Mapping GABAA Receptor Single Nucleotide Polymorphisms (SNPs) Linked to Epilepsy: Insights into the Receptor Gating and Assembly

Abstract

Missense polymorphisms or variants that affect function and/or expression of GABAA receptors (GABARs) have been associated with idiopathic generalized epilepsies (IGEs) in GABRA1, GABRA6, GABRB3, GABRG2 and GABRD genes. Three separate IGE-associated mutations were identified in GABAR subunits (β3(G32R), α6(Q237R) and γ2(K328M)), which represent a large range of GABARs in the nervous system. We sought to investigate the contributions of these mutations into the assembly and function of GABARs. Homology modeling suggested that G32R is located within the N-terminal α-helix β3 subunit domain, and Q237R and K328M are located within the pre-M1 segment of the N-terminal α6 subunit domain and the M2-M3 loop of the N-terminal γ2 subunit domain, respectively. We studied gating properties and surface expression of wild type (wt) α1β3γ2, α6β2γ2, α1β2γ2 and mutant α1β3(G32R)γ2, α6(Q237R)β2γ2, α1β2γ2(K328M) receptors expressed in HEK293T cells. We found that the mutations share common gating defects, but distinctive trafficking defects. Thus, mutant β3(G32R) subunits displayed a mixed profile, causing both gating and trafficking defects of α1β3γ2 receptors, whereas mutant α6(Q237R) and γ2(K328M) subunits caused exclusive channel gating defects of α6β2γ2 and α1β2γ2 receptors. Unexpected, homology modeling predicted that the β3(G32R) mutation affects a salt bridge across the γ2/β3 subunit interface, which underlies an assembly motif reported to be essential for inter-subunit interactions in assembled receptors. In contrast, α6(Q237R) and γ2(K328M) subunit mutations are predicted to interact with residues in Pre-M1 domain, M2-M3 loop and Cys-loop of α6 and γ2 subunits that are critical for desensitization-deactivation coupling of GABARs.NIH funding NS33300 to RLM.