From the
Active ion-coupled glutamate transport is of critical importance
for excitatory synaptic transmission, normal cellular function, and
epithelial amino acid metabolism. We previously reported the cloning of
the rabbit intestinal high affinity glutamate transporter EAAC1 (Kanai,
Y., and Hediger, M. A.(1992) Nature 360, 467-471), which
[Medline]
is expressed in numerous tissues including intestine, kidney, liver,
heart, and brain. Here, we report a detailed stoichiometric and kinetic
analysis of EAAC1 expressed in Xenopus laevis oocytes. Uptake
studies of
For glutamate to fulfill its diverse functions, the existence of
active cellular glutamate uptake systems with a high accumulative power
is of critical importance. The cDNAs encoding four different mammalian
high affinity glutamate transporter isoforms have been recently cloned
and characterized. These are the neuronal and epithelial glutamate
transporter EAAC1 (EAAT3)(1, 2) , the glial glutamate
transporters GLT-1 (EAAT2) (3, 4) and GLAST
(EAAT1)(4, 5) , and the cerebellar glutamate transporter
EAAT4(6) . These transporters exhibit 39-55% sequence
identities with each other.
Studies of glutamate transport in
salamander retina glial cells suggest that transport is electrogenic
and coupled to the cotransport of two Na
Recent electrophysiological studies of
electrogenic transporters provided important clues to how uphill solute
transport is coupled to downhill electrochemical ion gradients.
Experiments using the voltage jump paradigm to study the
Na
In the central nervous system,
the coupling stoichiometry of EAAC1 also has pathologic implications.
The reduced energy supply that occurs during ischemia after a stroke or
during anoxia is known to lead to reduced ATP levels and reduced
function of Na
In the present
study we investigated the electrogenic characteristics of EAAC1 using
the voltage jump technique. We determined the coupling of
EAAC1-mediated transport to inorganic ions, and we demonstrate that
EAAC1 runs in reverse if we mimic the ischemic condition.
The glutamate to charge flux ratio was determined by
comparing the initial rate of [
The uptake
of [
EAAC1-mediated glutamate transport in oocytes was associated with
intracellular pH (pH
Using the two-electrode voltage clamp method the reversed
operation of EAAC1 was observed as an outward current. This was
achieved in the presence of high extracellular K
This approach was used to study the presteady-state and
steady-state characteristics of EAAC1 in response to voltage jumps and
to determine the kinetic parameters of EAAC1-mediated glutamate
transport as a function of membrane potential.
The structure-function analysis of transport proteins
requires knowledge of their transport mechanisms and the molecular
rearrangements that occur during the transport process. Site-directed
mutagenesis has been a highly valuable approach in elucidating the
structure-function relationship of ion channels. The success of these
studies was enhanced by the availability of functional models
describing the transport process and the associated molecular events.
These models were introduced by Hodgkin, Huxley, and Katz and were
further refined by several investigators (see Ref. 22 for review). To
embark on structure-function analysis of ion-coupled solute
transporters, the availability of such models would be useful. To this
end, we performed a detailed analysis of the coupling stoichiometry and
transport kinetics of rabbit EAAC1, and we discuss a hypothetical
kinetic model of EAAC1-mediated glutamate transport.
EAAC1-mediated
transport was furthermore associated with a decrease in intracellular
pH (pH
Taken together, these data give an overall
stoichiometry of 1 glutamate: 2 Na
Most previous studies addressing the
stoichiometry of high affinity glutamate transporters were based on
kinetic analysis(8, 25) . However, as discussed above
(see Fig. 2), this technique can yield ambiguous results, in
particular in cases where complex cooperativities between different
substrate binding sites occur. Nevertheless, two reports were based on
the analysis of the fluxes of glutamate
([
Based on
this stoichiometry and the ionic concentration gradients which exist
across plasma membranes, the concentrating capacity of EAAC1 in neurons
and epithelial cells and the ratio of intra- and extracellular
glutamate concentrations which can be attained at equilibrium can be
estimated. Assuming that the intracellular glutamate concentration in
neurons is
The demonstration of EAAC1-mediated
reversed glutamate transport suggests that the disrupted ion gradients
which occur during pathologic conditions, such as ischemia after a
stroke or hypoxia, could cause EAAC1 to run in reverse resulting in a
non-vesicular release of glutamate into the synaptic cleft. The strong
and widespread expression of EAAC1 in neurons throughout the
CNS
At an
extracellular Na
Important new insights into mechanistic aspects of EAAC1 were
obtained from studies addressing the dependence of glutamate transport
on extracellular Na
Based on the Hill equation analysis of EAAC1, we
hypothesize that binding of L-glutamate increases the affinity
for Na
Our data provide new insights
into the molecular events that are associated with the transport
process of EAAC1. A central question that remains to be addressed is
which parts of EAAC1 are involved in glutamate translocation. The
extended hydrophobic stretch of EAAC1 (residues 357-439) is of
particular interest because it is highly conserved among all
prokaryotic and eukaryotic members of the EAAC1 family(36) .
This region is predicted to span the membrane at least three times
(putative transmembrane domains 8-10). Due to its hydrophobicity,
however, it is conceivable that it is largely embedded in the membrane
and forms a flexible structure which constitutes the charge
translocation pathway. An important role of putative membrane spanning
domains 1-7 may be to anchor this translocation pathway in the
membrane.
Na
and
[
C]glutamate, in combination with measurements
of intracellular pH with pH microelectrodes gave a glutamate to charge
ratio of 1:1, a glutamate to Na
ratio of 1:2, and a
OH
/H
to charge ratio of 1:1. Since
transport is K
dependent it can be concluded that
EAAC1-mediated glutamate transport is coupled to the cotransport of 2
Na
ions, the countertransport of one K
ion and either the countertransport of one OH
ion or the cotransport of 1 H
ion. We further
demonstrate that under conditions where the electrochemical gradients
for these ions are disrupted, EAAC1 runs in reverse, a transport mode
which is of pathologic importance.
Na
uptake studies revealed that there is a low level of
Na
uptake in the absence of extracellular glutamate
which appears to be analogous to the Na
leak observed
for the intestinal Na
/glucose cotransporter SGLT1. In
voltage clamp studies, reducing extracellular Na
from
100 to 10 mM strongly increased
K
and decreased I
. The data indicate that Na
binding at the extracellular transporter surface becomes
rate-limiting. Studies addressing the cooperativity of the
substrate-binding sites indicate that there are two distinct
Na
-binding sites with different affinities and that
Na
binding is modulated by extracellular glutamate. A
hypothetical ordered kinetic transport model for EAAC1 is discussed.
ions and the
countertransport of one K
and one OH
ion(7, 8) . The coupling pattern has not yet been
conclusively investigated for the recombinant high affinity glutamate
transporters. Knowledge of stoichiometry of cloned glutamate
transporters, however, is of critical importance to determine their
structure-function relationships and to understand their physiological
and pathophysiological roles.
/glucose cotransporter (SGLT1) (9) and the
Na
/Cl
/GABA (10) transporter
demonstrated current relaxation in response to voltage jumps similar to
gating currents observed in voltage-gated ion channels. These currents
were interpreted to reflect charge movement within the membrane
electric field that is associated with conformational changes of the
transporter molecule. It is likely that the identification of charged
residues in these transporters that undergo transient movements will
provide important information on the structure-function relationships
of ion-coupled solute transporters.
,K
-ATPase and to result
in rundown of electrochemical ion gradients(11) . The disrupted
ion gradients are thought to cause high affinity glutamate transporters
to run in reverse resulting in a non-vesicular release of glutamate
into the synaptic cleft. Since extracellular glutamate is highly toxic
to neurons, reversed glutamate transport likely results in neuronal
death as a result of these pathologic conditions.
Xenopus Oocyte Expression of EAAC1
cRNAs were in vitro transcribed from cDNAs in pSPORT1 using T7 RNA
polymerase (1). Xenopus oocyte expression studies were
performed as described previously (12, 13) using
collagenase-treated and manually defolliculated oocytes injected with
50 nl of water or cRNA (25 ng/oocyte). Oocytes were incubated in
modified Barth's medium (88 mM NaCl, 1 mM KCl,
0.33 mM Ca(NO)
, 0.41 mM CaCl
, 0.82 mM MgSO
, 2.4 mM NaHCO
, 10 mM HEPES, pH 7.4) supplemented with
gentamicin (50 µg/ml). Oocytes were used 3-6 days after
injection for electrophysiological analyses and flux studies.
Determination of the Glutamate to Charge
Stoichiometry
The glutamate to charge stoichiometry was
determined by comparing [C]glutamate uptake and
glutamate-induced inward current as described (14, 15) with some modifications. Five days after
injection, [
C]L-glutamate uptake (20
µM) by EAAC1 cRNA-injected oocytes and water-injected
control oocytes were measured in standard uptake solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl
, 1 mM MgCl
, 10 mM HEPES, 5 mM Tris, pH
7.4) for 1, 2, 3, 4, and 5 min. Immediately after completion of the
uptake measurements, two microelectrode voltage clamp experiments (2) were performed using the same batch of cRNA- or
water-injected oocytes. The oocytes were bathed in the standard uptake
solution, and the membrane potential was monitored after impaling
oocytes. The bath medium was then changed to the uptake solution
containing 20 µM of glutamate, and the membrane potential
was measured. After washing with uptake solution without glutamate,
each oocyte was clamped at this membrane potential. Inward currents
evoked by bath-applied 20 µM glutamate at the above
determined holding potentials were recorded, and the values were
converted to the rate of charge flux using Faraday's constant
(9.65
10
C/mol).
Analysis of Na
L-Glutamate evoked currents were measured
in uptake media containing n mM NaCl (where n was varied from 0 to 100 mM), 100 minus n mM choline chloride, 2 mM KCl, 1 mM CaCl Coupling Based on the
Hill Equation
, 1 mM MgCl
, 10 mM
HEPES, 5 mM Tris, pH 7.4. The currents were fitted by the Hill
equation, and the Hill coefficients were calculated for each glutamate
concentration.
Determination of the Na
To determine the Na to Glutamate
Coupling Ratio
to glutamate
stoichiometry, the uptakes of
Na
and
[
C]L-glutamate were compared in the
same batch of EAAC1 cRNA-injected oocytes. Groups of 8-10 oocytes
were preincubated for 30 min in 1 ml of uptake medium containing 40
mM NaCl, 60 mM choline-chloride, 2 mM KCl, 1
mM CaCl
, 1 mM MgCl
, 1 mM ouabain, 0.1 mM amiloride, 0.1 mM bumetanide, 10
mM HEPES, 5 mM Tris, pH 7.4. Glutamate uptake was
measured in the uptake solution containing 2 µCi of
[
C]L-glutamate and unlabeled L-glutamate to make up the final concentration of 1.0 and 0.2
mM. After 20 min of incubation, oocytes were washed with
ice-cold Na
-free uptake medium in which NaCl was
replaced with choline-Cl. In separate experiments Na
uptake was measured for 20 min in the uptake medium containing 20
µCi of
Na
and 0.2 or 1.0 mM of non-labeled L-glutamate.
Measurement of Intracellular pH Using pH
Microelectrodes
Intracellular pH (pH)
changes associated with EAAC1-mediated glutamate transport in oocytes
were measured by monitoring pH
of oocytes using a
pH-sensitive microelectrode filled with a hydrogen-selective
ionophore(2, 16, 17) . Oocytes were studied
3-7 days after injection of EAAC1 cRNA or water. Briefly, V
electrodes were pulled from 2 mm
diameter fiber-filled borosilicate capillaries (Warner Instruments).
These had resistances of 1-10 M
when back-filled with 3 M KCl. The pH electrodes were made from similar pipettes but
were silanized by adding 20 µl of tri-n-butyl-chlorosilane
to an enclosed container at 200 °C. Pipettes were cooled under
vacuum, their tips were filled with Fluka hydrogen ionophore I-mixture
B, and backfilled with 0.04 M KH
PO
,
0.023 M NaOH, 0.015 M NaCl, pH 7.0. The arrangement
of the electronics was as described previously (16, 17). Voltage due to
pH
was obtained by electronically subtracting the
signals from the pH and voltage electrodes. V
was obtained by subtracting the signals from the voltage
electrode and the external reference electrode. Electrometer outputs
were directed to an 80386-based computer via a 12-bit analog-to-digital
converter, and also to a strip chart recorder. Rates of pH
change (dpH
/dt) were
determined by fitting a line to the relevant portion of the
pH
versus time record. We determined the
portion of dpH
/dt due to the EAAC1
transporter by subtracting the dpH
/dt obtained during a control period from the
dpH
/dt obtained immediately thereafter in
the presence of L-glutamate. In these experiments, the chamber
was perfused with ND96, pH 7.40, followed by 100 µML-glutamate in ND96. We obtained the flux (expressed as
picomole cm
s
) due to the
transporter by multiplying the difference
dpH
/dt by the intracellular buffering
power
and the surface-to-volume ratio of the oocyte.
was
determined by measuring the dpH
due to the
addition of 1.5% CO
, 10 mM HCO
, ND96
at pH 7.4 and had a value of 13 ± 4 mM/pH unit (n = 18)(18) . The surface to volume ratio was obtained
from the oocyte diameter, assuming the cell is a sphere and the oocyte
volume is 0.25 µl(19) .
K
The K Dependence
dependence of the glutamate-evoked current was determined as
described previously (1) over a range of K
concentrations of 0-50 mM under voltage clamp
condition at holding potentials of -30 and -60 mV. Currents
induced by application of 50 µM glutamate were measured in
EAAC1 cRNA-injected oocytes.
Reversed Glutamate Transport
Oocytes expressing
EAAC1 were impaled in standard ND96 medium (96 mM NaCl, 2
mM KCl, 1.8 mM CaCl, 1 mM MgCl
, 5 mM HEPES, pH 7.4) and
voltage-clamped. After stabilization of the current at -60 mV,
oocytes were bathed in high K
/high glutamate/zero
Na
medium (98 mM KCl, 1.8 mM
CaCl
, 1 mM MgCl
, 10 mML-glutamate, 5 mM HEPES, pH 7.4). The bath
solution was then replaced by high K
medium without
glutamate to observe the outward current which is due to reversed
glutamate transport. After 45 s, the bathing solution was replaced by
high K
/high glutamate/zero Na
medium
to terminate the reversed glutamate transport. To determine the voltage
dependence of reversed transport, the outward currents were measured at
holding potentials between -100 mV and +30 mV. The reversed
transport of EAAC1 was also examined in voltage jump experiments (see
below). Oocytes expressing EAAC1 were clamped at -50 mV, and
voltage steps were applied as described below while bathed either in
the high K
, 10 mM glutamate, 0 mM Na
medium or in the high K
/0
mM glutamate, 0 mM Na
medium.
Voltage Jump Studies
Oocytes were subjected to
two-microelectrode voltage clamping and command potentials were applied
and controlled by an IBM compatible computer via the software CLAMPEX
from pCLAMP (version 5.5, Axon Instruments)(2) . The oocyte
membrane was held at -50 mV and pulsed to the test potential for
76 ms followed by a 1-s interpulse interval at the holding potential of
-50 mV before next pulse. Currents were low pass-filtered at 50
kHz, digitized at 200 µs (512 samples), and saved on computer.
Steady-state currents during the voltage jump were obtained by
averaging the current during the final 4 ms of the 76 ms jump (average
of 10 samples). Steady-state currents were measured for each test
potential in the presence and absence of L-glutamate, and the
glutamate-induced currents were taken as the difference between these
currents. The concentration dependence of L-glutamate evoked
currents at any given membrane potential and at different extracellular
Na concentrations
(Na
)
(
)were fitted
by the Michaelis-Menten equation. Apparent
K
and I
values were calculated and plotted as a function of membrane
potential.
Stoichiometry
C]glutamate
uptake and the net charge flux calculated from the glutamate-induced
inward current. We have used a similar approach to determine the
Na
- to glucose-coupling ratio of the high and low
affinity Na
/glucose cotransporters SGLT1 and
SGLT2(14) , and the H
- to peptide-coupling
ratio of the oligopeptide transporter PepT1(15) .
C]glutamate (20 µM) was linear
over the first 5 min (Fig. 1a). The slope of this curve
gave the glutamate influx, which was 293 ± 27 fmol/oocyte/s. The
charge flux computed from the amplitude of the inward currents induced
by application of 20 µM glutamate to oocytes injected with
EAAC1 cRNA, and Faraday's constant, was 350 ± 34
fmol/oocyte/s. The holding potential used to determine the inward
current for each oocyte corresponded to the steady-state potential in
the presence 20 µM glutamate in the bath and was
-38 mV. This gave a glutamate to charge flux ratio of 1:
1.19 (Fig. 1b). Transport of glutamate is therefore
associated with the translocation of one net positive charge.
Figure 1:
Glutamate to charge stoichiometry. The
initial rate of the [C]glutamate uptake (a) and the net charge flux calculated from the
glutamate-induced inward current are directly compared (b).
The glutamate-induced inward current was converted to the charge flux
using Faraday's constant. The holding potential was set at the
resting potential measured for each oocyte in the presence of 20
µM bath-applied glutamate. The
[
C]glutamate uptake studies and the
electrophysiological measurements were performed on the same batch of
EAAC1 cRNA-injected oocytes. The glutamate to charge flux ration was
1:1.19 (b). Glutamate translocation is therefore associated
with the translocation of one positive
charge.
The
Na- to glutamate-coupling ratio was initially studied
by measuring the Na
dependence of glutamate-induced
currents at a holding potential of -60 mV (Fig. 2a). Curve fits of the data by the Hill equation
showed that the Hill coefficients strongly depended on extracellular
glutamate concentration: the Hill coefficient was 1.2 at 1.0 mM glutamate and 2.0 at 0.2 mM glutamate. At lower glutamate
concentrations, the curves apparently display even larger Hill
coefficients.
Figure 2:
Na to glutamate
stoichiometry. a, Na
dependence of
glutamate-induced current. The data obtained for 0.2 and 1 mML-glutamate were fitted to the Hill equation. Hill
coefficients increased with decreasing glutamate concentrations. b,
Na
uptake (20 min) in the
presence and absence of L-glutamate. Oocytes expressing EAAC1
showed large glutamate-induced
Na
uptakes
in the presence of either 1 or 0.2 mML-glutamate. A
low level of
Na
uptake was also observed
in the absence of glutamate demonstrating the Na
leak
phenomenon of EAAC1. c, comparison of
Na
uptake and
[
C]glutamate uptake in the same batch of EAAC1
cRNA-injected oocytes. The Na
to glutamate flux ratios
were 2.1 to 1 for 1 mM glutamate and 2.3 to 1 for 0.2 mM glutamate. This demonstrates that two Na
ions are
cotransported with each glutamate molecule.
Na
and [
C]glutamate uptakes were measured in
the presence of 40 mM Na
, 1 mM ouabain, 0.1 mM amiloride, and 0.1 mM
bumetanide.
Because the Hill coefficient depended on extracellular
glutamate concentration (L-Glu), the Na
to glutamate coupling was reinvestigated using a different
approach. Briefly, the initial rates of the
[
C]glutamate and
Na
uptakes were directly compared. Ouabain, amiloride, and
bumetanide were added to the bath to inhibit endogenous oocyte
Na
transport. Oocytes injected with EAAC1 cRNA
exhibited large glutamate-induced
Na
uptakes (Fig. 2b). Water-injected control oocytes
did not show significant
Na
uptake. EAAC1
mediated a low level of
Na
uptake in the
absence of glutamate (Fig. 2b, right), which is
consistent with a Na
leak phenomenon, in analogy to
that of the Na
/glucose cotransporter(20) . The
difference between the
Na
uptake obtained
in the presence and absence of glutamate was taken as the
glutamate-induced
Na
uptake (Fig. 2c). The initial rates of
[
C]glutamate uptake obtained L-Glu
of 1 and 0.2 mM were determined on
the same batch of EAAC1 cRNA-injected oocytes using the approach
illustrated in Fig. 1a. The rates of the
Na
and
[
C]glutamate fluxes are compared in Fig. 2c. The ratios of the fluxes were 2.1:1 at 1.0
mML-Glu
and 2.3:1 at 0.2 mML-Glu
. The Na
to glutamate
coupling ratio is therefore 2:1 at both 1.0 and 0.2 mML-Glu
. This indicates that the Na
to glutamate coupling ratio does not depend on the extracellular
glutamate concentration as suggested by Hill equation analysis.
) changes as determined by
monitoring the pH
of oocytes using pH-sensitive
microelectrodes. In the experiment shown in Fig. 3a,
application of 100 µML-glutamate to EAAC1
cRNA-injected oocytes caused pH
to decrease from
7.58 to 7.50 and the oocyte membrane to depolarize. A similar result
was obtained using L-aspartate as a substrate, whereas leucine
which is not a substrate of EAAC1 did not result in intracellular
acidification. On average, the application of 100 µML-glutamate elicited a dpH
/dt of -10.0 ± 1.1
10
pH
units/s and a depolarization of 39.8 ± 2.4 mV (n = 25). These values allow the calculation of the
OH
/H
to charge coupling (Fig. 3b). The changes in pH and the membrane
depolarization were converted to currents by the following equations: I
=
[dpH
/dt]
F
z, and I
=
V
/R
, where F is the Faraday constant,
the buffering power (15
mM/pH unit), z = 1, and R
the input resistance of an oocyte which was (1 M
). Assuming
an oocyte volume of 0.25 µl, the result is an estimated
OH
/H
to charge coupling of 0.9
± 0.1:1. These values indicate that transport of 1 L-glutamate molecule is associated with the cotransport of
either 1 H
ion or the countertransport of 1
OH
ion.
Figure 3:
H/OH
coupling and K
dependence. a, acidic
amino acids transported by EAAC1 acidify and depolarize oocytes. The
effect of 100 µM of either L-glutamate, L-leucine, or L-aspartate on intracellular pH (pH)
and membrane potential (V) were measured using
microelectrodes. 100 µM of either L-glutamate or L-aspartate rapidly depolarize and acidify oocytes while L-leucine has no effect. b, calculated charge to
OH
or H
coupling. Plotted are the
currents calculated from either the dpH/dt or the
depolarization elicited by 100 µML-glutamate.
The OH
/H
to charge coupling ratio is
0.9 ± 0.1. c, inhibition of L-glutamate-induced current by external K
.
Currents evoked by 50 µML-glutamate were
measured at various K
concentration (bath medium 0
mM-50 mM) at holding potentials of -30 and
-60 mV and normalized to the current measured on the same oocyte
(the value at 0 mM K
is 100%). Each point
represents the mean ± S.E. from six oocytes. The abscissa indicates external K
concentration on a
logarithmic scale.
Extracellular K inhibited
glutamate-induced currents in voltage-clamped oocytes injected with
EAAC1 cRNA, as previously reported(1) . In Fig. 3c, the inhibition is compared at -60 and
-30 mV. It is greater at -30 mV than at -60 mV,
consistent with K
countertransport.
Reversed Glutamate Transport
and
by eliminating extracellular Na
. In control
experiments, glutamate-evoked inward currents were observed when using
the high Na
and low K
solution in the
perfusate (Fig. 4a). In the experiments addressing
reversed glutamate transport, it was assumed that the intracellular
glutamate concentration in oocytes is
10 mM, in analogy
to neurons(21) , and the experiment was therefore started by
counter-inhibition with 10 mML-Glu
(Fig. 4b). Removal of glutamate resulted in a
significant outward current, due to efflux of glutamate driven by the
outwardly directed electrochemical gradients of Na
and
glutamate. In support of this interpretation, the outward current was
enhanced by depolarization (Fig. 4b). No significant
currents were observed in water-injected control oocytes. Fig. 4c shows the current-voltage relationship
determined by stepping the voltage between -150 and +50 mV.
The figure further confirms that the current is strongly enhanced by
depolarization.
Figure 4:
Reversed transport of EAAC1. a,
``normal'' glutamate transport. Glutamate evoked currents
were determined in voltage-clamped oocytes injected with EAAC1 cRNA
under normal ion-gradient conditions (2 K and 96
mM Na
in the bath). b, reversed
transport under disrupted ion-gradient conditions (98 mM K
and no Na
in the bath). Due to
the coupling stoichiometry of EAAC1, the high extracellular potassium
and the absence of extracellular Na
causes EAAC1 to
run in reverse. The experiment was initiated by trans-inhibition with
bath applied 10 mM glutamate, assuming that the intracellular
glutamate concentration is
10 mM. Removal of glutamate
resulted in an outward current which is due to release of glutamate out
of the cell driven by the intracellular Na
concentration. The outward current was enhanced by
depolarization, consistent with Na
-driven reversed
transport. c, current-voltage relationship of reversed
glutamate transport based on voltage jump experiments. Oocytes
expressing EAAC1 were clamped at -50 mV, and voltage steps were
applied while bathed in medium containing either 96 mM K
and 10 mM glutamate and 0 mM
Na
or 96 mM K
without
glutamate and NaCl.
Voltage Jump Studies
Presteady State Currents
In contrast to the
Na/glucose cotransporter SGLT1(9) , no
significant presteady-state currents were observed in oocytes
expressing rabbit EAAC1 in the presence of Na
and in the absence of L-Glu
(data not
shown). A similar lack of presteady-state currents was observed for
human EAAC1 (2).
Steady-state Currents
The I-V curves for the
currents evoked by L-glutamate in the concentration range of 1
µM-1 mM demonstrated that the currents do not
reverse between -150 to + 50 mV (Fig. 5). The currents
saturated with hyperpolarization only at L-Glu below 40 µM whereas there was no saturation at
higher L-Glu
.
Figure 5:
Current-voltage relationship of
EAAC1-mediated glutamate uptake based on voltage jump studies. The
oocyte membrane was held at a holding potential of -50 mV and the
following six test potentials were applied: -150, -110,
-70, -30, +10, and +50 mV. Currents due to the
application of L-glutamate (1 µM to 1
mM) were recorded, and those currents obtained in the absence
of glutamate were subtracted. The extracellular concentrations of
K was 2 mM and of Na
98
mM.
Apparent
K and I
values obtained at different extracellular Na
(Na
) concentrations were plotted as
a function of membrane potential (Fig. 6). As previously observed
for human EAAC1, the K
and I
values determined at 100 mM Na
both increase with membrane
hyperpolarization (Fig. 6)(2) . In the present study we
have determined the voltage dependence of K
and I
at reduced
Na
. Fig. 6a shows that
re-ducing extracellular Na
reverses the voltage
dependence of K
and causes
it to increase to exceedingly high values with depolarization.
Figure 6:
Dependence of
K and I
on extracellular Na
concentration. The voltage
dependence of K
(a) and I
(b) were
determined at extracellular Na
-concentrations of 10,
30, 50, and 100 mM using the voltage jump
method.
Stoichiometry
The studies presented in Fig. 1reveal a glutamate to charge flux ratio of 1: 1.19 and
demonstrate that one net positive charge is translocated with each
glutamate molecule. Although the stoichiometry of
Na-coupled transporters is commonly determined based
on the Hill equation (see Ref. 23), this approach did not yield
reliable information on the coupling stoichiometry of EAAC1. Similar
problems were encountered when using this approach for the
determination of the stoichiometry of the Na
/glucose
cotransporter SGLT1(24) . Therefore, the Na
to
glutamate coupling ratio was determined by comparing the initial rates
of
Na
and
[
C]glutamate uptake. The Na
to
glutamate coupling ratio was close to 2:1 at extracellular glutamate
concentrations of either 1 or 0.2 mM.
) (Fig. 3a). The
OH
or H
to charge coupling ratio was
estimated from the rate of pH
decrease. This
calculation depends on the assumed oocyte volume of 0.25
µl(19) , an oocyte membrane resistance of 1 M
and a
buffering power (
) of 15 mM/pH unit(18) . The
computed coupling ratio of 0.9 ± 0.1 is consistent with a 1:1
ratio for the H
/OH
to charge
stoichiometry.
: 1
OH
/H
: 1 charge. Since we also
demonstrated that transport mediated by EAAC1 depends on K
and since Attwell and colleagues (8) proposed that high
affinity glutamate transport is coupled to the counter transport of
OH
or other pH-changing ions such as
HCO
, and not to the cotransport of
H
, we infer that EAAC1-mediated glutamate transport is
coupled to the cotransport of 2 Na
ions and the
countertransport of 1 OH
(or
HCO
) ion and 1 K
ion.
This stoichiometry is unlike that proposed by Stoffel and colleagues (25) for the glial glutamate transporter GLAST. Based on Hill
equation analysis, these investigators suggested that GLAST is coupled
to the cotransport of three Na
-ions. The overall
stoichiometry of EAAC1, however, corresponds to that proposed by
Attwell and co-workers (8) for high affinity glutamate transport
in salamander retia glia cells.
C]glutamate), Na
(
Na
), and K
(
Rb
)(37, 38) ,
yielding partial information on the stoichiometry. The present work
constitutes the first determination of the overall stoichiometry of a
high affinity glutamate transporter which is based solely on comparison
of the fluxes of the individual transporter substrates.
10 mM, the stoichiometry predicts an
equilibrium extracellular glutamate concentration of
0.6
µM(8) . This value is well within the range of the
prevalent glutamate concentration of the cerebrospinal fluid. Our
results therefore verify that EAAC1 has the capacity to contribute to
the maintenance of the low extracellular glutamate concentration in the
central nervous system.
Reversed Glutamate Transport
In facilitated
transporters such as the glucose transporter GLUT1 (26) and the
urea transporter UT2 (19), it is generally accepted that net transport
of substrates across cell membranes proceeds from the outside to the
inside or from the inside to the outside, depending on the
concentration gradients of the transport substrates across the cell
membrane. By contrast, in ion-coupled transporters, net transport is
considered to be unidirectional at given electrochemical ion gradients
that energize uphill solute transport. However, if the ionic gradients
are altered net transport of ion-coupled transporters can occur in the
opposite direction. This was demonstrated by Attwell and co-workers (27, 28) for glutamate transport in salamander retina
glia cells. In the present study, we show that EAAC1 expressed in Xenopus oocytes can run in the reversed direction. The I-V
relationships of the outward currents obtained by two different
procedures (Fig. 4, b and c) were essentially
identical and demonstrated that the currents are highly
voltage-dependent. Reversed glutamate transport currents become evident
at potentials above -70 mV and steadily increased with
depolarization. They did not saturate with increasing the membrane
potential up to +50 mV (Fig. 4c). This I-V curve,
when rotated by 180°, is similar to that of the
``forward'' glutamate transport (Fig. 5). This suggests
that the reversed mode of EAAC1-mediated transport is a true reversal
of the overall forward operation of transport and that the
stoichiometry, the basic mechanism of transport and the rate-limiting
step are likely to be the same.
(
)suggests that EAAC1 could contribute to the
rise in glutamate to neurotoxic levels during pathological conditions.
Glial-reversed glutamate transport may also contribute to the
extracellular rise, but the neuronal contribution is predicted to be
larger because neurons have a significantly higher content of glutamate
than astrocytes(29, 30) . Astrocytes convert glutamate
to glutamine by glutamine synthetase, an enzyme which is lacking in
neurons(31) . Thus, it is probable that reversed glutamate
transport mediated by neuronal EAAC1 appears to be of particular
pathologic consequence.
Charge Movement and Rate-limiting Steps of EAAC1-mediated
Glutamate Transport
EAAC1 did not exhibit significant current
relaxation in response to voltage jumps. A plausible interpretation of
this behavior is that the empty carrier [C]` (see Fig. 7) is electroneutral and that any conformational changes of
EAAC1 which occur during the transport process do not involve movement
of charged amino acid residues within the membrane electric field. This
would further suggest that the fully loaded carrier
[CNaGlu]` (intermediate 4 in Fig. 7) has
one positive charge and that its translocation is electrogenic and
thereby voltage-dependent.
Figure 7:
Hypothetical kinetic transport model of
EAAC1. The model predicts that the kinetics of transport are ordered
and that the transporter has a cation-binding site to which either
Na or K
binds and an anion-binding
site to which either Glu
or OH
binds. Loading of the carrier at the extracellular surface is
predicted to involve binding of the first Na
,
following glutamate binding which then allows binding of the second
Na
. The complex then translocates to the inside. This
process is called the charge translocation step (conversion of
intermediates 4-5) and is predicted to be rate limiting at an
extracellular Na
concentration of 100 mM. The
relocation step (translocation of intermediate 10) is predicted to be
accelerated by electroneutral countertransport to 1 OH
and 1 K
ion. Partial reactions such as the
Na
leak are not indicated in this
diagram.
In analogy to human EAAC1(2) ,
rabbit EAAC1 exhibited large steady-state currents in the presence of
extracellular L-glutamate (1 µM to 1 mM)
in response to voltage steps (Fig. 5), and currents were inwardly
directed and asymptotically approached zero near approximately +50
mV. Further in analogy to human EAAC1, the voltage dependence of the
currents was affected by the extracellular glutamate concentration. At
lower glutamate concentrations (below 30 µM), the current
saturated as the membrane hyperpolarized and exhibited a sigmoidal
dependence on membrane voltage. At higher glutamate concentrations, the
current did not saturate, even if the membrane was hyperpolarized to
-150 mV. This substrate-dependent current-voltage relationship is
distinct from that of other transporters such as SGLT1 (32) which was sigmoidal for all substrate concentrations
tested. This suggests that glutamate modulates the function of EAAC1, i.e. through a shift of the rate-limiting step.
concentration of 100 mM, the
apparent Michaelis constant for L-glutamate
(K
) and the maximal transport
rate I
of rabbit EAAC1 both increased with
hyperpolarization (Fig. 6). This result is congruent with
findings of human EAAC1 (2) according to which both
K
and I
increased with hyperpolarization and exhibited a linear
relationship. We previously concluded based on this observation that
K
and I
must be determined by a single, voltage-dependent step and that
this step must correspond to the charge translocation step, i.e. the translocation of the fully loaded carrier
[CNa
Glu]`(2). The strong voltage dependence of
the glutamate evoked current of rabbit EAAC1, the lack of saturation by
hyperpolarization at glutamate concentrations >40 µM (Fig. 5), and the simultaneous increase in both
K
and I
at 100 mM Na
(Fig. 6) all support the view that the rate-limiting step
of rabbit EAAC1 is also the voltage-dependent charge translocation
step. Thus, in both rabbit and human EAAC1, the
K
values appear to be determined
by the charge translocation step and not by glutamate binding.
concentration
(Na
). Reduction of
Na
changed the voltage
dependences of K
and I
entirely. At
Na
of 30 mM or less,
K
no longer decreased but
increased with depolarization to exceedingly high values, whereas I
became less voltage-dependent. This indicates
that, at reduced Na
, another step
which is less voltage-dependent becomes rate-limiting, e.g. binding of Na
or glutamate to the extracellular
surface.
Cooperativity of Na
The Hill equation gives information on the cooperativity
of the substrate-binding sites. For Na and Glutamate
Binding
-coupled solute
transporters, the Hill coefficient often reflects the Na
to substrate coupling ratio. However, as pointed out by Kimelberg et al.(33) the analysis is only valid if the
affinities for the different Na
-binding sites are
similar. If there is a coupling ratio of 2:1 and the second
Na
binds with an affinity 10-100 times greater
than that of the first Na
, the function becomes
essentially hyperbolic because it predominantly reflects the
Na
dependence of the low affinity
Na
-binding site. Fig. 2a demonstrates
that the Hill coefficient increase from 1.2 to >2 with decreasing
glutamate concentrations, whereas
Na
and
[
C]glutamate uptake studies reveal that the
increase in Hill coefficients with decreasing L-Glu
is not associated with a change in
the Na
to glutamate coupling ratio. The lack of
correlation between the Hill coefficients and the true coupling ratio
therefore indicates that EAAC1 has Na
-binding sites
with different affinities. The effect of L-Glu
on the sigmoidicity of the Na
dependence of
transport furthermore suggests that glutamate increases the affinity
for Na
, i.e. the second Na
(Fig. 7). Consistent with this, the effect on the
sigmoidicity was particularly strong when L-Glu
was near or below the apparent K
of L-glutamate (Fig. 2a) which was around 30
µM (Fig. 6a). The Na
leak
phenomenon which was demonstrated by a low level of
Na
uptake in the absence of L-Glu
(Fig. 2a) indicates
that even in the absence of L-Glu
, the
second Na
ion can bind with low affinity, resulting in
translocation of the putative [CNa
] complex (see Fig. 7).
binding (e.g. binding of the second
Na
ion). If L-Glu
indeed
modulates Na
binding, this would be consistent with
the finding that saturation of currents with hyperpolarization is only
observed at low L-Glu
(Fig. 5). At
low L-Glu
(<40 µM), it is
likely that the rate-limiting step is related to substrate binding, i.e. binding of Na
to the extracellular
surface, whereas at high L-Glu
, the
charge translocation step is rate limiting and the current steadily
increases with hyperpolarization.
Hypothetical Kinetic Model
A kinetic transport
model of EAAC1 which is based on the present data, previous studies of
human EAAC1 (2), and studies of brush border membrane vesicles (35) is presented in Fig. 7. The model includes ordered
transport kinetics. EAAC1 is predicted to have a cation-binding site
for either two Na ions or one K
ion
and an anion-binding site for either one glutamate
ion or one OH
ion. The cation-binding site is
proposed to have two Na
-binding sites with different
affinities for Na
. After binding of the first
Na
, glutamate binds, followed by binding of the second
Na
, in analogy to the model for the
Na
/glucose cotransporter proposed by Bennet and
Kimmich (34; see also ref. 23). The fully loaded carrier (intermediate
4) translocates and the substrates are released to the cytoplasm.
K
and OH
then bind to the cation-
and anion-binding sites, respectively, and the transporter recycles to
the outside, releasing K
and OH
to
the extracellular medium. As suggested by Kinne and
colleagues(35) , we hypothesize that the charge translocation
step (translocation of intermediate 4) is rate limiting and that the
relocation step (relocation of intermediate 10) is electroneutral. In
ion-coupled solute transporters, the relocation step of the empty
carrier is often considered to be rate-limiting (see Ref. 2). In EAAC1,
countertransport of K
and OH
is
predicted to serve to increase the rate of recycling of the transporter
to such an extent that the charge translocation step becomes
rate-limiting. In contrast, recycling of the empty carrier is thought
to be rate limiting for the Na
/glucose
cotransporter(9, 32) . However, to absorb glutamate
efficiently as an important metabolite in epithelial cells and to
terminate glutamate's neurotransmitter action at glutamatergic
synapses, glutamate transporters must have a high turnover rate. The
required speed may be obtained by electroneutral coupling of the
recycling step to energetically favorable K
and
OH
countertransport.
, extracellular Na
concentration; L-Glu, extracellular glutamate
concentration.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.