CELLBIOLOGICAL CONSEQUENCES OF Q40X MUTATION OF GABRG2 FOUND IN SEVERE MYOCLONIC EPILEPSY IN INFANCY (SMEI)

Objectives: To characterize cellbiological consequence of a nonsense mutation (c.118C>T, Q40X) of the gene encoding ã2 subunit of GABA (A) receptor. Heterozygous Q40X was found in individuals with severe myoclonic epilepsy in infancy (SMEI). Electrophysiological studies on GABA(A) receptor consisting of á1, â2 and mutant ã2 subunit with Q40X showed reduced GABA induced currents. Histochemical studies on brain specimen obtained from the autopsy for one of the individuals showed fine granules in neurons and neuropils.

Methods: Human embryonic kidney (HEK) cells were transfected with rat cDNA of GABRA1 and GABRB2, the genes encoding á1 and â2 subunits, respectively, and wild type or mutant GABRG2. GABRG2 cDNA and a fluorescent ER (endoplasmic reticulum) marker were co-expressed independently using the IRES expression system. Likewise, wild type GABRA2 cDNA with a myc epitope and GABRB2 cDNA with FLAG were co-expressed. Transfected cells were immunostained with the corresponding antibodies and fluorescence conjugated second antibodies followed by confocal microscopic analysis. Biochemical studies to determine intracellular localization of the receptor such as glycosylation analysis of subunits were also carried out. Results: GABA (A) receptors with wild type ã2 subunit were well expressed on the plasma membrane while those harboring the mutant ã2 subunit were found to be co-localized with the ER marker indicating that deficient receptors were retained in the ER. Biochemical experiments support ER retention of the receptor.

Conclusion: Our study demonstrated that Q40X retained GABA (A) receptor in the ER. It is suggested that granules observed in the brain of the patient were deficient receptors in the ER. Recently, certain mutations in GABRG2 and GABRA1 were found to lead intracellular trafficking defect and ER retention of the receptor. Such burden can induce ER stress and thereby apoptosis of neurons. These findings may provide us with a new paradigm in the athomechanisms underlying epilepsy resulting from channel abnormalities.