A GABA(A) receptor α1 subunit tagged with green fluorescent protein requires a β subunit for functional surface expression

Abstract

γ-Aminobutyric acid, type A (GABA(A)) receptors, the major inhibitory neurotransmitter receptors in the central nervous system, are heteropentameric proteins assembled from distinct subunit classes with multiple subtypes, α(1-6), β(1-4), γ(1-3), δ(1), and e(1). To examine the process of receptor assembly and targeting, we tagged the carboxyl terminus of the GABA(A) receptor α1 subunit with red-shifted enhanced green fluorescent protein (EGFP). Xenopus oocytes were injected with cRNA of this fusion protein, α1-EGFP, alone or in combination with cRNA of GABA(A) receptor β2, γ2, or β2+γ2 subunits. Within 72 h after injection, EGFP fluorescence was visible in all fusion protein-injected cells. The fluorescence was associated with the plasmalemma only when the β2 subunit was co-injected with α1-EGFP. Texas Red-conjugated immunolabeling of EGFP on nonpermeabilized cells demonstrated that EGFP was localized extracellularly. Hence, the COOH terminus of the α1 subunit is extracellular. Two-electrode voltage clamp of α1-EGFPβ2- and α1- EGFPβ2γ2-injected oocytes demonstrates that these cells express functional receptors, with EC50 values for GABA and diazepam similar to wild-type receptors. Thus, a COOH-terminal tag of the as subunit appears to be functionally silent, providing a useful marker for studies of GABA(A) receptor expression, assembly, transport, targeting, and clustering. Moreover, the β2 subunit is required for receptor assembly and surface expression.