Effect of alpha subunit on allosteric modulation of ion channel function in stably expressed human recombinant gamma-aminobutyric acid(A) receptors determined using Cl-36 ion flux

Inhibitory gamma -aminobutyric acid (GABA)(A) receptors are subject to modu lation at a variety of allosteric sites, with pharmacology dependent on rec eptor subunit combination. The influence of different alpha subunits in com bination with beta3 gamma 2s was examined in stably expressed human recombi nant GABA(A) receptors by measuring Cl-36 influx through the ion channel po re. Muscimol and GABA exhibited similar maximal efficacy at each receptor s ubtype, although muscimol was more potent, with responses blocked by picrot oxin and bicuculline. Receptors containing the alpha3 subunit exhibited sli ghtly lower potency. The comparative pharmacology of a range of benzodiazep ine site ligands was examined, revealing a range of intrinsic efficacies at different receptor subtypes. Of the diazepam-sensitive GABA(A) receptors ( alpha1, alpha2, alpha3, alpha5), alpha5 showed the most divergence, being discriminated by zolpidem in terms of very low affinity, and CL218,872 and CGS9895 with different efficacies. Benzodiazepine potentiation at alpha3 be ta3 gamma 2s with nonselective agonist chlordiazepoxide was greater than at alpha1, alpha2, or alpha5 (P < 0.001). The presence of an <alpha>4 subunit conferred a unique pharmacological profile. The partial agonist bretazenil was the most efficacious benzodiazepine, despite lower alpha4 affinity, an d FG8205 displayed similar efficacy. Most striking were the lack of affinit y/ efficacy for classical benzodiazepines and the relatively high efficacy of Ro15-1788 (53 +/- 12%), CGS8216 (56 +/- 6%), CGS9895 (65 +/- 6%), and th e weak partial inverse agonist Ro15-4513 (87 +/- 5%). Each receptor subtype was modulated by pentobarbital, loreclezole, and 5 alpha -pregnan-3 alpha -ol-20-one, but the type of a subunit influenced the level of potentiation. The maximal pentobarbital response was significantly greater at alpha4 bet a3 gamma 2s (226 +/- 10% increase in the EC20 response to GABA) than any ot her modulator. The rank order of potentiation for pregnanolone was alpha5 > alpha2 > alpha3 = alpha4 > alpha1, for loreclezole alpha1 = alpha2 = alpha 3 > alpha5 > alpha4, and for pentobarbital alpha4 = alpha5 = alpha2 > alpha 1 = alpha3.