PHARMACOLOGY OF THE HUMAN GAMMA-AMINOBUTYRIC ACID(A) RECEPTOR ALPHA-4SUBUNIT EXPRESSED IN XENOPUS-LAEVIS OOCYTES

The human gamma-aminobutyric acid(A) (GABA(A)) receptor alpha 4 subuni t was recently cloned and characterized pharmacologically using radiol igand binding techniques. These studies suggested that alpha 4 subunit s confer a novel diazepam-insensitive binding site. To further investi gate the pharmacology of the alpha 4 subunit, we expressed human alpha (4) beta 2 gamma L subunit combinations in oocytes and compared the ex pression and pharmacology of these receptors with alpha 1 beta 2 gamma 2L, beta 2 gamma 2L, and other possible binary subunit combinations. Apparent GABA affinity was 2-3-fold higher for alpha 4 beta 2 gamma 2L than for alpha 1 beta 2 gamma 2L receptors. Functional modulation of receptors by benzodiazepine-site ligands and other classes of alloster ic modulator were assayed over a broad concentration range (0.01-100 m u M) on currents that were 10% of the maximum GABA response. Diazepam (0.01-1 mu M) did not modulate GABA responses at alpha 4 beta 2 gamma 2L receptors, whereas it increased alpha 1 beta 2 gamma 2L responses b y similar to 110%. Bretazenil (0.01-1 mu M), a benzodiazepine partial agonist, induced higher efficacy modulation of alpha 4 beta 2 gamma 2L receptors (similar to 83%) than of alpha 1 beta 2 gamma 2L (similar t o 25%). The benzodiazepine antagonist flumazenil (0.1-10 mu M) unexpec tedly potentiated alpha 4 beta 2 gamma 2L responses up to similar to 4 1%, and the benzodiazepine partial inverse agonist Ro15-4513 (1 mu M) potentiated alpha 4 beta 2 gamma 2L responses by similar to 63%. Two o ther benzodiazepine-site ligands, CGS-9895 and ,7-dimethoxy-4-ethyl-be ta-carboline-3-carboxylate, had qualitatively similar effects at alpha 1 beta 2 gamma 2L and alpha 4 beta 2 gamma 2L. Modulators such as pen tobarbital, 3 alpha-hydroxy-5 alpha-pregnan-20-one, mefenamic acid, an d loreclezole also induced similar potentiation at both subtypes of re ceptor. The pharmacology conferred by the alpha 4 subunit was similar to that conferred by the alpha 6 subunit, to which it shows highest le vels of homology, but the two subunits differ in sensitivity to the be ta-carboline ,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate. Proper ties of the alpha 4-containing receptors are consistent with diazepam- insensitive binding sites found in cerebral cortex and other forebrain structures. Characterization of these receptors should further our un derstanding of mechanisms underlying the behavioral effects of GABA mo dulators and help in the design of drugs with improved, or novel, ther apeutic profiles.