The Juvenile Myoclonic Epilepsy GABA_AReceptor α1 Subunit Mutation A322D Produces Asymmetrical, Subunit Position-Dependent Reduction of Heterozygous Receptor Currents and α1 Subunit Protein Expression

Abstract

Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABA A receptor ␣1 subunit mutation (␣1A322D). GABA A receptor ␣␤␥ subunits are arranged around the pore in a ␤-␣-␤-␣-␥ sequence (counterclockwise from the synaptic cleft). Therefore, each ␣1 subunit has different adjacent subunits, and heterozygous expression of ␣1(A322D), ␤, and ␥ subunits could produce receptors with four different subunit arrangements:Expression of a 1:1 mixture of wild-type and ␣1(A322D) subunits with ␤2S and ␥2S subunits (heterozygous transfection) produced smaller currents than wild type and much larger currents than homozygous mutant transfections. Western blot and biotinylation assays demonstrated that the amount of total and surface ␣1 subunit from heterozygous transfections was also intermediate between those of wild-type and homozygous mutant transfections. ␣1(A322D) mutations were then made in covalently tethered triplet (␥2S-␤2S-␣1) and tandem (␤2S-␣1) concatamers to target selectively ␣1(A322D) to each of the asymmetric ␣1 subunits. Coexpression of mutant and wild-type concatamers resulted in expression of either Het ␤␣␤ or Het ␤␣␥ receptors. Het ␤␣␤ currents were smaller than wild type and much larger than Het ␤␣␥ and homozygous currents. Furthermore, Het ␤␣␤ transfections contained less ␤-␣ concatamer than wild type but more than both Het ␤␣␥ and homozygous mutant transfections. Thus, whole-cell currents and protein expression of heterozygous ␣1(A322D)␤2S␥2S receptors depended on the position of the mutant ␣1 subunit, and GABA A receptor currents in heterozygous individuals likely result primarily from wild-type and Het ␤␣␤ receptors with little contribution from Het ␤␣␥ and homozygous receptors.

The GABA A receptor α1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation

“…immunoblots of cellular lysates containing rat ␣1␤2␥2 or ␣1(A322D)␤2␥2 receptors stained with a polyclonal anti-rat ␣1 antibody (7). Previously, using quantitative fluorescence spectroscopy, we showed that the A322D mutation reduced ␣1(A322D)-GFP subunit fluorescence (10), and, using immunoblots with a monoclonal antibody, we showed that A322D reduced total cellular ␣1(A322D) subunit expression (9,10). Immunoblot experiments depicted in Fig.…”

“…The effect of the A322D mutation on total cellular ␣1 subunit expression is disputed. Immunoblots of lysates from cells transfected with rat ␣1␤2␥2 or ␣1(A322D)␤2␥2 receptors probed with a polyclonal anti-rat ␣1 subunit antibody did not differ by visual inspection (7), but immunoblots from cells transfected with human ␣1␤2␥2 and ␣1(A322D)␤2␥2 subunits and stained with a monoclonal antihuman ␣1 subunit antibody demonstrated that ␣1(A322D) subunit expression was reduced (9). To address this discrepancy, we transfected cells with cDNAs encoding rat ␣1␤2␥2, ␣1(A322D)␤2␥2, ␣1-FLAG␤2␥2, or ␣1(A322D)-FLAG␤2␥2 subunits and performed immunoblots with the same polyclonal ant-rat ␣1 antibody used by the investigators who found no difference in ␣1 and ␣1(A322D) expression (7) (Fig.…”

“…Electrophysiological experiments demonstrated that the A322D mutation altered GABA A receptor function (7)(8)(9). In addition, surface trafficking experiments demonstrated that the A322D mutation reduced receptor cell surface expression (9-11).…”

“…In addition, surface trafficking experiments demonstrated that the A322D mutation reduced receptor cell surface expression (9-11). Biochemical experiments revealed that the A322D mutation reduced the total cellular amount of the ␣1(A322D) subunit posttranslationally before subunit assembly and that the residual ␣1(A322D) subunits resided in the endoplasmic reticulum (ER) (9,10). These data suggested that the ␣1(A322D) subunit underwent ERassociated degradation (ERAD).…”

Expression of GABAA receptor α1 subunit mRNA and protein in rat neocortex following photothrombotic infarction

“…Interestingly, Raol et al (2006b) showed that an increased level of GABA A R α 1 subunits in the dentate gyrus can affect the development of spontaneous seizures after status epilepticus. In addition, recent studies have identified the α 1 (A294D) and α 1 (A322D) genetic mutations of the GABA A R α 1 subunit that are associated with juvenile myoclonic epilepsy (Cossette et al, 2002;Gallagher et al, 2004;Mizielinska et al, 2006). These mutations affect GABA A R gating, expression, and/or trafficking of the receptor to the cell surface -all pathophysiological mechanisms that result in neuronal disinhibition, cause hyperexcitability throughout the brain, and predispose to epileptic seizures (Fisher, 2004;Krampfl et al, 2005;Macdonald et al, 2006).…”