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.
Highlights
There has been ongoing debate in the GABAA receptor literature regarding what subunit cDNA ratios should be transfected in recombinant receptor studies [27,28,29], we chose to begin with equimolar ratios because this should approximate the relative gene dosage in vivo (␣1, 2, ␥2, and ␦ GABAA receptor subunit genes are autosomal and none has been shown to be imprinted)
Flow Cytometry Provided an Efficient Quantitative Method for Evaluating GABAA Receptor Subunit Expression—Among ion channels, GABAA receptors are remarkable for their complexity
Determining which GABAA receptor subunit combinations can traffic to the cell surface, and what the stoichiometries and arrangements are of these receptors, has presented a fascinating yet frustrating problem for investigators
Summary
To improve our understanding of GABAA receptor biogenesis, we compared the surface and total cellular expression profiles of human ␥2 and ␦ subunits permitted to freely assemble with ␣1 and 2 subunits in HEK293T cells using a multimodality approach that included flow cytometry, whole cell patch clamp recording (using both freely assembled and concatenated subunits), and traditional biochemistry techniques. By combining these methodologies, we deduced that ␣␥ and ␣␦ receptors have similar stoichiometries and arrangements. We found that ␣␥ and ␣␦ receptor composition depends on relative subunit availability, with alternative subunit arrangements and stoichiometries likely occurring with increasing levels of ␥ or ␦ subunit expression
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