A γ2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface

Genetic defects leading to epilepsy have been identified in gamma 2 GABA(A) receptor subunit. A gamma 2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a gamma 2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABAA receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant gamma 2 constructs and showed that the gamma 2(R43Q) mutation prevented surface expression of the subunit, unlike gamma 2(K289M) substitution. Several gamma 2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with alpha 3- and beta 3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the gamma 2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant gamma 2(R43Q) was retained within intracellular compartments, alpha beta complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that gamma 2(R43Q) was able to associate with alpha 3- or beta 3-subunits, although the stoichiometry of the complex with alpha 3 was altered. Our data show that gamma 2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABAA receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the gamma 2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous gamma 2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.