Protein interactions involving the γ2 large cytoplasmic loop of GABAA receptors modulate conductance

Native GABA(A) channels display a single-channel conductance ranging between similar to 10 and 90 pS. Diazepam increases the conductance of some of these native channels but never those of recombinant receptors, unless they are coexpressed 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 gamma 2 subunit (gamma(381-403)) of the GABA(A) receptor abrogates the potentiating effect of diazepam on both endogenous receptors and recombinant GABA(A) receptors coexpressed with GABARAP, by substantially reducing their conductance. The protein interaction disrupted by the peptide did not involve GABARAP, because a shorter peptide (gamma(386-403)) known to compete with the gamma 2-GABARAP interaction did not affect the conductance of recombinant alpha beta gamma receptors coexpressed with GABARAP. The requirement for receptor clustering and the fact that the gamma 2 MA helix is able to self-associate support a mechanism whereby adjacent GABA(A) receptors interact via their gamma 2-subunit MA helices, altering ion permeation through each channel. Alteration of ion-channel function arising from dynamic interactions between ion channels of the same family has not been reported previously and highlights a novel way in which inhibitory neurotransmission in the brain may be differentially modulated.-Everitt, A. B., Seymour, V. A. L., Curmi, J., Laver, D. R., Gage, P. W., Tierney, M. L. Protein interactions involving the gamma 2 large cytoplasmic loop of GABAA receptors modulate conductance. FASEB J. 23, 4361-4369 (2009). www.fasebj.org