Influence of GABA-A Receptor Intracellular Domain on Channel Activation and Desensitization

Abstract

Rapid opening and closing of GABA-A receptor (GABAR) channels regulate signaling in the brain. Mechanisms underlying channel open, closed, desensitized transitions remain unclear. GABAR cryo-EM structures provide details about its extracellular and transmembrane domains, but little information exists about its intracellular domain (ICD) and its role in regulating channel gating. We constructed a GABAR ICD homology model based on a cryo-EM 5HT3AR structure, which revealed a helix (MX) at the C-terminal end of M3 and a helix (MA) at the N-terminal end of M4 projecting intracellularly. Four residues with potential interactions between these helices were mutated to alanine (α1K327A in MX, α1K390A in MA, and γ2K409A and γ2Y413A in MA). Mutations effects on GABA-EC50 and current desensitization were measured using two-electrode voltage clamping of oocytes expressing wild-type (WT) and mutant GABARs. α1K327A increased GABA EC50 ∼6-fold compared to α1β2γ2 WT (35.98 ± 1.03 μM, n=9) and significantly reduced GABAR desensitization. Extent of current desensitization after 60 seconds in saturating GABA (10mM) was 30.16 ± 2.24 % (n=9) versus WT (62.96 ± 4.15 %, n=13). α1K390A had no effect on GABA EC50 or desensitization, and mutation of the aligned residue in γ2 subunit, γ2K409A, decreased GABA EC50 ∼2 fold without affecting desensitization. γ2Y413A significantly increased desensitization and increased GABA EC50 by 2-fold. Desensitization extent after 10 seconds was 76.57 ± 2.09 % (n=18) compared to WT (23.71 ± 2.30 %, n=10). These data indicate that structural perturbations of the α1MX and γ2MA regions in the GABAR ICD can influence macroscopic GABAR current desensitization. Interestingly, GABAR MX regions are lysine rich. We speculate that mechanisms underlying GABAR gating transitions may involve charged interactions between MX and other regions of the GABAR ICD (e.g. MA). Experiments are underway to test this idea.