GABAA Receptor α1 Subunit Mutation A322D Associated with Autosomal Dominant Juvenile Myoclonic Epilepsy Reduces the Expression and Alters the Composition of Wild Type GABAA Receptors

Abstract

A GABA A receptor (GABA A R) ␣1 subunit mutation, A322D (AD), causes an autosomal dominant form of juvenile myoclonic epilepsy (ADJME). Previous studies demonstrated that the mutation caused ␣1(AD) subunit misfolding and rapid degradation, reducing its total and surface expression substantially. Here, we determined the effects of the residual ␣1(AD) subunit expression on wild type GABA A R expression to determine whether the AD mutation conferred a dominant negative effect. We found that although the ␣1(AD) subunit did not substitute for wild type ␣1 subunits on the cell surface, it reduced the surface expression of ␣1␤2␥2 and ␣3␤2␥2 receptors by associating with the wild type subunits within the endoplasmic reticulum and preventing them from trafficking to the cell surface. The ␣1(AD) subunit reduced surface expression of ␣3␤2␥2 receptors by a greater amount than ␣1␤2␥2 receptors, thus altering cell surface GABA A R composition. When transfected into cultured cortical neurons, the ␣1(AD) subunit altered the time course of miniature inhibitory postsynaptic current kinetics and reduced miniature inhibitory postsynaptic current amplitudes. These findings demonstrated that, in addition to causing a heterozygous loss of function of ␣1(AD) subunits, this epilepsy mutation also elicited a modest dominant negative effect that likely shapes the epilepsy phenotype. GABA A Rs2 are ligand-gated ion channels that provide the major source of inhibitory control to the mammalian central nervous system. Each GABA A R is a pentamer whose five subunits arise from seven subunit families that contain multiple subtype isoforms. Neurons preferentially express GABA A Rs composed of distinct combinations of subunit isoforms in different brain regions at well defined times in development (1-3).At maturity, the most prevalent GABA A R throughout the brain consists of two ␣1 subunits, two ␤2 subunits, and one ␥2 subunit in a ␤2-␣1-␤2-␣1-␥2 assembly (4 -6). To date, 13 autosomal dominant mutations in GABA A R subunit genes have been associated with different epilepsy syndromes (7).The missense AD mutation in the GABA A R ␣1 subunit gene (GABRA1) causes ADJME (8), a monogenic form of a common epilepsy syndrome that begins at a distinct developmental time point (adolescence) and confers myoclonic, generalized tonicclonic, and absence seizures as well as neuropsychiatric comorbidities (9). We demonstrated previously that the AD mutation, which substitutes a negatively charged aspartate for a neutral alanine within the M3 transmembrane domain, causes the ␣1(AD) subunit to misfold with altered topology (10). Cells rapidly degrade the misfolded ␣1(AD) subunit through both proteasome-and lysosome-mediated processes (10, 11). Therefore, the ␣1(AD) subunit is expressed at substantially lower levels than the wild type ␣1 subunit. GABA A Rs that do incorporate the residual, nondegraded ␣1(AD) subunits exhibit substantially altered electrophysiological properties (8,12,13). Therefore, a major consequence of heterozygous expression of the AD mutation i...

Altered Cortical GABAA Receptor Composition, Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome

“…We replated the cells twice weekly. A description of plasmids expressing the ␣1, ␣3, ␤2, and ␥2 subunits was published previously (11). A plasmid expressing the ␤1 subunit was a generous gift from Dr. Robert Macdonald (Vanderbilt University).…”

The developmental evolution of the seizure phenotype and cortical inhibition in mouse models of juvenile myoclonic epilepsy

“…In vitro studies demonstrated that the A322D mutation causes α1(A322D) protein misfolding with altered transmembrane topology (Gallagher et al, 2007) and rapid elimination by endoplasmic reticulum associated degradation (Bradley et al, 2008;Gallagher et al, 2007). This causes a 92% reduction of total α1(A322D) subunit expression (Ding et al, 2010). In addition, when expressed in vitro , the residual α1(A322D) subunit protein causes a small, but significant reduction of wild type GABA A R expression.…”

Agonist-dependent Endocytosis of γ-Aminobutyric Acid Type A (GABAA) Receptors Revealed by a γ2(R43Q) Epilepsy Mutation

“…It must be also noted that a shortened lifetime caused by the epilepsy mutation A322D on ␣1-containing GABA A Rs has been proposed (74) (but also see Ref. 75). Interestingly, experiments on ␥2(R43Q) knock-in mice and transfected neurons or COS-7 cells have shown that ␣1 and ␣3 subunit surface expression was not reduced, despite a dramatic decrease in ␥2(R43Q) surface labeling (16,22).…”