Ethanol, Flunitrazepam, and Pentobarbital Modulation of GABAA Receptors Expressed in Mammalian Cells and Xenopus Oocytes

Abstract

GABA_A receptors composed of human α1β2γ2L, α1β2γ2S, α1 β3γ2S, α6β3γ2S, and αβ3γ3 subunits as well as bovine α1 β1 γ2L and α1β1 subunits were stably expressed in mammalian L(tk⁻) cells and transiently expressed in Xenopus oocytes. Effects of muscimol, ethanol, flunitrazepam, and pentobarbital on receptor function were compared for the two expression systems using a ³⁶CI⁻ flux assay for cells and an electrophysiological assay for oocytes. Muscimol activated all receptors in both expression systems but was more potent for L(tk⁻) cells than oocytes; this difference ranged from 2.6–to 26–fold, depending upon subunit composition. The most pronounced differences between receptors and expression systems were found for ethanol. In L(tk⁻) cells, low (5–50 mM) concentrations of ethanol potentiated muscimol responses only with receptors containing the γ2L subunit. In oocytes, concentrations of 30–100 mM produced small enhancements for most subunit combinations. Flunitrazepam enhanced muscimol responses for all receptors except α6β3γ2S and α1β1, and this enhancement was similar for both expression systems. Pentobarbital also enhanced muscimol responses for all receptors, and this enhancement was similar for L(tk⁻) cells and oocytes, except for α6β3γ2S where the pentobarbital enhancement was much greater in oocytes than cells. The α6β3γ2S receptors were also distinct in that pentobarbital produced direct activation of chloride channels in both expression systems. Thus, the type of expression/assay system markedly affects the actions of ethanol on GABA_A receptors and also influences the actions of muscimol and pentobarbital on this receptor. Differences between these expression systems may reflect posttranslational modifications of receptor subunits.