Residues in the extracellular loop 4 are critical for maintaining the conformational equilibrium of the gamma-aminobutyric acid transporter-1.

We mutated residues Met345 and Thr349 in the rat gamma-aminobutyric acid transporter-1 (GAT-1) to histidines (M345H and T349H). These two residues are located four amino acids apart at the extracellular end of transmembrane segment 7 in a region of GAT-1 that we have previously suggested undergoes conformational changes critical for the transport process. The two single mutants and the double mutant (M345H/T349H) were expressed in Xenopus laevis oocytes, and their steady-state and presteady-state kinetics were examined and compared with wild type GAT-1 by using the two-electrode voltage clamp method. Oocytes expressing M345H showed a decrease in apparent GABA affinity, an increase in apparent affinity for Na+, a shift in the charge/voltage (Q/Vm) relationship to more positive membrane potentials, and an increased Li+-induced leak current. Oocytes expressing T349H showed an increase in apparent GABA affinity, a decrease in apparent Na+ affinity, a profound shift in the Q/Vm relationship to more negative potentials, and a decreased Li+-induced leak current. The data are consistent with a shift in the conformational equilibrium of the mutant transporters, with M345H stabilized in an outward-facing conformation and T349H in an inward-facing conformation. These data suggest that the extracellular end of transmembrane domain 7 not only undergoes conformational changes critical for the translocation process but also plays a role in regulating the conformational equilibrium between inward- and outward-facing conformations.

The GABA 1 transporters belong to a large family of Na ϩ /Cl Ϫcoupled neurotransmitter transporters that includes the transporters for several other neurotransmitters such as serotonin, dopamine, noradrenaline, and glycine. These transporters are involved in re-uptake of the neurotransmitter at the synaptic terminals and thereby contribute to termination of the neuro-nal response. The transport proteins are predicted to contain 12 transmembrane domains (TMs) (1) with cytosolic amino and carboxyl termini (2). The GABA re-uptake is coupled to the cotransport of 2 Na ϩ and 1 Cl Ϫ , which renders the transport process electrogenic (3,4). Several electrophysiological studies of GAT-1 heterologously expressed in either mammalian cell lines or in Xenopus laevis oocytes have been carried out, and four current-generating modes of the transporter have been described; they are the Na ϩ -coupled substrate-induced current, capacitive Na ϩ -dependent presteady-state currents, a Li ϩ -induced leak conductance, and a not fully documented un-coupled substrate-induced channel activity (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).
It is a general assumption that the Na ϩ /Cl Ϫ -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 ϩ , and Cl Ϫ (19,20). Several residues in the transmembrane domains (6, 20 -25) and in the extracellular loops (26 -28) 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 (29 -31), 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 Zn 2ϩ to bidentate Zn 2ϩ 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 Zn 2ϩ 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 Zn 2ϩ binding sites from the dopamine transporter to GAT-1. Contrary to DAT (31), the combined mutation of the two residues Met 345 and Thr 349 to histidines did not result in Zn 2ϩ 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 confor-mational equilibrium of the transporter, possibly through intramolecular interactions.

EXPERIMENTAL PROCEDURES
Molecular Biology and Oocytes-The rat GAT-1 cDNA was mutated by PCR-derived site-directed mutagenesis and cloned into a vector optimized for oocyte expression (pNB1) as earlier described (14). The cDNA was linearized downstream of the poly(A) segment and in vitro transcribed with the T7 RNA polymerase using the mCAP mRNA capping kit (Stratagene, La Jolla, CA), and 50 ng cRNA was injected into defolliculated X. laevis oocytes (14). The oocytes were incubated in Kulori medium (90 mM NaCl, 1 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, pH 7.4) at 19°C for 3-7 days before experiments were performed.
Electrophysiology-The 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 (V m ) of the oocyte was held at Ϫ50 mV, and the experimental chamber was continuously perfused by a NaCl solution containing 100 mM NaCl, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, pH 7.4. In Na ϩ substitution experiments, Na ϩ was replaced by equimolar choline ions, and in the leak current experiments equimolar Li ϩ replaced Na ϩ . 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 0.5-2 megaohm and filled with 1 M KCl.
The transporter-specific GABA-induced current (I GABA ) was obtained by subtracting the current in NaCl solution from the current in NaCl solution ϩ GABA (I Na ϩ GABA Ϫ I Na ), whereas the leak current was obtained by subtracting the current in ChCl solution (100 mM ChCl, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, pH 7.4) from the current in LiCl solution (I Li Ϫ I Ch ). For steady-state kinetic analysis, the I GABA was measured at various membrane potentials and external substrate concentrations ([S]), and at each voltage, the I GABA versus the [S] relations were fitted to the Michaelis-Menten equation, I ϭ (I max ϫ [S])/([S] ϩ (K 0.5 )), where [S] is the substrate concentration, I max is the maximal current for saturating [S], and K 0.5 is the apparent affinity constant. The apparent GABA affinity was determined at 100 mM external Na ϩ by varying the GABA concentration, and Na ϩ activation was determined at a fixed (saturating) GABA concentration by varying the external Na ϩ concentration. For determination of the presteadystate transient currents, a pulse protocol was used where V m initially was held at Ϫ40 mV and then jumped to a series of test potentials (from ϩ40 to Ϫ160 mV with 20-mV increments) for 500 ms before returning to V m . At each test potential, the transporter-specific presteady-state transient currents (I Na Ϫ I Ch ) 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/V m ) relations were fitted to a Boltzmann function, (Q dep and Q hyp are the Q at depolarizing and hyperpolarizing limits), F is the Faraday constant, R is the gas constant, T is the absolute temperature, V 0.5 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 I tot (t) ϭ I 1 ϫ exp( 1 ϫ t) ϩ I 2 ϫ exp( 2 ϫ t) ϩ I ss , where I tot is the total current, I 1 is the transporter-specific current, I 2 is the capacitative current, I ss is the steady-state current, and t is the time. Calculations-The 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 Ϯ 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. 345 and Thr 349 , located at the extracellular end of TM 7 in GAT-1, were individually and together mutated to histidines, resulting in the mutants M345H, T349H, and M345H/T349H. All three mutants were functional as assessed upon expression in X. laevis oocytes and subsequent uptake experiments and two-electrode voltage clamp analysis. The apparent GABA affinity (K 0.5 ) was significantly altered by the histidine mutations ( Fig. 1, filled  symbols). In the T349H mutation, the K 0.5 decreased from 22 Ϯ 4 M in the WT (n ϭ 7) to 4 Ϯ 1 M (n ϭ 5) at a membrane potential of Ϫ60 mV, whereas M345H displayed a reduced apparent affinity as reflected in a K 0.5 value of 220 Ϯ 16 M (n ϭ 5). The double mutant T349H/M345H showed an apparent affinity intermediate of the two single mutations (K 0.5 ϭ 45 Ϯ 3 M, n ϭ 5). Contrary to M345H and T349H/M345H, the K 0.5 values of the WT and T349H were voltage-dependent, i.e. the apparent GABA affinity decreased upon hyperpolarization.

GABA Affinity and pH Dependence-Met
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 V m ϭ Ϫ60 mV (ϳ2-fold change as opposed to the ϳ10-fold in M345H, data not shown), whereas the apparent affinity of M345A was in between that of the WT and the M345H (ϳ6-fold change, data not shown). The apparent GABA affinity of the T349C mutant transporter was slightly decreased compared with that of the WT (ϳ2-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 Ϫ100 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.
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 Ϫ150 mV the current at pH 5.5 was 109 Ϯ 3% compared with that seen in the control solution of pH 7.4, n ϭ 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 K 0.5 for GABA was pH-dependent in both T349H and M345H/T349H. In T349H at pH 5.5, the K 0.5 was increased at hyperpolarized potentials compared with pH 7.4. Oppositely, the K 0.5(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 Ϫ150 mV the current for M345H at pH 5.5 was 148 Ϯ 6% (n ϭ 5) of that found at pH 7.4 and 157 Ϯ 18% (n ϭ 4) for T349H. An even larger effect was observed in the double mutant (258 Ϯ 20% of the current obtained at pH 7.4, n ϭ 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 The GABA-induced current (I Na ϩ GABA Ϫ I Na ) was measured as described in Fig. 1 (3 mM GABA) at pH 7.4 (black symbols) or pH 5.5 (white symbols). The data are presented as a representative example of five experiments. The data are summarized in the last panel as the GABA-induced current at pH 5.5 compared with the current obtained at pH 7.4 at a membrane potential of Ϫ150 mV, Student's t test; *, p Ͻ 0.05; ***, p Ͻ 0.001. above, in which the GABA-induced current was measured under voltage clamp (Ϫ150 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 Ϫ20 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 ϩ Affinity-The Na ϩ activation curves performed at Ϫ120 mV for the WT, M345H, T349H, and M345H/T349H are shown in Fig. 3, and the K 0.5 values obtained at different membrane potentials (V m s) are summarized in Table I. The apparent Na ϩ affinity for the WT was 44 Ϯ 6 mM (n ϭ 5) and was highly voltage-dependent, in agreement with previous studies (Table I and Refs. 15 and 16). Interestingly, the apparent Na ϩ 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 (K 0.5 ϭ 200 Ϯ 64 mM at Ϫ120 mV, n ϭ 4) and showed an even more pronounced voltage dependence. In contrast, M345H displayed increased apparent Na ϩ affinity (K 0.5 ϭ 6 Ϯ 1 mM, n ϭ 4). Similarly, the double mutant M345H/T349H displayed increased apparent affinity for Na ϩ (K 0.5 ϭ 6 Ϯ 1 mM, n ϭ 4). Note that the Na ϩ activation curve for the GABA-induced current in both the WT and the T349H mutant did not show saturation at potentials more depolarized than Ϫ80 and Ϫ120 mV, respectively, and could therefore not be fitted by the Michaelis-Menten equation. This indicates a strong voltage dependence of the Na ϩ binding and/or the associated conformational changes. On the contrary, the apparent Na ϩ affinity of the M345H and T349H/M345H transporters barely showed voltage dependence within our range of measurements (Table I).
Transient Currents-The 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 ϩ 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 Ϫ40 mV and jumped to the test potentials (from 40 mV to Ϫ160 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/V m relationships shown in the lower panel of Fig. 4 were obtained from the ON currents for WT, M345H, T349H, and M345H/T349H. The V 0.5 for the WT transporter was Ϫ36 Ϯ 2 mV (n ϭ 5), which is in agreement with previously published data (10,14,16). The Q/V m relationship was shifted markedly toward more negative membrane potentials in T349H, as reflected by a V 0.5 of Ϫ85 Ϯ 7 mV, n ϭ 5, whereas it was shifted toward more positive potentials in M345H (V 0.5 ϭ 17 Ϯ 3 mV, n ϭ 6). The V 0.5 of the double mutant (M345H/T349H) was in between that of the two single mutants (V 0.5 ϭ Ϫ10 Ϯ 6 mV, n ϭ 5). The time constants () for the relaxation of the ON currents were also examined. For the WT transporter, the max was 98 Ϯ 5 ms (n ϭ 4), which is in accordance with previously reported values (7,10). This time constant was increased significantly in T349H (230 Ϯ 17 ms, n ϭ 5, p Ͻ 0.001, Student's t test). We also observed an apparent, although not significant, increase in max for M345H and M345H/T349H (158 Ϯ 23 ms (n ϭ 6) and 132 Ϯ 15 ms (n ϭ 5), respectively).
Leak Current-The GAT-1 sustains substrate-independent leak currents at hyperpolarized potentials in the presence of Li ϩ (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 Ϯ 0.6 for M345H/T349H compared with 2.3 Ϯ 0.1 for the WT (at V m ϭ Ϫ150 mV), n ϭ 5. The leak:I GABA ratio of the single mutants approached that of the WT, although the ratio for M345H was significantly higher (3.0 Ϯ 0.2 for M345H and  (4) FIG. 3. Apparent Na ؉ affinity of GAT-1 and the mutant transporters. The Na ϩ activation of GABA-induced currents (I Na ϩ GABA Ϫ I Na ) was measured at a membrane potential of Ϫ120 mV with varying concentrations of Na ϩ (0 -100 mM with ChCl substitution), whereas the GABA concentration was kept constant and saturating at 3 mM. The GABA-induced current plotted as a function of the Na ϩ concentration was fitted to the Michaelis-Menten equation to derive the K 0.5 . The data are presented as the % of the current obtained at 100 mM Na ϩ , with an average of 4 -5 oocytes expressing each construct with a GABA-induced current (saturating GABA concentrations at Ϫ50 mV) of 180 -300 nA for the WT, 190 -600 nA for M345H, 50 -85 nA for T349H, and 110 -150 nA for M345H/T349H. When not visible, the error bars are within the symbol. The K 0.5 values are shown in Table I. 1.9 Ϯ 0.2 for T349H, n ϭ 5); see Fig. 5, lower panel. At a clamp potential of Ϫ90 mV, the GABA-induced current exceeded that of the Li ϩ -induced leak current in the WT (leak:GABA ratio ϭ 0.5 Ϯ 0.1) and in the T349H (0.3 Ϯ 0.1). The leak current of M345H and M345H/T349H was still larger than the GABAinduced current, with ratios of 1.6 Ϯ 0.1 and 1.8 Ϯ 0.2, respectively.

DISCUSSION
In the present paper we have identified two residues that may play an important role in the maintenance of the proper conformational equilibrium of the GABA transporter. Mutation of two residues (M345H or T349H) at the top of TM7 generated transporters with oppositely directed pre-steady-state and steady-state kinetics, which in the double mutant (M345H/ T349H) were sometimes additive and sometimes approached the kinetics of the WT transporter. In previous studies of the GAT-1 and the homologous DAT we have obtained evidence that this region of the transporter may undergo conformational changes during the substrate translocation process (14, 29 -31). As discussed below, the present data support that this part of the transporter, in addition to undergoing conformational changes during transport, also play a critical role in regulating the distribution between distinct structural states in the reaction cycle.
Introduction of histidines at residues Met 345 and Thr 349 caused changes in the apparent ligand affinity constants. M345H caused a reduction in apparent GABA affinity and an increase in apparent Na ϩ affinity and T349H caused an increase in apparent GABA affinity and a reduction in apparent Na ϩ 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 K 0.5 for Na ϩ should be obtained at completely saturating voltages and the K 0.5 for GABA should be obtained at completely saturating voltages and Na ϩ concentrations. We have applied the highest concentrations of Na ϩ (100 mM) and the most hyperpolarized potentials (Ϫ160 mV) at which the oocytes, at least in our hands, can survive repetitive recordings. These conditions (Ϫ160 mV/100 mM Na ϩ ) are saturating for M345H and M345H/T349H; the apparent Na ϩ affinities are virtually identical (4 versus 3 mM, Table I), the I/V curves approach saturation in both constructs (Fig. 2), but the K 0.5 for GABA is 220 versus 50 M (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), suggest- ing 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 (pK a 6 -7) 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 K 0.5 /V m 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 K 0.5(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 K 0.5(GABA) would be expected to give rise to a lower I max , which is opposite to the observed increase (Fig. 2).
The prevailing alternating access model for how the GAT-1 and other Na ϩ -coupled transporters operate is illustrated in Fig. 6. According to this model, the Na ϩ 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 ϩ to the outward-facing transporter (C 2 Na 2 ). Subsequently the substrate binds (C 3 Na 2 GABA), which triggers a conformational change that exposes the substrate binding site to the intracellular environment (C 4 Na 2 GABA). After the sequential release of substrate and Na ϩ , the empty transporter reverts to the extracellular side (C 6 3 C 1 ), and the cycle continues. Cl Ϫ 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 ϩ binding and unbinding (C 6 7 C 1 7 C 2 Na 2 ). 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.
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 ϩ affinity, which is reflected in the voltage dependence of the K 0.5 for Na ϩ . 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 ϩ 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 ϩ (C 6 7 C 1 7 C 2 Na 2 ); 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 K 0.5 for Na ϩ 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 ϩ -bound conformation (C 2 Na 2 ) and T349H shifted toward the inward-facing empty conformation (C 6 ) (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 ϩ affinities, the shift in V 0.5 points toward a general shift in the equilibrium of the mutants; the M345H is poised toward the outward-facing Na ϩ -bound conformation (C 2 Na 2 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 ϩ -bound conformation. The lack of voltage dependence of the apparent Na ϩ affinity and GABA-induced current may then signify that the voltage-dependent C 6 3 C 1 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 Ϫ40 mV. According to the model, the conformational equilibrium of the T349H mutant transporter would be shifted toward the inward-facing empty state (C 6 ). Thereby, the apparent Na ϩ affinity would be reduced, because the transporter is poised away from the Na ϩ -bound conformation (C 2 Na 2 ). The voltage dependence of this mutant transporter is increased compared with the WT (both for the GABA translocation and the apparent Na ϩ affinity). This may indicate that the voltage-dependent step (C 6 3 C 1 ) 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 (Ϫ160 mV). Once the transporter binds Na ϩ , 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 res- idues, 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 ϩ -induced, substrate-independent leak currents, although the ratio between the Li ϩ 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 ϩ binding to the transporter at the first cation binding site in the transporter cycle (15). As the C 6 3 C 1 3 C 2 Na 2 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 ϩ /Cl Ϫcoupled 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 (Tyr 335 ) 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).