Competitive antagonists facilitate the recovery from desensitization of α1β2γ2 GABAA receptors expressed in Xenopus oocytes

Abstract

Aim: The continuous presence of an agonist drives its receptor into a refractory state, termed desensitization. In this study, we tested the hypothesis that a competitive antagonist, SR95531, could facilitate the recovery of α1β2γ2 GABAA receptor from functional desensitization.

Methods: α1β2γ2 GABAA receptors were expressed in Xenopus oocytes. GABA-evoked currents were recorded using two-electrode voltage-clamp technique. Drugs were applied through perfusion.

Results: Long application of GABA (100 μmol/L) evoked a large peak current followed by a small amplitude steady-state current (desensitization). Co-application of SR95531 during the desensitization caused a larger rebound of GABA current after removal of SR95531. Furthermore, application of SR95531 after removal of GABA increased the rate of receptor recovery from desensitization, and the recovery time constant was decreased from 59±3.2 s to 33±1.6 s. SR95531-facilitated receptor recovery from desensitization was dependent on the perfusion duration of SR95531. It was also dependent on the concentration of SR95531, and the curve fitting with Hill equation revealed two potency components, which were similar to the two potency components in inhibition of the steady-state current by SR95531. Bicuculline caused similar facilitation of desensitization recovery.

Conclusion: SR95531 facilitates α1β2γ2 GABAA receptor recovery from desensitization, possibly through two mechanisms: binding to the desensitized receptor and converting it to the non-desensitized state, and binding to the resting state receptor and preventing re-desensitization.

Figure 1

SR95531-facilitated recovery from desensitization of the α1β2γ2 GABA receptor. (A) Co-application of SR95531 and GABA after the GABA-induced current had reached steady state (with desensitization) caused current rebound after removing SR95531. (B) The rebound current normalized by the initial peak current and compared to the normalized steady-state current (n=6).

Figure 2

SR95531-facilitated recovery of desensitization (in the presence of GABA) is time dependent. (A) Raw current traces of the GABA-induced current with co-application of SR95531 and GABA with varied durations of SR95531 application. (B) Normalized (to the initial peak) recovery of GABA-induced current after SR95531 application (n=6).

Figure 3

SR95531-facilitated recovery of desensitization in the absence of GABA. (A) Raw current traces of GABA response recovery in OR2 with different duration (n=5). (B) Raw current traces of GABA response recovery in SR95531 with different duration followed by a 2-s OR2 application to allow SR95531 dissociation before re-application of GABA (n=7). The last trace is the non-desensitization control with the initial current induced by a brief 2-s GABA application. (C) Normalized and averaged current recovery in the presence (normalized to the non-desensitization control) or absence of SR95531. The lines are fits of the data to a single exponential function. The resulting values of the time constant are plotted in D. (D) Time constants of GABA response recovery from desensitization in the presence or absence of SR95531.

Figure 4

SR95531-facilitated recovery of desensitization is concentration dependent. (A) Raw current traces of 100 μmol/L GABA-induced desensitization recovery induced by co-application of SR95531 with increasing concentrations of SR95531. (B) SR95531 concentration dependence of the normalized and averaged current recovery (n=5). The continuous line is the least-squares fit of the averaged recovery data to a double Hill equation. Due to an unsaturated low potency component, the fitting of data from individual oocytes resulted in a relatively large variability. Nevertheless, it is clear that a low potency component existed. The resulting EC₅₀ values are given in the text. The dashed line is the high potency component. Note that low potency component is not saturated for SR95531 at a concentration of 100 μmol/L.

Figure 5

The potency of SR95531 in inhibiting peak current and steady-state current. (A) Concentration-dependent inhibition of 100 μmol/L GABA-induced peak current (upper) or steady-state current (lower) by SR95531. (B) Normalized and averaged peak current (n=6) or steady-state current (n=5) inhibited by SR95531. The continuous lines are a least-squares fit of single (for peak current) or double (for steady-state current) Hill equations to the data. The resulting IC₅₀s were 0.39 μmol/L for the peak inhibition and 1.2 and 103 μmol/L for the steady-state inhibition. (C) Comparison of SR95531-induced steady-state current inhibition (inverted from B) and current recovery from desensitization (from Figure 4, re-normalized to 1).

Figure 6

Bicuculline facilitated desensitization recovery of the α1β2γ2 GABA receptor. (A) Raw traces of 100 μmol/L GABA-induced current and co-application of 100 μmol/L bicuculline with varied length of duration induced recovery of desensitization of the wild-type α1β2γ2 GABA receptor. (B) Normalized (to initial peak current) and averaged current rebound after termination of bicuculline co-application (n=5). The line is the least-squares fit of the data to a single exponential function.

Figure 7

Simulations of the concentration-dependent inhibition of the GABA-induced steady-state current by SR95531 and current rebound upon removal of SR95531 (with 40000 channels, 100 μmol/L GABA and varied SR95531 concentrations). (A) Upper current traces were simulated with the linear model from Scheme I, with the initial GABA-induced peak current omitted. Due to current fluctuation, the average current in the black box was used as the steady-state current level and the average current in the red box was used as current inhibition level. Note that although the extent of inhibition increased over time (waiting for recovery from desensitization), the concentration-dependent change in the antagonist potency remained similar. The lower current traces were simulated with the cyclic model from Scheme II, with the initial GABA-induced peak current omitted. Similar average currents were used to calculate the concentration dependency. Note that the major difference of this simulation from that with the linear model was that higher concentrations can change the time course of current block with a low-potency component. However, if the simulation duration is sufficiently long, when nearly all the receptors recover from desensitization, the two models would be similar. Arrows indicate the rebound currents in both model simulations. (B) Concentration-dependent inhibition from the measurements in A with non-linear fits of a single Hill equation for the results from the linear model and a double Hill equation for the results from the cyclic model.

Figure 8

100 and 1000 μmol/L GABA-induced desensitization of the α1β2γ2 GABA receptor. (A) Raw traces of current induced by 100 and 1000 μmol/L GABA. (B) Normalized (to peak current) and averaged steady-state desensitized current level at two GABA concentrations (n=10).

Figure 9

Comparison of the efficacy of bicuculline and SR95531 as inverse agonists in blocking the spontaneous current of the overexpressed wild type α1β2γ2 GABA receptor. (A) Raw traces of current show inhibition of the spontaneous current by a saturation concentration (to the wild-type α1β2γ2 GABA receptor) of SR95531, bicuculline, and picrotoxin. Note that it was not sensitive to 20 μmol/L ZnCl₂ inhibition. (B) Raw current traces of the homomeric β2 GABA receptor control. Its spontaneous current was sensitive to 20 mol/L ZnCl₂ inhibition. The spontaneous current was also inhibited by the other antagonists. (C) Normalized (to 100 μmol/L picrotoxin inhibitable current) and averaged inhibition of bicuculline and SR95531 for the α1β2γ2 heteromeric GABA_A receptor (n=9) or β2 homomeric GABA_A receptor.

Scheme I

Scheme II