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GABAA receptors increase their conductance through novel protein interactions

M.L. Tierney,1 A.B. Everitt,1 V.A.L. Seymour,1 J. Curmi1 and D.R. Laver,2 1JCSMR, Australian National University, ACT 0200, Australia and 2School of Biomedical Science, University of Newcastle, NSW 2308, Australia.

Native GABAA channels display a single-channel conductance ranging between ∼10-90 pS. Diazepam increases the conductance of some of these native channels but never those of recombinant receptors unless they are co-expressed with GABARAP. This trafficking protein clusters recombinant receptors in the membrane suggesting that high-conductance channels arise from receptors that are at locally high concentrations. The amphipathic (MA) helix that is present in the large cytoplasmic loop of every subunit of all ligand-gated ion channels mediates protein-protein interactions. Here we report that when applied to inside-out patches, a peptide mimicking the MA helix of the γ2 subunit (γ381-403) of the GABAA receptor abrogates the potentiating effect of diazepam on both endogenous receptors and recombinant GABAA receptors co-expressed with GABARAP, by substantially reducing their conductance. The protein interaction disrupted by the peptide did not involve GABARAP because a shorter peptide (γ386-403) known to compete with the γ2: GABARAP interaction did not affect the conductance of recombinant αβγ receptors co-expressed with GABARAP. The requirement for receptor clustering and the fact that the γ2 MA helix is able to self-associate support a mechanism whereby adjacent GABAA receptors interact via their γ2 subunit MA helices, altering ion permeation through each channel. This finding has important implications for understanding both the structural design of ligand-gated ion channels and the adaptive, dynamic means a cell invokes to amplify its signalling capacity.