title: 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.
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.
authors: From the Medical Scientist Training Program and.
authors: Emmanuel J
authors: EJ
authors: Botzolakis
authors: From the Medical Scientist Training Program and.
authors: Katharine N
authors: KN
authors: Gurba
authors: the Departments of Neurology, Pharmacology, and the Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37212, and.
authors: Andre H
authors: http://orcid.org/0000-0003-0147-7778
authors: AH
authors: Lagrange
authors: the Departments of Neurology.
authors: Hua-Jun
authors: HJ
authors: Feng
authors: the Department of Obstetrics and Gynecology, University of Wisconsin, Madison, Wisconsin 53792.
authors: Aleksandar K
authors: http://orcid.org/0000-0003-1946-6436
authors: AK
authors: Stanic
authors: the Departments of Neurology.
authors: Ningning
authors: N
authors: Hu
authors: the Departments of Neurology, Pharmacology, and Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37240-7915, robert.macdonald@vanderbilt.edu.
authors: Robert L
authors: http://orcid.org/0000-0002-9539-5556
authors: RL
authors: Macdonald
mesh_terms: D004827:Epilepsy / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology*; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics; D018079:Receptors, GABA / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D021122:Protein Subunits; D018079:Receptors, GABA
keywords: Cys loop; Forster; GABA receptor; electrophysiology; flow cytometry; fluorescence resonance energy transfer (FRET); ion channel; recombinant protein expression; stoichiometry