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

gamma-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, alpha(1-6), beta(1-4), gamma(1-3), delta(1), and epsilon(1). To examine the process of receptor assembly and targeting, we tagged the carboxyl terminus of the GABA(A) receptor alpha(1) subunit with red-shifted enhanced green fluorescent protein (EGFP). Xenopus oocytes were injected with cRNA of this fusion protein, alpha(1)-EGFP, alone or in combination with cRNA of GABA(A) receptor beta(2), gamma(2), or beta(2) + gamma(2) 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 beta(2) subunit was co-injected with alpha(1)-EGFP. Texas Red-conjugated immunolabeling of EGFP on nonpermeabilized cells demonstrated that EGFP was localized extracellularly. Hence, the COOH terminus of the alpha(1) subunit is extracellular. Two-electrode voltage clamp of alpha(1)-EGFP beta(2)- and alpha(1)-EGFP beta(2)gamma(2)-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 alpha(1) subunit appears to be functionally silent, providing a useful marker for studies of GABA(A) receptor expression, assembly, transport, targeting, and clustering. Moreover, the beta(2) subunit is required for receptor assembly and surface expression.