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Figure 1. Instrumentation for measuring fluorescence from voltage-clamped oocytes. The recording apparatus consists of a microscope (depicted by the objective and dichroic mirror), a photomultiplier tube attached to the side port of the microscope, and conventional two-electrode voltage-clamp instruments. The apparatus uses a stabilized 100-W Hg light source coupled to an inverted microscope (IX-70; Olympus Corp.), dichroic filter cubes for delivering the light, a 40Ã objective lens, and a photomultiplier tube to collect the fluorescence emission. The oocyte is placed on the microscope stage with its animal pole facing down and is visualized for electrophysiology by a separate stereomicroscope (not shown). The exciting beam is attenuated by factors approaching 300. The emission signal from the oocytes is collected by the objective lens and sent to the PMT. Appropriate amplifiers and electronic filters condition the signal. The command and output signals are interfaced with a computer through A/D and D/A converters.
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Figure 2. [3H]GABA uptake assayed in oocytes expressing WT-GAT17. The GABA uptake activity was measured in uninjected (â), GAT-1 mRNA-injected (âµ), GAT-1 mRNA-injected and TMRM-labeled (â´), and GAT-1 mRNA-injected and MTSET-treated (â¢) oocytes. Each data point is the mean value of measurements from three to four cells, and the error bars indicate SEM. The curves are the nonlinear fits to hyperbolic doseâresponse relations (Michaelis-Menten equation).
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Figure 3. Confocal microscopy of Xenopus oocytes labeled with tetramethylrhodamine as described in materials and methods. (Left) Injected with WT-GAT1, (right) uninjected. The oocytes are 0.9 mm in diameter. The more intense fluorescence is at the animal pole.
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Figure 4. Comparison of WT-GAT1 and C74A electrophysiology after labeling with TMRM. Oocytes expressing WT-GAT1 and C74A-GAT1 were exposed to TMRM as described in materials and methods. (A) GABA-induced current was measured at a holding potential of â60 mV during perfusion of 100 mM GABA for a period of 4 s. The traces are typical of eight cells. (B) The charge movement (ÎQ) was calculated by integrating the transient current induced by a voltage jump from a holding potential of â40 mV to various test potentials, as shown in Fig. 5, middle. Each data point shows the average value from 40 cells for WT-GAT1 (â¡) and 15 cells for C74A-GAT1 (âª). The error bars indicate SEM. The curve is a Boltzmann distribution with the indicated parameters.
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Figure 5. A survey of signals obtained with simultaneous recording of voltage-clamp current and fluorescence of TMRM-labeled GAT1 expressed in Xenopus oocytes. The voltage protocol is shown in A, top. The test potentials ranged from +60 to â140 mV in 20- mV increments. (Middle) The currents are shown. Arrows point to the transient currents that comprise the GAT1-specific charge movements. These transient currents far out last the endogenous capacitive currents of the oocyte membrane. The fluorescence signal is shown as the change of fluorescence intensity divided by the baseline fluorescence at the holding potential. Signals from an uninjected oocyte are shown in A. The current and fluorescence traces are the averaged signal from five cells for WT-GAT1 (B) and C74A-GAT1 (C).
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Figure 7. Tests for history dependence: effect of prepulse potential on the fluorescence relaxation. (A) Typical fluorescence traces from one WT-GAT1 cell. (Top) The voltage protocol is shown. The results from single-exponential fits to the rising phase of the fluorescence are superimposed on the traces. The amplitude (B) and time constant (C) from the fit are plotted as function of prepulse potential. Error bars indicate standard error of the fit using CLAMPFIT 8.
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Figure 6. Analysis of the fluorescence relaxations. The fluorescence from Fig. 5 is replotted for odd-numbered traces (test potentials between +60 and â140 mV in 40-mV increments). The fluorescence relaxations for WT-GAT1 (A) and C74AâGAT1 (B) were fit to single-exponential processes, which are superimposed on the traces. The amplitude (ÎF/F) and time constant t from the fits are plotted as functions of test potential in C and D for WT-GAT1 (â¡) and C74A-GAT1 (â¢). Error bars indicate standard error of the fits using CLAMPFIT 8.
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Figure 9. Comparison of the voltage dependence of the fluorescence relaxations and the charge movements. The normalized amplitude (A) and time constant (B) from single-exponential fits of the fluorescence change (â) and charge movement (âª). The charge movement data are those plotted in Fig. 4 B, inverted to simplify the comparison to fluorescence. Error bars indicate standard error of the fit using CLAMPFIT 8.
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Figure 8. Tests for history dependence: effect of test potential on the fluorescence relaxation. (A) Typical traces from the average of 100 sweeps. (Top) The voltage protocol is shown. The results from single-exponential fits to the falling phase of the fluorescence are superimposed on the traces. The amplitude (B) and time constant (C) are plotted as functions of the test potential. Error bars indicate standard error of the fit using CLAMPFIT 8.
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Figure 10. An alternative analysis of voltage dependence. (A) The fluorescence signal from an oocyte expressing TMRM-C74A-GAT1 was subtracted from the signal from another cell expressing TMRM-WT-GAT1. The curves are single-exponential fits to the fluorescence relaxations. (Top) The membrane potential was held at â40 mV and stepped to various test potentials. (B) The amplitudes of the fluorescence change were plotted as a function of membrane potential. (C) The time constants for the rising (â80, â100, â120, â140) or falling (â40 mV) phase of the fluorescence are plotted versus the membrane potential. Error bars indicate standard error of the fit.
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Figure 12. Effect of extracellular Na+ concentration on the fluorescence signal. (A) The fluorescence traces are the averaged traces from four cells. The curves show single-exponential fits to the traces. The membrane potential was held at â40 mV, stepped to a test potential of â140 mV for 800 ms, and then jumped back to the â40-mV holding potential. The recording solution was ND96, as described in the materials and methods, in which Na+ was substituted with various concentrations of NMDG. The residual small fluorescence at 0 [Na+] (â¼0.05%) was subtracted from each trace to show the Na+-dependent fluorescence signal. (B) The amplitude of the fluorescence change, plotted as a function of the Na+ concentration. The curve represents the equation ÎF/F = a [Na+]1.8. (C) The rate constants of single-exponential fits to the rising phase of the fluorescence relaxation are plotted as a function of Na+ concentration. The linear fit is superimposed on the data, and the slope equals 64 Mâ1 sâ1. (D) Rate constants from single-exponential fits to the falling phase of the fluorescence for the jump from â140 to â40 mV are plotted as function of Na+ concentration. The lines connect the data points. For all panels, data points are mean ± SEM (n = 4 cells).
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Figure 11. Effect of GAT1 substrates GABA, Clâ and Na+ on the fluorescence change. (Top) Typical fluorescence traces from one cell recorded in solutions containing various combinations of GABA, Clâ, and Na+. The membrane potential was held at â40 mV, stepped to a test potential of â140 mV for 550 ms, and then stepped back to â40 mV. (Bottom) Voltage dependence of the amplitude of the fluorescence change for the averaged data from five oocytes, ±SEM. The recording solution for the control traces was ND96, as described in materials and methods. (A) Effect of GABA. The fluorescence was recorded in the absence (light traces) and presence (heavy traces) of 100 mM GABA. (B) Effect of Clâ. The fluorescence was recorded in the presence (light traces) and absence (heavy traces) of 96 mM Clâ. Clâ was substituted with gluconate. (C) Effect of Na+. The fluorescence was recorded in the presence (light traces) and absence (heavy traces) of 96 mM Na+. Na+ was substituted with NMDG.
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Figure 13. Effect of GAT1 inhibitor, NO-711, on the fluorescence relaxations. (A) The structure of the compound. (B) Typical traces from one cell in the absence (light trace) and presence (heavy trace) of 3 mM NO-711. The voltage was held at â40 mV and stepped to a test potential of â140 mV. The cells were incubated in ND96 containing NO-711 for at least 5 min before recording. (C) Voltage dependence of the amplitude of the fluorescence relaxation, recorded in the absence (â¡) and presence (â¢) of 3 mM NO-711 (B2). Data points are mean ± SEM (n = 6 cells).
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Figure 14. The W68L-GAT1 mutant shows signals only at high positive potentials. The membrane potential was held at â40 mV and jumped to test potentials between +60 and â140 mV in 20-mV increments. The traces show the signals for the jumps to +60, +20, â20, â60, â100, and â140 mV, as in Fig. 6. Signals for six oocytes have been averaged. (A) Voltage-clamp currents. The arrows point to the trace for the jump to +60 mV; this is the only jump that evoked measurable charge movements. (B) Traces for the jumps from â40 to â140 mV and from â40 to +60 mV have been added to isolate the voltage-dependent capacitive currents for the jumps to and from +60 mV while subtracting the passive resistive and capacitive currents. (C) The fluorescence signals; only the jump to +60 mV evoked fluorescence relaxations large enough (approximately â0.2%) for kinetic analysis. The jumps to +40 mV are not shown, but also gave barely perceptible charge movements and fluorescence relaxations.
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Figure 15. The nature of the fluorescent state: a scheme that fits the available data. The scheme is superimposed on the state diagram accompanying Lu and Hilgemann (1999). The fluorescent state is pictured as a novel state, E*out-fluo between the E*out and Eout states, with characteristics intermediate to these states. Hilgemann and Lu 1999 concluded that the transition from the E*out to Eout is voltage dependent, occludes a Na+ onto the transporter, and constitutes the major rate-limiting step in the transport cycle. Because the transition is incomplete in the novel state, with Na+ only partially occluded, the Na+ concentration dependence and voltage dependence for the transition from E*out to E*out-fluo are less than those for the complete transition to Eout. The dashed oval is drawn to include the novel state and the immediate adjacent stable states; any agents that act within the oval would perturb the fluorescent state strongly enough to be detected in our experiments. The W68L mutant is thought to trap transporter in the state now characterized as Eout (Mager et al. 1996), which explains how this mutation eliminates the fluorescence relaxations at most potentials. NO-711 is thought to stabilize the Eout states as well (Mager et al. 1996), explaining how it blocks the relaxations. Agents that act outside the oval would perturb the fluorescent state too weakly to be detected in our experiments. The binding of both GABA and Clâ occur outside the oval, which explains their small effects on the fluorescence relaxations. Therefore, GABA binding and Clâ binding are shown in parentheses.
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