, unpublished observations). The
EAAT1 turnover rates were determined to be 4.8 ± 0.4 sec1 and 10.5 ± 1.3 sec1 at 30 mV for D-aspartate and
L-glutamate, respectively (n = 7). From the
voltage dependence of flux (e-fold 89.7 mV), the extrapolated turnover
rates at 80 mV were 7.3 and 16.0 sec1 for
D-aspartate and L-glutamate, respectively.
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Figure 6.
EAAT1 unitary current properties.
A, Semi-log plot of the charge movements for a group of
cells expressing EAAT1 (Qmax = 26.3 ± 1.2 nC). The data were fit to a Boltzmann function with a
V0.5 = 12.1 ± 3 mV and slope factor
74.3 ± 2 mV (z = RT/F * 74.3 = 0.34). The DHK
concentration dependence of the normalized charge movements
(EC50 for DHK block) is 1.43 ± 0.24 mM
(data not shown, n = 4). Inset,
Subtracted current record showing the voltage dependence of transient
currents blocked by 10 mM DHK. Voltage command pulses in 40 mV increments (+120 mV to 160 mV). B, Correlation
of transporter density with the D-aspartate-elicited anion
conductance per unit area (0 mV; , SCN; ,
Cl). The number of transporters was calculated by
dividing the charge blocked because of a saturating concentration of
DHK by the product of the Boltzmann function's effective valance and
the elementary charge (n = Qtotal/eoz = 1.6 × 1019 * 0.34). The anion conductance
in chloride (0 mV) was calculated by first subtracting the
D-aspartate-coupled transport current from the
D-aspartate dependent total current (as in Fig. 1). The
D-aspartate-dependent Cl and
SCN chord conductance per unit area at 0 mV (,
Erev = 22.3 mV; ,
Erev = 79.9 mV, respectively) was then
plotted as a function of transporter density. Linear regression of
these data yielded a slope of 1.37 × 1017
S/transporter and 2.65 × 1016 S/transporter
for Cl and SCN, respectively.
The average membrane area of oocytes was 2.85 × 107 ± 0.14 × 107
µm2 (Wadiche et al., 1995a). C,
Voltage dependence of the unitary current open probability
product (i * Po).
D-Aspartate-dependent currents from outside-out EAAT1
patches were recorded with symmetrical anions [(100 mM
NaSCN + 10 NaCl)out/(100 mM KSCN + 10 mM KCl)in]. The macroscopic current induced by
aspartate (NPoi) was divided
by the number of transporters in each patch based on a chord
conductance (+80 mV) of 1.69 × 1016
S/transporter (see Materials and Methods). The mean number of
transporters in these patches was 5.65 ± 0.37 × 105 transporters (n = 7).
D, Nonstationary noise analysis of EAAT1 currents.
Representative current trace (top) and variance
(bottom) resulting from 500 consecutive 625 msec
applications of 10 mM D-aspartate to an
EAAT1-expressing outside-out patch (0 mV). Middle traces
represent an enlarged 200 msec sub-record before, during, and after
agonist application. Recording solutions are the same as in Figure
4. E, Mean current and variance plot during current
deactivation (same patch as in C). Data were binned into
1000 points for clarity. The line drawn corresponds to the best fit to
the equation:  2 = Ii I2/N + C where N = 473962 and
i = 1.45 fA and C = 0.054 pA2. The number of transporters
(N) was determined as in B
given 2.65 × 1016 S/transporters at 0 mV.
F, Difference of the average spectra in the presence and
absence of 10 mM D-aspartate. Five hundred
sweeps (600 msec each) were acquired at 10 kHz and filtered at 5 kHz.
Same patch as in D.
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The transporter density values were used to estimate the anion
conductance of single channels, assuming a one-to-one correspondence of
transporters and channels. In seven oocytes expressing varying amounts
of EAAT1, the D-aspartate-induced chord conductance at 0 mV
was calculated after subtracting the coupled transport current from the
total current as described above. For each cell, the chord conductance
with external solutions containing either Cl or
SCN was plotted as a function of the number of
transporters. Least squares linear fits yielded slopes of 0.014 and
0.265 fS with Cl and SCN,
respectively (Fig. 6B). These values represent the
product of the unitary conductance and open probability for a single
transporter (Po
). The intrinsic voltage
dependence of the transporter anion conductance was also determined by
recording D-aspartate currents in symmetrical
SCN recording conditions. The
Po product at +80 mV in these conditions was
0.17 fS/transporter. The conductance exhibited a significant inward
rectification, with Po
100/Po+100 = 2.14 ± 0.2 (n = 7) (Fig. 6C).
To estimate the independent quantities Po and
, stationary and nonstationary noise analysis of currents induced by
D-aspartate in outside-out patches was performed. For these
analyses, we used data from patches with high seal resistances (>10
G) that exhibited no endogenous channel activity. Patches were held
at 0 mV and the pipette solutions contained SCN,
whereas the bath solution contained Cl. A
representative response to a 600 msec application of 10 mM D-aspartate to an outside-out patch is shown in Figure
6D. In this patch, the
D-aspartate-induced steady-state current was 12.7 pA at 0 mV, corresponding to a 125.6 pS macroscopic chord conductance (Erev = +101.1 mV). This macroscopic
conductance represents ~474,000 transporters (N = G/Po = 125.6 pS/0.265 fS). With
SCN in the recording pipette solution and
Cl in the bath solution, the current induced by
D-aspartate was consistently accompanied by a small
(~0.02 pA2) but significant increase in current
noise. This noise was not seen with injection of a similar current into
a test resistor (see Materials and Methods). The current induced by
pulses of D-aspartate to a patch held at 0 mV and the
ensemble variance are shown at the bottom of Figure
6D. Assuming that all the channels have a single open
state through which current i passes, and that the channels
open and close independently of each other, the binomial theorem
predicts that the current variance will change according to
I2 = Ii I2/N, where
I2 is the increase in variance
induced by D-aspartate. In seven patches, the transporter
number N was estimated from I, and the unitary
current i was then determined by measurement of the variance increases caused by D-aspartate application. This method
yielded a unitary current estimate of 1.9 ± 0.4 fA
(n = 7). Substituting this value of i into
the equation NPoi = I
results in a probability of channel opening
(Po) of 0.016 ± 0.002. As an
alternative method to investigate the unitary current properties,
nonstationary analysis was used. A plot of the macroscopic current
versus variance during the D-aspartate washout is shown in
Figure 6E. The relationship is approximately linear,
consistent with the probability of channel opening being very low even
at saturating (10 mM) concentrations of
D-aspartate. Fitting the nonstationary variance in patches containing a known number of transporters to
I2 = Ii I2/N + C resulted
in i = 1.2 ± 0.1 fA (Fig. 6E)
(n = 3). This corresponds to an open probability
(Po) of 0.022 ± 0.002 (n = 3). These results are thus consistent with a
unitary SCN conductance between 12 and 19 fS,
corresponding to a unitary Cl conductance between
0.63 and 1.0 fS.
Spectral analysis of the D-aspartate-induced fluctuations
was performed by subtracting the average power spectra of 600 msec control records from records during application of 10 mM
D-aspartate (filtered at 2 KHz and acquired at 5 kHz) (Fig.
6F). The power spectrum of the induced current did
not conform to a single Lorenztian function, unlike that of the
transporter current in salamander photoreceptors (Larsson et al.,
1996), suggesting that the kinetics of the unitary EAAT1 currents are
more complex.
Glutamate-independent conductance
To determine whether the EAAT1 transporter channel could
open in the absence of L-glutamate or
D-aspartate, we examined the action of the nontransported
glutamate analog DHK on background currents in outside-out patches.
With SCN in the recording pipette,
D-aspartate currents were measured with either
Cl or SCN present
extracellularly. As expected for these ionic conditions, the currents
induced by amino acid were inward at potentials up to +60 mV with
extracellular Cl and reversed at 0 mV in
symmetrical SCN solutions (Fig.
7A1,B). In
contrast, application of 10 mM dihydrokainate resulted in a
decrease of a conductance with the same properties as that activated by
D-aspartate (Fig. 7A2,B).
Furthermore, the magnitude of the current blocked by dihydrokainate was
directly proportional to the magnitude of the current induced by
D-aspartate. The conductance decrease at 80 mV
represented 17% of the conductance activated by
D-aspartate (Fig. 7C). These results indicate
that the anion conductance is partially active in the absence of amino acid, similar to conclusions reached by Bergles and Jahr (1997) .
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Figure 7.
Agonist-independent EAAT1 anion currents.
A1, Representative outside-out patch recording
of steady-state D-aspartate (10 mM)-induced
currents from an EAAT1-expressing oocyte. A2,
Record of the DHK blocked current (10 mM) from the same
patch as in A1. Records of both currents were
measured in asymmetrical anion solutions (see below). B,
Current-voltage plots of steady-state difference currents induced or
blocked by application of D-aspartate (10 mM;
filled symbols; n = 4) or DHK (10 mM; open symbols; n = 4). Outside-out patches expressing EAAT1 were recorded in asymmetrical
anionic solutions [(110 mM Cl)out and (100 mM SCN + 10 mM Cl)in].
C, Current-voltage plots (as in B), but
with symmetrical anionic solutions [(100 mM SCN + 10 mM Cl)out and (100 mM SCN + 10 mM Cl)in]. Current amplitude has been
normalized to D-aspartate-dependent currents measured at
100 mV. D, Correlation of the current induced by 10 mM D-aspartate and the current blocked by 10 mM DHK at 80 mV. Squares represent data
obtained in asymmetrical anionic solutions (as in B) and
circles represent data obtained in symmetrical anion
solutions (as in C). The slope of this line is
0.17.
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Channel gating and the transport cycle
A four-state alternating access model with two states
corresponding to open channel states (Larsson et al., 1996) was
initially used to simulate currents observed during applications of
L-glutamate or D-aspartate to outside-out EAAT1
patches. For simplicity, the Na+/H+/Glu
bound states were collapsed in the model (represented as TGlu), and the
K+ binding and countertransport steps were omitted.
It was necessary to add two branching anion conducting states to the
cyclical four-state model to adequately fit the data (Fig.
8A). The two extra
states correspond to open channel states for the liganded and
unliganded transporter. The output of the model is the probability of
channel opening, which is equal to the sum of the probabilities that
the transporter is in one of these two open states. Several parameters were constrained in the model. (1) The ratio of states
Tout and Tin in the
absence of glutamate was fixed to 0.8:0.2 based on the Boltzmann
equilibrium (80 mV), which suggests that 80% of the transporters are
bound with sodium and ready to bind glutamate (Fig.
6A). (2) The turnover rate (
) was assigned to the
rate constant k1. (3) The glutamate-independent probability of
channel opening was constrained to be 0.17 of the probability of
channel opening in saturating glutamate (Fig. 7C). (4) The
binding rate constant of amino acid was fixed to 6.8 × 106 M1
sec1. The remaining free parameters in the kinetic
model were allowed to vary, and the output of the model was fitted by
least squares to patch data representing normalized average responses
to D-aspartate or L-glutamate.
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Figure 8.
Computer simulation. A, Kinetic
model of L-glutamate and D-aspartate transport
and anion conductance. Model parameters were obtained by least squares
fitting of data and fit an average pulse of L-glutamate of
D-aspartate. The microscopic rates were as follows:
konout = 6.8 × 106 M1
sec1,
koffout = 30.6 sec1; k1 = 16.0 sec1; k1 = 2.9 sec1;
konin = 6.8 × 106 M1
sec1;
koffin = 37.2 sec1; k2 = 885 sec1; k2 = 200 sec1;  1 = 8094 sec1;  1 = 100 sec1; 2 = 1260 sec1; 2 = 70 sec1. D-Aspartate data were fit with
identical rates for agonist independent states and
koffout = 7.6 sec1; k1 = 7.3 sec1; k1 = 1.0 sec1;
koffin = 165 sec1; 2 = 978 sec1, and 2 = 70 sec1. DHK binding was assigned as 6.8 × 106 M1
sec1, whereas DHK unbinding
(kdhkout) = 97 sec1.
B, Probability of occupancy for each state in the
kinetic scheme shown in A during a 250 msec pulse of 10 mM L-glutamate. The top traces show
the nonconducting states: the unliganded states are represented by
dashed lines (Tout and
Tin; bold), whereas the liganded
states are represented by a solid line
(ToutGlu; bold and
TinGlu). The bottom traces show the
occupancy of the anion conducting states. Note the different scale
bars. C, Simulation of a 250 msec pulse of 10 mM L-glutamate or D-aspartate
(A). The channel's steady-state open probability
was determined from nonstationary noise analysis (Fig. 4) and the
DHK-blocked currents (Fig. 6). The fraction of transporters in either
conducting state TGluopen or
Topen are plotted as a function of time.
D, Concentration dependence of the time constant for
activation of L-glutamate currents for the kinetic scheme
shown in A. The time constants for the activation and
deactivation were calculated by fitting the current records to a single
exponential. The limiting slope for the activation rate equals 6.8 × 106 M1
sec1. Inset,
L-Glutamate concentration dependence of the open
probability (1 µM, 10 µM, 100 µM, and 1 mM). The model's apparent affinity
at steady state is 7 µM.
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A summary of the kinetic parameters of the simulated and experimental
data is given in Table 2, and the output
of the simulation for a pulse of L-glutamate or
D-aspartate is shown in Figure 8B. The
principal macroscopic kinetic features of the simulated currents and
their amino acid dependence were similar to the experimental data. The
open channel probability increases rapidly (activation = 0.9 msec) in response to a high concentration of
L-glutamate (10 mM). Lower
L-glutamate concentrations (1-30 µM) elicit
currents that rise more slowly, with less inactivation during the
agonist pulse (Fig. 8C). A plot of the time constant of
activation as a function of the concentration of
L-glutamate results in a relationship similar to
experimental results presented in Figure 7B. Linear regression yielded a limiting slope at low L-glutamate
concentrations of 6.8 × 106
M1 sec1, the same value
as the model's association rate constant for L-glutamate.
This suggests that for this kinetic scheme the rising rate of the
current at low agonist concentrations is a good approximation of the
binding rate of L-glutamate.
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DISCUSSION |
The glutamate transporter anion channel
Data have accumulated showing that glutamate transport activates
an anion conductance both in situ (Grant and Dowling, 1995; Picaud et al., 1995b; Billups et al., 1996; Eliasof and Jahr, 1996;
Bergles and Jahr, 1997; Bergles et al., 1997; Otis et al., 1997) and in
exogenous expression systems (Fairman et al., 1995; Wadiche et al.,
1995a,b). There is an important distinction between the
flux of chloride, which is not stoichiometrically coupled to flux of glutamate (Wadiche et al., 1995b; Billups et al., 1996), and
the fluxes of sodium, potassium, and protons, which are tightly coupled
to glutamate flux (Kanner and Sharon, 1978; Stallcup et al., 1979;
Erecinska et al., 1983; Nelson et al., 1983; Zerangue and Kavanaugh,
1996a). The glutamate transporter-associated flux of anions
occurs through a pathway that is gated and selective, hallmarks of ion
channel permeation. The relative permeabilities of different anions
exhibited a remarkably wide range but fit well with Eisenman's first
anion selectivity sequence (Eisenman, 1965) (Table 1). The
channel pore diameter was at least 5 Å. No evidence was found for
multiple occupancy or interaction between anions in the pore; the anion
concentration dependence of the channel conductance and lack of
anomalous mole fraction behavior are consistent with the permeating
anion binding to a single site (Fig. 2). In addition, the sequence of
permeabilities measured by reversal potentials was the same as the
sequence of relative conductances. The conductance in the absence of
glutamate (Fig. 7) demonstrates that channel gating and ion selectivity
do not require the amino acid substrate (Bergles and Jahr, 1997).
A critical property distinguishing flux of glutamate and chloride was
temperature dependence (Fig. 2A,B). In general, flux through a channel is relatively insensitive to temperature, with Q10 values typically <1.5, because of the
low-energy barriers associated with ionic diffusion (Hodgkin et al.,
1952; Miller, 1987; Hille, 1992). The Q10 value
for the chloride current (~1) is consistent with such a mechanism,
whereas the Q10 for the coupled uptake current
(~3) is consistent instead with energy requirements for large
conformational transitions that occur during each transport cycle
(Grunewald and Kanner, 1995).
Application of D-aspartate or L-glutamate
to the intracellular surface of patches containing tr
Top
Abstract
Introduction
