Three epilepsy-associated GABRG2 missense mutations at the γ plus /β - interface disrupt GABAA receptor assembly and trafficking by similar mechanisms but to different extents

We compared the effects of three missense mutations in the GABA(A) receptor gamma 2 subunit on GABAA receptor assembly, trafficking and function in HEK293T cells cotransfected with alpha 1, 32, and wildtype or mutant gamma 2 subunits. The mutations R82Q and P83S were identified in families with genetic epilepsy with febrile seizures plus (GEFS+), and N79S was found in a single patient with generalized tonic-clonic seizures (GTCS). Although all three mutations were located in an N-terminal loop that contributes to the gamma + /beta - subunit-subunit interface, we found that each mutation impaired GABAA receptor assembly to a different extent. The gamma 2(R82Q) and gamma 2(P83S) subunits had reduced alpha 1 beta 2 gamma 2 receptor surface expression due to impaired assembly into pentamers, endoplasmic reticulum (ER) retention and degradation. In contrast, gamma 2(N79S) subunits were efficiently assembled into GABAA receptors with only minimally altered receptor trafficking, suggesting that N79S was a rare or susceptibility variant rather than an epilepsy mutation. Increased structural variability at assembly motifs was predicted by R82Q and P83S, but not N79S, substitution, suggesting that R82Q and P83S substitutions were less tolerated. Membrane proteins with missense mutations that impair folding and assembly often can be "rescued" by decreased temperatures. We coexpressed wildtype or mutant gamma 2 subunits with alpha 1 and beta 2 subunits and found increased surface and total levels of both wildtype and mutant gamma 2 subunits after decreasing the incubation temperature to 30 C for 24 h, suggesting that lower temperatures increased GABAA receptor stability. Thus epilepsy-associated mutations N79S, R82Q and P83S disrupted GABAA receptor assembly to different extents, an effect that could be potentially rescued by facilitating protein folding and assembly. (C) 2014 Elsevier Inc All rights reserved.