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

The subunit stoichiometry and arrangement of synaptic GABA(A) receptors are generally accepted as 2:2:1 with a counterclockwise configuration, respectively. Whether extrasynaptic receptors adopt the analogous subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant 2 and subunits when co-transfected with 1 and/or 2 subunits in HEK293T cells. Nearly identical patterns of 2 and subunit expression were observed as follows: both required co-transfection with 1 and 2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of 2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of subunit degradation, 10-fold less subunit cDNA was required to recapitulate 2 subunit expression patterns and to eliminate the functional signature of 12 receptors. Interestingly, titrating 2 or subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both and receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical receptors, including receptors at lower levels of 2/ expression and receptors at higher levels of expression. These findings provide important insight into the role of GABA(A) receptor subunit under- or overexpression in disease states such as genetic epilepsies.