Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αβγ and αβδ Expression Using Flow Cytometry and Electrophysiology

Abstract

The subunit stoichiometry and arrangement of synaptic αβγ GABAA receptors are generally accepted as 2α:2β:1γ with a β-α-γ-β-α counterclockwise configuration, respectively. Whether extrasynaptic αβδ receptors adopt the analogous β-α-δ-β-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 β2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and β2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of β2 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 α1β2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αβγ and αβδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αβγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical β-α-γ/δ-β-α receptors, including β-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and β-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.

Etomidate Effects on Desensitization and Deactivation of α4β3δ GABAA Receptors Inducibly Expressed in HEK293 TetR Cells

“…Etomidate evokes apparently divergent effects on GABA concentration responses in a1b3d and a1b3g2 receptors; however, quantitative modeling analysis indicates that etomidate exerts similar effects on channel gating in both receptor isoforms (Feng et al, 2014). Because etomidate selectively binds to b 1 /a 2 transmembrane intersubunit sites (Forman and Miller, 2016), these data support the idea that the stoichiometry and subunit arrangement of a1bd receptors are similar to those of a1bg2 receptors (Botzolakis et al, 2016). Etomidate reduces the extent of desensitization of concatenated b3-a1-d/b3-a1 receptors, which have the same stoichiometry and subunit arrangement as a1b3g2 receptors (Liu et al, 2015).…”

“…Possible Stoichiometry and Subunit Arrangement of a4b3d Receptors. Although multiple stoichiometries of free recombinant a1b3d receptors have been proposed , studies suggest that the predominant receptor isoform in free a1b3d receptors is b3-a1-d-b3-a1, which shares stoichiometry and subunit arrangement with a1b3g2 receptors (Feng et al, 2014;Botzolakis et al, 2016). However, anesthetic photolabeling of a4b3d receptors inducibly expressed in HEK293 TetR cells suggests that b3-b3 interfaces are present.…”

Low Expression in Xenopus Oocytes and Unusual Functional Properties of α1β2γ2 GABAA Receptors with Non-Conventional Subunit Arrangement

“…[19]), as their Hill coefficient is > 1. In addition to the consensus receptor, Fig 1 shows three unconventional receptors [17] with one predicted agonist site each, two of them with one, one of them with two predicted benzodiazepine binding sites. All these unconventional receptors do not conform to the well-established subunit stoichiometry of 2:2:1 for α 1 , β 2 and γ 2 subunits [4,5].…”

“…The stoichiometry of non-conventional GABA A receptors β 2 α 1 γ 2 α 1 α 1 , β 2 α 1 γ 2 α 1 γ 2 and β 2 α 1 γ 2 β 2 γ 2 would be predicted to be 1:1, 0.5:1 and 1:1, respectively [17] and that of conventional GABA A receptors 2:1. The subunit stoichiometries of non-conventional GABA A receptors clearly do not agree to the reported stoichiometry.…”

“…Subunit stoichiometry and arrangement are important as they govern functional properties [16]. Recently, unconventional subunit arrangements of α 1 β 2 γ 2 receptors have been proposed [17]. Some of these receptors do not conform to the above stoichiometry as they contain 2γ 2 subunits.…”