ENHANCEMENT BY GABA OF THE ASSOCIATION RATE OF PICROTOXIN AND TERT-BUTYLBICYCLOPHOSPHOROTHIONATE TO THE RAT CLONED ALPHA-1-BETA-2-GAMMA-2 GABA(A) RECEPTOR SUBTYPE

I We examined how gamma-aminobutyric acid (GABA) influences interactio n of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with rec ombinant rat alpha 1 beta 2 gamma 2 GABA(A) receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes i n Cl- currents measured by the whole-cell patch clamp technique. 2 Dur ing application of GABA (5 mu M) for 15 s, picrotoxin and TBPS dose-de pendently accelerated the decay of inward GABA-induced currents (a hol ding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial curre nt amplitude in a preincubation time and concentration-dependent manne r. This indicates their interaction with both GABA-bound and resting r eceptors. 3 The half maximal inhibitory concentration for picrotoxin a nd TBPS at the beginning of a 15 s GABA (5 mu M) pulse was several tim es greater than that obtained at the end of the pulse. GABA thus appea rs to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 mu M. Preincubation of the receptors with the drugs in the presence o f GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable c hange in potency of picrotoxin or TBPS. This indicates that they prefe rentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has high er affinity to both mono- and multiliganded receptors than resting rec eptors. 4 The time-dependent decay and preincubation time-dependent re duction of initial amplitude of GABA-induced Cl- currents followed mon oexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data us ing a kinetic model with a single drug site showed that GABA (5 mu M) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtai ned from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis. 5 We conclude that picrotoxin and TBPS interact with both resting and GABA-bound receptors, but the ir affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the m ultiliganded receptors (but not monoliganded ones). This and our earli er study with U-93631 improves our understanding of functional couplin g between GABA. and picrotoxin sites, which appears to be useful in ch aracterizing the mode of interaction for various picrotoxin site ligan ds.