A GABA(A) RECEPTOR ALPHA(1) SUBUNIT TAGGED WITH GREEN FLUORESCENT PROTEIN REQUIRES A BETA-SUBUNIT FOR FUNCTIONAL SURFACE EXPRESSION

gamma-Aminobutyric acid, type A (GABA(A)) receptors, the major inhibit ory neurotransmitter receptors in the central nervous system, are hete ropentameric proteins assembled from distinct subunit classes with mul tiple subtypes, alpha(1-6), beta(1-4), gamma(1-3), delta(1), and epsil on(1). To examine the process of receptor assembly and targeting, we t agged the carboxyl terminus of the GABA(A) receptor alpha(1) subunit w ith red-shifted enhanced green fluorescent protein (EGFP). Xenopus ooc ytes were injected with cRNA of this fusion protein, alpha(1)-EGFP, al one 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 flu orescence was visible in all fusion protein-injected cells. The fluore scence was associated with the plasmalemma only when the beta(2) subun it was co-injected with alpha(1)-EGFP. Texas Red-conjugated immunolabe ling of EGFP on nonpermeabilized cells demonstrated that EGFP was loca lized extracellularly. Hence, the COOH terminus of the alpha(1) subuni t is extracellular. Two-electrode voltage clamp of alpha(1)-EGFP beta( 2)- and alpha(1)-EGFP beta(2)gamma(2)-injected oocytes demonstrates th at these cells express functional receptors, with EC50 values for GABA and diazepam similar to wild-type receptors. Thus, a COOH-terminal ta g 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 i s required for receptor assembly and surface expression.