Abstract

We discovered the ability of U-93631 (4-dimethyl-3-t-butylcarboxyl-4,5- dihydro[1,5-a]imidazoquinoxaline) to accelerate decay of gamma-aminobutyric acid (GABA)-induced currents, and we explored its mechanism in human embryonic kidney cells (HEK-293) stably expressing the alpha 1 beta 2 gamma 2 subtype of GABAA receptors. Inward currents (Cl- efflux) induced by 5 microM GABA at the holding potential of -60 mV (under a symmetrical Cl- gradient) decayed with an exponential time course with a mean time constant (tau) of 222 +/- 25 sec, as examined with the whole-cell configuration of the patch-clamp technique. The monoexponential decay was greatly accelerated in the presence of U-93631 at 5 microM, with the mean tau value being 5.2 +/- 0.5 sec. The tau values were dependent on the concentration of U-93631, with an estimated Kd of approximately 2 microM. Outward currents at the holding potential of +60 mV decayed with a similar tau value in the presence of the drug, suggesting the voltage independence of the drug action. The initial amplitude of the GABA (5 microM)-induced Cl- current was not affected by preincubation with U-93631 (5 microM) or GABA (200 nM) alone but was reduced by preincubation with the combination of the two. In the presence of U-93631 at 5 microM, the peak amplitude decreased as a function of GABA concentration, with the half-maximal inhibitory concentration being approximately 100 nm, which is close to the Kd for the high affinity GABA site (85 nM). It appears that the drug interacts with GABA-bound receptors (at least monoliganded) and accelerates receptor desensitization, rather than acting as an open channel blocker. The binding site for U-93631 on GABAA receptors seems not to overlap with GABA, barbiturate, or benzodiazepine sites, because the drug effect persisted in the presence of excess ligands for those sites. With cloned GABAA receptors composed of only alpha 1 beta 2, beta 2 gamma 2, or alpha 1 gamma 2 subunits, U-93631 also accelerated the decay rate. This lack of subtype selectivity raises the possibility that the compound interacts with a region common among the three subunits, probably a novel modulatory site, which can possibly be exploited as a novel therapeutic target.