A γ2(R43Q) Mutation, Linked to Epilepsy in Humans, Alters GABAA Receptor Assembly and Modifies Subunit Composition on the Cell Surface

• Published in: The Journal of biological chemistry Vol. 282; no. 6; p. 3819
• Main Authors: Guillaume Frugier, Françoise Coussen, Marie-France Giraud, Marie-Françoise Odessa, Michel B. Emerit, Eric Boué-Grabot, Maurice Garret
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 09.02.2007
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M608910200

Genetic defects leading to epilepsy have been identified in γ2 GABA A receptor subunit. A γ2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a γ2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABA A receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant γ2 constructs and showed that the γ 2(R43Q) mutation prevented surface expression of the subunit, unlike γ2(K289M) substitution. Several γ2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with α3- and β3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the γ2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant γ2(R43Q) was retained within intracellular compartments, αβ complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that γ2(R43Q) was able to associate with α3- or β3-subunits, although the stoichiometry of the complex with α3 was altered. Our data show that γ2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABA A receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the γ2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous γ2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.