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Figure 2. Definition of GAT1-mediated currents and IâV relations. (A) Chart record of membrane current at 0 mV: extracellular and cytoplasmic solutions were as indicated in millimolar. Outward GAT1 current was activated by applying 20 mM cytoplasmic GABA in the presence of 120 mM cytoplasmic NaCl. After washing off all cytoplasmic substrates with 120 mM NMG-MES, extracellular solution was changed to 120 mM NaCl via pipette perfusion. Inward GAT1 current was then turned on and off by adding and removing 0.2 mM GABAo from the pipette solution. (B) IâV protocol and the corresponding membrane current responses at the time points indicated in A. (C) IâV relations of GABA-defined outward and inward GAT1 current. The median current amplitudes during the last 3 ms of each voltage step in B are plotted against membrane potential.
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Figure 1. Perspectives on the minimum reactions involved in forward and reverse GAT1 transport. (A) Forward (GABA uptake) transport mode. Starting in the upper left, one Na+ can be occluded from the extracellular side in a slow reaction that is strongly electrogenic (i.e., a large lightning bolt). Thereafter, Clâ, GABA, and a second Na+ bind and are translocated to the cytoplasmic side in a âfastâ reaction that is electroneutral. Finally, transporter binding sites reorient to the outside-open position when no substrates are bound on the cytoplasmic side. This reaction is fast and weakly voltage dependent (i.e., small lightning bolt). (B) Reverse (GABA extrusion) transport mode. Starting in the lower left, Clâ and Na+ can bind from the cytoplasmic side. One Clâ and one Na+ are occluded in a slow reaction with weak voltage dependence (i.e., small lightning bolt). Thereafter, one Na+ and one GABA bind and are translocated in a fast electroneutral reaction. When empty binding sites are open to the extracellular side, one Na+ ion is still occluded in the transporter. This Na+ ion is released to the outside in a fast, strongly voltage-dependent reaction (i.e., large lightning bolt), and, in close association with this reaction, binding sites rearrange to the cytoplasmic side-open configuration.
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Figure 6. Reversal potential measurements of GAT1 current. (A) GAT1 transport current with all three substrates on both membrane sides was defined by pipette perfusion of NO-711 (13 μM) to the extracellular side. The pipette (extracellular) solution contained 0.4 mM GABA and 120 mM NaCl; the bath (cytoplasmic) solution contained 2 mM GABA, 60 mM Na+, and 6 mM Clâ. (B) NO-711oâdefined reversal potentials are plotted against the theoretical values for various conditions listed below the graph. All reversal potentials were measured at 35°C. The dotted line represents the values predicted for perfect 2Na+:1Clâ:1GABA stoichiometry.
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Figure 3. Outward GAT1 transport current defined by cytoplasmic GABA and NO-711. Outward GAT1 transport current activated by 20 mM cytoplasmic GABA (120 mM NaCl on both membrane sides) was defined by removing GABA with substitution by aspartate (âª) and by applying 0.13 mM NO-711 in the continued presence of GABA (â¡) on the cytoplasmic side. Note that the two subtraction protocols give very similar results. The results were therefore pooled and fitted by the Boltzmann equation described in materials and methods (solid line; equivalent charge, 0.63). (â¢) Subtraction of an IâV relation with GABA from one without GABAi in the presence of 0.13 mM cytoplasmic NO-711.
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Figure 4. Activation and deactivation of reverse GAT1 current by rapid solution changes. (A) Application and removal of 20 mM cytoplasmic GABA. (B) Application and removal of 120 mM cytoplasmic Clâ. (C) Application and removal of 120 mM cytoplasmic Na+. Fast changes of cytoplasmic solutions were achieved by moving the recording pipette, which was attached to a computer-controlled piezoelectric device, between two steady streams of different cytoplasmic solutions. The extracellular solution contained 20 mM Clâ and no Na+. See text for further explanations.
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Figure 5. IâV relations of GABA-activated currents in the presence and nominal absence of extracellular Clâ. The patch was obtained using a pipette solution with 120 mM extracellular Na-MES and 0 Clâo, whereby a 300-mM KCl âagar bridgeâ was used in the pipette. Outward transport current was defined by substituting 20 mM GABA for 20 mM aspartate in the presence of 120 mM NaCl on the cytoplasmic side. After replacing the cytoplasmic solution with 120 mM NMG-MES, inward current was activated with extracellular GABA in the presence of Na+o. Both were defined with a 0.4 mM GABAo subtraction. The extracellular solution in b contained 120 mM Na-MES and was Clâ free; in c the extracellular solution contained 120 mM NaCl, which was applied by pipette perfusion. The voltage steps were 35 ms in duration. Data points represent the average of current magnitudes at the specified membrane potential during the cumulative voltage protocol.
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Figure 9. Cytoplasmic substrate dependencies of outward GAT1 transport current at 0 mV. (A) Cytoplasmic GABA dependence with 120 and 30 mM cytoplasmic Na+ (upper graph) and with 60 and 3 mM cytoplasmic Clâ (lower graph). (B) Cytoplasmic Na+ dependence with 20 and 2 mM cytoplasmic GABA (upper graph) and with 50 and 5 mM cytoplasmic Clâ (lower graph). (C) Cytoplasmic Clâ dependence with 100 and 20 cytoplasmic Na+ (upper graph) and with 20 and 2 mM cytoplasmic GABA (lower graph). Solid lines are predicted results from fitting an entire data base on GAT1 function by an alternating access model (Hilgemann and Lu 1999).
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Figure 7. Comparison of predictions from a âmulti-substrate single-file modelâ and a two-state alternating access model. (A) Multi-substrate single-file transport model for GAT1 (Su et al. 1996). The channel contains three single-file binding sites that can accommodate either GABA or Na+, and the substrates âhopâ between them. (B) Simulation of the âtrans-effectâ of Na+o on the GABAi dependence of outward GAT1 transport current. (C) Simulation of the âcis-effectâ of Na+i on the GABAi dependence of outward GAT1 transport current in the presence of Na+o. In both cases, [Na+]i is not limiting and [GABA]o is zero. (D) A two-state alternating-access model for a simulated âXY-cotransporter,â where efflux of X and Y generates outward transport current. The two time-dependent steps (arrow pairs) of the transport cycle are the rearrangement of binding sites in the empty (1) and fully loaded (2) transporters. The equations employed were equivalent to those used for more extensive simulations (Hilgemann and Lu 1999). The intrinsic transition rates for fully loaded and empty binding sites were equal. Double-headed arrows indicate âinstantaneousâ association/dissociation of the substrates. (E) Comparison of the measured effect of extracellular Na+ on outward GAT1 current with the simulated trans-effect of Xo. Cytoplasmic GABA concentration dependence of the outward GAT1 current with 120 mM cytoplasmic NaCl at 0 mV was measured in the absence (â¢) or presence (â) of 100 mM Na+o. Solid lines represent simulated Yi dependence of outward transport current when [X]i is not limiting and [Y]o is zero, where the fraction of transporters with Xo bound (fxo) is 0.6 (upper curve) or 1 (lower curve). (F) Comparison of the measured effect of lowering cytoplasmic Clâ on outward GAT1 current with the simulated cis-effect of Xi. Cytoplasmic GABA dependence of the outward GAT1 current with 120 mM extracellular NaCl at 0 mV was measured with 120 mM (â¢) or 30 mM (â) cytoplasmic Clâ. Solid lines represent the simulated Yi dependence of outward transport current in the presence of Xo (fxo = 0.8) and non-limiting Xi, where the fraction of transporters with Xi bound (fxi) is 1 (upper curve) or 0.4 (lower curve).
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Figure 8. Cytoplasmic substrate dependencies of outward GAT1 transport current. (AâC) Cytoplasmic GABA (A), cytoplasmic Na+ (B), and cytoplasmic Clâ (C) dependencies of GAT1 outward transport current at 0 mV. Half-maximal substrate concentrations (K1/2s) were obtained from fits by the Hill equation (solid lines). (DâE) Voltage dependencies of Imaxs, K1/2s, and nHs from the Hill fits for cytoplasmic GABA, Na+, and Clâ. The ImaxâV relations were fit by the Boltzmann equation given in materials and methods. The slope coefficients are 0.3, 0.36, and 0.29 in DâF, respectively. The fits of GABAi dependencies (D) assumed a Hill coefficient of 1. Results in A and D are from one patch, and results in B, C, E, and F are from another patch.
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Figure 10. Effects of cytoplasmic substrates on IâV relations of the inward GAT1 current. Inward GAT1 current is defined as the current activated by 0.2 mM extracellular GABA via pipette perfusion. (A) IâV relations of the inward GAT1 current in the absence of cytoplasmic substrates (â¡), in the presence of 20 mM cytoplasmic GABA alone (âª), in the presence of 120 mM cytoplasmic Na+ alone (â´), and in the presence of 20 mM cytoplasmic GABA and 120 mM cytoplasmic Na+ (âµ). (B) Effect of cytoplasmic Clâ on the IâV relation of the inward GAT1 current. (C) Inhibition of the inward GAT1 current by cytoplasmic Clâ at 0 mV. Half-inhibition occurs at 12.4 mM. See text for further details. The voltage protocol used in A and B was identical to that in Fig. 2. Data points represent the average of current magnitudes at the specified membrane potential during the cumulative voltage protocol.
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Figure 11. GAT1 capacitance changes in response to rapid changes of the cytoplasmic Clâ concentration. (A) Membrane capacitance changes in response to removing cytoplasmic Clâ were measured in the absence (left) or presence (right) of both cytoplasmic GABA (20 mM) and cytoplasmic Na+ (120 mM). The Clâi concentration is changed rapidly by an automated solution switcher. (C) Cytoplasmic Clâ concentration dependence of the membrane capacitance change. Solid lines represent fits by the Hill equation with a fixed slope factor (nH) of 1.
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Scheme S1.
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