Originally published as Biophys J. BioFAST on April 22, 2005.
doi:10.1529/biophysj.105.061002
Biophysical Journal 89:211-231 (2005)
© 2005 The Biophysical Society
Rapid Substrate-Induced Charge Movements of the GABA Transporter GAT1
Ana Bicho * and
Christof Grewer * 
* Max-Planck-Institut für Biophysik, Frankfurt, Germany; and University of Miami School of Medicine, Miami, Florida
Correspondence: Address reprint requests to Christof Grewer, PhD, Dept. of Physiology and Biophysics, University of Miami School of Medicine, 1600 NW 10th Ave., Miami, FL 33136. Tel.: 305-243-1021; Fax: 305-243-5931; E-mail: cgrewer{at}med.miami.edu.
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ABSTRACT
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The GABA transporter GAT1 removes the neurotransmitter GABA from the synaptic cleft by coupling of GABA uptake to the co-transport of two sodium ions and one chloride ion. The aim of this work was to investigate the individual reaction steps of GAT1 after a GABA concentration jump. GAT1 was transiently expressed in HEK293 cells and its pre-steady-state kinetics were studied by combining the patch-clamp technique with the laser-pulse photolysis of caged GABA, which allowed us to generate GABA concentration jumps within <100 µs. Recordings of transport currents generated by GAT1, both in forward and exchange transport modes, showed multiple charge movements that can be separated along the time axis. The individual reactions associated with these charge movements differ from the well-characterized electrogenic "sodium-occlusion" reaction by GAT1. One of the observed electrogenic reactions is shown to be associated with the GABA-translocating half-cycle of the transporter, in contradiction to previous studies that showed no charge movements associated with these reactions. Interestingly, reactions of the GABA-bound transporter were not affected by the absence of extracellular chloride, suggesting that Cl– may not be co-translocated with GABA. Based on the results, a new alternating access sequential-binding model is proposed for GAT1's transport cycle that describes the results presented here and those by others.
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INTRODUCTION
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During synaptic transmission, high-affinity neurotransmitter transporters are thought to play a fundamental role in regulating the concentration of neurotransmitter present in the synaptic cleft as a function of space and time (1
–4). At GABAergic synapses, reuptake of the inhibitory neurotransmitter 
-aminobutyric acid (GABA) is performed by specific high-affinity secondary transporters, which are expressed in neurons, presynaptic terminals, and glial cells surrounding the synapse (5). Active GABA transport by GAT1 (GABA transporter 1) is driven electrochemically by the concentration gradient of the co-transported ions across the cell membrane and the transmembrane potential. It is generally assumed that GABA uptake is accompanied by the co-transport of two sodium ions and one chloride ion per GABA molecule transported (6–10). However, both the stoichiometry of transport and the involvement of chloride in substrate translocation are still controversial. Although experiments under steady-state conditions showed that extracellular Cl– is not absolutely required for the transport of GABA by GAT1 (11,12), it was also observed that the presence of chloride increases the turnover rate of GAT1 and increases the apparent affinities for the other two co-substrates (13–15). In particular, Loo et al. (12) established a stoichiometry of 1GABA:2Na+ in the presence and absence of chloride that led to the proposal of a Cl–/Cl– exchange mechanism during the GAT1 transport cycle.
Independent of the stoichiometry of chloride co-transport, GABA transport is electrogenic and, thus, associated with inwardly-directed transport currents, which can be studied by using electrophysiological techniques (16). Transport currents can be used as a highly sensitive assay for the function of secondary transporters in general, allowing one to elucidate individual steps in the transport cycle caused by ion binding, ion translocation, and/or associated protein configuration changes that produce electrical signals (see, for example, 16–22).
In the past, transient currents mediated by GAT1 have been detected in response to steps of the transmembrane potential (13,16,20,23) and were attributed to specific partial reactions of the transport cycle. One of these reactions, which was observed in the absence of transported substrates such as GABA, was found to be dependent on the extracellular sodium concentration and has been linked to the binding of sodium ion(s) to an extracellular binding site on GAT1, or conformational changes of the transporter protein linked to Na+ binding. Sodium ions were proposed to bind to the empty transporter, before the interaction with GABA (13,16,20). This Na+-binding reaction is associated with the inward movement of 0.8–1 apparent positive charge, and it is characterized by a time constant in the range of 10–100 ms, its value being strongly dependent on the membrane potential (10,20,24). Therefore, it was proposed that electrogenic Na+ binding accounts for most of the total charge of +1 moved inwardly during a transport cycle and that Na+ binding governs the voltage-dependence of GABA transport at positive and intermediate negative membrane potentials. Additionally, measurements of current relaxations after voltage jumps, applied to giant membrane patches from Xenopus oocytes expressing GAT1 in the nominal absence of substrates on both sides of the membrane, have revealed distinct fast transient currents (<1 ms). It was suggested that these transient currents are generated by the reorientation of the empty transporter substrate binding sites to the outside to allow the rebinding of sodium ions from the extracellular side (20,25). These reactions are inhibited by cytoplasmic chloride and presumably never become rate-limiting for GABA transport.
So far, little is known about the electrogenic properties of reactions that follow GABA binding, such as GABA translocation. Attempts were made by Lu and Hilgemann (20) to restrict GAT1 reactions to the GABA-translocating limb of the transport cycle by applying equilibrium conditions with all three substrates in saturating concentrations present on both sides of the membrane (the so-called exchange transport mode; see Refs. 26 and 27). However, Lu and Hilgemann (20) failed to detect transient currents under these conditions after applying voltage jumps to giant patches of membrane from GAT1-expressing Xenopus oocytes. Accordingly, they postulated an electroneutral reaction for the translocation of substrates across the membrane.
The aims of this work were to investigate in detail individual reaction steps of the transport cycle of GAT1, such as GABA binding and translocation, and ionic movements triggered by or associated with these reactions, and to elucidate the role of chloride in GABA transport by GAT1. To achieve these aims, we applied GABA concentration jumps to the transporter within <100 µs, thus perturbing the pre-existing steady-state, and we analyzed the currents associated with the subsequent relaxations to a new steady-state. Rapid GABA concentration jumps were generated by photolytic release of GABA from 
-carboxy-o-nitrobenzyl (CNB)-caged GABA (28), a nontransportable precursor of GABA. These were combined with patch-clamp measurements to obtain new kinetic information about GABA-induced individual reaction steps in GAT1's transport cycle not yet detected by means of voltage-jump methods (13,14,16). The application of this technique allowed us to detect two GABA-induced steps of the transport cycle and to investigate possible reactions corresponding to the translocation of substrates across the membrane. Using this approach, we demonstrate that the GABA-induced reactions are fast and contribute no more than 
10% of the charge moved per transport cycle. The results obtained, together with data from voltage-jump experiments, are well described by a sequential alternating access model in which sodium ions and GABA are transported across the membrane without the participation of chloride. It is proposed that Cl– is transported through a different mechanism that simultaneously involves the relocation of the empty binding sites for sodium and GABA of the transporter across the membrane.
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MATERIALS AND METHODS
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Transient expression of GAT1 and electrophysiological recording
Mouse brain GAT1 (23) subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) or pBK-CMV-
-(1098–1300) lacking the T3 promoter preceding the multiple cloning site (a kind gift by Dr. Thomas Rauen, from Westfälische-Wilhelms-Universität, Münster, Germany) were used for transient transfection of subconfluent human embryonic kidney cells (HEK293, ATCC No. CRL 1573) with the Effectene transfection method (Qiagen, Hilden, Germany), or the calcium phosphate transfection method, respectively. Both constructs contain the CMV promoter necessary for efficient expression in mammalian cells. Green fluorescent protein (pGreenLantern, Life Technologies, Gaithersburg, MD), was coexpressed for positive selection of transfected cells. The Effectene transfection method was performed with modifications from the supplied protocol: The DNA complexes were formed with 1.8 µg GAT1 cDNA, 0.15 µg green fluorescent protein cDNA, 20 µl enhancer, 50 µl buffer EC, and 25 µl Effectene, total incubation time of 2 h. The calcium phosphate-mediated transfection was performed as described previously (21,29). Electrophysiological recordings were performed 24 h after transfection, for 6–8 h, with an Adams and List EPC7 amplifier (Instrutech, Port Washington, NY) under voltage-clamp conditions in the whole-cell current recording configuration (30). Cells were typically held at –40 or 0 mV during photolysis experiments in the forward or the exchange transport modes, respectively. Recording electrodes were pulled from Clark borosilicate glass and had typical open-tip resistances of 2–4 M
. For the study of the sodium-dependent transient currents, voltage jumps with a duration of 300 ms were applied to the cells from a holding potential of 0 mV to transmembrane potentials ranging from –120 to +90 mV (30 mV increments) and back to the holding potential. Data were recorded with a digitizer board, which was controlled by the pClamp6 software (Axon Instruments, Foster City, CA), digitized with a sampling rate of 1 kHz (solution exchange) or 25 kHz (laser-pulse photolysis), and lowpass-filtered at 250 Hz or 3–10 kHz, respectively.
Composition of solutions in the different transport modes employed
Unless stated otherwise, typical bath solutions contained 140 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, and 30 mM HEPES (pH 7.4/NaOH). For ion substitution experiments sodium was replaced by N-methyl-D-glucamine (NMG) or tetramethylammonium (TMA), and chloride was replaced with methanesulfonic acid (MES) or gluconate. Voltage-jump experiments were performed in a bath solution containing 60 mM NaCl, 90 mM TMA-Cl, 2 mM MgCl2, 2 mM CaCl2, and 5 mM MOPS (pH 7.4/NaOH). The blocker SKF-89976A was kindly provided by SmithKline Beecham (Munich, Germany). All the experiments were performed at room temperature.
An illustration of the three transport modes used in this study, namely forward transport, inward exchange mode, and outward exchange mode, is shown in Scheme 1. For the study of the forward transport mode (Scheme 1, left panel), recording pipettes were filled typically with 140 mM KAspartate, 1 mM MgCl2, 10 mM EGTA, and 10 mM HEPES (pH 7.2/KOH, zero-trans conditions). The voltage dependence of the currents was studied with a CsMES-based solution to avoid the activation of HEK293-native ionic channels by voltage (31); under both conditions the currents showed the same general characteristics and similar apparent affinity for GABA. For the electrophysiological investigation of the inward exchange mode the pipette solution contained 140 mM NaCl, 20 mM GABA, 1 mM MgCl2, 10 mM EGTA, and 20 mM HEPES (pH 7.2/NaOH). The bath solution contained 140 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 30 mM HEPES, and 1 mM caged GABA (pH 7.4/NaOH). In this transport mode, similar concentrations of Na+ were used on both sides of the membrane, and concentrations of GABA were used on the intra- and extracellular side that saturate their respective binding site, thus preventing substrate dissociation on the intracellular side of the membrane. The affinity of GAT1 for cytoplasmic GABA is 2 mM (14). Therefore, we used 20 mM cytoplasmic GABA to ensure saturation of the intracellular binding site (10-fold Km). In contrast, the affinity for extracellular GABA is 12 µM (see Results) and 100–200 µM extracellular GABA is sufficient for saturation of this binding site. Initially, GABA was omitted from the extracellular solution. Under these conditions, the high intracellular Na+ and GABA concentrations will drive the majority of GABA binding sites to face the extracellular side, ready to accept extracellular GABA. Subsequently, rapid application of extracellular GABA resulted in a redistribution of these binding sites to reach a new steady state. According to the nature of the inward exchange mode it is not associated with steady-state transport current. However, inwardly-directed transient transport currents were observed when GABA was applied to the extracellular side of the membrane, due to the electrogenic redistribution of Na+ and GABA binding sites within the membrane (shaded arrow in Scheme 1, middle panel). Experiments in the inward exchange mode were also performed in the total absence of chloride on either side of the membrane (replacement with MES).
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SCHEME 1 Illustration of the transport modes employed in this study. A and B represent GAT1 with the substrate binding sites exposed to the extracellular and intracellular side, respectively. G represents GABA and N a sodium ion. Chloride has been neglected for clarity. The shaded arrows show the major direction of the reaction in each transport mode.
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Alternatively, in the outward exchange mode, GABA was initially omitted from the cytoplasmic solution, in the presence of saturating concentrations of extracellular GABA and Na+. The majority of the binding sites for GABA will be driven to face the cytoplasm under these conditions. Rapid application of GABA by photolysis of 5 mM CNB-caged GABA (Molecular Probes, Karlsruhe, Germany) (28) in the intracellular solution (intracellular photolytic release) leads again to a redistribution of the translocation equilibrium, but this time in the opposite direction (Scheme 1, right panel). In this case, the pipette solution contained 140 mM NaCl, 5 mM caged GABA, 1 mM MgCl2, 10 mM EGTA, and 20 mM HEPES (pH 7.2/NaOH). The bath solution contained 140 mM NaCl, 0.5 mM GABA, 2 mM MgCl2, 2 mM CaCl2, and 30 mM HEPES (pH 7.4/NaOH).
Solution exchange and laser-pulse photolysis
Rapid solution exchange and delivery of substrates was performed by means of a quartz tube (350 µm I.D.) positioned at 
0.2 mm away from the cell with a linear flow rate of 5–10 cm/s. Laser-pulse photolysis experiments were performed as described previously (21,32). Briefly, 1.2 mM CNB-caged GABA was delivered to the cells in the bath solution and the photolysis of the caged GABA was initiated with a light flash (340 nm, 15-ns pulse duration, excimer laser pumped dye laser, Lambda Physik, Göttingen, Germany) 3 s after cell equilibration with the caged compound. For the ion substitution experiments the concentration of caged GABA was increased to 5 mM to account for the decreased GABA affinity of GAT1 in the presence of low Na+ or Cl– concentrations. For intracellular photolytic release experiments, photolysis was performed after a 15-min waiting period, after establishing the whole-cell configuration to allow complete diffusive equilibration of caged GABA between the recording pipette and the cell interior. The waiting period between photolysis trials was 3 min. The laser light was coupled into a quartz fiber (365 µm in diameter) positioned at 200 µm distance from the cell. The laser energy (maximum of 400 mJ/cm2) was adjusted with neutral density filters (Andover, Salem, MA). The released GABA concentration was estimated by comparison of the steady-state current with that generated by rapid perfusion of the same cell with 200 µM GABA (inward transport mode), by using the known GABA dose response curve (see Fig. 1 B). In exchange mode experiments the concentration of photolytically released GABA was saturating, as compared to the forward currents from another cell.
Data evaluation
The forward transport pre-steady-state currents were fitted with a sum of three exponential functions and a steady-state current component (Iss): I(t) = I1* exp (–t/
1) + I2* exp (–t/2) + I3 (–t/3) + Iss. Under exchange conditions the transient currents were fitted with one exponential function (characterized by the time constant H when elicited by extracellular, or HI by intracellular [GABA]-jumps, respectively) and Iss became zero. Transient currents obtained after voltage jumps were fitted with a single exponential function that decayed with a characteristic time constant of VM. Experimental values obtained for the voltage dependence of the maximum moved charge were fitted with the Boltzmann-like relationship Q(Vm) = Q1 + Q2 [1 + e(zQ(Vm–VQ) F/(RT))]–1, respectively, where F, R, and T have their usual meaning, VQ is the midpoint potential of the charge movement, zQ is the apparent valence, and Q1 and Q2 are the minimum and the maximum values of the charge movement, respectively. The moved charge was calculated by integrating the transient components of the measured currents. The intrinsic inhibition constant for caged GABA, Ki, was estimated from the competitive inhibition curve (Fig. 3 B) by taking into account that the Km for GABA in the presence of the inhibitor, I, is increased by a factor (1 + [I]/[Ki]); a value of the fractional inhibition, i, can be calculated by using the formula i = [I]/{[I] + Ki(1+[S]/Km)} (e.g., 33). The apparent rate k*GABA of GABA binding to GAT1 was obtained from the slope of the linear dependence of 1/3 versus [GABA] at low GABA concentrations where binding is rate-limiting for transport (<10 µM). The intrinsic binding rate of GABA to the transporter, kGABA, was determined assuming a rapid pre-equilibrium approach (34) according to the expression k*GABA = kGABA · {Ki/(Ki + [I])}. Linear and nonlinear regression fits of experimental data were performed with Origin (Microcal, Northampton, MA) or Clampfit (Axon Instruments, Foster City, CA) software. When indicated, the data shown are mean ± SE (n), where n is the number of cells used for a particular experiment.
Simulations
Simulations of steady-state and pre-steady-state currents were performed similar to the kinetic analysis by Parent et al. (35) for the sodium/glucose transporter, as pioneered by Läuger and co-workers (36). The transitions occurring between states were modeled according to the Eyring transition-state theory (37) assuming symmetrical energy barriers for each transition. For any given transition between states i and j involving an apparent valence zij, the first-order forward and backward rate constants are given by kij = k°ij · exp (–zijFV/2RT) and kji = k°ji · exp (zijFV/2RT), respectively. The values k°ij and k°ji are the forward and backward rate constants at 0 mV, and V is the membrane potential; F, V, and T have their usual meaning. For transitions involving substrate binding the pseudo-first order rate constants were scaled by the concentration of reactants to the power of the number of molecules involved in the binding reaction. The time-dependent, net transmembrane current in response to GABA is given by the sum of all charge movements that occur within the transmembrane electrical field, which depends on the population of transporter states as a function of time. Accordingly, the current was calculated using the equation I = –F 
zij · (Pikij – Pjkji), where kij and kji are the forward and the backward reaction rate constants for the transition between any two given states with the fractional population Pi and Pj. The variation of the population of each carrier state as a function of time was calculated by numerically integrating the set of differential equations describing the cyclic reaction scheme (see Fig. 8). For numerical integration of the set of differential equations the Berkeley Madonna software (University of California at Berkeley, CA) was used.
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FIGURE 8 State diagram for an ordered kinetic model that can account for the kinetic properties deduced for GAT1. A and B represent transporter states with the binding sites for GABA and sodium facing the extracellular or the intracellular side, respectively. The extracellular binding of the first sodium to the empty transporter (A  AN) is assumed to increase the apparent affinity for GABA. This reaction accounts for most of the charge transported per forward cycle, it is strongly voltage-dependent and responsible for the voltage-dependence of the steady-state currents at intermediate negative and positive membrane potentials. Under forward transport conditions, the transport cycle proceeds clockwise around the loop with the binding of the second sodium ion (AN AN2). After the rapid and almost electroneutral binding of one GABA molecule (AN2 AN2G), the loaded transporter is able to translocate across the membrane to expose sodium and GABA binding sites to the intracellular side (AN2G BN2G). The results do not provide information about the sequence of intracellular release/binding of substrates; a simplification of these reactions was made in which they were lumped together as a single transition that includes the release/binding of two sodium ions and one GABA molecule to the cytosol (BN2G B). At very negative potentials this reaction is assumed to become rate-limiting for the steady-state turnover. The transport cycle proceeds via two different pathways: through the reactions leading to the fast translocation of chloride across the membrane (BC AC) and/or by the slow reorientation of the empty carrier (B A). In the presence of extracellular chloride the equilibrium (A B BC) is rapidly shifted to form the intermediate BC. In the absence of extracellular chloride, with saturating GABA and sodium concentrations, the reorientation of the empty transporter in the membrane is assumed to become the rate-limiting step for forward transport. The translocation of chloride to the intracellular side of the membrane occurs via a reaction independent of the GABA- and Na+-induced reactions (BC AC) that simultaneously exposes the biding sites for sodium and GABA to the extracellular side. It is here proposed that chloride binds to the transporter before the other substrates. In this interpretation chloride is not directly involved in the reaction steps that the protein undergoes to translocate the substrate across the membrane, but the external binding of a chloride ion facilitates the relocation of the empty transporter and increases the number of proteins ready to bind sodium (and subsequently GABA), increasing in this way the apparent affinity of GAT1 for sodium and GABA. k12, k21..., are the rate constants for the different transitions; z12, z23..., are the corresponding apparent valence values. The experimental data could be well described with the following set of parameters: k120 = 0.5 M–1 ms–1, k210 = 0.01 ms–1, z12 = 0.9, k230 = 10 M–1 ms–1, k320 = 0.1 ms–1, z23 = 0.1, k340 = 10,000 M–1 ms–1, k430 = 1 ms–1, z34 = 0, k450 = 1 ms–1, k540 = 1 ms–1, z45 = 0.2, k560 = 0.3 ms–1, k650 = 1e+6 M–3 ms–1, z56 = 0.2, k160 = 0.02 ms–1, k610 = 0.005 ms–1, z61 = –0.215, k670 = 200 M–1 ms–1, k760 = 10 ms–1, z67 = 0.01, k780 = 0.4 ms–1, k870 = 0.02 ms–1, z78 = 0.005, k810 = 50 ms–1, k180 = 150 M–1 ms–1, and z81 = –0.2. The total concentration of the transporter in the membrane was considered constant (A + AN + ANG + AN2G + BN2G + B + AC + BC = 1) and as starting conditions it was assumed that A = AN = AC = 0.33 (for both forward and inward exchange transport modes). However, to achieve a real pre-steady-state, the populations of A, AN, and AC were allowed to equilibrate before the virtual GABA concentration jump was applied.
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RESULTS
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Steady-state GAT1 currents in the forward transport mode
Steady-state currents of the GABA transporter GAT1 were studied by means of a rapid solution exchange method. Although nontransfected cells showed no current signals in response to the application of extracellular GABA, GAT1 transfected cells exhibited GABA-induced currents as shown in Fig. 1 A, which could be fully inhibited by co-application of 100 µM SKF-89976A, a specific GAT1 blocker (38). In the presence of infinite concentration gradients of the substrates across the membrane (forward transport mode, zero-trans conditions) these currents were inwardly directed, as expected from the proposed GAT1 stoichiometry (2Na+:1Cl–:1GABA) (7,8). The currents were dependent on the GABA concentration and saturated with an apparent Km value of 12.1 ± 1.1 µM (n = 7, Fig. 1 B), an indication that GABA is transported by GAT1 with high affinity. This Km value is in the same range as those previously described for GAT1 by others (4.7, 3, 15 µM) (8,9,12). Reducing the external sodium concentration from 140 to 20 mM caused a decrease in the apparent affinity of the transporter for GABA, the apparent Km value becoming 66.4 ± 12.7 µM (n = 4, Fig. 1 B). This effect of the Na+ concentration on GABA affinity has been previously reported by others (12,16). In addition, the reduced extracellular sodium concentration resulted in a reduction of the maximum current amplitude at saturating GABA concentrations (1 mM, Fig. 1 A).
Next, we determined the steady-state current-voltage (Iss/Vm) relationship of GAT1 in the forward transport mode. The averaged data from five cells are shown in Fig. 1 D. The steady-state current increases with increasingly negative membrane potential and does not reverse at positive potentials, as expected for the zero-trans conditions applied here. At the most negative membrane potentials used (Vm < –100 mV), the Iss curve begins to level off, as previously observed for GAT1 expressed in Xenopus oocytes (8,20). Taken together, the data obtained at steady-state conditions agree well with data previously published for GAT1 expressed in other expression systems, such as Xenopus oocytes and HeLa cells.
Pre-steady-state GAT1 currents in the forward transport mode
GABA-induced pre-steady-state currents catalyzed by GAT1 were studied by means of GABA concentration jumps generated within 100 µs via the laser-induced photolysis of CNB-caged GABA. The photolysis reaction that generates free GABA with a time constant of 28 µs is shown in the top panel of Fig. 2 (28). The photolytic release of GABA from 1.2 mM caged GABA generates a whole-cell current of the same steady-state amplitude as that activated by flow application of 200 µM GABA (Fig. 2, A and B), suggesting that saturating substrate concentration jumps can be generated by using this method. Due to the at-least 100-fold higher time-resolution compared to the flow method (21,28) and the presence of a competing nontransported substrate (caged GABA), the photolysis-induced current shows a different time dependence and reveals additional information not seen in the solution exchange experiment, in particular the early reactions of the transport cycle initiated by GABA binding to the transporter (notice the different timescales in Fig. 2, A and B). The effects of caged GABA competition on the time dependence of the transport currents will be discussed in the next section. The pre-steady-state currents show a very fast outward transient current component (phase a), which is not resolved by the method's time resolution, followed by a rapid inwardly-directed phase (phase b). Finally, a slower relaxation process (phase c) to a new steady-state level (Iss) of the current is observed, that is characterized by a current rise. The time course of the pre-steady-state currents of the transporter (corresponding to the currents assigned to phases b and c) could be well described by a sum of three exponential functions and a steady-state current component (see Materials and Methods) and the values found for the respective time constants were 1 = 0.27 ± 0.1 ms, 2 = 2.8 ± 0.4 ms, and 3 = 11.4 ± 0.8 ms (n = 9) at –40 mV transmembrane potential and at saturating GABA concentration.
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FIGURE 2 Typical whole-cell currents induced by GABA in HEK293 cells expressing GAT1, at Vm = –40 mV in the forward transport mode. (A) Steady-state currents measured in response to the application of 200 µM external GABA (rapid solution exchange) at t = 0. Currents were measured with internal 140 mM KAsp and external 140 mM NaCl buffers. (B) Typical pre-steady-state currents induced by the photolytic release of GABA (170 µM, t = 0) from 1.2 mM CNB-GABA (h , arrowhead) triggered a fast outward transient current (a), followed by a rapid inwardly directed component (b), and a slow relaxation component (c) to the steady-state level of current (Iss). The time constants 1, 2, and 3 indicated were obtained from fitting the data relative to b and c with a sum of three exponential functions (1 = 0.27 ± 0.1 ms, 2 = 2.8 ± 0.4, and 3 = 11.4 ± 0.8, n = 9 cells, solid line). The upper panel shows the photolytic release reaction of GABA from its caged precursor. The laser energy was 400 mJ/cm2. The cells were equilibrated for 3 s with the CNB-GABA bath solution before the laser flash. The leak current was subtracted. (C) Photolysis control experiments. Effect of a laser flash on the activation of the pre-steady-state currents in a nontransfected cell (in the presence of CNB-GABA, upper trace) and in transfected cells in the absence of CNB-GABA (control), and in the presence of 1 mM GABA or 100 µM SKF-89976A (co-delivered with the CNB-GABA), as compared with a positive control (three lower traces).
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To exclude the possibility that the electrical signal obtained upon photolysis of caged GABA could be due to an unspecific response of the cell to the caged substrate or photolysis by-products, control photolysis experiments were performed on nontransfected cells in the presence of caged GABA (n = 2), without any currents being elicited (Fig. 2 C, upper trace). To further eliminate possible artifacts due to the interaction of the transporter molecule itself with the light flash, the transfected cells were submitted to laser flashes in the absence of caged GABA, and did not show a light-induced signal (n = 6, Fig. 2 C). Furthermore, both laser flash-induced transient currents and Iss were fully inhibited by pre-applying 100 µM of the specific GAT1 inhibitor SKF-89976A (38) together with caged GABA, as expected from the competitive inhibition mechanism (n = 4, Fig. 2 C). A similar experiment was performed by pre-equilibrating the cell with a super-saturating concentration of GABA (1 mM) together with the caged-GABA (n = 2, Fig. 2 C). Again, photolysis-induced currents were completely abolished. This result is in agreement with the idea that under these experimental conditions most GAT1 molecules have their GABA-binding sites occupied by substrate and should not respond to an additional release of GABA after photolysis. Together, the results presented in Fig. 2 C demonstrate that the observed pre-steady-state currents are due to the presence of GAT1 transporters in the membrane of the HEK293 cells.
Biological properties of CNB-caged-GABA
The CNB-caged GABA was used in this study for the first time to investigate GAT1 reactions elicited by GABA concentration jumps generated by a laser flash. There are several examples reported of competitive inhibition of the biological system under investigation by the respective caged compound (39–41). In particular, CNB-GABA is known to inhibit ionotropic GABA receptors (42). For this reason, the possibility that caged GABA binds to GAT1, thus acting as a competitive inhibitor, was investigated. The application of 200 µM GABA in either the absence or the presence of 1.2 mM caged GABA (a typical concentration used in the experiments) elicited the same steady-state current amplitude (Fig. 3 A). To test whether the absence of an inhibitory effect under these conditions may be due to the high concentration of free GABA (200 µM) preventing caged GABA from binding to GAT1, steady-state experiments were performed in which the concentration of caged compound was increased and the free GABA concentration was varied. A bath solution containing 5 mM caged GABA was co-applied with free GABA at different concentrations, ranging from 100 to 500 µM. Under these conditions, an inhibitory effect by the caged compound on the currents was observed, in particular in the presence of lower concentrations of free GABA. An inhibition curve is shown in Fig. 3 B. The data (n = 5) were fitted with a Michaelis-Menten function from which the Km for GABA in the presence of inhibitor, 
for competitive inhibition was found. An intrinsic inhibition constant, Ki, of 225 µM for caged CNB-GABA (in the absence of free GABA) was estimated from these data (see Materials and Methods). The thermodynamic constants for GABA binding and caged GABA binding to GAT1 are summarized in Table 1. According to a competitive inhibition mechanism it is expected to have populations of transporter molecules before the photolytic release of GABA with and without caged compound bound to the substrate binding site, in the presence of inhibitory concentrations of caged GABA. Accordingly, values of fractional inhibition can be calculated (see Materials and Methods). With a Ki of 225 µM, 84% of the transporters are bound to the inhibitor (1.2 mM caged GABA) before a laser pulse. After equilibration after a 200 µM GABA concentration jump only 23% of the transporters are expected to be in the inhibitor-bound form. This estimation shows that under the experimental conditions used here most of the caged compound is removed from the binding site after photolysis. At any fixed nonsaturating concentration of inhibitor (caged-GABA) it should be possible to drive all the transporter molecules to the GABA-bound form by applying higher GABA concentrations and to achieve the same current as in the absence of the inhibitor. It is expected that the released GABA concentration is large enough to compete off the inhibitory effect of the caged GABA to obtain saturated transporter steady-state current. This idea holds even in the presence of high caged GABA concentrations (5 mM) when the estimated concentrations of photolytically released GABA are 0.5–1 mM.
Although dissociation of caged GABA from the transporter is most likely fast, because of its relatively low affinity, the data presented so far do not exclude the possibility that caged GABA dissociates slowly, thus retarding GABA binding to GAT1 after photolytic release. Therefore, it was tested whether the displacement of the caged GABA molecules from the GAT1-caged GABA complex by free GABA causes a delay of the current rise, thus influencing the shape of the observed pre-steady-state currents as a function of time. The interaction of CNB-caged GABA with GAT1 and its effect on the transient currents was studied by comparing the currents obtained from cells to which two different concentrations of caged GABA (1 and 0.5 mM) were applied and the laser light intensity was varied by means of different neutral density filters in such a way as to cause the release of similar concentrations of free GABA (80 µM). Under these conditions, it would be expected that the time course of the current rise would be slowed when the same concentration of GABA is released from a higher concentration of caged precursor. In contrast to this expectation, there were no observable differences between the time courses of the pre-steady-state currents generated by release from 0.5 or 1 mM CNB-GABA, indicating that they depend only on the amount of available free GABA. With 250 µM CNB-GABA and using the light energy levels tolerated by GAT1-HEK293 cells without losing the giga-seal, smaller amounts of free GABA were released but all transient current steps were present (n = 4, not shown). Together, these experiments demonstrate that, although the CNB-GABA acts as a competitive inhibitor of GAT1 activity, the released GABA concentrations rapidly displace caged GABA from its binding site. Therefore, this caged compound can be used for the study of GAT1's GABA-induced pre-steady-state kinetics. In agreement with this conclusion, pre-steady-state currents with the same characteristics and time dependency were obtained after the photolysis of a newly developed caged GABA derivative, ,4-DCNB (,4-dicarboxy-o-nitrobenzyl) caged-GABA (K. Schaper, S. Madani Mobarekeh, P. Doro, A. Bicho, and C. Grewer, unpublished results). ,4-DCNB-caged GABA does not inhibit GABA-induced GAT1 currents.
Additional control experiments were performed by generating rapid GABA concentration jumps by means of a fast perfusion system. By flowing a supersaturating concentration of GABA (50 mM 4200 x Km) over the cell it is possible to reduce the apparent time necessary to apply a saturating concentration to the GABA transporters in the cell membrane, thus increasing the apparent time resolution of the flow system. A similar approach was detailed by Dilger and co-workers (43) for studying the kinetics of acetylcholine receptor activation. After jumping the GABA concentration to 50 mM a rapid rise of the current was observed, followed by a minor transient current that decays exponentially with a time constant >100 ms to the steady-state current level (n = 3, Fig. 3 D). This minor transient current was absent in the experiments with the lower GABA concentration of 200 µM (Fig. 3 D, inset). An important feature of the current trace obtained with 50 mM GABA by means of fast solution exchange methods is that the transient current is not much larger than the steady-state current, the maximum peak current being 20% bigger than the maximum steady-state current level (n = 3 cells). This result is in agreement with the photolysis experiments, indicating that no large transient component, which may have been missed in the photolysis experiments because of inhibition by the caged compound, is induced in GAT1 in response to the GABA concentration jump.
[GABA]-dependence of the pre-steady-state currents
Next, we investigated the effect of the concentration of photolytically released GABA on the different phases of the GAT1 current. The concentration of released neurotransmitter was controlled by using different laser energies for photolysis, adjusted with neutral density filters, and the released GABA concentration was subsequently estimated by comparison of the steady-state current induced by photolysis with that generated by the rapid perfusion of the same cell with 200 µM saturating external GABA, and by using the known GABA dose-response curve of Fig. 1 B (see Materials and Methods). The pre-steady-state currents of a typical cell induced by the photolytic release of different concentrations of GABA are shown in Fig. 4 A. Rapid application of a GABA concentration of 4 µM, which is at the low end of the range of concentrations investigated, resulted in a slow rise of the current to a steady-state level characterized by a time constant of 25–20 ms (data from five cells), whereas the two fast phases were absent. This result indicates that GABA binding becomes rate-limiting at these low extracellular GABA concentrations. The dependence of the decay time constants of the pre-steady-state currents on the GABA concentration is shown in Fig. 4 B. In contrast to 3, which displayed a pronounced GABA concentration-dependence, 2 was practically unchanged by increasing the GABA concentration. The Michaelis-Menten-like saturation behavior of 3 is indicative of a two-step process underlying this apparent time constant: a rapid GABA binding process followed by a rate-limiting reaction, possibly a conformational change. Assuming that GABA binding is rate-limiting at low GABA concentrations, the slope of the linear-dependence of 3 versus [GABA] < 10 µM gives an estimation of the apparent rate constant for the binding of GABA to the transporter, k*GABA. A value of 0.9 ± 0.1 µM–1 s–1 (n = 5 lower concentrations) at –40 mV was found. As shown above (Fig. 3), caged GABA is a competitive inhibitor of GABA-induced transport by GAT1. Therefore, the value found for k*GABA is a lower limit and the GABA binding reaction to the transporter and the intrinsic rate constant for GABA binding to the free transporter is expected to be faster. After the laser flash, caged GABA has to first dissociate from its binding site before GABA can bind to the empty transporter to allow further reactions in the transport cycle to take place, as illustrated in Scheme 2:
Assuming that caged GABA (cGABA) is in rapid equilibrium with the transporter, the apparent rate of GABA binding can be expressed as k*GABA = kGABA Ki/([caged GABA] + Ki).
A similar rapid pre-equilibrium approach was used by Fendler and co-workers (34) to account for the inhibitory effect of caged-ATP on ATP-induced NaK-ATPase currents. Using the known value of Ki (see above), an intrinsic bimolecular rate constant (kGABA) of 5.9 µM–1 s–1 for the binding of GABA to the transporter was calculated (Table 1).
The concentration-dependence of the maximum current amplitudes of the different phases is shown in Fig. 4 C. The values Iss and I3 saturate with comparable apparent Km values of 12.1 ± 1.1 µM (n = 7) and 8.1 ± 1.1 µM (n = 11), respectively. In contrast, I2 is associated with a higher apparent Km of 245 ± 53 µM (n = 11 cells). This result suggests that the Km determined at steady state may not be a good measure of the real affinity of the transporter for GABA, which is most likely lower than previously assumed.
Voltage jump-induced pre-steady-state currents
To estimate the amount of charge moved in the GABA-induced reactions, the charge was calibrated in parallel experiments by using the voltage-dependent Na+ binding reaction to the GABA-free form of GAT1, a process that has been well established and is associated with the inward movement of 0.8–1 apparent positive charge (10,20,24,44). Applying rectangular voltage jumps from a holding potential of 0 mV to –90 and +90 and back to 0 mV to GAT1-expressing HEK293 cells generates transient currents that can be inhibited by 100 µM SKF-89976A delivered in the bath solution. The current traces recorded in the presence of SKF-89976A were subtracted from those in the absence of the drug to isolate GAT1-specific currents. Fig. 5 A shows the difference current obtained from a typical GAT1-expressing cell in the presence and in the absence of inhibitor. To obtain the corresponding amount of charge moved, the OFF transient currents (which exhibited a better signal/noise ratio than the ON signals) were fitted to a single exponential decaying function to obtain the rate constant for the current decay and were subsequently integrated over time to obtain the amount of moved charge. Since the transient current was shown to be capacitive in nature (13), the current integral of the OFF should be identical to that of the ON signal. Although the membranes of GAT1-transfected HEK293 cells became slightly unstable at jumps to +90 mV, as evident by the noisy steady-state component of the current, the current integrals of the ON and OFF signals were generally similar after correcting for the steady-state component. The voltage dependence of the charge movement is shown in Fig. 5 B. It follows a Boltzmann-like charge-voltage relationship, as expected for the Na+-binding reaction (24). The experiments were performed in the presence of 60 mM external sodium. In this way, it was possible to attain an almost symmetric charge distribution with respect to the holding potential of 0 mV, as indicated by the midpoint potential value (VQ) of –9.9 mV found, and the saturation behavior at both hyper- and depolarizing extremes of the Q versus Vm curve. The maximum normalized charge Qmax can be estimated as 79 fC/pA steady-state current. The estimated apparent valence, zQ, is 0.94, which is in good agreement with previous results (20). In the presence of 140 mM external sodium and at a transmembrane potential of 0 mV the first sodium binding site is close to being saturated (13,15,45), as was noticed from the absence of transient currents within the experimental membrane potential range applicable to HEK293 cells. Using the value of Qmax from the Na+ binding reaction for calibration, values for the ratio Q/Qmax of 3 · 10–3 ± 1 · 10–3 for the inwardly-directed phase (I2) and 3 · 10–2 ± 4 · 10–3 for the rising phase of the GABA-induced transient current signal (I3) were calculated (n = 3, Fig. 5 C). These values demonstrate that the GABA-induced charge movements detected here with saturating GABA concentration jumps are small compared to those found for the Na+ occlusion reaction.
[Na+]- and voltage-dependence of the pre-steady-state currents
The sodium concentration-dependence of the GABA-induced pre-steady-state currents was studied by performing photolysis experiments in the forward transport mode at three different extracellular sodium concentrations (140, 20, and 5 mM, NMG-substitution), as shown in Fig. 6 A. At low sodium concentrations, saturating concentrations of GABA (500 µM, Ref. 12) were photolytically released from 5-mM caged GABA, instead of the typical 1.2 mM used in the control experiments in the presence of 140 mM extracellular Na+. At 20-mM external sodium, the GABA-induced pre-steady-state currents are similar to the control currents (at 140 mM Na+), with all the three phases present, albeit with reduced amplitude. A value of 0.45 ± 0.03 and 0.34 ± 0.07 was found for I2 and I3, respectively (n = 4 cells), relative to the control currents at 140 mM Na+. In the presence of 5 mM external sodium (n = 4 cells) and in a nominally sodium-free bath solution (n = 2 cells, not shown) no observable currents were evoked by the photolytic release of GABA. The [Na+]-dependence of the amplitude of the steady-state component of the currents follows the same trend, showing a reduction at low external sodium concentration and being abolished in the absence of Na+. The time constants of the three phases of the pre-steady-state current were quantified in the presence of 20 mM extracellular sodium. No significant changes compared to the time constants observed at 140 mM Na+ were observed for 1 and 2 (1 = 0.27 ± 0.1 ms, n = 4, and 2 = 3.4 ± 1.3 ms, eight trials from three cells). In contrast, the reaction step characterized by 3 became slower. Values ranging from 20 to 98 ms were determined for this time constant from three cells.
Additional relevant information about the electrogenic reactions of transporter proteins can be obtained by comparing the characteristics of these reactions at different transmembrane potentials (17,21,22,35,46). Therefore, the effect of voltage on GAT1 pre-steady-state currents was investigated in the forward transport mode. Fig. 6 B shows typical currents obtained after the photolytic release of GABA as a function of the transmembrane potential. Although the amplitude of the steady-state component (Iss) of the GABA-induced currents is still increasing with hyperpolarization between –40 and –60 mV (compare also with Fig. 1 D), the values for the time constants show little variation over the entire range of negative potentials. At positive potentials the currents become too small (or alternatively too slow), to be fitted with accuracy. The relaxation rate constants 1/2 and 1/3 are shown at different membrane potentials in Fig. 6 C. The voltage dependence of the logarithm of these rate constants can be quantitatively described by linear regression analysis with slopes of 2.4 x 10–3/mV and –2.0 x 10–3/mV for 1/2 and 1/3, respectively. This demonstrates that the electrogenic reactions causing the respective currents are either only very weakly electrogenic (consistent with the respective moved charge found above from the calibration with the Na+ binding transient) or that they may be rate-limited by an electroneutral reaction. The corresponding fraction of the transmembrane electric field sensed by these processes, if a total of +1 charge was moved, is 0.29 (1/2) and 0.24 (1/3). Additionally, as can be seen in Fig. 6 B, the peak current value I2 corresponding to the individual reaction characterized by 2, reaches its maximum absolute value at potentials less negative than the steady-state current (n = 4; compare also with Fig. 1 D). This result, together with the weak voltage-dependence of the corresponding time constant, indicates that, even when increasing the rate of sodium binding by changing the membrane potential to more negative values, this individual reaction would be unlikely to become slow enough to be rate-limiting for the transport cycle.
Pre-steady-state currents in the absence of extracellular chloride
It has been proposed previously that extracellular chloride is required for GABA transport by GAT1 and that translocation of one GABA molecule is linked to the co-transport of one Cl– ion (8–10). In contrast, in another report it was observed that no net inward transport of Cl– takes place when GAT1 is activated by GABA, but that chloride is transported in a Cl–/Cl– exchange mechanism (12). In an attempt to understand the requirement of chloride for GABA transport, the effect of the extracellular chloride concentration on the GABA-induced pre-steady-state currents was investigated. In analogy to the experiments at low sodium concentration described above, GABA was photolytically released from 5 mM caged GABA, instead of the typical 1.2 mM to counterbalance the decrease of GAT1's affinity for GABA in the absence of external chloride (12). The results are presented in Fig. 6 D, which shows the currents obtained in nominally Cl–-free bath solution after the photolytic release of GABA in comparison with the signal in the presence of chloride (140 mM external Cl–). Unexpectedly, in the nominal absence of external chloride GABA can still bind to the transporter and evoke transient current. In contrast, the steady-state (Iss) component of the current is negligible, as expected in the absence of chloride (11,12), and the slow rise to the steady-state characterized by 3 is no longer detectable, being either slowed down or absent (n = 5). Furthermore, the inset in Fig. 6 D shows that the photolytic release of nonsaturating GABA concentrations (25 µM) does not evoke pre-steady-state currents (n = 3). These experiments show that GABA can bind to the transporter in the absence of extracellular chloride but that chloride modulates GAT1's responses to GABA concentration jumps.
Pre-steady-state currents in the inward and outward exchange transport modes
To assign the GABA-induced transient currents to specific reactions in the GAT1 transport cycle, photolysis experiments were performed in the inward exchange transport mode. In this transport mode, the population of transporter states can be restricted to the substrate-translocation limb of the transport cycle. Before photolysis, Na+ and Cl– were present on both the intracellular and extracellular sides at concentrations of 140 mM, and 20 mM GABA was present at only the intracellular side, saturating the cytosolic GABA binding site of GAT1. Under these conditions, the absence of extracellular GABA should result in a translocation equilibrium and a GABA binding equilibrium shifted to a form in which the majority of GABA binding sites are exposed to the extracellular side, due to the law of mass action (Scheme 1, middle panel). Subsequently, the cells were equilibrated with extracellular caged-GABA and an external GABA concentration jump by photolysis initiated the re-equilibration of the GABA binding sites to face either the extracellular side or the intracellular side of the membrane without completing a full transport cycle. While this re-equilibration takes place, it is possible to observe charge movements that are associated with the GABA translocation branch of the transport cycle. The results from this experimental strategy are shown in Fig. 7 A. In HEK293 cells expressing GAT1, the photolytic release of 100 µM GABA at 0 mV (calibrated from the forward transport currents measured from another cell at the same conditions) induced an inwardly-directed transient current, which was inhibited by 100 µM SKF-89976A (n = 2), and which decayed with a single-exponential behavior with a characteristic time constant of H = 2.9 ± 0.6 ms (n = 7, Fig. 7 A). As expected from the inward exchange conditions, there is no directional cycling of the transporter (Scheme 1, middle panel) and, therefore, no steady-state (Iss) current was observed. Accordingly, the slow rising phase (I3) observed in the forward transport mode (see Fig. 2 B) was absent from the recordings under exchange conditions. To test whether true exchange conditions were established, we repeated the experiment shown in Fig. 7 A, but in the sole presence of 20 mM GABA at the intracellular side (0 mM NaCl internal, replaced with NMG-MES). It had been shown by Lu and Hilgemann (14) that internal GABA does not trans-inhibit forward GABA transport in the absence of cytosolic sodium. Consistently, application of 100 µM GABA to GAT1 under these conditions resulted in a current signal with large steady-state component (41 ± 20 pA, n = 6) and a time-course indistinguishable from that observed under forward transport conditions in the absence of intracellular GABA, demonstrating that the sole presence of GABA at the intracellular side is not sufficient to attain exchange conditions.
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FIGURE 7 Pre-steady-state currents recorded in the exchange transport modes. Typical whole-cell transient currents elicited at t = 0 by the photolytic release of GABA from the extracellular (A, inward exchange mode, 140 mM NaCl and 20 mM GABA internal, 140 mM NaCl external) or intracellular (B, outward exchange mode, 140 mM NaCl and 0.5 mM GABA external, 140 mM NaCl internal) sides, respectively. The corresponding time constants H = 2.98 ± 0.6 ms (n = 7, solid line) and HI = 5.8 ± 1.0 ms (n = 5, solid line) were obtained from the best single-exponential fit to the data in A and B. (C) [GABA]-dependence of currents recorded in the inward exchange mode: two trials from the same cell with extracellular release of 100 or 40 µM GABA. (D) Effect of extracellular chloride concentration on currents in the inward exchange mode: two trials from the same cell in the presence or nominal absence of external Cl– (the internal solution was Cl–-free). (E) Effect of [Na+] and membrane potential on inward exchange currents. Several trials from the same cell with external sodium concentrations of 20 mM and 140 mM are shown. The membrane potential was typically held at 0 mV and with low [sodium] it was varied between 0 and –80 mV. The pipette solution was chloride-free (MES substitution) and contained 20 mM GABA. (F) Ratio Q20 (20 mM Na+)/Q140 (140 mM Na+) at different transmembrane potentials (data from five cells, shaded bars) in the inward exchange mode. The dotted line marks the ratio value at 0 mV in comparison with the charge obtained with 140 mM external Na+ at the same transmembrane potential (0 mV, white bar). (G) Voltage-dependence of the normalized charge with 140 mM ( ) (n = 5 cells) or 20 mM extracellular Na+ ( ) (n = 7 cells) in the inward exchange mode. All values were normalized to the charge obtained with 140 Na+ at 0 mV. The error bars are means ± SE. The internal solution contained 140 mM NaCl (or MES as Cl– substitute in D) and 20 mM GABA. The external NaCl solution (NMG-substitution at low sodium) contained 500 µM GABA. GABA was typically released from 1.2 mM caged GABA or from 5 mM caged GABA (intracellular GABA, 20 Na+, 0 Cl–). The concentration of photolytically released GABA was saturating (300–400 mJ/cm2, as compared to the forward transport currents from another cell). Unless otherwise indicated, the membrane potential was 0 mV.
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The transient currents observed after extracellular GABA application in the inward exchange transport mode were further investigated by varying the concentrations of the substrates GABA, Na+ and Cl–. Lowering the extracellular GABA concentration from 140 µM to a concentration of 40 µM caused a reduction of the amplitude of the transient currents without a clear effect on H (n = 2). Two trials from the same cell are shown in Fig. 7 C; the amplitude of the currents obtained with 40 µM released GABA were decreased approximately threefold when compared to those with 140 µM GABA. This result is consistent with the one found in the forward transport mode for the GABA concentration dependence of I2 and 2, where the amplitude of the peak current was dependent of the GABA concentration but not the time constant. The currents recorded in the inward exchange transport mode were also investigated in the nominal absence of extra- and intracellular chloride (MES substitution). Fig. 7 D compares two traces from the same cell in the presence and in the absence of chloride. The time constant found in the nominal absence of chloride, 3.5 ± 0.9 ms (n = 5 cells), was similar to the one found in its presence, indicating that chloride is not directly involved in generating the electrogenic steps associated with the translocation branch of the GAT1 transport cycle. The currents observed in the absence of chloride were also completely inhibited by 100 µM SKF-89976A (n = 2, data not shown).
The effect of the extracellular Na+ concentration on the pre-steady-state currents in the inward exchange transport mode is shown in the two upper traces of Fig. 7 E. With saturating release of GABA, lowering the typical 140 mM extracellular Na+ concentration to 20 mM (n = 8) caused a reduction of the amplitude and the charge associated with the inward transient current (Q20/Q140 = 0.49 ± 0.01 at 0 mV, n = 4 cells, Fig. 7 F), without an apparent modification of the time constant H. Furthermore, the voltage de
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), with apparent Km values of 8.1 ± 1.7 µM and 12.1 ± 1.1 µM for I3 and Iss, respectively. The dotted line shows a Michaelis-Menten relationship representing I2 (
). I2 and I3 do not correspond to fitted values, but to the maximum current amplitudes of the different phases. The released GABA concentration was estimated by comparison of Iss with that generated by rapid perfusion of the same cell with 200 µM external GABA, by using the known GABA dose response curve of 
