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

Abstract

We compared the effects of three missense mutations in the GABAA receptor γ2 subunit on GABAA receptor assembly, trafficking and function in HEK293T cells cotransfected with α1, β2, and wildtype or mutant γ2 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 γ+/β− subunit-subunit interface, we found that each mutation impaired GABAA receptor assembly to a different extent. The γ2(R82Q) and γ2(P83S) subunits had reduced α1β2γ2 receptor surface expression due to impaired assembly into pentamers, endoplasmic reticulum (ER) retention and degradation. In contrast, γ2(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 γ2 subunits with α1 and β2 subunits and found increased surface and total levels of both wildtype and mutant γ2 subunits after decreasing the incubation temperature to 30 °C for 24 hours, 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.

Rare variants in γ‐aminobutyric acid type A receptor genes in rolandic epilepsy and related syndromes

“…As surface levels of a 1 and b 2 subunit-containing receptors were unaffected by c 2 -G257R, we assume that GABA A -R complexes with different stoichiometries (eg, a 1 b 2 dimeric receptors) were formed. Similarly, ab heteropentamers have been reported for other GABRG2 mutations, 48,49 which affected the channel properties of GABA A -Rs in these patients. 50,51 Considering that the c 2 subunit is critical for postsynaptic clustering and maintenance of abundant subtypes of GABA A -Rs receptors in vivo, 22,52,53 decreased levels of surface c 2 -G257R might affect the architecture of postsynaptic GABAergic synapses and may consequently impair GABAergic inhibition with a lowering of the seizure threshold.…”

Epileptic encephalopathy de novo GABRB mutations impair γ‐aminobutyric acid type A receptor function

“…These mutations could be more disruptive to channel function than the IS-associated mutations at the α+/β− and γ+/β− interfaces (that are indirectly coupled by rearrangements throughout the β-sheets/α-helices of the receptor 26 ). For the mutations located in the signal peptide, GABRB3 (P11S, S15F) 6 , and at the γ+/β− interface, GABRB3(G32R) 7 and GABRG2(R82Q, P83S) 32 , the reductions in GABA A receptor currents were smaller (reduced to ~42, ~48, ~50–62, ~34 and ~12% of the wt currents, respectively) than those caused by the LGS-associated GABRB3(D120N, E180G, Y302C) mutations (reduced to ~24, ~1, ~5% of the wt currents, respectively) located at the β+/α− interface. Similarly, we found small (reduced to ~75% of the wt current) or no effects on current amplitudes for the IS-associated mutations at the γ+/β− interface.…”

Molecular Pathogenic Basis forGABRG2Mutations Associated With a Spectrum of Epilepsy Syndromes, From Generalized Absence Epilepsy to Dravet Syndrome

“…There mutations include N79S, R82Q, P83S, R177G and K328M 19;32,33,34 . Four of the five missense mutations are located in the extracellular N-terminus (N79S, R82Q, P83S, R177G), and one is located in the middle of the short TM2-TM3 extracellular linker (K328M).…”