From the Max-Planck-Institut für Biophysik,
Kennedyallee 70, Frankfurt D-60596 and
Westfälische
Wilhelms Universität Münster, Institut für Biochemie
Wilhelm-Klemm-Strasse 2, Münster D-48149, Germany
Received for publication, August 5, 2002, and in revised form, September 25, 2002
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ABSTRACT |
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Glutamate transport by the neuronal excitatory
amino acid carrier (EAAC1) is accompanied by the coupled movement of
one proton across the membrane. We have demonstrated previously that
the cotransported proton binds to the carrier in the absence of
glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here,
we used site-directed mutagenesis together with a rapid kinetic
technique that allows one to generate sub-millisecond glutamate
concentration jumps to locate possible binding sites of the glutamate
transporter for the cotransported proton. One candidate for this
binding site, the highly conserved glutamic acid residue Glu-373 of
EAAC1, was mutated to glutamine. Our results demonstrate that the
mutant transporter does not catalyze net transport of glutamate,
whereas Na+/glutamate homoexchange is unimpaired.
Furthermore, the voltage dependence of the rates of Na+
binding and glutamate translocation are unchanged compared with the
wild-type. In contrast to the wild-type, however, homoexchange of the
E373Q transporter is completely pH-independent. In line with these
findings the transport kinetics of the mutant EAAC1 show no deuterium
isotope effect. Thus, we suggest a new transport mechanism, in which
Glu-373 forms part of the binding site of EAAC1 for the cotransported
proton. In this model, protonation of Glu-373 is required for
Na+/glutamate translocation, whereas the relocation of the
carrier is only possible when Glu-373 is negatively charged.
Interestingly, the Glu-373-homologous amino acid residue is glutamine
in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently,
our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes.
The uphill transport of negatively charged amino acid substrates
catalyzed by high affinity plasma membrane glutamate transporters is
driven by the coupled downhill movement of three sodium ions and one
potassium ion across the membrane (1-3). In addition, one proton is
cotransported with glutamate into the cell (4, 5). Because of this
stoichiometry, glutamate transport is electrogenic with a total of two
positive charges being translocated to the intracellular side during
each completed transport cycle. Whereas glutamate, sodium ions, and the
proton are cotransported together in one branch of the reaction cycle
(6), the potassium ion is countertransported in the
glutamate-independent relocation step of the carrier (7, 8).
Recently, we have investigated the mechanism of proton transport by
EAAC1 (excitatory amino acid
carrier
1),1 a neuronal
subtype of the glutamate transporter family (9, 10). We (6) and others
(11, 12) have demonstrated previously that protonation of EAAC1 creates
a high affinity binding site for the amino acid substrate on the
transporter. In addition, protonation is required for substrate
translocation across the membrane, whereas the relocation of the
glutamate-free transporter form requires dissociation of the proton
from EAAC1 (6). However, no information was obtained about the
molecular nature of the proton binding site on EAAC1 in this previous
study. On the basis of the apparent pKa of
the ionizable residue(s) of 6.5-8, and according to a previous
interpretation (13), it was hypothesized that the protonation might
involve a histidine residue. However, not only histidine residues, but
also acidic amino acid residues within the transmembrane domain, appear
to be important for proton translocation in other proton transporters
and pumps (14-17).
In high affinity glutamate transporters at least three acidic amino
acid residues are located in putative membrane-spanning regions. These
amino acid residues, which are highly conserved in the EAAT family
(Asp-398, Glu-404, and Asp-470 in GLT1, which correspond to Glu-367,
Glu-373, and Asp-443 in EAAC1, Fig. 1), are critical for the functioning of the transporter as demonstrated by
Pines et al. (18). Neutralizing the putative negative charge of these amino acid residues of EAATs by site-directed mutagenesis severely impaired glutamate uptake by the mutant transporters (18). The
mutant transporters D398N and D470N are non-functional, whereas Glu-404
mutants showed residual activity that was finally attributed to an
electroneutral substrate homoexchange reaction suggesting that the
potassium-induced relocation of the transporter is impaired (19).
Consequently, the acidic amino acid residue in position 404 appears to
be important for the relocation step but not for the initial glutamate
translocation reaction of GLT1. Therefore, Glu-404 in GLT1 and
homologous amino acid residues in other EAATs could be possible
candidates for the proton binding site in glutamate transporters.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence alignment of the highly conserved
putative transmembrane-spanning region around Asp-367 and Glu-373 of
the EAA and ASC transporters belonging to the EAAT family. Acidic
amino acids are shown in red, and the
arrow indicates the position of the mutation.
In this study, the charge translocation reaction by the mutant E373Q
glutamate transporter EAAC1 (corresponding to E404N in GLT1) was
investigated in detail and correlated with the proton transport by
EAAC1. Furthermore, it was determined whether Glu-373 contributes to
the charge neutralization provided by the empty EAAC1 cation binding
sites, and whether its charge moves across the electric field during
the charge translocating conformational change of the transporter.
Based on the facts that glutamate binding to EAAC1E373Q is
completely pH-independent and homoexchange is unimpaired, we propose
that Glu-373 is part of the proton-translocating machinery of
EAAC1.
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EXPERIMENTAL PROCEDURES |
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Molecular Biology and Transient Expression-- Wild-type EAAC1 cloned from rat retina was subcloned into pBK-CMV (Stratagene) as described previously (20, 21) and was used for site-directed mutagenesis according to the QuikChange protocol (Stratagene, La Jolla, CA) as described by the supplier. The primers for mutation experiments were obtained from MWG Biotech (Ebersberg, Germany). The complete coding sequences of mutated EAAC1 and ASCT2 clones were subsequently sequenced. Rat ASCT2 (22, 23) was subcloned into the EcoRI site of the pBK-CMV vector (Stratagene) for mammalian expression.
Wild-type and mutant EAAC1 and ASCT2 constructs were used for transient transfection of sub-confluent human embryonic kidney cell (HEK293, ATCC number CGL 1573) cultures using the calcium phosphate-mediated transfection method as described previously (24). Electrophysiological recordings were performed between days 1 to 3 post-transfection.
Immunofluorescence--
Immunostaining of EAAC1-expressing cells
was performed as described (25). In brief, transfected HEK293 cells
plated on poly-D-lysine-coated coverslips were fixed in 5%
acetic acid in methanol for 4 min at 20 °C. After several washing
steps with phosphate-buffered saline (PBS), they were incubated
overnight at 4 °C in 0.1% (v/v) Triton X-100 in PBS in the presence
of 0.01 mg/ml affinity-purified EAAC1 antibody (Alpha Diagnostics).
Following primary antibody incubation, the cells were rinsed and
incubated (1 h) with anti-rabbit IgG conjugated to Cy3 (1:500, Dianova,
Germany) in PBS containing 0.1% (v/v) Triton X-100. After washing with
PBS and water the cells were affixed with coverslips in Mowiol
(Hoechst, Germany). The Cy3 immunofluorescence was excited with a
mercury lamp, visualized with an inverted microscope (Zeiss) by using a
TMR filterset (Omega) and photographed with a digital camera (Sony).
Electrophysiology--
Glutamate-induced EAAC1 currents were
recorded with an Adams & List EPC7 amplifier under voltage-clamp
conditions in the whole-cell current-recording configuration (26). The
typical resistance of the recording electrode was 2-3 M; the series
resistance was 5-8 M
. Because of the small glutamate-induced
membrane conductance changes (typically <5 nS), series resistance
(RS) compensation had no effect on the magnitude
of the observed currents. Therefore, RS was not
compensated. Two different pipette solutions were used depending on
whether mainly the non-coupled anion current (with thiocyanate) or the
coupled transport current (with chloride) was investigated. These
solutions contained (in mM): 130 KSCN or KCl, 2 MgCl2, 10 TEACl, 10 EGTA, and 10 HEPES (pH 7.4/KOH). Thiocyanate was used because it enhances glutamate transporter associated currents and allows the detection of the EAAC1
anion-conducting mode (21, 27). For the electrophysiological
investigation of the Na+/glutamate homoexchange mode the
pipette solution contained (in mM) 130 mM
NaCl/NaSCN, 2 MgCl2, 10 TEACl, 10 EGTA, 10 glutamate, and
10 HEPES (pH 7.4/NaOH). The currents were amplified with an Adams & List EPC-7 amplifier, low pass filtered at 1-10 kHz (Krohn-Hite 3200),
and digitized with a digitizer board (Axon, Digidata 1200) at a
sampling rate of 10-50 kHz, which was controlled by software (Axon
PClamp). All the experiments were performed at room temperature.
Laser-pulse Photolysis and Rapid Solution Exchange--
The
rapid solution exchange was performed as described previously (8, 21).
Briefly, substrates were applied to the EAAC1-expressing cell by means
of a quartz tube (opening diameter, 350 µm) positioned at a distance
of ~0.5 mm to the cell. The linear flow rate of the solutions
emerging from the opening of the tube was ~5-10 cm/s, resulting in
typical rise times of the whole-cell current of 30-50 ms (10-90%).
Laser-pulse photolysis experiments were performed according to previous
studies (21, 28). CNB-caged glutamate (Molecular Probes (29)), in
concentrations of 1 mM or free glutamate were applied to
the cells and photolysis of the caged glutamate was initiated with a
light flash (340 nm, 15 ns, excimer laser pumped dye laser, Lambda
Physik, Göttingen, Germany). The light was coupled into a quartz
fiber (diameter, 365 µm) that was positioned in front of the cell in
a distance of 300 µm. The laser energy was adjusted with neutral
density filters (Andover Corp.). With maximum light intensities of
500-600 mJ/cm2 saturating glutamate concentrations could
be released, which was tested by comparison of the steady-state current
with that generated by rapid perfusion of the same cell with 1 mM glutamate.
Data Evaluation and Terminology--
For simplicity, the
following terminology was used: The glutamate-induced coupled transport
current was termed
I
Non-linear regression fits of experimental data were performed with
Origin (Microcal, Northampton, MA) or Clampfit (Axon Instruments, Foster City, CA) by the use of the following equations: The
pre-steady-state currents of the anionic current
I) were fitted with a sum of two exponential
functions and a steady-state current component
(Iss): I = I1·exp(
t/
rise) + I2·exp(
t/
decay) + Iss. The pre-steady-state transport currents
I
) were fitted with a sum of three
exponential functions and a stationary current component:
I = I1·exp(
t/
rise) + I2·exp(
t/
decay1) + I3·exp(
t/
decay2) + Iss. Under homoexchange conditions
Iss became zero. The observed time constants of
rise of
I
decay1 of
I
fast. A similar time dependence with
~8 ms was
found for the time constants
decay of
I
decay2 of
I
slow. Dose-response data were fitted with the Hill equation: I = Imax([Glu]/([Glu] + Km))n, with n being the Hill
coefficient. The Km as a function of pH was
calculated with the following equation: Km = KS(KH + [H+])/[H+], where KS
and KH are the apparent affinities for the
substrate and the proton.
Each experiment was repeated at least five times with at least three
different cells. Error bars represent the error of a single
measurement (mean ± S.D.), unless stated otherwise. For some
kinetic constants a Student's t test analysis was performed to test for significance of the comparison of WT and mutant transporter data.
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RESULTS |
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The mutant glutamate transporter EAAC1E373Q expressed
in HEK293 cells was functionally analyzed by whole-cell current
recording experiments. Typical current measurements of wild-type EAAC1
(EAACWT) and EAAC1E373Q after application of
saturating concentrations of glutamate by using a rapid solution
exchange method are shown in Fig. 2. No
steady-state transport currents
I35 pA (Fig. 2B, n = 20, 6 cells), in line with previous observations (2). These observations are consistent
with results obtained for the homologous mutation of GLT1 (E404N) under
steady-state conditions (18). Although glutamate-induced steady-state
currents are abolished by the mutation, a rapid inwardly directed
transient EAAC1E373Q current is observed at the time of the
glutamate application (Fig. 2A) that is absent in
non-transfected control cells (Fig. 2C). This result
indicates that EAAC1E373Q is functionally expressed and
mediates glutamate-induced charge movements. Expression of
EAAC1E373Q was confirmed by immunocytochemistry as shown in
Fig. 2D, demonstrating the membranous localization of the
mutant transporter. The time dependence of the rapid charge movements
is too fast to be resolved by the rapid solution exchange method with a
maximum time resolution of about 50 ms. Therefore, we used laser-pulse
photolysis of caged glutamate, allowing us to generate glutamate
concentration jumps within less than 100 µs (21) to study the
function of EAAC1E373Q in more detail.
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Pre-steady-state Kinetics--
EAACWT exhibits
inwardly directed pre-steady-state currents upon applying a rapid
concentration jump of extracellular glutamate ICNB-caged glutamate. The current rises very rapidly with an average time constant
of
rise = 0.3 ± 0.1 ms (n = 8; WT,
0.5 ± 0.1 ms). The decay consists of two components. The time
constant for the rapidly decaying component is
fast = 1.2 ± 0.3 ms (n = 4; WT, 0.9 ± 0.1 ms).
Thus, the kinetics of the current rising phase and the rapid component
of the decaying phase are unchanged compared with the EAACWT (p > 0.15).
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The second component of the current decay, slow, was
slower than that observed in EAACWT. This decay process was
previously assigned to the electrogenic glutamate translocation
reaction across the membrane. Therefore, the existence of the slow
current component indicates that glutamate translocation is still
functional in the mutated transporter but slowed compared with
EAAC1WT. Because EAACE373Q-mediated currents
were generally smaller than those observed for the wild-type, the
slowly decaying component could not be quantitatively evaluated for the
I
Glutamate-induced Anion Currents--
To obtain quantitative
information about slow, we performed experiments in the
anion-conducting mode of EAAC1. It was previously shown for
EAAC1WT that (i) the current mediated by glutamate-induced SCN
outward movement
I
I
100 pA, whereas the
stationary current component, Iss, is only
10
pA (Iss/Ips = 0.04 ± 0.01, n = 3). In contrast, the
Iss/Ips ratio for
EAAC1WT is significantly larger (0.46, p = 0.004, Fig. 4B) (8, 30). The decay of the transient current
could be represented with a monoexponential decaying function, within
experimental error. The average time constant for this decay of
EAACE373Q (
slow = 20 ± 2 ms,
n = 6) was significantly slower than the decay of the EAAC1WT (
slow = 7.4 ± 1 ms,
n = 6, p < 0.0001). The
Iss/Ips ratio recorded in
the anion-conducting mode is a measure for the turnover rate of the
transporter (Iss/Ips = kb·
slow and
kt = kb (1
kb·
slow)) (8, 30). Here,
kb is the rate constant for relocation of the
glutamate-free transporter. From the
Iss/Ips ratio, together with the time constant
slow, a maximum steady-state
turnover rate constant (kt) for
EAACE373Q of about 1.9 s
1 was calculated.
This low value, which is almost 20-fold lower than the one determined
for EAACWT (35 s
1), explains why no
steady-state transport currents can be recorded for
EAACE373Q.
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Voltage Dependence of EAACE373Q Kinetics--
The
voltage dependence of the steady-state and pre-steady-state properties
of EAACWT and EAACE373Q are shown in Fig.
5. The rate constant for the current rise
of EAACE373Q, 1/fast, increases slightly
with increasing transmembrane potential (Fig. 5A). The slope
of the log(1/
fast) versus
Vm relationship of (0.5 ± 0.3) V
1 is within the experimental error identical to that
found for EAACWT (1.2 ± 0.3) V
1. In
contrast, the voltage dependence of the rate constant for the decaying
phase of the current is reversed and about 4-fold stronger, as shown in
Fig. 5A. A similar behavior is well documented for the
EAACWT (8). The slope of the log(1/
slow)
versus Vm relationship is (3.9 ± 0.4) V
1, which agrees well with that of
EAACWT (
4.0 ± 0.3 V
1, Fig.
5A).
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The voltage dependence of steady-state currents is shown in Fig.
5B. Because EAACE373Q does not catalyze
steady-state transport, these experiments could not be performed for
I I
60
mV (Fig. 5B). As demonstrated in Fig. 5A,
wild-type EAAC1 shows the same saturation of currents at
Vm <
60 mV under homoexchange conditions.
Such a saturating behavior has not been found for previous
EAACWT experiments using the forward transport mode
(8).
EAACE373Q Currents Are pH-independent--
The
experiments described above demonstrate that the E373Q mutation in EAAC
only affects the rate of glutamate translocation but not its voltage
dependence, suggesting that translocation can also occur in the absence
of an acidic amino acid side chain at position 373. To test this
hypothesis, we determined the pH dependence of EAACE373Q
transport kinetics. Fig. 6A
shows the dose-response relationship of EAACE373Q currents
at different extracellular pH values between 7.3 and 10.0. In this pH
range EAACWT exhibits a 100-fold change in its apparent
glutamate affinity (Fig. 6B) (6). In contrast, the apparent
affinity of EAACE373Q for glutamate is pH-independent. This
effect is not caused by an altered intrinsic glutamate affinity of
EAACE373Q, because at pH 7.3 the Km of
10 ± 5 µM is not significantly different from that
determined for EAACWT (Km = 13 ± 2 µM, p = 0.58). The 100-fold change in
Km has been determined under conditions of
EAACWT forward transport, whereas the Km value for EAACE373Q was determined under homoexchange
conditions. Therefore, we tested if under homoexchange conditions the
Km of EAACWT for glutamate becomes
pH-independent. At pH 10.0 the Km value of
EAACWT increases to 700 ± 250 µM, about
55 times the Km measured at pH 7.3, indicating that
glutamate transport by wild-type EAAC1 is also pH-dependent
under homoexchange conditions.
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In addition to the pH effect on the Km value, we determined the maximum glutamate-induced current (Imax) at saturating glutamate concentrations (1 mM) as a function of extracellular pH. As shown in Fig. 6C, the Imax of EAACE373Q decreases by 8% at pH 10.0, compared with pH 7.3. A similar slight decrease (25%) is observed for EAACWT and was previously attributed to a pH effect on the anion conductance of EAAC1 but not on the transport rate of glutamate, because the maximum transport currents were unaffected by the external pH (6).
As a further control, the pH effect on the neutral amino acid
transporter ASCT2 was investigated. In analogy to EAACE373Q studied here, the rat ASCT2 contains a glutamine residue in position 392, which is homologous to the glutamate in position 373 of EAAC1 (22). Application of the neutral amino acid L-alanine to
ASCT2-expressing HEK293 cells elicited concentration-dependent
uncoupled anion currents with an average amplitude of 250 pA (0 mV,
n = 8) in the presence of intracellular thiocyanate,
sodium ions, and neutral amino acid substrate
(I
Generation of a pH-sensitive ASC Transporter-- To test if the glutamine residue in position 392 of ASCT2 is responsible for the pH independence of alanine transport, we generated the reverse mutation of EAAC1E373Q in this transporter by replacing Gln-392 with a glutamate residue. The pH dependence of the Km for alanine of ASCT2Q392E is shown in Fig. 6C (open triangles). In contrast to ASCT2WT, the Km of the mutant transporter strongly increases at pH values > 8. The Km change is 18-fold from pH 7.3 to 10. This Km increase is reminiscent of that found for EAACWT, although the apparent pKa value is shifted to a slightly more basic value of 9.0 compared with 8.0 found for EAAC.
Absence of Deuterium Isotope Effect on EAACE373Q
Kinetics--
To further demonstrate the proton independence of
EAACE373Q function, we performed pre-steady-state kinetic
experiments in the presence D2O (Fig.
7). Clearly, D2O substitution
has no effect on the amplitude of the glutamate-induced current of
EAACE373Q (Fig. 7, A and C), as well
as on the time constant for the rise of the current
(1/fast, Fig. 7, A and B),
consistent with data previously obtained for the EAACWT
(6). However, a 2-fold decrease of the rate constant for the current
decay (1/
slow) was observed after the substitution of
H2O by D2O for EAACWT, but this
effect was absent for EAACE373Q, as demonstrated in Fig. 7
(A and B). For EAACE373Q an average
value for
slow of 35 ± 2 ms in D2O and 31 ± 7 ms in H2O-based buffer solution was observed
(n = 10, 3 cells, p = 0.49). These data
demonstrate the absence of a significant kinetic isotope effect on
EAACE373Q.
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Inhibition of EAACE373Q by Extracellular Potassium
Ions--
So far, the data suggest that the residue Glu-373 is
involved in proton binding in the glutamate translocation step of
EAAC1. To further elucidate the transport mechanism, we asked the
question: Does Glu-373, after dissociation of the proton, coordinate a
potassium ion? To test this hypothesis, the effect of potassium ion
concentration on the transport kinetics of EAACE373Q was
determined. As shown in Fig.
8A, the addition of 130 mM K+ to the extracellular side of the
transporter inhibits glutamate-induced inward currents
I
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DISCUSSION |
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In a previous study, we have demonstrated that the proton that is cotransported with glutamate by EAAC1 associates with a binding site on the glutamate-free form of the transporter (6). However, no information was obtained in that study concerning which amino acid residues in the EAAC1 sequence are involved in the binding of the proton. Here, we addressed this question by using site-directed mutagenesis, focusing on the EAAC1 glutamic acid residue 373, which is highly conserved in the EAAT family, but not in the related H+-independent ASC transporters that are selective for neutral amino acids. The central result of this study is that the substitution of Glu-373 of EAAC1 with a non-ionizable glutamine residue leads to a transporter that apparently does not interact with protons. Although slowed by a factor of about 2.5 compared with wild-type EAAC1, glutamate translocation catalyzed by the mutant transporter is otherwise unaffected by the E373Q amino acid substitution. The simplest interpretation of these results is that the binding site for protons, which modulates the affinity of EAAC1WT for glutamate, is permanently occupied in EAACE373Q. Therefore, proton release on either the intra- or the extracellular side of the membrane cannot occur, because the glutamine side chain cannot be deprotonated. Because this proton release is essential for the completion of the transport cycle, steady-state turnover of the transporter is impaired. This interpretation is in good agreement with previous reports, demonstrating that the potassium-induced relocation of the glutamate-free transporter is not functional in GLT1 when the EAAC1-homologous glutamate residue Glu-404 was replaced by amino acids with non-ionizable side chain (18, 19). Our results not only underscore the importance of these studies but provide additional information about the molecular mechanism of glutamate transport by investigating partial reaction steps of the transport cycle using pre-steady-state kinetic methods (21, 34).
A Molecular Model for Proton Cotransport by EAAC1--
In the
light of the new results we propose a more detailed model for proton
cotransport by glutamate transporters. This model is illustrated in
Fig. 9 and is based on a cyclic reaction
mechanism that incorporates independent reaction steps for
glutamate-induced translocation and K+-induced relocation
of the transporter (7). To create the model as simple as possible, it
is assumed that Glu-373 provides the sole binding site for the proton.
However, our data do not exclude the possibility that the proton, once
bound to the transporter, is shared by several amino acid residues that
provide a proton binding network. Such a model was recently proposed
for the proton binding to the lac-permease of Escherichia
coli (16).
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In our model, Glu-373 has to be protonated to permit glutamate binding
that is followed by a conformational change of the transporter and the
final exposure of the glutamate-proton binding sites to the cytoplasm.
To complete the reaction cycle and to relocate the binding sites back
to the extracellular side, Glu-373 has to be deprotonated and,
therefore, negatively charged. Therefore, the transport direction is
based on the glutamate concentration- and K+
concentration-dependent modulation of the affinity of EAAC1
for protons. Thus, it is essential that the pKa of
Glu-373 be higher than 7 to ensure that the H+ binding site
is always occupied under physiological conditions when its
-carboxylate is accessible from the external side of the membrane.
In fact, the apparent pKa was previously determined
to be about 8.0 for EAAC1 (6). Thus, the pKa of the
-carboxylate of glutamate 373 in EACC1 appears to be highly altered
compared with the pKa of glutamate in aqueous solution (~4.2). Consistent with this observation, highly perturbed pKa values are assumed to be common in catalytically active amino acid residues (35) and are found in membrane proteins that
mediate proton transfer, such as bacteriorhodopsin (36, 37), the
bacterial reaction center (38) and the F1F0-ATP
synthase (14). In analogy to these systems, we observed a
conformation-dependent modulation of the
pKa of the proton acceptor of EAAC1. In this case,
when the binding site for glutamate is exposed to the cytoplasm, the
apparent pKa is shifted to ~6.5 as shown in Fig. 9
(6). Such a pKa switch is important for the
functioning of the EAAC1, because it promotes association of glutamate
on the extracellular side and its dissociation on the intracellular
side and therefore establishes favorable conditions for inward
transport of glutamate (6).
An alternative model for proton transport by native brain glutamate transporters, proposed by Auger and Attwell (39), incorporates separate transport steps for glutamate and the proton suggesting that protons are countertransported in the potassium-driven relocation step of the glutamate transporter (39). Obviously, such a model would explain the absence of an effect of the E373Q amino acid substitution on the glutamate translocating branch of the half cycle, assuming that Glu-373 is always protonated independent of the state of the transporter. However, this model is not consistent with some of the experimental data. First, the authors assumed that changing the external pH has no effect on the transporter kinetics when homoexchange conditions are applied. This assumption is in clear contrast to the data presented here, showing that the extracellular proton concentration strongly modulates glutamate affinity in both the forward and the homoexchange mode. Second, no effect of proton concentration on the amplitude of synaptically evoked transport and anion currents was found (39), which was taken as further evidence that [H+] does not affect glutamate translocation. Considering the model shown in Fig. 9, this result is not surprising because reduced proton concentration can be compensated by increased glutamate concentration. Therefore, at saturating [glutamate], Imax becomes pH-independent (6). The glutamate concentration in the synaptic cleft after release is not precisely known, but probably is high enough to saturate the glutamate binding site on EAATs even at elevated extracellular pH. Third, Watzke et al. (6) demonstrated recently the existence of a deuterium isotope effect on the glutamate translocation rate of EAAC1, which indicates that the glutamate-translocating reaction branch of EAAC1 is, in fact, involved in proton transport. For these reasons, the H+ exchange model presented in Ref. 39 should be re-evaluated.
At present, two models of the transmembrane topology of EAATs are
discussed in the literature. One model proposed for the EAAT sequence
region around Glu-373 a membrane-spanning -helix structure (40),
whereas the other model suggests a pore-loop-like structure that dips
into the membrane from the extracellular surface (41, 42). In both
topology models Glu-373 is located in a fairly hydrophobic, yet
somewhat water accessible segment of the transporter that has been also
implicated to contribute to the binding of cotransported ions other
than the proton. For example, the amino acid residues Asp-367
(highlighted in Fig. 1) and Tyr-372 (Asp-398 and Tyr-403 in
GLT1) were proposed to participate in binding of Na+ and
K+, respectively (18, 43). In addition, some cysteines that substitute amino acid residues close to Glu-373 in the EAAT sequence can be protected from sulfhydryl-reactive reagent labeling by EAAT
substrates and competitive inhibitors (41). Taken together, these data
suggest that the highly conserved stretch of amino acids shown in Fig.
1 may form part of the permeation pathway for the substrate and co- or
countertransported ions. In agreement with these structural
interpretations, we found a highly perturbed pKa of
Glu-373, indicating that this amino acid residue is physically located
in an environment that is much different from a free carboxyl group in
aqueous solution. It can be speculated that Glu-373 is partially buried
in the low dielectric interior of the transport protein, as illustrated
in Fig. 9, but still accessible for proton binding from the
extracellular water phase. Consistently, EAAC1 in which glutamate in
position 373 is substituted by a cysteine residue is unaffected by
extracellularly applied positively and negatively charged sulfhydryl
reagents (see Ref. 44)2 and
becomes only accessible when extracellular Na+ is removed
(44), supporting the view that in the presence of Na+ this
position is not easily accessible from the aqueous phase. Furthermore,
the neighboring amino acid residue Tyr-372 (Tyr-403 in GLT1) was
proposed to be alternately accessible to either side of the membrane,
depending on the conformation of the carrier (44), consistent with the
alternate accessibility model for Glu-373 proposed here.
Interaction of EAAC1E373Q with Potassium Ions-- Our model discussed above for the E373Q mutation does not exclude the possibility that Glu-373 binds H+ in the glutamate translocation reaction and K+ in the relocation reaction of the glutamate-free form of EAAC1. In fact, it was previously reported that the EAACE373Q-analogous GLT1E404D mutant is defective in potassium ion binding and/or potassium ion countertransport (19), supporting the idea of an alternating H+/K+ binding. This interpretation was based on the observation that GLT1E404D is not able to catalyze reverse transport induced by application of extracellular K+ (19). However, using this experimental approach it is not possible to rule out that K+ still binds to the transporter but does not induce the conformational change associated with the relocation reaction. Here, we tested the latter possibility by determining if Na+/glutamate homoexchange is inhibited by increasing external [K+]. Inhibition by potassium ions was found, indicating that K+ can still bind to the mutant transporter. In contrast, K+-induced relocation of the EAAC1 binding sites is impaired, which is in line with the observations made by the Kavanaugh and Kanner groups (19). Our results suggest that the acidic amino acid residue Glu-373 is not essential for the binding of K+ to EAAC1. The negative charge on Glu-373, however, controls the rate of the K+-dependent conformational change of the K+ branch of the transport cycle. It can be speculated that Glu-373 is important for counterbalancing the positive charge of the potassium ion to facilitate its movement across the low dielectric membrane barrier. Therefore, Glu-373 may provide one of the two negative charges that are involved in this reaction (21). These negative charges are essential for the functioning of the transporter, because they also electrostatically compensate the three positive charges moved along with glutamate in the substrate translocation reaction.
Recently, a new model was proposed to explain the apparently defective
K+ interaction of mutant glutamate transporters (45). This
model is based on the finding that the arginine residue in position 446 of EAAC1 is involved in binding of the -carboxylate of glutamate (46). In this report, it was suggested that Glu-373 forms an ion pair
with Arg-446 in the absence of potassium ions, whereas it complexes
K+ in its presence. The results presented here support this
model, showing that Glu-373 is important for the K+-induced
relocation reaction of EAAC1. Extending this model to the glutamate
translocating half-cycle of EAAC1, one can speculate that Glu-373 forms
an ion pair with Arg-446 only in the absence of protons and glutamate.
To allow glutamate binding, the ion pair must be destabilized, which is
accomplished by binding of H+ to Glu-373, thus making
Arg-446+ available for associating with glutamate. This
model would also explain the sequential binding order for
H+ and glutamate to EAAC1 that was reported earlier
(6).
Importance of Acidic Amino Acid Side Chains for Proton-transporting Systems-- The results presented here emphasize the general importance of acidic amino acid residues for proton transport across membranes. There is compelling evidence for the direct involvement of aspartate and glutamate residues in proton transport by bacteriorhodopsin (15), lac permease (16), F1F0-ATP synthase (14), the bacterial reaction center (38), the multidrug resistance transporter EmrE (17), and, presumably, the Ca2+ ATPase (47). In all of these transmembrane proteins the important acidic amino acids are localized in the hydrophobic environment of the membrane-spanning part of the protein, and they are characterized by strongly perturbed pKa values. For the lac permease it has been shown that protonation of Glu-325, which is facilitated by extracellular substrate binding, induces its partitioning into the low dielectric membrane phase (16). The protonated complex is therefore able to undergo the conformational change that leads to exposure of the proton and substrate binding sites to the cytoplasm and subsequent intracellular dissociation of H+ and the substrate. This mechanism is remarkably similar to the model that we propose here for proton translocation by EAAC1. In contrast to EAAC1 and lac permease, the transport mechanism of the Ca2+ ATPase is based on proton countertransport (47, 48). Protonation of the ATPase occurs most likely at the same acidic amino acid residues that participate in complexing the two calcium ions (47). Although different with regard to the directionality of the proton transport, this mechanism is similar to the mechanism proposed here for EAAC1, because release of the proton is controlled by a pKa switch that is brought about by conformational changes of the protein. The involvement of acidic amino acid side chains as proton acceptors is critical for such mechanisms, because it ensures the neutrality of the protonated translocation complex.
Mechanism of Amino Acid Transport by ASCT--
Our results also
have implications for the understanding of the mechanism of neutral
amino acid transport by ASCT2. In agreement with previous suggestions
we show that amino acid transport by ASCT2 is associated with a much
simpler mechanism than glutamate transport by the EAA transporter
family (23). As demonstrated here, changes in the extracellular pH
between 7.3 and 10.0 affect neither the translocation rate of alanine
across the membrane nor the affinity of the transporter for the
substrate. These results indicate that, in contrast to EAAC1, no
protons are cotranslocated together with the neutral amino acid
substrate. It is, therefore, likely that the proton binding site that
is present in EAAC1 is permanently protonated in ASCT2, thus preventing
directional proton cotransport. This interpretation agrees well with
former reports that demonstrated that the transport rate of glutamine
by ASCT is not affected by changes in the extracellular proton
concentration at pH values greater than 7 (23, 33), whereas ASCT
glutamate transport strongly increases with decreasing pH, suggesting
that acidic amino acids are translocated in their protonated, neutral form (33). Furthermore, in contrast to glutamate transport by EAAC1,
ASCT transport of neutral amino acids is not associated with
intracellular acidification (23). Together, these data demonstrate that
ASCT is functionally not affected by the extracellular pH.
Interestingly, pH sensitivity can be engineered in ASCT2 by generating
the reverse mutation at the homologous position, Q392E. We, therefore,
suggest that the distinct difference in transport mechanisms of EAATs
and ASCTs is, at least in part, mediated by a simple acidic-to-neutral
amino acid exchange in position 373.
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ACKNOWLEDGEMENTS |
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We thank S. Bröer for kindly providing ASCT2 cDNA, E. Bamberg and H.-J. Galla for constant encouragement and support, E. Grabsch and B. Legrum for help in molecular biology, and A. Becker for subcloning of the ASCT2 cDNA.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft (Grants GR 1393/2-2 (to C. G.) and RA 753/1-1 (to T. R.)) and the Fundação para a Ciência e a Tecnologia (PRAXIS XXI/BD/18095/98, fellowship (to A. B.)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: IonGate Biosciences GmbH, Paul-Ehrlich-Strasse 17, Frankfurt/Main D-60596, Germany.
§ To whom correspondence should be addressed. Tel.: 49-69-6303-336; Fax: 49-69-6303-305; E-mail: grewer@mpibp-frankfurt.mpg.de.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M207956200
2 C. Grewer, N. Watzke, T. Rauen, and A. Bicho, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
EAAC1, excitatory
amino acid carrier 1;
CMV, cytomegalovirus;
PBS, phosphate-buffered
saline;
WT, wild-type;
CNB, -carboxy-O-nitrobenzyl;
ASC, alanine-serine-cysteine.
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