title: Influence of subunit configuration on the interaction of picrotoxin-site ligands with recombinant GABA(A) receptors.
abstract: We have assessed the interaction of picrotoxin and a putative picrotoxin-site ligand [4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1, 5-a) quinoxaline] (U-93631) with varying configurations of recombinant GABA(A) receptors, using the whole-cell patch clamp technique. In alpha2beta2gamma2 GABA(A) receptors, coapplication of picrotoxin with GABA had minimal effects on initial GABA-activated Cl(-) current amplitude, and subsequently enhanced decay of GABA-activated Cl(-) currents. The half-maximal inhibitory concentration (IC(50)) for picrotoxin in alpha2beta2gamma2 receptors was 10.3+/-1.6 microM. The alpha subunit isoform did not affect picrotoxin-induced inhibition, as IC(50) values for alpha3beta2gamma2 (5.1+/-0.7 microM) and alpha6beta2gamma2 receptors (7.2+/-0.4 microM) were comparable to those obtained in alpha2beta2gamma2 receptors. Interestingly, in receptors lacking an alpha subunit (beta2gamma2 configuration), picrotoxin had a markedly lower IC(50) (0.5+/-0.05 microM) compared to alpha-containing receptors. The inhibitory profile was generally similar for the presumed picrotoxin-site ligand U-93631, i.e., IC(50) values were comparable in all alphabetagamma-containing receptors, but the IC(50) in beta2gamma2 receptors was greater than 10-fold lower. In addition, a modest but significant initial stimulation of GABA-activated current by U-93631 was observed in alpha2beta2gamma2 and beta2gamma2 receptors. A mutation in the second transmembrane domain, shown previously to abolish picrotoxin sensitivity, also greatly attenuated sensitivity to U-93631. Moreover, incubation of receptors with excess U-93631 hindered picrotoxin's ability to gain access to its binding site; both results indicate that U-93631 interacts at the picrotoxin site of the receptor. Our results indicate the presence of an alpha subunit hinders the ability of picrotoxin to block the GABA(A) receptor, and thus provides additional insight into the site of action of picrotoxin. In addition, we have shown that domains important for the actions of picrotoxin also affect U-93631. Thus, this compound should prove to be a useful ligand for analysis of the convulsant site of this receptor.
authors.affiliation: Department of Pharmacology, University of North Texas Health Science Center at Forth Worth, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Bell-Horner
authors.forename: M
authors.initials: M
authors.lastname: Dibas
authors.forename: R Q
authors.initials: RQ
authors.lastname: Huang
authors.forename: J A
authors.initials: JA
authors.lastname: Drewe
authors.forename: G H
authors.initials: GH
authors.lastname: Dillon
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D003292:Convulsants / Q000378:metabolism*; D058787:GABA-A Receptor Antagonists; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010852:Picrotoxin / Q000378:metabolism*; D011810:Quinoxalines / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D003292:Convulsants; D058787:GABA-A Receptor Antagonists; D008024:Ligands; D010852:Picrotoxin; D011810:Quinoxalines; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; C083857:U 93631