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The Journal of Neuroscience, April 15, 2000, 20(8):2749-2757
Isolation of Current Components and Partial Reaction Cycles in
the Glial Glutamate Transporter EAAT2
Thomas S.
Otis1 and
Michael P.
Kavanaugh2
1 Department of Neurobiology, University of California,
Los Angeles Medical Center, Los Angeles, California 90095-1763, and
2 Vollum Institute, Portland, Oregon 97202
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ABSTRACT |
The kinetic properties of the excitatory amino acid transporter
EAAT2 were studied using rapid applications of L-glutamate to outside-out patches excised from transfected human embryonic kidney
293 cells. In the presence of the highly permeant anion SCN , pulses of glutamate rapidly activated
transient anion channel currents mediated by the transporter. In the
presence of the impermeant anion gluconate, glutamate pulses activated
smaller currents predicted to result from stoichiometric flux of
cotransported ions. Both anion and stoichiometric currents displayed
similar kinetics, suggesting that anion channel gating and
stoichiometric charge movements are linked to early transitions in the
transport cycle. Transporter-mediated anion currents were recorded with
ion and glutamate gradients favoring either unidirectional influx or
exchange. Analysis of deactivation and recovery kinetics in these two
conditions suggests that, after binding, translocation of substrate is
more likely than unbinding under physiological conditions. The kinetic properties of EAAT2, the dominant glutamate transporter in brain astrocytes, distinguish it as an efficient sink for synaptically released glutamate.
Key words:
GLT1; glutamate uptake; anion conductance; EAAT2; astrocyte; glutamate transporter
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INTRODUCTION |
Clearance of neurotransmitter
released by glutamatergic excitatory synapses is accomplished by the
activity of sodium-dependent transporters located on glial and neuronal
cell membranes. Five distinct members [excitatory amino acid
transporter EAAT1 (GLAST), EAAT2 (GLT-1), EAAT3 (EAAC1),
EAAT4, and EAAT5] of a gene family encoding glutamate
(Glu) transporters have been identified to date (Kanai and
Hediger, 1992 ; Pines et al., 1992; Storck et al., 1992; Fairman et al.,
1995; Arriza et al., 1997). As expected, expression of each of the
resulting gene products confers the ability to accumulate radiolabeled
glutamate. Expression also gives rise to an anion conductance (Fairman
et al., 1995; Wadiche et al., 1995a) consisting of a small anion leak
in the absence of substrate and a larger anion conductance linked to
substrate binding (Bergles and Jahr, 1997; Otis and Jahr, 1998; Wadiche and Kavanaugh, 1998). For this reason, electrophysiological
measurements of transporter currents typically monitor the sum of two
currents, a relatively large anion current, and a smaller current
generated by stoichiometric movement of cotransported ions and substrate.
The present study uses rapid solution exchange techniques applied to
outside-out patches containing EAAT2 transporters to determine the
kinetic relationship between the two components of glutamate
transporter current. Understanding the link between gating of the anion
conductance and the transport cycle is important in several respects.
Although anion currents can be readily detected, it is difficult to
measure the smaller stoichiometric transporter currents in
physiological contexts, such as in native cells engaged in excitatory
synaptic transmission (Otis et al., 1997). To estimate parameters of
glutamate transport at functioning synapses based on measurements of
the larger anion currents, it is important to establish the
relationship between transporter current and glutamate flux. Transport
is a multistep process involving the binding of substrate and ions,
translocation, and the unbinding of substrate and ions to the
intracellular space. During this process, net charge is moved across
the membrane field; this charge movement generates the stoichiometric
current. Kinetic analysis of these currents is necessary to establish
the precise timing of individual steps in the transport cycle,
information critical for evaluating the role of transporters in
buffering and sequestering synaptically released glutamate (Diamond and
Jahr, 1997; Mennerick et al., 1999).
Our results on EAAT2 transporters identify a tight linkage between the
two components of glutamate transporter current. Furthermore, they
suggest that both currents arise from closely connected states occupied
at early steps in the transport cycle, a proposal supported by an
extremely simple mathematical model. Recordings with ion gradients
designed to compare presteady-state kinetics of glutamate exchange with
those of unidirectional influx suggest that, after binding, glutamate
is rapidly translocated by EAAT2. Moreover, binding and translocation
steps take place on a much faster time scale than does a complete
transport cycle. Finally, recombinant EAAT2 shows very similar kinetics
to transporter currents evoked in patches from hippocampal astrocytes
(Bergles and Jahr, 1997), consistent with the proposal that EAAT2
(GLT1) is responsible for the majority of glutamate transport across
astrocyte plasma membranes (Rothstein et al., 1996).
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MATERIALS AND METHODS |
Human embryonic kidney 293 cells. Human embryonic
kidney HEK 293 (HEK 293) cells stably transfected with an
ecdysone-inducible plasmid (Invitrogen, Carlsbad, CA) containing
the human EAAT2 cDNA were a generous gift from Dr. John Dunlop
(Wyeth-Ayerst, Princeton, NJ). Cells were cultured in DMEM
(catalog #10569-010; Life Technologies, Gaithersburg,
MD) supplemented with 10% fetal bovine serum, 100 U/ml
penicillin-streptomycin (catalog #15140-122; Life Technologies), 0.4 mg/ml G418 (Geneticin, catalog #10131-019; Life Technologies), and 0.2 mg/ml Zeocin (catalog #45-0430; Invitrogen). EAAT2 expression was
induced by exposure of the cells to 5-10 µg/ml of the
ecdysone analog ponasterone A (Sigma, St. Louis, MO) for 24-72 hr.
Peak expression, assayed by uptake of radiolabeled glutamate, occurred
24-48 hr after addition of hormone to the culture medium.
Electrophysiology. All recordings were made with an Axopatch
200B amplifier (Axon Instruments, Foster City, CA) and at
temperatures of 21-23 °C. Signals were filtered at 2-5 kHz and
digitized at 10-20 kHz. In whole-cell recordings, pipette solutions
consisted of (in mM): 135 CsNO3, 10 HEPES, 10 tetraethylammonium
(TEA)-Cl, 10 EGTA, and 1 MgCl2, adjusted
to pH 7.3 with CsOH.
Outside-out patch recordings and rapid solution exchange.
Pipettes glass (World Precision Instruments, Sarasota, FL) was pulled to make electrodes with resistances of 1-3 M in the bath solution. Three different pipette solutions were used. To record
transporter-associated currents under conditions that maximized net
transport of glutamate, pipettes contained (in
mM): either 135 KSCN or 135 K-gluconate. To
record transporter-associated currents under conditions that ensured
homoexchange of glutamate, pipettes contained (in
mM): 130 NaSCN and 10 Na-glutamate. All pipette
solutions also contained (in mM): 10 HEPES, 10 TEA-Cl, 10 EGTA, and 1 MgCl2, adjusted to pH 7.3 with either KOH or NaOH as appropriate.
Rapid solution exchange to outside-out patches was accomplished with a
two-barreled application pipette attached to a piezoelectric bimorph.
Each barrel of this application pipette was connected to a four- or
six-way manifold allowing the application of multiple combinations of
solutions to the same patch. At the end of each experiment, the
recording pipette tip was cleared with positive pressure, and a
solution of reduced ionic strength (diluted 50%) was allowed to flow
through the glutamate-containing barrel. Jumps of the application
pipette were then delivered, and the change in holding current was
recorded. The resulting "open-tip" currents are displayed above
each set of experimental traces and represent the approximate time
course of solution exchange across the patch. A more complete
description of these methods has been described previously (Otis and
Jahr, 1998).
The control extracellular solution contained (in mM):135
NaCl, 5.4 KCl, 1.8 CaCl2, 1.3 MgCl2, and 5 HEPES, adjusted to pH 7.4 with NaOH.
For the current versus voltage analyses in different anion gradients,
some of the patches were also exposed to (in mM):
135 NaSCN, 5.4 KSCN, 1.8 Ca gluconate2, 1.3 Mg
gluconate2, and 5 HEPES, adjusted to pH 7.4 with
NaOH. All chemicals were purchased from Sigma.
Data analysis. Data analysis was performed with pClamp 6.0 (Axon Instruments), Origin 5.0 (Microcal, Northampton, MA), and Igor
(Wavemetrics, Lake Oswego, OR). Artifacts caused by the voltage pulse applied to the bimorph have been removed (Otis and Jahr, 1998).
Error bars represent ±1 SEM. Student's t test was used to
determine confidence intervals.
Simulations. Simulations were performed using ScoP 3.51 (Simulation Resources Inc.; http://www.simresinc.com/menu.html).
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RESULTS |
Whole-cell currents
Whole-cell recordings were made from HEK 293 cells stably
transfected with a ponasterone-inducible promoter driving expression of
EAAT2 (see Materials and Methods). Pipettes containing
CsNO3 solution were used to voltage-clamp cells,
and whole-cell currents were recorded in response to application of 250 µM L-Glu. After induction, the cells
exhibited large inward currents in response to Glu (Fig.
1A). Current-voltage
(I-V) relationships were measured by
delivering a series of voltage steps [ 90 to +50 mV;  V
of 10 mV; holding potential of 50 mV] in the absence and presence of
Glu. Families of subtracted responses (Glu control) are shown for a cell exposed to the inducing hormone (Fig. 1A)
and for an untreated control cell (Fig. 1B). In
Figure 1C, mean I-V curves for three
ponasterone-treated cells (filled circles)
and for five untreated cells (open circles) show that
hormone treated cells develop a glutamate-dependent conductance that is
inward at all membrane potentials tested, as expected given a high
permeability to NO3 anions (Wadiche et al.,
1995a; Levy et al., 1998; Wadiche and Kavanaugh, 1998). By comparison,
there was no measurable conductance activated by glutamate in the
untreated cells, demonstrating that, if HEK cells have endogenously
expressed glutamate transporters, they are undetectable with these
methods.
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Figure 1.
Whole-cell currents recorded in HEK 293 cells
stably transfected with EAAT2 under the control of an inducible
promoter. Application of the hormone ponasterone A for >24 hr causes
the appearance of transporter currents activated by 250 µM Glu. A, A family of voltage steps (90
to +50 mV; V of 10 mV; holding potential of 50 mV)
imposed on a hormone-treated cell in the presence of Glu. Currents
recorded in the absence of Glu have been subtracted. B,
A transfected cell that has not been exposed to hormone but was
subjected to the same voltage-clamp protocol as in A.
C, Mean I-V relationships for untreated
transfected cells ( ; n = 5) and for cells
treated with ponasterone A ( ; n = 4). Pipettes
contained CsNO3-based solutions.
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Currents in outside-out patches
The large whole-cell currents elicited by Glu indicated that it
might be possible to record transporter responses in cell-free patches.
To measure rapid dynamics of EAAT2 glutamate transporters in response
to Glu concentration jumps, patches in the outside-out configuration
were removed from cells that had been stimulated with ponasterone A for
24-72 hr. At negative holding potentials, patch currents evoked by a
jump into 10 mM Glu showed rapid inward transients,
followed by sustained current components (Fig.
2A). Outward current
shifts were elicited by jumps from control solution into solution
containing 500 µM kainate, an EAAT2-selective
competitive antagonist (Fig. 2B). The time constants
of activation and deactivation of the outward current evoked by kainate
were 1.17 ± 0.1 (n = 5) and 6.2 ± 0.4 (n = 5) msec, respectively. Consider the two-state reaction, the simplest mechanism by which kainate may block the leak
current:
The experimentally measured time constants predict
kon to be 1.7 × 106
M/sec1, and
koff equal to 161 sec1. These rate constants yield an
affinity constant of 95 µM, in line with
previous estimates (15-60 µM) of kainate
affinity (Arriza et al., 1994; Wadiche et al., 1995b).
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Figure 2.
Transporter currents in an outside-out patch in
response to agonist and antagonist concentration jumps.
A, Transporter anion current in response to a 100 msec
duration, 10 mM pulse of Glu. The time course of the
concentration jump in this and all subsequent figures is displayed
directly above the response. B, A pulse
of the EAAT2 antagonist kainate (100 msec, 500 µM)
elicits an outward current because of the blockade of a persistent
inward anion current observed in the absence of substrate.
C, The response to 10 mM Glu in the
continuous presence of 500 µM kainate. Note the outward
shift in the current trace (dotted trace
indicates the current level before kainate application). The strong
antagonism and slowing of the transient component is expected for a
competitive antagonist that is not transported. Holding potential
(Vh), 88 mV.
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In the continuous presence of 500 µM kainate, responses
to Glu jumps showed blunted peaks and larger steady-state components measured from the prejump baseline (Fig. 2C). These
responses also showed a maintained outward shift in current caused by
kainate, visible in Figure 2B as the shift of
baseline current level from the prekainate level indicated by the
dotted line. Average results for kainate application to six
patches are presented in Table 1, along
with kinetic data measured from other patches exposed only to Glu. In
general, EAAT2 responses appear qualitatively similar to those reported
previously for other native and recombinant glutamate transporters in
that (1) Glu jumps elicit a transient followed by a smaller
steady-state component and (2) kainate or its analog dihydrokainate
induces an apparent antagonism of a leak current (Bergles and Jahr,
1997; Otis and Jahr, 1998; Wadiche and Kavanaugh, 1998). The
pharmacology and kinetics of EAAT2 were very similar to those reported
for glutamate transporters present in patches from hippocampal
astrocytes (Bergles and Jahr, 1997, 1998). Table 1 indicates that patch
data from hippocampal astrocytes and from EAAT2 are statistically
indistinguishable. The comparison suggests that the glutamate
transporter currents described previously in CA1 astrocytes result
either from GLT1 (EAAT2) or a functionally identical isoform.
Patch currents under conditions favoring either transport or
"exchange mode"
Glutamate transporters are powered by transmembrane gradients of
Na+, K+,
H+, and substrate (Kanner and Sharon,
1978; Stallcup et al., 1979; Erecinska et al., 1983; Zerangue and
Kavanaugh 1996b); therefore, changes in internal
[Na+] and [Glu] are expected to alter
transporter currents (Zerangue and Kavanaugh, 1996a; Levy et al.,
1998). Figure 3 shows transporter current
elicited by a jump into 10 mM Glu in two different patches, one with no internal [Na+] or [Glu]
(Fig. 3A) and another with a pipette solution containing 140 mM Na+ and 10 mM Glu substituted for
K+ (Fig. 3B). On average, high
[Na+] and [Glu] in the pipette caused
Glu-elicited currents to have a significantly larger steady-state
component compared with control (control
Iss/Ipeak,
0.2 ± 0.02; n = 14; high
Na+, 0.68 ± 0.06; n = 5; p < 0.0005). This internal solution also converted the fast double-exponential decay observed at the end of the
Glu pulse in control into a slower, single-exponential decay (control,
 1 of 1.3 ± .11 msec;
2 of 18.7 ± 2 msec; 71 ± 3% fast;
n = 12; high Na+,
21.6 ± 2.7 msec; n = 5). This change in kinetics
is consistent with the idea that a complete cycle of transport is
highly unfavorable because of the high internal
[Na+] and [Glu] and because of the
absence of internal K+. With these ionic
gradients, it is believed that the transporter operates as an
exchanger, engaging in a futile shuttle in which the transporter brings
Glu and coupled ions in and is forced by the high internal
[Na+] and [Glu] to reverse and carry
Glu and ions out. Indeed, measurements of substrate and ion fluxes
support the idea no net Na+ or Glu flux is
accomplished under these conditions (Kanner and Sharon, 1978; Kanner
and Bendahan, 1982; Kavanaugh et al., 1997).
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Figure 3.
The kinetics of the transporter anion current are
altered by high [Na+]in and
[Glu]in. A, B, Responses in
two different outside-out patches to a 100 msec pulse of 10 mM Glu. In A, the pipette contained 140 mM KSCN and in B, 130 mM NaSCN
plus 10 mM NaGlu. Vh,
87 mV for both patches.
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The differences in decay kinetics after removal of external Glu (Fig.
3) may reflect two different fates of bound Glu. When operating as an
exchanger, the current decay is expected to predominantly reflect rates
of transition leading to Glu unbinding to the extracellular space,
whereas with 0 internal [Na+] and
[Glu], the decay can reflect an additional path: forward movement
through a complete transport cycle. To explore this hypothesis, the
voltage dependence of current decay was examined under conditions of 0 internal Na+ and high internal
Na+ and Glu. A family of responses to 10 mM Glu jumps recorded at different membrane potentials
(100 to +40; V of 10 mV) are displayed for two such
patches (Fig.
4A,B).
Scaling the steady-state components for the two patches shows that,
when operating with high internal Na+
(Fig. 4B2), current decay is
markedly voltage-dependent, becoming faster at positive potentials. In
contrast, internal solutions favoring net uptake (0 internal
Na+) yield currents with little change in
the decay rate over the same range of membrane voltages (Fig.
4A2). Mean times to
1/2-decay after the offset of the Glu step were measured as a
function of voltage and are displayed for comparison in Figure
4C. A simple explanation for these results is that, upon
removal of 10 mM Glu, the transporter current
deactivates by different routes in the two experimental conditions. In
the high internal Na+/Glu condition, upon
removal of external Glu, relaxation to steady state occurs as
transporters reverse and Glu dissociates into the extracellular space.
However, with 0 internal Na+, complete
forward cycles of transport occur. In this situation, transporters
rarely reverse and allow Glu to unbind to the outside. The sign of the
voltage dependence when the transporters are operating as exchangers
(faster deactivation at positive potentials) suggests that net positive
charge is being expelled from the cell during deactivation. This is the
opposite of the voltage dependence of normal uptake (slower forward
rates at positive potentials) and is consistent with reversal of the
transporter and movement of the positively charged
Na+ ions outward through the membrane
voltage field.
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Figure 4.
The voltage dependence of the decay after the end
of the Glu pulse depends on the pipette solution.
A1, A family of transporter currents
elicited with 10 mM Glu steps recorded at different
membrane potentials (98 to +32 mV; V, 10 mV). A KSCN
solution was in the pipette. A2 shows the
normalized decays at the end of the Glu pulse.
B1, A similar voltage-clamp protocol
(97 to +33 mV; V, 10 mV) applied to a different patch
recorded with 140 mM Na+ and 10 mM Glu in the pipette.
B2, Normalized decay phases.
C, Mean 1/2-decay times as a function of membrane
potential for patches recorded with pipette solutions containing 140 mM KSCN (; n = 14) or 140 mM Na+ and 10 mM
Glu (; n = 5). Note the lack of voltage
dependence to the decay with high [K+] and the
marked voltage dependence with high [Na+] and
[Glu] in the pipette.
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Under 0 internal Na+ conditions, if most
transporters successfully complete cycles, then the average time
required for a single cycle of transport will dictate the rate of
recovery from the steady-state current level (Otis and Jahr, 1998). On
the other hand, under exchange conditions, rates of reversal and/or
unbinding will set the recovery time course. Figure
5 compares recovery time courses with 0 internal Na+ and high internal
Na+/Glu by delivering pairs of pulses of
Glu separated by various intervals. As is evident from the single
patches (Fig. 5A,B) and from the
group data (Fig. 5C), recovery is slower with high
Na+/Glu pipette solutions. Average
single-exponential fits to recovery data from individual patches with 0 internal Na+ (18.4 ± 2.8 msec;
n = 9) and with high internal
Na+/Glu (38.7 ± 10.3 msec;
n = 5) are superimposed on the data in Figure
5C. Interestingly, under exchange conditions, the rate of
decay upon Glu removal and the rate of recovery are more similar (21.6 and 38.7 msec, respectively) than the same rates with 0 internal
Na+ (1.3 and 18.4 msec, respectively).
This can be explained as follows: with high internal
Na+/Glu, the anion current deactivates and
recovers by the same route (unbinding to the outside), but with 0 internal Na+, anion current deactivates by
forward movement into nonconducting states further along in the cycle
(a fast process) and recovers only upon completion of an entire cycle
(a slower process).
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Figure 5.
The rate of recovery from depression of the
transporter current depends on the pipette solution. A,
B, Responses to pairs of 10 msec steps into 10 mM Glu with the indicated pipette solutions. Intervals of
5, 10, 15, 20, 30, 40, 50, 60, 80, 100, and 150 msec are displayed. A
response to a single pulse of Glu has been subtracted from each
displayed response. The dotted trace indicates the mean
peak current of the response to a single pulse.
Vh, 88 mV in A and
101 mV in B. C, Mean amount of recovery
(peak of the 2nd response/peak of the 1st) plotted against the interval
between pulses (note the log scale). indicates 140 mM
[KSCN]in (n = 9), and indicates
140 mM [Na+]in and 10 mM [Glu] (n = 5).
Lines display the means of single-exponential fits to
the individual patches with values of 18.4 and 38.7 msec for the
K+ and high Na+ solutions,
respectively.
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Different anion gradients isolate the stoichiometric and anion
current components
The conditions used to record currents in the previous experiments
(140 mM SCN in the pipette)
cause the anion current component to dominate the total transporter
current. Many of the interpretations in this study are based on the
premise that the anion current is tightly linked to the transport cycle
and that conducting states are accessible only from limited regions of
that cycle. This premise predicts that the kinetics of the two
components of transporter current should be correlated.
To isolate the different components of transporter current,
I-V relationships in response to 10 mM Glu jumps were measured using four different
anion gradients:
SCNin/Clout,
SCNin/SCNout,
gluconatein/SCNout,
and
gluconatein/Clout
in which the indicated anion is present at a concentration of 135-150
mM. Previous estimates of permeability have
established that SCN is ~ 70 times more permeable than Cl and that
gluconate is impermeable (Wadiche and
Kavanaugh, 1998). Figure
6A1-D1
displays families of superimposed responses recorded at different
membrane potentials (100 to +50 mV; V of 10 mV). These
traces were recorded from three different patches
(C1 and D1
are from the same patch) exposed to the anion gradients listed above.
Mean I-V relationships are shown to the right.
As expected, the reversal potentials
(Erev values) shift with changes
in the anion gradient. With an
SCNin/Clout
gradient, Erev was more than
+40 mV (n = 11) and thus not determinable; with
SCNin/SCNout,
Erev was +3 ± 2 mV
(n = 4); with
gluconatein/SCNout,
Erev was 59 ± 2 mV
(n = 8); and with
gluconatein/Clout,
Erev was more than +50 mV and not
measurable (n = 7). How do responses dominated by
anion current (Fig. 6A) compare in size with
responses dominated by stoichiometric current (Fig.
6D)? Tested in two different groups of patches, the
mean peak current at 100 mV was 255 ± 84 pA
(n = 11) for the
SCNin/Clout
condition and 18 ± 4 pA (n = 7) for the
gluconatein/Clout
gradient. This is consistent with previous estimates of the contribution of anion current to transporter currents from hippocampal astrocytes in physiological chloride gradients (Table 1).
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Figure 6.
Different anion gradients can be used to
discriminate the stoichiometric and the anion components of transporter
current. A1-D1
display families of traces recorded at different membrane potentials
(100 to +50; V, 10 mV) using the following gradients of
major anions SCNin/Clout,
SCNin/SCNout,
gluconatein/SCNout, and
gluconatein/Clout for A -D,
respectively. C1 and
D1 show the same patch.
A2-D2 represent
mean I-V relationships for these same combinations of
solutions. The data were taken from n = 11, 4, 8, and 7 patches for A-D. For comparison, responses in
A are dominated by the anion current, and responses in
D are dominated by stoichiometric component of the
transporter current.
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To more closely examine the relationship between coupled charge flux
and the anion conductance, the kinetics of responses dominated by anion
current
(SCNin/Clout
gradient) and by stoichiometric current
(gluconatein/Clout
gradient) were compared. On average, the 1/2-decay time in
gluconatein/Clout
measured between 80 and 100 mV was 0.82 ± 0.11 msec
(n = 7), whereas in the
SCNin/Clout
gradient, it was 1.07 ± 0.06 msec (n = 14). This
suggests that stoichiometric current develops and subsides slightly
faster than anion conductance (although not significantly;
p > 0.025). Moreover, anion and stoichiometric
components slowed in parallel at positive potentials (Fig.
6A1,D1),
perhaps reflecting voltage dependence of the early events in the
transport cycle. These results can be generally interpreted to reflect
fast charge translocation steps (giving rise to the stoichiometric
current) and slightly slower anion conducting steps, which are confined
to a region at the beginning of a multistep cycle. This region of the
cycle is rapidly and transiently occupied by most of the transporters after nearly synchronous binding. Thereafter, transporters
"desynchronize" and equilibrate throughout the cycle, their
desynchronous cycling giving rise to steady-state current (Bergles et
al., 1997; Otis et al., 1997).
We designed a more sensitive protocol to compare the kinetics of the
anion and stoichiometric currents. Each patch was exposed to two
different anion gradients. In
gluconatein/SCNout
(Fig. 7A), both current
components are apparent; inward currents represent stoichiometric
current, whereas outward currents represent SCN currents. Replacing
SCNout with
Clout (Fig.
7B) causes the outward anion currents to disappear, leaving
inward currents at all potentials. These responses are dominated by the
stoichiometric component of the transporter current. Net outward
current is minimal in this condition because of the relatively small
contribution of anion current in physiological chloride (Arriza et al.,
1994; Wadiche et al., 1995a; Levy et al., 1998). Figure 7C
shows a subtraction of the stoichiometric currents (Fig. 7B)
from the mixed currents (Fig. 7A) yielding pure
SCN currents. From these comparisons, it
is apparent that the stoichiometric current activates and deactivates
slightly faster than the anion conductance. When both components are
present, the slight mismatch in time course gives rise to an inward
transient, followed by a delayed trough in the responses recorded at
negative potentials (Fig. 7A, arrow).
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Figure 7.
Stoichiometric current activates slightly faster
than anion current. A, Patch currents at a series of
membrane potentials (100 to +20; V, 30 mV) in
response to 10 mM Glu steps with
gluconatein/SCNout. Under these
conditions, stoichiometric current is inward and anion current is
outward. B, In the same patch and at the same holding
potentials, responses with Clout. Inward stoichiometric
current dominates the small outward anion current. C,
Subtraction of traces in B from those in
A, yielding pure anion currents carried by
SCN. Note that the outward currents are slightly
slower than the inward currents. With both current components present,
this is evident as an overshoot occurring after the fast inward
transient (arrow in A).
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Many of the measurements and conclusions in this study depend on
achieving a high level of expression of the EAAT2 protein. The density
of expression can be estimated from responses using gluconate as the internal anion. The
number of transporters per patch can be estimated by integrating
currents over 3 msec intervals, beginning at the Glu pulse onset, and
then applying the equation n = Q/z eo, where Q is the charge;
z = 3, the number of charges translocated in the first
part of the transport cycle; and eo is
the charge per elementary particle, 1.6 × 1019 C (Bergles and Jahr, 1997). Average
charge transfer measured in this way was 29.4 ± 6.9 fC
(n = 7), yielding an estimate of 61,000 ± 14,000 transporters per patch. Assuming the same mean surface area for the
patches as was measured in a previous study (~ 7 µm2; Bergles and Jahr, 1997), the
density of transporters in our experiments is high
(~9000/µm2). Although this estimate is
higher than the density estimated for CA1 astrocytes
(~2000/µm2; Bergles and Jahr, 1997),
it is similar to the density estimate of
8500/µm2 based on quantitative
immunoblotting in hippocampus (Lehre and Danbolt, 1998). The estimate
is also comparable with the density estimates for EAAT1 transporters
expressed in Xenopus oocytes (17,000/µm2; Wadiche and Kavanaugh,
1998).
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DISCUSSION |
Recombinant EAAT2 transporter currents are similar to transporter
currents in hippocampal astrocytes
EAAT2 (GLT1) is the predominant glial cell transporter in the CNS,
accounting for most of the glutamate clearance in the brain (Rothstein
et al., 1994, 1996; Tanaka et al., 1997). However, many astrocytic
glia, such as those located in the CA1 region of the hippocampus, also
express EAAT1 (GLAST) at lower levels (Rothstein et al., 1994; Lehre et
al., 1995). Comparing the results in this study with those of EAAT1
(GLAST) (Wadiche and Kavanaugh, 1998) and to transporter currents from
rat hippocampal astrocytes (Bergles and Jahr, 1997, 1998) supports the
proposal that glutamate transport by astrocytes is dominated by EAAT2
(GLT1). Moreover, if functional transporters have a multimeric
structure (Haugeto et al., 1996; Dehnes et al., 1998), these results
also imply that EAAT2 operates as a homomultimer. Lastly, the data
argue that the high degree of homology observed between rodent and
human EAAT2 sequences (Arriza et al., 1994) dictates similar functional properties. However, these preliminary conclusions must be strengthened by studies that further examine the properties of individual
transporters and combinations of coexpressed transporters.
To date, rapid solution exchange techniques have uncovered distinct
kinetic properties for different transporter isoforms. Recombinant
EAAT1 shows a much larger
Iss/Ipeak
ratio (0.64) and a much more slowly decaying transient ( of 14 msec)
than those measured in this study (Wadiche and Kavanaugh, 1998). On
Bergmann glial cells, a native transporter that is insensitive to
dihydrokainate (and thus is not EAAT2/GLT1), also shows a larger
Iss/Ipeak
ratio and slower transients (Bergles et al., 1997). Finally, Purkinje neuron glutamate transporters exhibit a smaller
Iss/Ipeak
ratio (0.14) and a more slowly decaying transient ( of 8 msec) (Otis and Jahr, 1998).
A simple kinetic model can explain both components of the
transporter current
All glutamate transporters tested to date show a transient
followed by a steady-state current in response to rapid applications of
Glu. Although previous measurements concentrated on anion currents, in
CA1 astrocytes, small stoichiometric currents were described (Bergles
and Jahr, 1997). The high level of expression in this study allows a
direct comparison of both components of the transporter current. To
test hypotheses about how charge translocation is related to the anion
conductance, we used a simple model of the transport cycle. The cycle
was divided into three collections of states as shown in Figure
8: nonconducting states with substrate binding sites facing the extracellular space, designated
[nonconductingout]; anion
conducting states, designated [conducting]; and
nonconducting states with substrate binding sites facing the
intracellular space, designated
[nonconductingin]. In the model,
conducting transporters are formed after the binding of substrate and
cotransported ions (Na+ and
H+). The small conductance seen in the
absence of substrate (shown in Fig.
2B,C) has been omitted for
simplicity. As an additional simplification, individual binding steps
are not explicit in the model (none of the rate constants are
concentration-dependent). Forward rate constants (i.e., those leading
to Glu uptake) are denoted by positive subscripts, whereas backward
rate constants have negative subscripts.
View larger version (13K):
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|
Figure 8.
A simple kinetic model that accounts for the two
components of transporter current. A, The model consists
of three sets of states connected by the following rate constants:
k1 = 0 or 2000/sec;
k1 = 4/sec;
k2 = 480/sec *
exp[(VF)/(2RT)];
k2 = 400/sec *
exp[(VF)/(2RT)];
k3 = 10/sec *
exp[(VF)/(2RT)];
and k3 = 1/sec *
exp[(VF)/(2RT)].
V of 90 mV for simulations in B and
C and 100 mV in D. B,
Simulated stoichiometric current (the sum of the net fluxes for the two
voltage-dependent transitions [conducting]  [nonconductingin] and
[nonconductingin] [nonconductingout]) in
response to a 100 msec duration jump in the value of
k1 from 0 to 2000/sec (to simulate the Glu
pulse). C, Simulated anion current (the occupancy of
[conducting]) in response to the Glu pulse.
D, Anion current in response to the Glu pulse with the
effects of high [Na]in and [Glu]in
simulated by increasing the rate constant factor in
k2 from 400/sec to 6000/sec.
|
|
Two forward and two backward rate constants have voltage dependence of
the form
where kf0 and
kb0 are the forward and backward rate
constants at 0 mV; z = 1, the valence of the charge
movement; V is the membrane potential; T = 298 K; and F and R are
Faraday's constant and the gas constant, respectively. Charge
movements were assumed to traverse symmetric energy barriers and to
cross the entire field (Läuger, 1991). Reflecting the established
stoichiometry for the transport cycle (1 Glu/3 Na+/1
H+ cotransported and 1 K+ counter-transported) (Zerangue and
Kavanaugh, 1996a; Levy et al., 1998), the total charge movement per
cycle (i.e., the sum of z values for a complete cycle) was
constrained to two. Stoichiometric current was simulated by summing the
net charge flux that occurs as a result of the two voltage-dependent
steps, [conducting] [nonconductingin] and
[nonconductingin] [nonconductingout].
Simulations of the anion current and stoichiometric charge flux in
response to a [Glu] jump are shown in Figure 8. Many features of both
components of transporter current are captured by the model. These
include a rapid rise-to-peak, a quickly decaying transient
(1/2-decay times for stoichiometric and anion currents is 0.8 msec), a double-exponential deactivation, and a steady-state level of
~15% of the peak. Simulated effects of high
[Na+]in and
[Glu]in
(Iss/Ipeak,
0.72 at 100 mV) (Fig. 8D) are similar to the data.
Although not shown, the model also reproduces other behaviors of the
transporter such as the rate of recovery ( of 17.4 msec) and the
slowing of the transient at positive membrane potentials.
Several general insights can be drawn from this simple model. First,
the model provides an explanation for why large transient and smaller
steady-state responses are observed for both the anion and
stoichiometric components of the transporter current. This results from
movement through a multistate cycle in which only a subset of states
are experimentally detectable (the [conducting] states).
To generate the transient, movement through these states must be rapid
and synchronous. This is accomplished by trapping transporters in the
absence of Glu in the
[nonconductingout] states. Glu
binding can then trigger nearly synchronous entry into the [conducting] collection of states. Transporters eventually
become desynchronized relative to one another because, in the presence of Glu, they have access to all states in the cycle.
A second prediction is that the steps generating stoichiometric and
anion components of the current occur at very similar points in the
transport cycle. This provides support for physical models in which the
transporter protein adopts a configuration that is leaky to anions
during the translocation of glutamate and cotransported ions (Wadiche
et al., 1995a).
Last, the model explains why the relative contribution of the
steady-state anion current is larger under exchange conditions (Billups
et al., 1996; Otis and Jahr, 1998). Simulations of high [Glu] and
[Na+] on both sides of the membrane
confines transporters to the [conducting] collection of
states, making it unlikely that transporters remain unbound or enter
other states in the cycle.
This is a simplified version of previous models that we and others have
developed (Billups et al., 1996; Larsson et al., 1996; Otis and Jahr,
1998; Wadiche and Kavanaugh, 1998). Although the present model fails to
describe all aspects of the data, including the conductance observed in
the absence of substrate (Fig. 2) and the concentration dependencies of
transitions, the simplifications introduced result in fewer free
parameters and more easily understood dynamic behavior. More detailed
models that explicitly incorporate substrate and ion binding are
essential for a complete description of the transporter.
Different deactivation pathways suggest that transporters have
high efficiency
Efficient glutamate transport requires Glu binding in the
extracellular space, dissociation into the intracellular compartment, and recycling of the empty binding site. In contrast, in ionic gradients favoring exchange, complete cycles of transport are believed
to be extremely rare. In the exchange mode, transporters bind Glu,
activate an anion conducting state, and deactivate by unbinding Glu
into the extracellular space. Independent experimental evidence
supporting the existence of exchange modes of operation has been
provided by biochemical measurements of substrate and ion flux (Kanner
and Sharon, 1978; Kanner and Bendahan, 1982; Kavanaugh et al., 1997;
Zhang et al., 1998).
Different fates for Glu under conditions of high internal
Na+/Glu (exchange) or 0 internal
Na+/Glu are reflected in different time
courses of deactivation and recovery of the anion conductance (Figs. 4,
5). Under exchange conditions, both deactivation and recovery must
occur by reversal and unbinding to the extracellular space. Glu pulses
with 0 internal Na+ elicit faster,
voltage-independent deactivation kinetics, suggesting that forward
transitions away from anion conducting states rapidly sequester
transmitter and thereby prevent the voltage-dependent route of reversal
and unbinding. This dichotomy in deactivation behavior supports the
proposal that EAAT2 efficiently translocates Glu under physiological
conditions. This may not be the case for all transporter subtypes;
significantly different kinetics have been observed in concentration
jump experiments on other glutamate transporter subtypes (Wadiche and
Kavanaugh, 1998; A. Tzingounis and M. P. Kavanaugh,
unpublished observations). In contrast to EAAT2, kinetic modeling of
EAAT1/GLAST suggests that it is more probable for glutamate to unbind
to the outside than to be transported (Wadiche and Kavanaugh, 1998).
The importance of translocation efficiency is highlighted by recent
experiments on hippocampal neurons cocultured with glia (Mennerick et
al., 1999). Glial cell depolarization was shown to slow excitatory
synaptic currents. However, depolarization did not affect glutamate
affinity for the transporter, implying that depolarization prolongs the
lifetime of glutamate without reducing binding but by slowing the
translocation of glutamate after binding. These results argue that key
steps after binding, perhaps encompassing translocation of substrate
and ions, are critical for determining the functional properties of
glutamate transporters. The present data are consistent with rapid and
efficient sequestration of glutamate by EAAT2 during synaptic
transmission. Furthermore, the data provide strong confirmation
that anion conductance is tightly associated with the transport
cycle and that it can be used to estimate glutamate transport in
situ.
| |
FOOTNOTES |
Received Oct. 25, 1999; revised Jan. 24, 2000; accepted Jan. 31, 2000.
This work was supported by National Institutes of Health Grant NS33270.
We are grateful to D. Bergles, C. Jahr, and J. Wadiche, as well as A. Tzingounis for discussions during this work. We thank Dr. J Dunlop for
the generous gift of the HEK 293 cell line.
Correspondence should be addressed to Thomas S. Otis, Department of
Neurobiology, University of California, Los Angeles Medical Center, 650 Charles Young Drive, Box 951763, Los Angeles, CA 90095-1763. E-mail:
otist{at}ucla.edu.
| |
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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]
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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]
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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]
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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]
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B. Barbour
An Evaluation of Synapse Independence
J. Neurosci.,
October 15, 2001;
21(20):
7969 - 7984.
[Abstract]
[Full Text]
[PDF]
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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]
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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]
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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]
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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]
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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]
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TOP
ABSTRACT
INTRODUCTION
![k<SUB><UP>forward</UP></SUB>=k<SUB><UP>f0</UP></SUB> <UP>exp</UP>[<UP>−</UP>(zVF)/(2RT)] <UP>and</UP> k<SUB><UP>backward</UP></SUB>=k<SUB><UP>b0</UP></SUB> <UP>exp</UP>[(zVF)/(2RT)],](/content/vol20/issue8/fulltext/2749/img002.gif)
