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, 20 (3), 899-907

Effects of Halothane on GABA(A) Receptor Kinetics: Evidence for Slowed Agonist Unbinding

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Effects of Halothane on GABA(A) Receptor Kinetics: Evidence for Slowed Agonist Unbinding

X Li et al. J Neurosci.

Abstract

Many anesthetics, including the volatile agent halothane, prolong the decay of GABA(A) receptor-mediated IPSCs at central synapses. This effect is thought to be a major factor in the production of anesthesia. A variety of different kinetic mechanisms have been proposed for several intravenous agents, but for volatile agents the kinetic mechanisms underlying this change remain unknown. To address this question, we used rapid solution exchange techniques to apply GABA to recombinant GABA(A) receptors (alpha(1)beta(2)gamma(2s)) expressed in HEK 293 cells, in the absence and presence of halothane. To differentiate between different microscopic kinetic steps that may be altered by the anesthetic, we studied a variety of measures, including peak concentration-response characteristics, macroscopic desensitization, recovery from desensitization, maximal current activation rates, and responses to the low-affinity agonist taurine. Experimentally observed alterations were compared with predictions based on a kinetic scheme that incorporated two agonist binding steps, and open and desensitized states. We found that, in addition to slowing deactivation after a brief pulse of GABA, halothane increased agonist sensitivity and slowed recovery from desensitization but did not alter macroscopic desensitization or maximal activation rate and only slightly slowed rapid deactivation after taurine application. This pattern of responses was found to be consistent with a reduction in the microscopic agonist unbinding rate (k(off)) but not with changes in channel gating steps, such as the channel opening rate (beta), closing rate (alpha), or microscopic desensitization. We conclude that halothane slows IPSC decay by slowing dissociation of agonist from the receptor.

Figures

Fig. 1.
Fig. 1.
Effect of halothane on deactivation.A, Response to a brief pulse of GABA (1 mm, 5 msec). Halothane (0.43 mm) slowed deactivation and reduced the peak response. Both effects were reversed after washout of anesthetic. Top trace shows the junction current recorded at the end of the experiment. B, Currents were normalized to peak amplitude for comparison of the time course of deactivation. C, Graphical summary of the effect on the weighted decay time constant. **p < 0.01;n = 16; paired t test.
Fig. 2.
Fig. 2.
Effect of halothane on the GABA concentration–response relationship. Peak currents, normalized to the 10 mm peak response, are plotted as a function of GABA concentration, normalized to EC50(Control). Data are from three different cells, represented by different symbols. Open symbols, control; filled symbols, halothane.Insets show responses of one cell to 10 μm, 30 μm, 100 μm, 1 mm, and 10 mm GABA. Calibration: 200 msec, 500 pA. Solid lines are best fits of normalized data to the Hill equation [control, EC50 (normalized) of 1.0;n = 2.23; halothane, EC50 (normalized) of 0.40; n = 2.34].
Fig. 3.
Fig. 3.
Computer simulations with altered kinetic parameters. U0, Unbound (resting) state; B1, monoliganded state;B2, double liganded state;D2, desensitized state;O2, open conducting state; see Materials and Methods for kinetic rate constants used for simulations.A, Effects on deactivation after a brief pulse of GABA (1 mm, 1 msec). Control, τdecay(area/peak) = 44.3 msec; 1/3 koffτdecay = 118 msec; 1/3 α τdecay = 116 msec; 3β τdecay, 103 msec. B, Effects on agonist sensitivity. Control, EC50 of 16.3 μm; n = 1.13; 1/3koff, EC50 of 7.7 μm; n = 0.89; 1/3 α, EC50 of 6.2 μm; n = 1.36; 3β, EC50 of 7.7 μm;n = 1.25.
Fig. 4.
Fig. 4.
Effect of halothane on macroscopic desensitization: response to a high-agonist concentration.A, Response of two cells (i,ii) with different desensitization kinetics, during application of a high concentration of GABA (1 mm, 100 msec). Currents have been normalized to the peak of the response. Halothane slowed deactivation after agonist removal but did not alter desensitization. B, Computer simulations of desensitization. Although altering the closing rate (α), opening rate (β), or microscopic unbinding rate (koff) all slowed deactivation, only the change in koff did not alter desensitization.
Fig. 5.
Fig. 5.
Effect of halothane on macroscopic desensitization: influence of agonist concentration. Left panels, Experimental data. Right panels, Computer simulations. GABA was applied at low (3 μm, 500 msec) (A), intermediate (10 μm, 300 msec) (B), and high (1 mm, 100 msec) (C) concentrations. Data are from three separate experiments. For the 1 mm data, responses have been normalized (traces reproduced from Fig.4Aii). Calibration:Ai, 100 pA, 200 msec; Aii, 0.1, 200 msec; Bi, 500 pA, 200 msec;Bii, 0.1, 200 msec; Ci, 200 msec,Cii, 200 msec.
Fig. 6.
Fig. 6.
Effect of halothane on the activation rate. A, Current rising phase, normalized to the peak amplitude, in response to a step application of GABA. Solid lines are best fits of monoexponential functions to the rising phase. Inset shows responses to the highest concentrations (100 μm to 10 mm) on an expanded time scale. B, Activation rate (1/τ) as a function of agonist concentration, under control conditions (filled symbols) and in the presence of halothane (open symbols). Solid lines are best fits of the data to a logistic equation (see Results for values).n = 4 for all points. C, Computer simulations of activation rate. Increasing the opening rate (β) led to a large increase in the maximal activation rate and an increase in the concentration required for half-maximal activation. Other changes had much smaller effects.
Fig. 7.
Fig. 7.
Effect of halothane on the response to the low-affinity agonist taurine (20 mm). A, Halothane increased peak amplitude and accentuated desensitization (i). Currents normalized to the beginning of deactivation (ii) show little effect on deactivation, which was extremely rapid compared with deactivation from GABA.Inset shows the deactivation phase on an expanded time scale. B, Graphical summary of effect of halothane on deactivation (i) and normalized to control (ii). Halothane produced a significantly smaller increase in the deactivation time constant after taurine application (**p < 0.01; n = 16 for GABA;n = 8 for taurine). C, Computer simulation of the response to taurine. i, Reduction inkoff increased peak current amplitude and accentuated desensitization. ii, Responses normalized to the amplitude at the beginning of deactivation show that there was little effect on the rapid deactivation.
Fig. 8.
Fig. 8.
Effect of halothane on paired-pulse depression.A, Brief pulses of GABA (1 mm, 5 msec) were applied with variable interpulse intervals. Percent recovery, [(peak2 − onset2)/(peak1 − onset2)] × 100, is plotted as a function of interpulse interval and fitted to a monoexponential function. Halothane depressed the amplitude of the second response and delayed its recovery (n = 5). Inset shows an example from an individual experiment. B, Computer simulations of paired-pulse depression. Reduction of the agonist unbinding rate led to more pronounced depression and slowed recovery, but alteration of opening and closing rates had little or opposite effects. For these simulations, d2 = 0.4 msec−1, r2 = 0.1 msec−1, to more closely match observed rates of desensitization and recovery. Using the rates used for other simulations (d2 = 0.2 msec−1, r2 = 0.02 msec−1), the results were qualitatively the same, with increases in both the amplitude and time constant of recovery (control τrecovery, 99.0 msec; 40.0%; 1/3koff τrecovery, 172.3 msec; 64.0%).

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