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

Abstract

A form of autosomal dominant juvenile myoclonic epilepsy is caused by a nonconservative missense mutation, A322D, in the GABA A receptor ␣1 subunit M3 transmembrane helix. We reported previously that the A322D mutation reduced total and surface ␣1(A322D) subunit protein and that residual ␣1(A322D) subunit resided in the endoplasmic reticulum. Here, we demonstrate that the reduction in ␣1(A322D) expression results from rapid endoplasmic reticulum-associated degradation of the ␣1(A322D) subunit through the ubiquitin-proteasome system. We provide direct evidence that the ␣1(A322D) subunit misfolds and show that in at least 33% of ␣1(A322D) subunits, M3 failed to insert into the lipid bilayer. We constructed a series of mutations in the M3 domain and empirically determined the apparent free energy cost (⌬G app) of membrane insertion failure, and we show that the ⌬G app correlated directly with the recently elucidated transmembrane sequence code (⌬G Lep). These data provide a biochemical mechanism for the pathogenesis of this epilepsy mutation and demonstrate that ⌬G Lep predicts the efficiency of lipid partitioning of a naturally occurring protein's transmembrane domain expressed in vivo. Finally, we calculated the ⌬⌬G Lep for 277 known transmembrane missense mutations associated with Charcot-Marie-Tooth disease, diabetes insipidus, retinitis pigmentosa, cystic fibrosis, and severe myoclonic epilepsy of infancy and showed that the majority of these mutations also are likely to destabilize transmembrane domain membrane insertion, but that only a minority of the mutations would be predicted to be as destabilizing as the A322D mutation.ion channel ͉ peripheral myelin protein ͉ protein folding ͉ sodium channel ͉ translocon G ABA A receptors, the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are pentameric ligand-gated ion channels whose five subunits originate from seven families containing multiple subtypes but most commonly assemble with two ␣1 subunits, two ␤2 subunits, and one ␥2 subunit (1-3). In mammalian cells, expression of an ␣-subunit is required to form functional receptors (4). GABA A receptors are homologous to other types of ligand-gated ion channels in the cys-loop receptor ion channel superfamily, which also includes glycine, serotonin type three, and nicotinic acetylcholine receptors (AChR). Cryoelectron microscopy experiments elucidated the three-dimensional structure of the transmembrane domains of the Torpedo AChR to a resolution of 4Å (5), and it is assumed that the GABA A receptor structure is similar (6). Each GABA A receptor subunit contains four transmembrane helices (M1-M4). One M2 domain from each subunit lines the ion channel pore and is surrounded by M1, M3, and M4 helices.A nonconservative missense mutation (A322D) in the M3 domain of the GABA A receptor ␣1 subunit gene (GABRA1) causes autosomal dominant juvenile myoclonic epilepsy (ADJME) (7). Electrophysiological experiments demonstrated that the A322D mutation altered GABA A receptor function (7-9)....

Ubiquitin-dependent lysosomal targeting of GABA A receptors regulates neuronal inhibition

“…Therefore, these experiments provide compelling evidence that the lysosomal targeting of GABA A Rs depends on the ubiquitination of lysine residues between residues 317-338 of the ␥2 subunit. Ubiquitination of lysine residues within the intracellular loops of the GABA A R ␣1 and ␤3 subunits can target unassembled subunits within the endoplasmic reticulum (ER) for ER associated degradation by the proteosome, but does not appear to be involved in GABA A R endocytic trafficking (17,18). Moreover, this proteosomal degradation of GABA A R subunits within the secretory pathway regulates synaptic inhibition over long time scales (days) (17).…”

“…A number of studies on the role of ubiquitination in regulating synaptic function under normal conditions and in pathological conditions such as ischemia have focused on the ubiquitin-proteasome system (14)(15)(16)(17)(18). However, within the endocytic pathway, mono-ubiquitination (or the addition of short ubiquitin chains) is a key signal that results in the specific targeting of cargo to late endosomes for subsequent lysosomal degradation (12,19,20).…”

Combining Valosin-containing Protein (VCP) Inhibition and Suberanilohydroxamic Acid (SAHA) Treatment Additively Enhances the Folding, Trafficking, and Function of Epilepsy-associated γ-Aminobutyric Acid, Type A (GABAA) Receptors

“…The ␣1(A322D) subunits cannot efficiently form the TM3 ␣ helix in the ER membrane, resulting in its misfolding (40). Misfolded ␣1(A322D) subunits are recognized by the ER quality control machinery, polyubiquitinated, extracted from the ER membrane to the cytosol, and targeted to the proteasome for degradation.…”

“…35 S]methionine chase result (39,40). EerI treatment (5 M, 6 h) increased the half-life of the ␣1(A322D) subunit from 45 to 82 min, and, remarkably, coapplication of SAHA (2.5 M, 24 h) further extended its half-life to 130 min (Fig.…”

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

“…In addition, the study by Sanders and Myers (2004) revealed that many diseaselinked mutations result from loss of protein function and from protein misfolding, which alters protein function, assembly, or subcellular trafficking, but not protein topology. Gallagher et al (2007) showed that the GABA A R α 1 epilepsy mutation A322D reduced total α 1 (A322D) subunit expression, altered α 1 subunit topology, inhibited transmembrane helix formation, and caused proteasomal degradation.…”