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J. Biol. Chem., Vol. 282, Issue 6, 3819-3828, February 9, 2007

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A 2(R43Q) Mutation, Linked to Epilepsy in Humans, Alters GABA_A Receptor Assembly and Modifies Subunit Composition on the Cell Surface^*

Guillaume Frugier, Françoise Coussen, Marie-France Giraud^¶, Marie-Françoise Odessa, Michel B. Emerit^||, Eric Boué-Grabot, and Maurice Garret¹

From the Laboratoire de Neurophysiologie, CNRS-UMR 5543, Université de Bordeaux II, 33076 Bordeaux, UMR 5091 Institut F. Magendie, 33077 Bordeaux, ^¶Institut de Biochimie et de Génétique Cellulaires du CNRS, 33077 Bordeaux, and ^||Institut National de la Santé et de la Recherche Médicale U288, Pitié-Salpêtrière, 75013 Paris, France

Genetic defects leading to epilepsy have been identified in 2 GABA_A receptor subunit. A 2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a 2(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 2 constructs and showed that the 2(R43Q) mutation prevented surface expression of the subunit, unlike 2(K289M) substitution. Several 2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with 3- and 3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the 2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant 2(R43Q) was retained within intracellular compartments, complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that 2(R43Q) was able to associate with 3- or 3-subunits, although the stoichiometry of the complex with 3 was altered. Our data show that 2(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 2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous 2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.

Received for publication, September 18, 2006 , and in revised form, November 10, 2006.

^* This work was supported by Centre National de la Recherche Scientifique, Université Bordeaux2, and Conseil Régional Aquitaine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental movie S1 and Fig. S1.

¹ To whom correspondence should be addressed: CNRS UMR 5543, Université de Bordeaux II, Laboratoire de Neurophysiologie, 146 Rue Léo Saignat, 33076 Bordeaux, France. Tel.: 33-557571686; Fax: 33-556901421; E-mail: maurice.garret{at}u-bordeaux2.fr.

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