 |
 Previous Article | Next Article 
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
 |
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
| |
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.
| |
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.
| |
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.
View larger version (34K):
[in this window]
[in a new window]
|
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).
|
|
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.
View larger version (21K):
[in this window]
[in a new window]
|
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).
|
|
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.
View larger version (32K):
[in this window]
[in a new window]
|
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).
|
|
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).
View larger version (21K):
[in this window]
[in a new window]
|
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).
|
|
View larger version (17K):
[in this window]
[in a new window]
|
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.
|
|
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.
View larger version (34K):
[in this window]
[in a new window]
|
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.
|
|
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) .
View larger version (21K):
[in this window]
[in a new window]
|
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.
|
|
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.
View larger version (22K):
[in this window]
[in a new window]
|
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.
|
|
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.
| |
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 transporters show
that the anion current can be activated by reverse transport [also see
Billups et al. (1996); Kavanaugh et al. (1997)]. The apparent affinity of EAAT1 for D-aspartate on the internal side of the
membrane was ~10-fold lower than at the external binding site with
the same ionic conditions. Activation of the anion current by
intracellular substrate was dependent on the trans cations
present (Fig. 3C). Reverse transport currents were seen when
K+ but not choline was present as the external
cation. A small but significant current was also observed when
K+ was substituted by Na+.
Predicted unitary properties of the anion channel
Evidence that glutamate transporter substrates induce
channel-like current fluctuations (Tachibana and Kaneko, 1988; Picaud et al., 1995b; Larsson et al., 1996) suggests that the
Cl conductance is stochastically gated. Unitary
events and current noise have also been associated with monoamine
transporters (Cammack and Schwartz, 1996; Galli et al., 1996; Lin et
al., 1996). No glutamate-dependent unitary events were directly
resolved in membrane patches containing EAAT1 transporters. Noise
analysis of glutamate transporter currents in photoreceptors indicates
that glutamate activates a channel with a unitary conductance of
0.5-0.7 pS (Tachibana and Kaneko, 1988; Picaud et al., 1995b; Larsson
et al., 1996). However, glutamate induced much less current noise in
EAAT1 patches. Stationary and nonstationary analysis were consistent
with a chloride channel conductance approximately three orders of
magnitude smaller than predicted in photoreceptors. The reason for this
difference is unclear, but it may be attributable to differences
between EAAT1 and the excitatory amino acid transporter subtypes found in photoreceptors. Although all cloned glutamate transporters examined
to date mediate Cl flux, its magnitude relative to
glutamate flux varies (Wadiche et al., 1995b; Eliasof et al., 1998). In
oocytes expressing EAAT5, a transporter that appears to be abundantly
expressed in retinal photoreceptor terminals, glutamate activates
currents carried predominantly by chloride (Arriza et al., 1997;
Eliasof et al., 1998). The unitary properties of the cloned EAAT5
transporter expressed in oocytes have not yet been examined, but its
conductance may be much larger than that of EAAT1. It is also possible
that the open time of the EAAT1 channel is much briefer, resulting in
an underestimate of the conductance caused by bandwidth limitation.
Transporter and channel kinetics
When outside-out patches of excised membranes were exposed to
rapid pulses of glutamate, anion currents activated rapidly and
partially inactivated during the glutamate pulse (Fig. 4) [also see
Otis et al. (1997); Bergles et al. (1997); Otis and Jahr
(1998)]. In the context of a cyclic transport scheme such as
the Na-K pump, transient currents induced by concentration jumps are
presumed to reflect early charge translocating steps in the cycle
(Borlinghaus et al., 1987). The EAAT1 current observed after a
glutamate pulse indicates that channel activation occurs soon after a
rapid glutamate binding step. The cyclic kinetic model predicts a
nearly synchronous channel opening followed by relaxation (or
desynchronization) to a steady-state distribution of open and closed
states. The rate constants in the transport cycle, together with the
rates leading into and out of the open channel states, determine the
kinetics of the current after a concentration jump. There was a twofold
difference in turnover rates between L-glutamate and
D-aspartate, and striking differences in macroscopic anion
current kinetics were observed. Because L-glutamate and
D-aspartate are transported at different rates, it may be inferred that the rate-limiting step in transport is an amino acid-bound state transition. We tentatively assign this step to a
gating event involved in amino acid translocation across the membrane.
The simulation of a cyclical transport scheme with branching open
channel states demonstrates how the difference in transport rates can
account for differences in anion current activation and deactivation as
well as differences in peak/steady-state anion current ratios (Fig. 8).
Furthermore, the model predicts that at very low concentrations of
amino acid, the activation rate of the anion current reflects the
binding rate of the amino acid to the transporter (Figs. 5B,
8D). At synaptic time scales (Clements et al., 1992),
transporter density and glutamate binding rates will be critical
determinants of the transporters' roles in buffering synaptically
released glutamate (Diamond and Jahr, 1997). The experimentally
measured limiting slope of the anion current activation rate by low
concentrations of glutamate was 6.8 × 106
M1 sec1. This rate is
similar to the estimated rate of glutamate binding to AMPA receptors
(Jonas et al., 1993). The predicted glutamate unbinding rate at 25°C
leads to the somewhat paradoxical result that after binding to the
transporter, a molecule of glutamate has a significantly higher
probability of unbinding than of being transported
(Punbind/Ptransport > 30.6 sec1/16 sec1) (Fig.
8, legend). This suggests that a molecule of glutamate may bind to and
unbind from multiple transporters, and perhaps receptors, before being
removed from the extracellular space. In some conditions, synaptically
released glutamate can diffuse away from the cleft and activate
presynaptic receptors (Scanziani et al., 1997). Furthermore,
temperature effects on transport appear to influence the extent to
which glutamate can diffuse to neighboring synapses (Asztely et al.,
1997). If a significant fraction of released glutamate is bound by
transporters near the synaptic cleft (Otis et al., 1997), an increase
in temperature may reduce the escape of glutamate primarily by
decreasing the relative probability of unbinding before transport.
In summary, transporter currents recorded in patches are consistent
with the occurrence of rapid state transitions, including glutamate
binding and channel opening, within a relatively slow overall transport
cycle. Further work to precisely correlate open channel states to
states in the transport cycle will provide a powerful technique for
monitoring transporter function with high time resolution.
| |
FOOTNOTES |
Received May 11, 1998; revised July 6, 1998; accepted July 16, 1998.
This work was supported by National Institutes of Health. We
thank D. Bergles, J. Diamond, J. Dzubay, S. Eliasof, and M. Jones for
discussions. We thank T. Otis and C. Jahr for discussions and sharing
unpublished data, and J. Arriza and S. Amara for the EAAT1 cDNA.
Correspondence should be addressed to Michael Kavanaugh, Vollum
Institute, L-474, Oregon Health Sciences University, Portland, OR
97201.
| |
REFERENCES |
-
Anderson CR,
Stevens CF
(1973)
Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction.
J Physiol (Lond)
235:655-691[Abstract/Free Full Text].
-
Arriza JL,
Kavanaugh MP,
Fairman WA,
Wu YN,
Murdoch GH,
North RA,
Amara SG
(1993)
Cloning and expression of a human neutral amino acid transporter with structural similarity to the glutamate transporter gene family.
J Biol Chem
268:15329-15332[Abstract/Free Full Text].
-
Arriza JL,
Eliasof S,
Kavanaugh MP,
Amara SG
(1997)
Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance.
Proc Natl Acad Sci USA
94:4155-4160[Abstract/Free Full Text].
-
Asztely F,
Erdemli G,
Kullmann DM
(1997)
Extrasynaptic glutamate spillover in the hippocampus: dependence on temperature and the role of active glutamate uptake.
Neuron
18:281-293[Web of Science][Medline].
-
Barbour B,
Brew H,
Attwell D
(1988)
Electrogenic glutamate uptake in glial cells is activated by intracellular potassium.
Nature
335:433-435[Medline].
-
Bergles DE,
Jahr CE
(1997)
Synaptic activation of glutamate transporters in hippocampal astrocytes.
Neuron
19:1297-1308[Web of Science][Medline].
-
Bergles DE,
Dzubay JA,
Jahr CE
(1997)
Glutamate transporter currents in bergmann glial cells follow the time course of extrasynaptic glutamate.
Proc Natl Acad Sci USA
94:14821-14825[Abstract/Free Full Text].
-
Billups B,
Rossi D,
Attwell D
(1996)
Anion conductance behavior of the glutamate uptake carrier in salamander retinal glial cells.
J Neurosci
16:6722-6731[Abstract/Free Full Text].
-
Borlinghaus R,
Apell HJ,
Läuger P
(1987)
Fast charge translocations associated with partial reactions of the Na,K-pump. I. Current and voltage transients after photochemical release of ATP.
J Membr Biol
97:161-178[Web of Science][Medline].
-
Cammack JN,
Schwartz EA
(1996)
Channel behavior in a -aminobutyrate transporter.
Proc Natl Acad Sci USA
93:723-727[Abstract/Free Full Text].
-
Clements JD,
Lester RA,
Tong G,
Jahr CE,
Westbrook GL
(1992)
The time course of glutamate in the synaptic cleft.
Science
258:1498-1501[Abstract/Free Full Text].
-
Diamond JS,
Jahr CE
(1997)
Transporters buffer synaptically released glutamate on a submillisecond time scale.
J Neurosci
17:4672-4687[Abstract/Free Full Text].
-
Eisenman G (1965) Some elementary factors involved in
specific ion permeation. In: International Congress of Physical
Science, pp 489-506. Tokyo.
-
Eliasof S,
Jahr CE
(1996)
Retinal glial cell glutamate transporter is coupled to an anionic conductance.
Proc Natl Acad Sci USA
93:4153-4158[Abstract/Free Full Text].
-
Eliasof S,
Arriza JL,
Leighton BH,
Kavanaugh MP,
Amara SG
(1998)
Excitatory amino acid transporters of the salamander retina: identification, localization, and function.
J Neurosci
18:698-712[Abstract/Free Full Text].
-
Erecinska M,
Wantorsky D,
Wilson DF
(1983)
Aspartate transport in synaptosomes from rat brain.
J Biol Chem
258:9069-9077[Abstract/Free Full Text].
-
Fairman WA,
Vandenberg RJ,
Arriza JL,
Kavanaugh MP,
Amara SG
(1995)
An excitatory amino-acid transporter with properties of a ligand-gated chloride channel.
Nature
375:599-603[Medline].
-
Galli A,
Blakely RD,
DeFelice LJ
(1996)
Norepinephrine transporters have channel modes of conduction.
Proc Natl Acad Sci USA
93:8671-8676[Abstract/Free Full Text].
-
Grant GB,
Dowling JE
(1995)
A glutamate-activated chloride current in cone-driven ON bipolar cells of the white perch retina.
J Neurosci
15:3852-3862[Abstract].
-
Grunewald M,
Kanner B
(1995)
Conformational changes monitored on the glutamate transporter GLT-1 indicate the existence of two neurotransmitter-bound states.
J Biol Chem
270:17017-17024[Abstract/Free Full Text].
-
Halm DR,
Frizzell RA
(1992)
Anion permeation in an apical membrane chloride channel of a secretory epithelial cell.
J Gen Physiol
99:339-366[Abstract/Free Full Text].
-
Hille B
(1992)
In: Ionic channels of excitable membranes, Ed 2. Sunderland, MA: Sinauer.
-
Hodgkin AL,
Huxley AF,
Katz B
(1952)
Measurement of current-voltage relations in the membrane of the giant axon of Loligo.
J Physiol (Lond)
116:424-448.
-
Jonas P,
Major G,
Sakmann B
(1993)
Quantal components of unitary EPSCs at the mossy fibre synapse on CA3 pyramidal cells of rat hippocampus.
J Physiol (Lond)
472:615-663[Abstract/Free Full Text].
-
Kanner BI,
Sharon I
(1978)
Active transport of L-glutamate by membrane vesicles isolated from rat brain.
Biochemistry
17:3949-3953[Medline].
-
Kavanaugh MP
(1993)
Voltage dependence of facilitated arginine flux mediated by the system y+ basic amino acid transporter.
Biochemistry
32:5781-5785[Medline].
-
Kavanaugh MP,
Bendahan A,
Zerangue N,
Zhang Y,
Kanner BI
(1997)
Mutation of an amino acid residue influencing potassium coupling in the glutamate transporter GLT-1 induces obligate exchange.
J Biol Chem
272:1703-1708[Abstract/Free Full Text].
-
Klamo EM,
Drew ME,
Landfear SM,
Kavanaugh MP
(1996)
Kinetics and stoichiometry of a proton/myo-inositol cotransporter.
J Biol Chem
271:14937-14943[Abstract/Free Full Text].
-
Larsson HP,
Picaud SA,
Werblin FS,
Lecar H
(1996)
Noise analysis of the glutamate-activated current in photoreceptors.
Biophys J
70:733-742[Web of Science][Medline].
-
Läuger P
(1991)
In: Electrogenic ion pumps. Sunderland, MA: Sinauer.
-
Lehre KP,
Levy LM,
Ottersen OP,
Storm-Mathisen J,
Danbolt NC
(1995)
Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations.
J Neurosci
15:1835-1853[Abstract].
-
Lin F,
Lester HA,
Mager S
(1996)
Single channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation.
Biophys J
71:3126-3135[Web of Science][Medline].
-
Maconochie DJ,
Knight DE
(1989)
A method for making solution changes in the sub-millisecond range at the tip of a patch pipette.
Pflügers Arch
414:589-596[Web of Science][Medline].
-
Mager S,
Naeve J,
Quick M,
Labarca C,
Davidson N,
Lester HA
(1993)
Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes.
Neuron
10:177-188[Web of Science][Medline].
-
Malandro MS,
Kilberg MS
(1996)
Molecular biology of mammalian amino acid transporters.
Annu Rev Biochem
65:305-336[Web of Science][Medline].
-
Miller C
(1987)
How ion channel proteins work.
In: Neuromodulation (Kaczmarek LK,
Levitan IB,
eds), pp 39-61. New York: Oxford.
-
Nelson PJ,
Dean GE,
Aronson PS,
Rudnick G
(1983)
Hydrogen ion cotransport by the renal brush border glutamate transporter.
Biochemistry
22:5459-5463[Medline].
-
Otis TS, Jahr CE (1998) Anion currents and predicted
glutamate flux through a neuronal glutamate transporter. J Neurosci, in
press.
-
Otis TS,
Kavanaugh MP,
Jahr CE
(1997)
Postsynaptic glutamate transport at the climbing fiber-Purkinje cell synapse.
Science
277:1515-1518[Abstract/Free Full Text].
-
Picaud SA,
Larsson HP,
Wellis DP,
Lecar H,
Werblin FS
(1995a)
Cone photoreceptors respond to their own glutamate release in the tiger salamander.
Proc Natl Acad Sci USA
92:9417-9421[Abstract/Free Full Text].
-
Picaud SA,
Larsson HP,
Grant GB,
Lecar H,
Werblin FS
(1995b)
Glutamate-gated chloride channel with glutamate transporter-like properties in cone photoreceptors of the tiger salamander.
J Neurophysiol
74:1760-1771[Abstract/Free Full Text].
-
Rothstein JD,
Martin L,
Levey AI,
Dykes-Hoberg M,
Jin L,
Wu D,
Nash N,
Kuncl RW
(1994)
Localization of neuronal and glial glutamate transporters.
Neuron
13:713-725[Web of Science][Medline].
-
Sarantis M,
Everett K,
Attwell D
(1988)
A presynaptic action of glutamate at the cone output synapse.
Nature
332:451-453[Medline].
-
Scanziani M,
Salin PA,
Vogt KE,
Malenka RC,
Nicoll RA
(1997)
Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors.
Nature
385:630-634[Medline].
-
Sigworth FJ
(1980)
The variance of sodium current fluctuations at the node of ranvier.
J Physiol (Lond)
307:97-129[Abstract/Free Full Text].
-
Stallcup WB,
Bulloch K,
Baetge EE
(1979)
Coupled transport of glutamate and sodium in a cerebellar nerve cell line.
J Neurochem
32:57-65[Web of Science][Medline].
-
Szatkowski M,
Barbour B,
Attwell D
(1990)
Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake.
Nature
348:443-446[Medline].
-
Tachibana M,
Kaneko A
(1988)
L-Glutamate-induced depolarization in solitary photoreceptors: a process that may contribute to the interaction between photoreceptors in situ.
Proc Natl Acad Sci USA
85:5315-5319[Abstract/Free Full Text].
-
Wadiche JI,
Arriza JL,
Amara SG,
Kavanaugh MP
(1995a)
Kinetics of a human glutamate transporter.
Neuron
14:1019-1027[Web of Science][Medline].
-
Wadiche JI,
Amara SG,
Kavanaugh MP
(1995b)
Ion fluxes associated with excitatory amino acid transport.
Neuron
15:721-728[Web of Science][Medline].
-
Yamada K,
Watanabe M,
Shibata T,
Tanaka K,
Wada K,
Inoue Y
(1996)
EAAT4 is a post-synaptic glutamate transporter at Purkinje cell synapses.
NeuroReport
7:2013-2017[Web of Science][Medline].
-
Zampighi GA,
Kreman M,
Boorer KJ,
Loo DD,
Bezanilla F,
Chandy G,
Hall JE,
Wright EM
(1995)
A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes.
J Membr Biol
148:65-78[Web of Science][Medline].
-
Zerangue N,
Kavanaugh MP
(1996a)
Flux coupling in a neuronal glutamate transporter.
Nature
383:634-637[Medline].
-
Zerangue N,
Kavanaugh MP
(1996b)
ASCT-1 is a neutral amino acid exchanger with chloride channel activity.
J Biol Chem
271:27991-27994[Abstract/Free Full Text].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18197650-12$05.00/0
This article has been cited by other articles:
|
|
|
|
 
S. Broer, H.-P. Schneider, A. Broer, and J. W. Deitmer
Mutation of Asparagine 76 in the Center of Glutamine Transporter SNAT3 Modulates Substrate-induced Conductances and Na+ Binding
J. Biol. Chem.,
September 18, 2009;
284(38):
25823 - 25831.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Teichman, S. Qu, and B. I. Kanner
The equivalent of a thallium binding residue from an archeal homolog controls cation interactions in brain glutamate transporters
PNAS,
August 25, 2009;
106(34):
14297 - 14302.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. M. Ryan, E. L. R. Compton, and J. A. Mindell
Functional Characterization of a Na+-dependent Aspartate Transporter from Pyrococcus horikoshii
J. Biol. Chem.,
June 26, 2009;
284(26):
17540 - 17548.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. C Holley and M. P Kavanaugh
Interactions of alkali cations with glutamate transporters
Phil Trans R Soc B,
January 27, 2009;
364(1514):
155 - 161.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-H. Kim, S. Uehara, A. Muroyama, B. Hille, Y. Moriyama, and D.-S. Koh
Glutamate Transporter-Mediated Glutamate Secretion in the Mammalian Pineal Gland
J. Neurosci.,
October 22, 2008;
28(43):
10852 - 10863.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Torres-Salazar and C. Fahlke
Neuronal Glutamate Transporters Vary in Substrate Transport Rate but Not in Unitary Anion Channel Conductance
J. Biol. Chem.,
November 30, 2007;
282(48):
34719 - 34726.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Zhang, Z. Tao, A. Gameiro, S. Barcelona, S. Braams, T. Rauen, and C. Grewer
Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1
PNAS,
November 13, 2007;
104(46):
18025 - 18030.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. P. Koch, J. M. Hubbard, and H. P. Larsson
Voltage-independent Sodium-binding Events Reported by the 4B-4C Loop in the Human Glutamate Transporter Excitatory Amino Acid Transporter 3
J. Biol. Chem.,
August 24, 2007;
282(34):
24547 - 24553.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Weerachayaphorn and A. M. Pajor
Sodium-dependent Extracellular Accessibility of Lys-84 in the Sodium/Dicarboxylate Cotransporter
J. Biol. Chem.,
July 13, 2007;
282(28):
20213 - 20220.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Teichman and B. I. Kanner
Aspartate-444 Is Essential for Productive Substrate Interactions in a Neuronal Glutamate Transporter
J. Gen. Physiol.,
June 1, 2007;
129(6):
527 - 539.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Lisk, S. Scott, T. Solomon, A. D. Pillai, and S. A. Desai
Solute-Inhibitor Interactions in the Plasmodial Surface Anion Channel Reveal Complexities in the Transport Process
Mol. Pharmacol.,
May 1, 2007;
71(5):
1241 - 1250.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Dallas, H. E. Boycott, L. Atkinson, A. Miller, J. P. Boyle, H. A. Pearson, and C. Peers
Hypoxia Suppresses Glutamate Transport in Astrocytes
J. Neurosci.,
April 11, 2007;
27(15):
3946 - 3955.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Tao and C. Grewer
Cooperation of the Conserved Aspartate 439 and Bound Amino Acid Substrate Is Important for High-Affinity Na+ Binding to the Glutamate Transporter EAAC1
J. Gen. Physiol.,
March 26, 2007;
129(4):
331 - 344.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. P. Leary, E. F. Stone, D. C. Holley, and M. P. Kavanaugh
The Glutamate and Chloride Permeation Pathways Are Colocalized in Individual Neuronal Glutamate Transporter Subunits
J. Neurosci.,
March 14, 2007;
27(11):
2938 - 2942.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. P. Koch, R. Lane Brown, and H. P. Larsson
The Glutamate-Activated Anion Conductance in Excitatory Amino Acid Transporters Is Gated Independently by the Individual Subunits
J. Neurosci.,
March 14, 2007;
27(11):
2943 - 2947.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H.-P. Schneider, S. Broer, A. Broer, and J. W. Deitmer
Heterologous Expression of the Glutamine Transporter SNAT3 in Xenopus Oocytes Is Associated with Four Modes of Uncoupled Transport
J. Biol. Chem.,
February 9, 2007;
282(6):
3788 - 3798.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. G. Owe, P. Marcaggi, and D. Attwell
The ionic stoichiometry of the GLAST glutamate transporter in salamander retinal glia
J. Physiol.,
December 1, 2006;
577(2):
591 - 599.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Rosental, A. Bendahan, and B. I. Kanner
Multiple Consequences of Mutating Two Conserved beta-Bridge Forming Residues in the Translocation Cycle of a Neuronal Glutamate Transporter
J. Biol. Chem.,
September 22, 2006;
281(38):
27905 - 27915.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Glowatzki, N. Cheng, H. Hiel, E. Yi, K. Tanaka, G. C. R. Ellis-Davies, J. D. Rothstein, and D. E. Bergles
The glutamate-aspartate transporter GLAST mediates glutamate uptake at inner hair cell afferent synapses in the mammalian cochlea.
J. Neurosci.,
July 19, 2006;
26(29):
7659 - 7664.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Torres-Salazar and C. Fahlke
Intersubunit interactions in EAAT4 glutamate transporters.
J. Neurosci.,
July 12, 2006;
26(28):
7513 - 7522.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Tao, Z. Zhang, and C. Grewer
Neutralization of the Aspartic Acid Residue Asp-367, but Not Asp-454, Inhibits Binding of Na+ to the Glutamate-free Form and Cycling of the Glutamate Transporter EAAC1
J. Biol. Chem.,
April 14, 2006;
281(15):
10263 - 10272.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. I. Wadiche, A. V. Tzingounis, and C. E. Jahr
Intrinsic kinetics determine the time course of neuronal synaptic transporter currents
PNAS,
January 24, 2006;
103(4):
1083 - 1087.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Linsdell
Mechanism of chloride permeation in the cystic fibrosis transmembrane conductance regulator chloride channel
Exp Physiol,
January 1, 2006;
91(1):
123 - 129.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Mim, P. Balani, T. Rauen, and C. Grewer
The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism
J. Gen. Physiol.,
November 28, 2005;
126(6):
571 - 589.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Oshiro and A. M. Pajor
Functional characterization of high-affinity Na+/dicarboxylate cotransporter found in Xenopus laevis kidney and heart
Am J Physiol Cell Physiol,
November 1, 2005;
289(5):
C1159 - C1168.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Obermeyer and S. D. Tyerman
NH4+ Currents across the Peribacteroid Membrane of Soybean. Macroscopic and Microscopic Properties, Inhibition by Mg2+, and Temperature Dependence Indicate a SubpicoSiemens Channel Finely Regulated by Divalent Cations
Plant Physiology,
October 1, 2005;
139(2):
1015 - 1029.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. I. Melendez, J. Vuthiganon, and P. W. Kalivas
Regulation of Extracellular Glutamate in the Prefrontal Cortex: Focus on the Cystine Glutamate Exchanger and Group I Metabotropic Glutamate Receptors
J. Pharmacol. Exp. Ther.,
July 1, 2005;
314(1):
139 - 147.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. S. Diamond
Deriving the Glutamate Clearance Time Course from Transporter Currents in CA1 Hippocampal Astrocytes: Transmitter Uptake Gets Faster during Development
J. Neurosci.,
March 16, 2005;
25(11):
2906 - 2916.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Ma, R. Kotaria, J. A. Mayor, S. Remani, D. E. Walters, and R. S. Kaplan
The Yeast Mitochondrial Citrate Transport Protein: CHARACTERIZATION OF TRANSMEMBRANE DOMAIN III RESIDUE INVOLVEMENT IN SUBSTRATE TRANSLOCATION
J. Biol. Chem.,
January 21, 2005;
280(3):
2331 - 2340.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Carvelli, P. W. McDonald, R. D. Blakely, and L. J. DeFelice
Dopamine transporters depolarize neurons by a channel mechanism
PNAS,
November 9, 2004;
101(45):
16046 - 16051.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Funicello, P. Conti, M. De Amici, C. De Micheli, T. Mennini, and M. Gobbi
Dissociation of [3H]L-Glutamate Uptake from L-Glutamate-Induced [3H]D-Aspartate release by 3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic Acid and 3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic Acid, Two Conformationally Constrained Aspartate and Glutamate Analogs
Mol. Pharmacol.,
September 1, 2004;
66(3):
522 - 529.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Grewer and E. Grabsch
New inhibitors for the neutral amino acid transporter ASCT2 reveal its Na+-dependent anion leak
J. Physiol.,
June 15, 2004;
557(3):
747 - 759.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. M. Ryan, A. D. Mitrovic, and R. J. Vandenberg
The Chloride Permeation Pathway of a Glutamate Transporter and Its Proximity to the Glutamate Translocation Pathway
J. Biol. Chem.,
May 14, 2004;
279(20):
20742 - 20751.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Brasnjo and T. S. Otis
Isolation of glutamate transport-coupled charge flux and estimation of glutamate uptake at the climbing fiber-Purkinje cell synapse
PNAS,
April 20, 2004;
101(16):
6273 - 6278.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. P. Larsson, A. V. Tzingounis, H. P. Koch, and M. P. Kavanaugh
Fluorometric measurements of conformational changes in glutamate transporters
PNAS,
March 16, 2004;
101(11):
3951 - 3956.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Borre and B. I. Kanner
Arginine 445 Controls the Coupling between Glutamate and Cations in the Neuronal Transporter EAAC-1
J. Biol. Chem.,
January 23, 2004;
279(4):
2513 - 2519.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Melzer, A. Biela, and C. Fahlke
Glutamate Modifies Ion Conduction and Voltage-dependent Gating of Excitatory Amino Acid Transporter-associated Anion Channels
J. Biol. Chem.,
December 12, 2003;
278(50):
50112 - 50119.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Wei, N. Zhang, Z. Peng, C. R. Houser, and I. Mody
Perisynaptic Localization of {delta} Subunit-Containing GABAA Receptors and Their Activation by GABA Spillover in the Mouse Dentate Gyrus
J. Neurosci.,
November 19, 2003;
23(33):
10650 - 10661.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Umesh, B. N. Cohen, L. S. Ross, and S. S. Gill
Functional characterization of a glutamate/aspartate transporter from the mosquito Aedes aegypti
J. Exp. Biol.,
July 1, 2003;
206(13):
2241 - 2255.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Palmer, H. Taschenberger, C. Hull, L. Tremere, and H. von Gersdorff
Synaptic Activation of Presynaptic Glutamate Transporter Currents in Nerve Terminals
J. Neurosci.,
June 15, 2003;
23(12):
4831 - 4841.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. C. Mathews and J. S. Diamond
Neuronal Glutamate Uptake Contributes to GABA Synthesis and Inhibitory Synaptic Strength
J. Neurosci.,
March 15, 2003;
23(6):
2040 - 2048.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. E. Bergles, A. V. Tzingounis, and C. E. Jahr
Comparison of Coupled and Uncoupled Currents during Glutamate Uptake by GLT-1 Transporters
J. Neurosci.,
December 1, 2002;
22(23):
10153 - 10162.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. H. Leighton, R. P. Seal, K. Shimamoto, and S. G. Amara
A Hydrophobic Domain in Glutamate Transporters Forms an Extracellular Helix Associated with the Permeation Pathway for Substrates
J. Biol. Chem.,
August 9, 2002;
277(33):
29847 - 29855.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. MacAulay, U. Gether, D. A Klaerke, and T. Zeuthen
Passive water and urea permeability of a human Na+-glutamate cotransporter expressed in Xenopus oocytes
J. Physiol.,
August 1, 2002;
542(3):
817 - 828.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. A. Clark and S. G. Cull-Candy
Activity-Dependent Recruitment of Extrasynaptic NMDA Receptor Activation at an AMPA Receptor-Only Synapse
J. Neurosci.,
June 1, 2002;
22(11):
4428 - 4436.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. M. Ryan and R. J. Vandenberg
Distinct Conformational States Mediate the Transport and Anion Channel Properties of the Glutamate Transporter EAAT-1
J. Biol. Chem.,
April 12, 2002;
277(16):
13494 - 13500.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Borre, M. P. Kavanaugh, and B. I. Kanner
Dynamic Equilibrium between Coupled and Uncoupled Modes of a Neuronal Glutamate Transporter
J. Biol. Chem.,
April 12, 2002;
277(16):
13501 - 13507.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-S. Yuan, L. Dey, J.-T. Xie, and H. H. Aung
Pharmacology and toxicology of astrocyte-neuron glutamate transport and cycling.
J. Pharmacol. Exp. Ther.,
April 1, 2002;
301(1):
1 - 6.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Brocke, A. Bendahan, M. Grunewald, and B. I. Kanner
Proximity of Two Oppositely Oriented Reentrant Loops in the Glutamate Transporter GLT-1 Identified by Paired Cysteine Mutagenesis
J. Biol. Chem.,
February 1, 2002;
277(6):
3985 - 3992.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. P. Seal, Y. Shigeri, S. Eliasof, B. H. Leighton, and S. G. Amara
Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance
PNAS,
December 18, 2001;
98(26):
15324 - 15329.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Lopez-Corcuera, E. Nunez, R. Martinez-Maza, A. Geerlings, and C. Aragon
Substrate-induced Conformational Changes of Extracellular Loop 1 in the Glycine Transporter GLYT2
J. Biol. Chem.,
November 9, 2001;
276(46):
43463 - 43470.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Zhu, J. S. Duerr, H. Varoqui, J. R. McManus, J. B. Rand, and J. D. Erickson
Analysis of Point Mutants in the Caenorhabditis elegans Vesicular Acetylcholine Transporter Reveals Domains Involved in Substrate Translocation
J. Biol. Chem.,
November 2, 2001;
276(45):
41580 - 41587.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. S. Diamond
Neuronal Glutamate Transporters Limit Activation of NMDA Receptors by Neurotransmitter Spillover on CA1 Pyramidal Cells
J. Neurosci.,
November 1, 2001;
21(21):
8328 - 8338.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Zhou and H. K. Kimelberg
Freshly Isolated Hippocampal CA1 Astrocytes Comprise Two Populations Differing in Glutamate Transporter and AMPA Receptor Expression
J. Neurosci.,
October 15, 2001;
21(20):
7901 - 7908.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Barbour
An Evaluation of Synapse Independence
J. Neurosci.,
October 15, 2001;
21(20):
7969 - 7984.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. V Poulsen and R. J Vandenberg
Niflumic acid modulates uncoupled substrate-gated conductances in the human glutamate transporter EAAT4
J. Physiol.,
July 1, 2001;
534(1):
159 - 167.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Watzke, E. Bamberg, and C. Grewer
Early Intermediates in the Transport Cycle of the Neuronal Excitatory Amino Acid Carrier Eaac1
J. Gen. Physiol.,
June 1, 2001;
117(6):
547 - 562.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. MacAulay, U. Gether, D. A Klaerke, and T. Zeuthen
Water transport by the human Na+-coupled glutamate cotransporter expressed in Xenopus oocytes
J. Physiol.,
February 1, 2001;
530(3):
367 - 378.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. J. Mitchell and R. A. Silver
GABA Spillover from Single Inhibitory Axons Suppresses Low-Frequency Excitatory Transmission at the Cerebellar Glomerulus
J. Neurosci.,
December 1, 2000;
20(23):
8651 - 8658.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Watzke, T. Rauen, E. Bamberg, and C. Grewer
On the Mechanism of Proton Transport by the Neuronal Excitatory Amino Acid Carrier 1
J. Gen. Physiol.,
November 1, 2000;
116(5):
609 - 622.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Grewer, N. Watzke, M. Wiessner, and T. Rauen
Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds
PNAS,
August 6, 2000;
(2000)
160170397.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. Eskandari, M. Kreman, M. P. Kavanaugh, E. M. Wright, and G. A. Zampighi
Pentameric assembly of a neuronal glutamate transporter
PNAS,
July 18, 2000;
97(15):
8641 - 8646.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. S. Diamond and C. E. Jahr
Synaptically Released Glutamate Does Not Overwhelm Transporters on Hippocampal Astrocytes During High-Frequency Stimulation
J Neurophysiol,
May 1, 2000;
83(5):
2835 - 2843.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. S. Otis and M. P. Kavanaugh
Isolation of Current Components and Partial Reaction Cycles in the Glial Glutamate Transporter EAAT2
J. Neurosci.,
April 15, 2000;
20(8):
2749 - 2757.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Z. Han, K. Grant, and C. C. Bell
Rapid Activation of GABAergic Interneurons and Possible Calcium Independent GABA Release in the Mormyrid Electrosensory Lobe
J Neurophysiol,
March 1, 2000;
83(3):
1592 - 1604.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. P. Koch, M. P. Kavanaugh, C. S. Esslinger, N. Zerangue, J. M. Humphrey, S. G. Amara, A. R. Chamberlin, and R. J. Bridges
Differentiation of Substrate and Nonsubstrate Inhibitors of the High-Affinity, Sodium-Dependent Glutamate Transporters
Mol. Pharmacol.,
December 1, 1999;
56(6):
1095 - 1104.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. Mennerick, W. Shen, W. Xu, A. Benz, K. Tanaka, K. Shimamoto, K. E. Isenberg, J. E. Krause, and C. F. Zorumski
Substrate Turnover by Transporters Curtails Synaptic Glutamate Transients
J. Neurosci.,
November 1, 1999;
19(21):
9242 - 9251.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. P. Kavanaugh
Neurotransmitter transport: Models in flux
PNAS,
October 27, 1998;
95(22):
12737 - 12738.
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. D. Mitrovic, F. Plesko, and R. J. Vandenberg
Zn2+ Inhibits the Anion Conductance of the Glutamate Transporter EAAT4
J. Biol. Chem.,
July 6, 2001;
276(28):
26071 - 26076.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Borre and B. I. Kanner
Coupled, but Not Uncoupled, Fluxes in a Neuronal Glutamate Transporter Can Be Activated by Lithium Ions
J. Biol. Chem.,
October 26, 2001;
276(44):
40396 - 40401.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. MacAulay, A. Bendahan, C. J. Loland, T. Zeuthen, B. I. Kanner, and U. Gether
Engineered Zn2+ Switches in the gamma -Aminobutyric Acid (GABA) Transporter-1. DIFFERENTIAL EFFECTS ON GABA UPTAKE AND CURRENTS
J. Biol. Chem.,
October 26, 2001;
276(44):
40476 - 40485.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Grewer, N. Watzke, M. Wiessner, and T. Rauen
Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds
PNAS,
August 15, 2000;
97(17):
9706 - 9711.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
Compound via MeSH
