Identification of Residues within GABAAReceptor α Subunits That Mediate Specific Assembly with Receptor β Subunits

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Abstract
GABAA receptors can be constructed from a range of differing subunit isoforms: α, β, γ, δ, and ε. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of α and β subunits. The expression of a γ subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58–67 within α subunit isoforms are important in the assembly of receptors comprised of αβ and αβγ subunits. Deletion of these residues from the α1 or α6 subunits results in retention of either α subunit isoform in the endoplasmic reticulum on coexpression with the β3, or β3 and γ2 subunits. Immunoprecipitation revealed that residues 58–67 mediated oligomerization of the α1 and β3 subunits, but were without affect on the production of α/γ complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional α1β3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of α1β3 receptors and the resulting expression of β3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S α1β3 complex representing functional GABAA receptors. Therefore, our studies detail residues that specify GABAA receptor αβ subunit interactions. This domain, which is conserved in all α subunit isoforms, will therefore play a critical role in the assembly of GABAA receptors composed of αβ and αβγ subunits.