Abstract
gamma-Aminobutyric acid, type A (GABAA) 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 GABAA receptor alpha1 subunit with red-shifted enhanced green fluorescent protein (EGFP). Xenopus oocytes were injected with cRNA of this fusion protein, alpha1-EGFP, alone or in combination with cRNA of GABAA receptor beta2, gamma2, or beta2+gamma2 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 beta2 subunit was co-injected with alpha1-EGFP. Texas Red-conjugated immunolabeling of EGFP on nonpermeabilized cells demonstrated that EGFP was localized extracellularly. Hence, the COOH terminus of the alpha1 subunit is extracellular. Two-electrode voltage clamp of alpha1-EGFPbeta2- and alpha1-EGFPbeta2 gamma2-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 alpha1 subunit appears to be functionally silent, providing a useful marker for studies of GABAA receptor expression, assembly, transport, targeting, and clustering. Moreover, the beta2 subunit is required for receptor assembly and surface expression.
Highlights
Our results provide evidence that ␣1 subunits are not localized to nor inserted into the surface membrane in oocytes expressing ␣1 subunits alone or ␣1 ϩ ␥2 subunits
We show that ␣1 subunits expressed alone are not targeted to nor inserted into the oocyte surface membrane and that the 2 subunit is required for targeting and assembling the ␣1 subunit into a functional cell surface receptor
Our data unequivocally demonstrate that COOH-terminal tagging of the ␣1 subunit of the GABAA receptor with EGFP yields fully functional ion channels in Xenopus oocytes when expressed with 2 or 2␥2 subunits
Summary
Construction of ␣1-EGFP Fusion cDNA—By recombinant polymerase chain reaction, the stop codon of ␣1 was changed to proline, which created a new AgeI site, using the oligonucleotide 5Ј-CTAAAAGAAC-. The ␣1 fragment was digested with AgeI and BamHI and subcloned into the pEGFP-N1 vector (CLONTECH) which fused the EGFP gene, in frame, to the COOH terminus of the ␣1 fragment This recombinant plasmid was digested with BamHI and XbaI, and the resulting ␣1-EGFP fragment was subcloned into a pGH19 vector, which contained the entire wild-type ␣1 cDNA (19) for expression in Xenopus oocytes. All images were recorded at the same adjustments of laser power and photomultiplier sensitivity and were later processed by using Adobe Photoshope software (ADOBE Systems, Mountain View, CA) with identical values for contrast and brightness
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