Influences on blockade by t-butylbicyclo-phosphoro-thionate of GABA(A) receptor spontaneous gating, agonist activation and desensitization

Picrotoxin and t-butylbicyclophosphorothionate (TBPS) are GABA(A) receptor (GABA(A)R) open channel blockers. However, picrotoxin displaceable [S-35]TBPS binding to alpha 1 beta 2 gamma 2 GABA(A)Rs occurs in the absence of GABA, suggesting that access to the binding site is independent of activation. Alternatively, spontaneous gating may provide access to the channel. In the absence of episodic GABA application, picrotoxin and TBPS blocked (by 91 +/- 3% and 85 +/- 5%, respectively) GABA-evoked currents mediated by alpha 1 beta 2 gamma 2 receptors. We used two approaches to inhibit spontaneous GABA(A)R gating, bicuculline, which inhibits spontaneous current in the absence of exogenous agonist and the alpha 1(K278M) mutant subunit. Whole-cell patch-clamp recordings demonstrated that alpha 1(K278M)beta 2 gamma 2 receptors have negligible spontaneous gating. Application of bicuculline to alpha 1 beta 2 gamma 2 receptors in the absence of exogenous GABA caused a 35% reduction of current blockade by TBPS and reduced [S-35]TBPS binding by 25%. Consistent with this, in the absence of exogenous GABA, alpha 1(K278M)beta 2 gamma 2 receptors exhibited reduced blockade by TBPS current compared to wild-type receptors. These data suggest that a decrease in spontaneous gating reduces accessibility of TBPS to its binding site. GABA application during picrotoxin or TBPS administration enhanced alpha 1 beta 2 gamma 2 receptor blockade (to 98% in both cases). The GABA-dependent component of TBPS blockade accounts for the stimulation of [S-35]TBPS binding to alpha 1 beta 2 gamma 2 receptors seen with GABA (1 mu M) application. Moreover, application of GABA at concentrations that cause significant steady-state desensitization reduced [S-35]TBPS binding. The alpha 1(K278M) subunit slowed desensitization kinetics and increased the rate of deactivation of GABA-evoked currents. Furthermore, there was a marked increase in the GABA EC50 for desensitization of alpha 1(K278M)beta 2 gamma 2 receptors associated with a large increase in the GABA-dependent stimulation of [S-35]TBPS binding. These data establish a relationship between GABA(A)R function and the three phases of [S-35]TBPS binding seen in the absence and the presence of GABA.