Influence of subunit configuration on the interaction of picrotoxin-site ligands with recombinant GABA(A) receptors

We have assessed the interaction of picrotoxin and a putative picrotoxin-site ligand [4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1,5-a) quinoxaline] (U-93631) with varying configurations of recombinant GABA(A) receptors, using the whole-cell patch clamp technique. In α2β2γ2 GABA(A) receptors, coapplication of picrotoxin with GABA had minimal effects on initial GABA-activated Cl^- current amplitude, and subsequently enhanced decay of GABA-activated Cl^- currents. The half-maximal inhibitory concentration (IC₅₀) for picrotoxin in α2β2γ2 receptors was 10.3±1.6 μM. The α subunit isoform did not affect picrotoxin-induced inhibition, as IC₅₀ values for α3β2γ2 (5.1±0.7 μM) and α6β2γ2 receptors (7.2±0.4 μM) were comparable to those obtained in α2β2γ2 receptors. Interestingly, in receptors lacking an α subunit (β2γ2 configuration), picrotoxin had a markedly lower IC₅₀ (0.5±0.05 μM) compared to α-containing receptors. The inhibitory profile was generally similar for the presumed picrotoxin-site ligand U-93631, i.e., IC₅₀ values were comparable in all αβγ-containing receptors, but the IC₅₀ in β2γ2 receptors was greater than 10-fold lower. In addition, a modest but significant initial stimulation of GABA-activated current by U-93631 was observed in α2β2γ2 and β2γ2 receptors. A mutation in the second transmembrane domain, shown previously to abolish picrotoxin sensitivity, also greatly attenuated sensitivity to U-93631. Moreover, incubation of receptors with excess U-93631 hindered picrotoxin's ability to gain access to its binding site; both results indicate that U-93631 interacts at the picrotoxin site of the receptor. Our results indicate the presence of an α subunit hinders the ability of picrotoxin to block the GABA(A) receptor, and thus provides additional insight into the site of action of picrotoxin. In addition, we have shown that domains important for the actions of picrotoxin also affect U-93631. Thus, this compound should prove to be a useful ligand for analysis of the convulsant site of this receptor. Copyright (C) 2000 Elsevier Science B.V.