A γ2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface
A γ2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface
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DOI：
10.1074/jbc.m608910200
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发表时间：
2007-02-09
影响因子：
4.8
通讯作者：
Garret, Maurice
中科院分区：
文献类型：
--
作者：
Frugier, Guillaume;Coussen, Francoise;Garret, Maurice
遗传缺陷导致癫痫已确定在γ - 2 GABA(A)受体亚基。伽玛2（R43Q）突变与儿童期缺失性癫痫和热性发作有关，伽玛2（K289M）突变与全身性癫痫伴热性发作增加有关。为了了解这些突变的影响，活细胞亚单位特异性免疫荧光标记分析了GABAA受体的表面靶向性。我们首先用重组γ - 2构建体转染培养的海马神经元，结果表明，与γ - 2（K289M）替代不同，γ - 2（R43Q）突变阻止了亚基的表面表达。在具有α 3-和β 3-亚基的COS-7细胞中表达了一些在Arg-43结构域内携带点突变的γ 2亚基构建物。R43Q和R43A取代显著降低了γ 2亚基的表面表达，而R43K、P44A和D39A取代的影响较小，但仍然显著，而K289M取代没有影响。而突变体γ - 2（R43Q）保留在细胞内隔室中，α - β复合物仍然靶向细胞膜。共免疫沉淀实验表明，gamma 2（R43Q）能够与α 3或β 3亚基结合，尽管与α 3的配合物的化学计量改变了。我们的数据显示，伽马2（R43Q）不是显性负基因，突变导致细胞表面GABAA受体亚基组成的修饰，从而损害神经元的突触靶向。本研究揭示了γ - 2- arg -43结构域参与受体组装的控制，这可能与杂合γ - 2（R43Q）突变导致儿童失神性癫痫和热性癫痫的影响有关。
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.