BICUCULLINE AND GABAZINE ARE ALLOSTERIC INHIBITORS OF CHANNEL OPENINGOF THE GABA(A) RECEPTOR

Anesthetic drugs are known to interact with GABA(A) receptors, both to potentiate the effects of low concentrations of GABA and to directly gate open the ion channel in the absence of GABA; however, the site(s) involved in direct gating by these drugs is not known. We have studie d the ability of alphaxalone (an anesthetic steroid) and pentobarbital (an anesthetic barbiturate) to directly activate recombinant GABA(A) receptors containing the alpha 1, beta 2, and gamma 2L subunits, Stero id gating was not affected when either of two mutated beta 2 subunits [beta 2(Y157S) and beta 2(Y205S)] are incorporated into the receptors, although these subunits greatly reduce the affinity of GABA binding. These observations indicate that steroid binding and subsequent channe l gating do not require these particular residues, as already shown fo r barbiturates. Bicuculline or gabazine (two competitive antagonists o f GABA binding) reduced the currents elicited by alphaxalone and pento barbital from wild-type GABA(A) receptors; however, gabazine produced only a partial block of responses to pentobarbital or alphaxalone, and bicuculline only partially blocked responses to pentobarbital. These observations indicate that the blockers do not compete with alphaxalon e or pentobarbital for a single class of sites on the GABA(A) receptor . Finally, at receptors containing alpha 1 beta 2(Y157S)gamma 2L subun its, both bicuculline and gabazine showed weak agonist activity and ac tually potentiated responses to alphaxalone. These observations indica te that the blocking drugs can produce allosteric changes in GABA(A) r eceptors, at least those containing this mutated beta 2 subunit. We co nclude that the sites for binding steroids and barbiturates do not ove rlap with the GABA-binding site. Furthermore, neither gabazine nor bic uculline competes for binding at the steroid or barbiturate sites. The data are consistent with a model in which both gabazine and bicuculli ne act as allosteric inhibitors of channel opening for the GABA(A) rec eptor after binding to the GABA-binding site.