Endoplasmic reticulum retention and associated degradation of a GABA A receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the α1 subunit

Abstract

A GABAA receptor α1 subunit epilepsy mutation (α1(A322D)) introduces a negatively charged aspartate residue into the hydrophobic M3 transmembrane domain of the al subunit. We reported previously that heterologous expression of α1(A322D)β2γ2 receptors in mammalian cells resulted in reduced total and surface α1 subunit protein. Here we demonstrate the mechanism of this reduction. Total α1(A322D) subunit protein was reduced relative to wild type protein by a similar amount when expressed alone (86 ± 6%) or when coexpressed with β2 and γ2S subunits (78 ± 6%), indicating an expression reduction prior to subunit oligomerization. In α1β2γ2S receptors, endoglycosidase H deglycosylated only 26 ± 5% of α1 subunits, consistent with substantial protein maturation, but in α1(A322D)β2γ2S receptors, endoglycosidase H deglycosylated 91 ± 4% of α1(A322D) subunits, consistent with failure of protein maturation. To determine the cellular localization of wild type and mutant subunits, the al subunit was tagged with yellow (α1-YFP) or cyan (α1-CFP) fluorescent protein. Confocal microscopic imaging demonstrated that 36 ± 4% of α1-YFPβγ2 but only 5 ± 1% α1(A322D)- YFPβ2γ2 colocalized with the plasma membrane, whereas the majority of the remaining receptors colocalized with the endoplasmic reticulum (55 ± 4% α1-YFPβ2γ2S, 86 ± 3% α1(A322D)-YFP). Heterozygous expression of α1-CFPβ2γ2S and α1(A322D)- YFPβ2γ2S or α1-YFPβ2γ2S and α1(A322D)- CFPβ2γ2S receptors showed that membrane GABAA receptors contained primarily wild type α1 subunits. These data demonstrate that the A322D mutation reduces α1 subunit expression after translation, but before assembly, resulting in endoplasmic reticulum-associated degradation and membrane α1 subunits that are almost exclusively wild type subunits. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.