An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence - PubMed (original) (raw)

An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence

M Li et al. J Gen Physiol. 2000 Apr.

Abstract

The rat gamma-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5-maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at -140 mV was approximately 0. 8% greater than at +60 mV. The time course of the fluorescence relaxations was mostly described by a single exponential with voltage-dependent but history-independent time constants ranging from approximately 20 ms at +60 mV to approximately 150 ms at -140 mV. The fluorescence did not saturate at the most negative potentials tested, and the midpoint of the fluorescence-voltage relation was at least 50 mV more negative than the midpoint of the charge-voltage relation previously identified with Na(+) binding to GAT1. The presence of gamma-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na(+) concentration with a Hill coefficient approaching 2. Increasing Cl(-) concentration modestly increased and accelerated the fluorescence relaxations for hyperpolarizing jumps. The fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that this mutant is trapped in one conformational state except at these potentials. These observations suggest that the fluorescence signals monitor a novel state of GAT1, intermediate between the E*(out) and E(out) states of Hilgemann, D.W., and C.-C. Lu (1999. J. Gen. Physiol. 114:459-476). Therefore, the study provides verification that conformational changes occur during GAT1 function.

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Figures

Figure 1

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 40× 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.

Figure 2

Figure 2

[3H]GABA uptake assayed in oocytes expressing WT-GAT17. The GABA uptake activity was measured in uninjected (○), GAT-1 mRNA-injected (▵), GAT-1 mRNA-injected and TMRM-labeled (▴), and GAT-1 mRNA-injected and MTSET-treated (•) 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 dose–response relations (Michaelis-Menten equation).

Figure 3

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.

Figure 4

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 −60 mV during perfusion of 100 mM GABA for a period of 4 s. The traces are typical of eight cells. (B) The charge movement (ΔQ) was calculated by integrating the transient current induced by a voltage jump from a holding potential of −40 mV to various test potentials, as shown in Fig. 5, middle. Each data point shows the average value from 40 cells for WT-GAT1 (□) and 15 cells for C74A-GAT1 (▪). The error bars indicate SEM. The curve is a Boltzmann distribution with the indicated parameters.

Figure 5

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 −140 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).

Figure 7

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.

Figure 6

Figure 6

Analysis of the fluorescence relaxations. The fluorescence from Fig. 5 is replotted for odd-numbered traces (test potentials between +60 and −140 mV in 40-mV increments). The fluorescence relaxations for WT-GAT1 (A) and C74A–GAT1 (B) were fit to single-exponential processes, which are superimposed on the traces. The amplitude (Δ_F_/F) and time constant t from the fits are plotted as functions of test potential in C and D for WT-GAT1 (□) and C74A-GAT1 (•). Error bars indicate standard error of the fits using CLAMPFIT 8.

Figure 9

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 (○) and charge movement (▪). 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.

Figure 8

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.

Figure 10

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 −40 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 (−80, −100, −120, −140) or falling (−40 mV) phase of the fluorescence are plotted versus the membrane potential. Error bars indicate standard error of the fit.

Figure 12

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 −40 mV, stepped to a test potential of −140 mV for 800 ms, and then jumped back to the −40-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+] (∼0.05%) 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 Δ_F_/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 M−1 s−1. (D) Rate constants from single-exponential fits to the falling phase of the fluorescence for the jump from −140 to −40 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).

Figure 11

Figure 11

Effect of GAT1 substrates GABA, Cl− and Na+ on the fluorescence change. (Top) Typical fluorescence traces from one cell recorded in solutions containing various combinations of GABA, Cl−, and Na+. The membrane potential was held at −40 mV, stepped to a test potential of −140 mV for 550 ms, and then stepped back to −40 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 Cl−. The fluorescence was recorded in the presence (light traces) and absence (heavy traces) of 96 mM Cl−. Cl− 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.

Figure 13

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 −40 mV and stepped to a test potential of −140 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 (□) and presence (•) of 3 mM NO-711 (B2). Data points are mean ± SEM (n = 6 cells).

Figure 14

Figure 14

The W68L-GAT1 mutant shows signals only at high positive potentials. The membrane potential was held at −40 mV and jumped to test potentials between +60 and −140 mV in 20-mV increments. The traces show the signals for the jumps to +60, +20, −20, −60, −100, and −140 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 −40 to −140 mV and from −40 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 −0.2%) for kinetic analysis. The jumps to +40 mV are not shown, but also gave barely perceptible charge movements and fluorescence relaxations.

Figure 15

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 E out states, with characteristics intermediate to these states. Hilgemann and Lu 1999 concluded that the transition from the E*out to E out 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 E out. 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 E out (Mager et al. 1996), which explains how this mutation eliminates the fluorescence relaxations at most potentials. NO-711 is thought to stabilize the E out 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 Cl− occur outside the oval, which explains their small effects on the fluorescence relaxations. Therefore, GABA binding and Cl− binding are shown in parentheses.

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