Abstracts

Rationale: We demonstrated previously that the A322D mutation in the GABA_A receptor (GABA_AR) α1 subunit associated with juvenile myoclonic epilepsy resulted in α1(A322D) subunit misfolding and degradation, and that residual α1(A322D) subunits were retained the endoplasmic reticulum (ER). These data suggested that this mutation caused a heterozygous loss of functional α1 subunits. Recent studies demonstrated that α1(A322D) subunits oligomerized with γ2 subunits, a finding that suggested that α1(A322D) subunits could entrap wild type GABA_AR subunits in the ER and thus cause a dominant effect. Here we determined if the α1(A322D) subunit altered the surface trafficking of wild type subunits or altered synaptic GABA_AR currents. Methods: We expressed β2 and γ2S subunits and either α1, α1(A332D), or a mixture of α1 and α1(A322D) subunits in HEK293T cells and measured subunit expression by immunoblot and flow cytometry and determined subunit association by co-immunoprecipitation and FRET assays. We next transfected rat cortical neurons with either a control plasmid, or α1 or α1(A322D) subunit cDNA. We performed confocal immunofluorescence studies using antibodies specific to either the recombinant or the endogenous α1 subunits. Finally, we performed patch-clamp electrophysiology studies on transfected neurons and measured the miniature inhibitory post synaptic current (mIPSC) peak amplitudes and decay time constants. Results: In HEK293T cells, expression of the α1(A322D) subunit inhibited formation of α1γ2 oligomers, an assembly incapable of trafficking to the cell surface. In contrast, α1(A322D) expression did not inhibit formation of the trafficking-competent α1β2γ2 or α1β2 receptors. In cultured cortical neurons, surface expression of recombinant α1(A322D) subunit was significantly reduced compared to that of wild type α1 subunit. Overexpression of wild type α1 subunit, but not α1(A322D) subunit, reduced surface expression of endogenous α1 subunits, a result that demonstrated that recombinant wild type α1 subunits, but not α1(A322D) subunits were capable of replacing endogenous α1 subunits. In addition, overexpression of the wild type α1 subunit, but not the α1(A322D) subunit, reduced the time constant of mIPSC decay, a result consistent with recombinant α1 subunit replacement of endogenous α1 and non-α1 subunit isoforms. Finally, overexpression of the α1(A322D) subunit caused only a modest reduction in the median mIPSC amplitude (53 pA to 47 pA) while expression of the wild type α1 subunit reduced the median mIPSC amplitude to 41 pA. Conclusions: The α1(A322D) subunit associates with wild type β2 and γ2 subunits and reduces the formation of αγ oligomers, but does not alter the expression of αβ or αβγ GABA_AR. These data suggest that the formation of trafficking-competent GABA_AR protect cells from dominant effects of the α1(A322D) subunit and that the seizure phenotype arises predominantly from heterozygous loss of the α1 subunit.