From the
Department of Medical Physiology and the
||Department of Pharmacology, The Panum Institute,
University of Copenhagen, DK-2200 Copenhagen N, Denmark
Received for publication, December 20, 2002 , and in revised form, April 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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It is a general assumption that the Na+/Cl-dependent transporters operate by an alternating access mechanism, where the transporter interchanges between a series of "outward-facing" conformations, in which the substrate binding sites are accessible to the extracellular medium, and a series of "inward-facing" conformations, in which the binding sites are accessible to the intracellular environment. However, relatively little is known about the conformational changes associated with the binding and translocation of GABA, Na+, and Cl (19, 20). Several residues in the transmembrane domains (6, 2025) and in the extracellular loops (2628) have been proposed to be involved in substrate binding and/or translocation in GAT and the monoaminergic transporters. Previously, we have obtained evidence in both GAT-1 (14) and the homologous dopamine transporter, DAT (2931), that the external ends of TM 7 and 8 undergo conformational changes that are critical for the translocation process. This conclusion was based on the observation that binding of Zn2+ to bidentate Zn2+ binding sites engineered between TM 7 and 8 in both GAT-1 and DAT results in potent non-competitive inhibition of substrate transport. This is consistent with the ability of Zn2+ to restrain movements between the two helices and/or of the connecting fourth extracellular loop.
In this study we have investigated two mutants in the GAT-1 situated only four residues apart at the external end of TM 7. The residues were initially mutated during our attempt to transfer engineered Zn2+ binding sites from the dopamine transporter to GAT-1. Contrary to DAT (31), the combined mutation of the two residues Met345 and Thr349 to histidines did not result in Zn2+ sensitivity of GAT-1 (data not shown). Instead, we show here that the individual mutation of these two residues results in characteristic and distinct phenotypes that are consistent with oppositely directed shifts in the conformational equilibrium. It is therefore proposed that the TM 7/8 microdomain in this class of transporters not only may undergo conformational changes during the transport process but also may play a role in maintaining an appropriate conformational equilibrium of the transporter, possibly through intramolecular interactions.
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EXPERIMENTAL PROCEDURES |
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ElectrophysiologyThe two-electrode voltage clamp method was used to control the membrane potential and monitor the current in oocytes expressing WT and mutant transporters, as earlier described (14). Generally, the membrane potential (Vm) of the oocyte was held at 50 mV, and the experimental chamber was continuously perfused by a NaCl solution containing 100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4. In Na+ substitution experiments, Na+ was replaced by equimolar choline ions, and in the leak current experiments equimolar Li+ replaced Na+. Two-electrode voltage clamp recordings were performed at room temperature with a Dagan Clampator interfaced to an IBM-compatible PC using a DigiData 1200 A/D converter and pCLAMP 6.0/8.0 (Axon Instruments). Currents were low pass-filtered at 500 Hz and sampled at 2 kHz. Electrodes were pulled from borosilicate glass capillaries to a resistance of 0.52 megaohm and filled with 1 M KCl.
The transporter-specific GABA-induced current (IGABA)
was obtained by subtracting the current in NaCl solution from the current in
NaCl solution + GABA (INa + GABA
INa), whereas the leak current was obtained by subtracting
the current in ChCl solution (100 mM ChCl, 2 mM KCl, 1
mM CaCl2, 1 mM MgCl2, 10
mM HEPES, pH 7.4) from the current in LiCl solution
(ILi ICh). For steady-state
kinetic analysis, the IGABA was measured at various
membrane potentials and external substrate concentrations ([S]), and at each
voltage, the IGABA versus the [S] relations were
fitted to the Michaelis-Menten equation, I =
(Imax x [S])/([S] + (K0.5)),
where [S] is the substrate concentration, Imax is the
maximal current for saturating [S], and K0.5 is the
apparent affinity constant. The apparent GABA affinity was determined at 100
mM external Na+ by varying the GABA concentration, and
Na+ activation was determined at a fixed (saturating) GABA
concentration by varying the external Na+ concentration. For
determination of the presteady-state transient currents, a pulse protocol was
used where Vm initially was held at 40 mV
and then jumped to a series of test potentials (from +40 to 160 mV with
20-mV increments) for 500 ms before returning to
Vm. At each test potential, the
transporter-specific presteady-state transient currents
(INa ICh) were obtained by
subtraction of the current traces in ChCl solution from those obtained in NaCl
solution. The charge movement, Q, was obtained by integrating the ON
transient currents at different potentials with respect to time. The
charge-voltage (Q/Vm) relations were
fitted to a Boltzmann function, Q
Qhyp/Qmax = 1/[1 +
exp(z(Vt
V0.5)F/RT)], where Qmax =
Qdep Qhyp
(Qdep and Qhyp are the Q at
depolarizing and hyperpolarizing limits), F is the Faraday constant,
R is the gas constant, T is the absolute temperature,
V0.5 is the membrane potential where there is 50% charge
transfer, and z is the apparent valence of the movable charge
(32). The time constants,
, were obtained by fitting the current traces to the equation
Itot(t) = I1 x
exp(
1 x t) + I2 x
exp(
2 x t) + Iss, where
Itot is the total current, I1 is the
transporter-specific current, I2 is the capacitative
current, Iss is the steady-state current, and t
is the time.
[3H]GABA Uptake Experiments in OocytesThe uptake experiments were performed in 24-well plates with 100 µM GABA (RBI, Natick, MA) and 50 nM 4-amino-n-[2,3-3H]butyric acid ([3H]GABA), 88 Ci/mmol (Amersham Biosciences) added to a total of 400 µl of uptake buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4). Oocytes were incubated for 30 min at room temperature, washed 3 times with 1 ml of wash buffer with choline chloride (100 mM ChCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4), and dissolved in 200 µl of 10% SDS. Before counting, 2 ml of scintillation fluid (Opti-fluor, PerkinElmer Life Sciences) was added to the samples.
CalculationsThe data were analyzed by nonlinear regression analysis using Prism 3.0 (GraphPad Software, San Diego, CA), Clampfit (Axon Instruments Inc., Union City, CA), and SigmaPlot (Version 6.0, SPSS Inc., Chicago, IL). All numbers are given as means ± S.E., with n equal to the number of oocytes tested. The current range of the oocytes expressing the various constructs was taken as a measure of the range in expression level and is stated in the figure legends and in the table.
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RESULTS |
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The effect of histidine substitutions were compared with substitutions with
an alanine and a cysteine in position 345 and a cysteine in position 349. The
effects of these substitutions were less pronounced and are, therefore, not
dealt with in detail; M345C showed slightly reduced apparent GABA affinity at
Vm = 60 mV (2-fold change as opposed
to the
10-fold in M345H, data not shown), whereas the apparent affinity
of M345A was in between that of the WT and the M345H (
6-fold change, data
not shown). The apparent GABA affinity of the T349C mutant transporter was
slightly decreased compared with that of the WT (
2-fold change), which
was exactly the opposite effect of the T349H (data not shown).
From the I/V plots in Fig. 2 (filled symbols), it is apparent that the voltage dependence of the GABA-induced current was changed in the mutant transporters; T349H showed increased voltage dependence compared with the WT, as seen from the steep slope in the I/V relation. The current does not saturate at the clamp potentials employed in these experiments, and very little current is seen at potentials more depolarized than 100 mV. In contrast, M345H and M345H/T349H displayed less voltage dependence than the WT. This is indicated by the early saturation of the I/V relation as the membrane potential of the oocytes is increasingly hyperpolarized and by the presence of a substrate-induced current even at 0 mV, especially for M345H.
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We analyzed the effect of lowering the pH from 7.4 to 5.5 (open symbols in Figs. 1 and 2). Such a pH change would be expected to cause protonation of the inserted histidines and thereby introduce a positive charge. In the WT we observed no change in the voltage-dependent apparent GABA affinity upon lowering the pH (Fig. 1), and the magnitude of the current and the shape of the I/V-relation in the presence of saturating GABA concentration was not affected at the potentials tested. For example, at a membrane potential of 150 mV the current at pH 5.5 was 109 ± 3% compared with that seen in the control solution of pH 7.4, n = 5 (Fig. 2). This is in agreement with previous studies (10). In the mutants, however, we found a different pattern. For M345H, the reduced pH caused an increase in the apparent affinity for GABA (Fig. 1), which was completely voltage-insensitive at pH 5.5 as well as at pH 7.4. The K0.5 for GABA was pH-dependent in both T349H and M345H/T349H. In T349H at pH 5.5, the K0.5 was increased at hyperpolarized potentials compared with pH 7.4. Oppositely, the K0.5(GABA) at pH 5.5 in M345H/T349H decreased at depolarized potentials and also became voltage-dependent. For all three mutant transporters the magnitude of the GABA-induced currents was markedly increased at pH 5.5 (open symbols in Fig. 2). At a membrane potential of 150 mV the current for M345H at pH 5.5 was 148 ± 6% (n = 5) of that found at pH 7.4 and 157 ± 18% (n = 4) for T349H. An even larger effect was observed in the double mutant (258 ± 20% of the current obtained at pH 7.4, n = 5), suggesting an additive effect on the transport-associated current upon protonation of both M345H and T349H. This increase in GABA-induced current at lower pH did not reflect an increase in an uncoupled transport-associated current as shown by uptake experiments with [3H]GABA. The [3H]GABA uptake of WT was reduced to 90 ± 3% at pH 5.5 compared with that at pH 7.4 (n = 4), whereas the [3H]GABA uptake of M345H/T349H was 128 ± 5 (n = 5), p < 0.001, Student's t test. The uptake experiments were performed under unclamped conditions, in which the membrane potential approaches the reversal potential of the transporter; therefore, the numbers are not directly comparable with those above, in which the GABA-induced current was measured under voltage clamp (150 mV). As seen From Fig. 2, the percentage increase in GABA-induced current at low pH increases with more negative clamp potentials. At a membrane potential of 20 mV (which is a feasible membrane potential during an uptake experiment), the pH-induced increase in current is comparable with that obtained in the uptake experiments.
Na+ AffinityThe Na+ activation curves performed at 120 mV for the WT, M345H, T349H, and M345H/T349H are shown in Fig. 3, and the K0.5 values obtained at different membrane potentials (Vms) are summarized in Table I. The apparent Na+ affinity for the WT was 44 ± 6 mM (n = 5) and was highly voltage-dependent, in agreement with previous studies (Table I and Refs. 15 and 16). Interestingly, the apparent Na+ affinity of the two individual mutants (M345H and T349H) were affected in opposite directions. In T349H the apparent affinity was reduced compared with the WT (K0.5 = 200 ± 64 mM at 120 mV, n = 4) and showed an even more pronounced voltage dependence. In contrast, M345H displayed increased apparent Na+ affinity (K0.5 = 6 ± 1 mM, n = 4). Similarly, the double mutant M345H/T349H displayed increased apparent affinity for Na+ (K0.5 = 6 ± 1 mM, n = 4). Note that the Na+ activation curve for the GABA-induced current in both the WT and the T349H mutant did not show saturation at potentials more depolarized than 80 and 120 mV, respectively, and could therefore not be fitted by the Michaelis-Menten equation. This indicates a strong voltage dependence of the Na+ binding and/or the associated conformational changes. On the contrary, the apparent Na+ affinity of the M345H and T349H/M345H transporters barely showed voltage dependence within our range of measurements (Table I).
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Transient CurrentsThe GAT-1 and related transporters give
rise to substrate-independent capacitive transient currents upon shifts in the
membrane potential (10,
14,
16,
33,
34). These currents are
thought to arise from conformational changes associated with Na+
binding and dissociation and may, therefore, be used as a functional read-out
of the partial reactions of the transport cycle involving these steps. To
perform these experiments, the membrane potential was held at 40 mV and
jumped to the test potentials (from 40 mV to 160 mV with intervals of
20 mV) for 500 ms and returned to the holding potential for another 500 ms
before the next test potential was applied
(Fig. 4, upper
panels). Because these currents are generally entirely capacitive
(16,
33,
34), the time integral of the
transient OFF current is identical to that of the ON current (data not shown).
The Q/Vm relationships shown in the
lower panel of Fig. 4
were obtained from the ON currents for WT, M345H, T349H, and M345H/T349H. The
V0.5 for the WT transporter was 36 ±
2mV(n = 5), which is in agreement with previously published data
(10,
14,
16). The
Q/Vm relationship was shifted markedly
toward more negative membrane potentials in T349H, as reflected by a
V0.5 of 85 ± 7 mV, n = 5, whereas
it was shifted toward more positive potentials in M345H
(V0.5 = 17 ± 3 mV, n = 6). The
V0.5 of the double mutant (M345H/T349H) was in between
that of the two single mutants (V0.5 = 10 ±
6 mV, n = 5). The time constants () for the relaxation of the ON
currents were also examined. For the WT transporter, the
max
was 98 ± 5 ms (n = 4), which is in accordance with previously
reported values (7,
10). This time constant was
increased significantly in T349H (230 ± 17 ms, n = 5,
p < 0.001, Student's t test). We also observed an
apparent, although not significant, increase in
max for M345H
and M345H/T349H (158 ± 23 ms (n = 6) and 132 ± 15 ms
(n = 5), respectively).
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Leak CurrentThe GAT-1 sustains substrate-independent leak currents at hyperpolarized potentials in the presence of Li+ (6, 14, 15, 17). As seen from Fig. 5, upper panel, the clamp potential at which the leak current commences differed for the WT and the mutants. The T349H mutant required increased hyperpolarization (compared with that of the WT) in order for the leak conductance to initiate, whereas the M345H mutant supported leak current at a more depolarized potential than the WT. The double mutant (M345H/T349H) behaved more or less like the WT with the requirement of an intermediate hyperpolarization. A remarkable feature of the M345H/T349H mutant was the significantly increased ratio between the magnitude of the leak current and the GABA-induced current: 6.7 ± 0.6 for M345H/T349H compared with 2.3 ± 0.1 for the WT (at Vm = 150 mV), n = 5. The leak:IGABA ratio of the single mutants approached that of the WT, although the ratio for M345H was significantly higher (3.0 ± 0.2 for M345H and 1.9 ± 0.2 for T349H, n = 5); see Fig. 5, lower panel. At a clamp potential of 90 mV, the GABA-induced current exceeded that of the Li+-induced leak current in the WT (leak:GABA ratio = 0.5 ± 0.1) and in the T349H (0.3 ± 0.1). The leak current of M345H and M345H/T349H was still larger than the GABA-induced current, with ratios of 1.6 ± 0.1 and 1.8 ± 0.2, respectively.
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DISCUSSION |
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Introduction of histidines at residues Met345 and Thr349 caused changes in the apparent ligand affinity constants. M345H caused a reduction in apparent GABA affinity and an increase in apparent Na+ affinity and T349H caused an increase in apparent GABA affinity and a reduction in apparent Na+ affinity. It is important to note that the shift in voltage dependence of the mutant transporters may contribute to the change in apparent affinities, since the GABA-induced currents do not always fully saturate at the most negative clamp potentials applied in this study. Ideally, the K0.5 for Na+ should be obtained at completely saturating voltages and the K0.5 for GABA should be obtained at completely saturating voltages and Na+ concentrations. We have applied the highest concentrations of Na+ (100 mM) and the most hyperpolarized potentials (160 mV) at which the oocytes, at least in our hands, can survive repetitive recordings. These conditions (160 mV/100 mM Na+) are saturating for M345H and M345H/T349H; the apparent Na+ affinities are virtually identical (4 versus 3 mM, Table I), the I/V curves approach saturation in both constructs (Fig. 2), but the K0.5 for GABA is 220 versus 50 µM (Fig. 1).
The apparent GABA affinity was not as drastically changed in constructs with more conservative amino acid substitutions, such as M345A, M345C, and T349C (data not shown), suggesting that mainly the bulky histidine residue led to alterations in transporter function. It has been suggested earlier that the external loops in this region of the transporter are involved in the initial GABA binding (27). Therefore, a change in the charge of a nearby residue might interfere with the interaction with the zwitterionic substrate. At pH 5.5, externally located histidines (pKa 67) should become fully protonated and thereby positively charged, as opposed to their prevalent neutral state at pH 7.4. The introduction of a positive charge by lowering the extracellular pH did not alter the apparent GABA affinity of the WT but did affect the mutant transporters. Upon lowering the pH, the apparent GABA affinity of all three constructs changed in the direction of that of the WT, and the voltage dependence of the GABA affinity increased for T349H and M345H/T349H, shifting the K0.5/Vm curve of these transporters toward the WT. The mutants did not completely revert to the phenotype of the WT transporter upon protonation, as seen from the I/V curves in Fig. 2. Because the K0.5(GABA) for T349H was increased by lowering the pH, it seems unlikely that the residues are directly involved in GABA binding, since an increase of K0.5(GABA) would be expected to give rise to a lower Imax, which is opposite to the observed increase (Fig. 2).
The prevailing alternating access model for how the GAT-1 and other
Na+-coupled transporters operate is illustrated in
Fig. 6. According to this
model, the Na+ and substrate binding sites of the transporter
alternately face the extracellular and the intracellular side (referred to as
outward-facing and inward-facing conformations). The initial event in the
transport cycle is binding of Na+ to the outward-facing transporter
(C2Na2). Subsequently the substrate binds
(C3Na2GABA), which triggers a conformational change that
exposes the substrate binding site to the intracellular environment
(C4Na2GABA). After the sequential release of substrate
and Na+, the empty transporter reverts to the extracellular side
(C6 C1), and the cycle continues.
Cl has been omitted for clarity. The overall kinetics of the
transport cycle can be described by rate constants for the transition between
the different conformations. According to the model of Wright and co-workers
(33), the pre-steady-state
currents represent the conformational changes associated with the
re-orientation of the empty transporter and the Na+ binding and
unbinding (C6
C1
C2Na2). A prerequisite for an optimal transport function
within the framework of this model is an intrinsic ability of the transporter
to maintain an appropriate equilibrium between outward- and inward-facing
conformations in the translocation cycle.
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The voltage dependence of the substrate-induced current is a result of
voltage-dependent rate-limiting steps within the overall transport cycle. In
GAT-1, this voltage dependence is most likely associated with the poor
apparent Na+ affinity, which is reflected in the voltage dependence
of the K0.5 for Na+. This is consistent with
our finding that the WT and, even more pronounced, T349H both had highly
voltage-dependent GABA-induced currents and relatively low apparent
Na+ affinities, which also showed strong voltage dependence. The
rate-limiting step in the transport cycle of these transporters probably
involves voltage-dependent reorientation of the empty transporter and binding
of Na+ (C6 C1
C2Na2); see Fig.
6. The opposite was the case for M345H and M345H/T349H. For these
two mutants, the substrate-induced current was independent of the voltage at
the most hyperpolarized potentials tested, and the voltage dependence of the
K0.5 for Na+ was negligible, suggesting that
the rate-limiting step is now somewhere else in the reaction cycle.
In this study, presteady-state analysis demonstrated a shift in the
steady-state conformational equilibrium for the mutant transporters, with the
equilibrium of M345H being shifted toward the outward-facing
Na+-bound conformation (C2Na2) and T349H
shifted toward the inward-facing empty conformation (C6) (according
the model of Wright and co-workers
(33)). In the double mutant,
we observed characteristics of the WT transporter. The conformational
equilibrium of the double mutant is most likely affected equally by the two
oppositely directed shifts in the equilibrium observed in the two single
mutants. Taken together with the altered apparent GABA and Na+
affinities, the shift in V0.5 points toward a general
shift in the equilibrium of the mutants; the M345H is poised toward the
outward-facing Na+-bound conformation (C2Na2
in Fig. 6), and the apparent
GABA affinity of this mutant transporter may then be reduced because the
protein is reluctant to leave the Na+-bound conformation. The lack
of voltage dependence of the apparent Na+ affinity and GABA-induced
current may then signify that the voltage-dependent C6
C1 step is no longer the rate-limiting step in the forward
transport cycle, as supported by the Q/V relationship, in
which the charge movement was complete at potentials more hyperpolarized than
40 mV. According to the model, the conformational equilibrium of the
T349H mutant transporter would be shifted toward the inward-facing empty state
(C6). Thereby, the apparent Na+ affinity would be
reduced, because the transporter is poised away from the Na+-bound
conformation (C2Na2). The voltage dependence of this
mutant transporter is increased compared with the WT (both for the GABA
translocation and the apparent Na+ affinity). This may indicate
that the voltage-dependent step (C6
C1) is
increasingly rate-limiting in this mutant, as seen from the
Q/V relationship, in which the charge movement is not
completed even at the most hyperpolarized potentials (160 mV). Once the
transporter binds Na+, it may readily bind and translocate GABA in
order to return to its "favored" inward-facing conformation, and
thereby, the apparent GABA affinity could appear to be increased. Although we
cannot rule out that the true substrate affinities have been affected by the
introduction of histidines in this extracellular region of the transporter,
our data point toward an alteration of apparent substrate affinities as a
secondary effect of a shift in the conformational equilibrium.
The increase in GABA transport at low pH caused by the single mutations was additive in the double mutant. One explanation could be that protonation of the histidines would increase the rate of the forward conformational change from the outward-facing conformations to the inward-facing conformations for M345H, and for T349H, from the inward-facing conformations to the outward-facing conformations. Thereby, the rate of both steps could be increased in the double mutant, causing M345H/T349H to considerably increase its turnover at low pH. Several studies point to this region as being involved in conformational changes associated with substrate translocation (10, 14, 19, 26, 27). Amino acid residues may interact with and/or repulse each other depending on the charge of the residues, and these interactions may affect the interhelical movements underlying the conformational changes taking place in the reaction cycle. The external loops in the C-terminal part of the transporters have been proposed earlier to be pH-sensing; acidic pH increased serotonin-induced current in rat serotonin transporter, whereas human serotonin transporter was unaffected. Residues 490 and 493 in the external loop 5 in rat serotonin transporter were responsible for this effect of pH (35). Mutating K448E in the homologous loop in rat GAT-1 conferred pH sensitivity to the otherwise insensitive GABA-induced current (10). Altogether these findings suggest that conformational changes underlying substrate translocation in transport proteins are sensitive to the charge of the residues residing in the loop regions of the transporters.
The mutant transporters and the WT supported Li+-induced,
substrate-independent leak currents, although the ratio between the
Li+ leak current and the GABA-induced current was altered in the
mutants. The leak current mode of the double mutant was increased severalfold
over that of the GABA-induced current, which is probably partly because of its
low GABA translocation turnover rate and partly because of its increased leak
current. M345H also had an increased ratio compared with the WT, although not
as pronounced as M345H/T349H. According to our recent study, the leak current
takes place after Li+ binding to the transporter at the first
cation binding site in the transporter cycle
(15). As the C6
C1
C2Na2 steps are less
voltage-dependent in M345H and M345H/T349H and the M345H mutation causes a
poise of the transporters toward the outward-facing conformations, these two
transporters would more readily enter into the cation-bound leak current
conformation. Thereby these transporters (compared with the WT) sustain an
increased leak current, which initiates at more depolarized potentials
(Fig. 5). T349H, on the other
hand, favors the inward-facing conformation, and it takes increased
hyperpolarization for the transporter to enter into the cation-bound state.
Therefore, the leak current represents a smaller component than the
GABA-induced current in this mutant transporter.
In conclusion, our study provides further support that the TM 7 region plays a critical role for the function of Na+/Cl-coupled neurotransmitter transporters. In this study, we identify two positions in this part of the transporter that upon mutation cause opposite shifts in the steady-state conformational equilibrium. This occurs most likely via disruption of particular amino acid interactions in this micro-domain after introduction of bulky histidine side chains. Obviously, we cannot in the absence of high resolution structural information deduce the specific structural correlates reflecting the observed phenotypes. However, the distinct effects on the steady-state equilibrium by mutation of two residues situated four amino acids apart in a conformationally sensitive region of the transporter support a hypothesis where a domain around the external end of TM 7 serves a critical role in controlling conformational changes and the conformational equilibrium of the transporter. It is obvious, however, that other domains within the transporter molecule also may contribute to the control of these processes. In the homologous dopamine transporter, we have recently shown evidence that a tyrosine (Tyr335) situated in the intracellular loop connecting TM 6 and 7 might be indispensable for maintaining the transporter in a conformation in which extracellular substrates can bind and initiate transport (36).
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FOOTNOTES |
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Contributed equally to this paper.
¶ To whom correspondence should be addressed: Division of Cellular and Molecular Physiology, Dept. of Medical Physiology 12-5, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark. Tel.: 45-3532-7467; Fax: 45-3532-7555; E-mail: nmacaulay{at}mfi.ku.dk.
1 The abbreviations used are: GABA, -aminobutyric acid; GAT-1, GABA
transporter-1; DAT, dopamine transporter; TM, transmembrane domain; WT, wild
type; ChCl, choline chloride; V0.5, the membrane potential
where there is 50% charge transfer; Vm, membrane
potential; K0.5, the apparent affinity constant;
,
relaxation time constant.
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ACKNOWLEDGMENTS |
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REFERENCES |
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