Abstracts

Rationale: Although uncommon, monogenic forms of idiopathic generalized epilepsy (IGE) syndromes provide invaluable models for the elucidation of the pathogenic mechanisms of the corresponding polygenic and/or sporadic IGE syndrome. A single missense mutation (A322D) in the GABA_A receptor α1 subunit causes an autosomal dominant form of juvenile myoclonic epilepsy. We demonstrated previously that the A322D mutation reduced α1 subunit expression and that residual α1 subunit localized to the endoplasmic reticulum. Here we determined the mechanism by which a single missense mutation substantially reduced α1 expression.Methods: We transfected heterologous mammalian cells with wild type or mutant α1 subunits and performed [³⁵S]methionine pulse-chase experiments to determine the rates of α1 subunit degradation and biosynthesis. We elucidated the role of the ubiquitin-proteasome system in α1 degradation by performing pulse-chase experiments in the presence or absence of proteasome inhibitors and also by directly identifying polyubiquitinated α1 subunits via immunoblot. We determined the effect of the A322D mutation as well as the effects of a series of other α1 mutations on α1 subunit folding by performing glycosylation topology assays. Finally, to determine the proportion of surface GABA_A receptors in heterozygous cells that contained the α1(A322D) subunit, we transfected cells with wild type and mutant subunits that contained different epitope tags and quantified the surface wild type and mutant subunits by flow cytometry. Results: The A322D mutation caused rapid α1 subunit degradation (t_1/2= 23 min) through the ubiquitin proteasome system. Because of their altered hydrophobicity, mutant α1 subunits misfold by adopting an aberrant transmembrane topology. Because of this rapid degradation, in heterozygous expression, only 6 ± 1% of surface α1 subunits contained the A322D mutation.Conclusions: The A322D mutation decreases the hydrophobicity of an α1 subunit transmembrane domain causing misfolding and rapid α1 subunit degradation through the ubiquitin proteasome system resulting in haploinsufficiency in heterozygous expression. Thus, the A322D mutation provides the first example of a protein folding disease that produces an IGE syndrome.