Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αßγ and αßδ Expression Using Flow Cytometry and Electrophysiology: EVIDENCE FOR ALTERNATIVE SUBUNIT STOICHIOMETRIES AND ARRANGEMENTS.

The subunit stoichiometry and arrangement of synaptic αßγ GABAA receptors are generally accepted as 2α:2ß:1γ with a ß-α-γ-ß-α counterclockwise configuration, respectively. Whether extrasynaptic αßδ receptors adopt the analogous ß-α-δ-ß-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or ß2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and ß2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of ß2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1ß2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αßγ and αßδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αßγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical ß-α-γ/δ-ß-α receptors, including ß-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and ß-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies.