Three epilepsy-associated GABRG2 missense mutations at the gamma+beta -interface disrupt GABA_A receptor assembly and trafficking by similar mechanisms but to different extents

摘要

We compared the effects of three missense mutations in the GABA_A receptor gamma2 subunit on GABA_A receptor assembly, trafficking and function in HEK293T cells cotransfected with alpha1, beta2, and wildtype or mutant gamma2 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 GABA_A receptor assembly to a different extent. The 72(R82Q.) and 72(P83S) subunits had reduced alphadbeta2gamma2 receptor surface expression due to impaired assembly into pentamers, endoplasmic reticulum (ER) retention and degradation. In contrast, 72(N79S) subunits were efficiently assembled into GABA_A 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 gamma2 subunits with al and beta2 subunits and found increased surface and total levels of both wildtype and mutant gamma2 subunits after decreasing the incubation temperature to 30 °C for 24 h, suggesting that lower temperatures increased GABA_A receptor stability. Thus epilepsy-associated mutations N79S, R82Q. and P83S disrupted GABA_A receptor assembly to different extents, an effect that could be potentially rescued by facilitating protein folding and assembly.