Properties of putative cerebellar gamma-aminobutyric acid A receptor isoforms.

Abstract

Analysis of the composition of cerebellar gamma-aminobutyric acidA (GABAA) receptors (GABARs) with in situ hybridization of GABAR subunit subtype mRNAs [J. Neurosci 12:1063-1076 (1992)] and Western blot analysis and quantitative binding of radioligands to immunopurified receptors from the rat cerebellum [J. Biol. Chem. 269:16020-16028 (1994)] have suggested that GABAR isoforms likely to occur in the cerebellum of adult rats are alpha1betaxgamma2, alpha6betaxgamma2, and alpha6betaxdelta isoforms. Based on these data, GABARs composed of different combinations of rat alpha1, alpha6, beta2, beta3, gamma2L, and delta subunits, corresponding to the three putative cerebellar GABAR isoforms, were transiently expressed in mouse fibroblast cells (L929 cells). Whole-cell currents were recorded from acutely transfected cells to determine whether the alpha1beta2/3gamma2L, alpha6beta2/3gamma2L, and alpha6beta2/3delta GABAR isoforms could form functional receptor channels in L929 cells and to compare their electrophysiological and pharmacological properties. All three putative cerebellar GABAR isoforms showed a high efficiency of expression of functional GABARs. We chose to study the beta3 and gamma2L subtypes as major representatives of the native subunit subtype proteins. The recombinant alpha1beta3gamma2L, alpha6beta3gamma2L, and alpha6beta3delta GABAR isoforms displayed different affinities (EC50 values) for GABA, differential sensitivity to block by the divalent cation zinc and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, and differences in enhancement by diazepam. Our results provide an initial characterization of the electrophysiological and pharmacological properties of possible in vivo cerebellar GABAR isoforms and demonstrate that subunit compositions of different GABAR isoforms play a crucial role in determining their properties.