Abstract
GABAA receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit results in its excessive endoplasmic reticulum-associated degradation at the expense of plasma membrane trafficking, leading to autosomal dominant juvenile myoclonic epilepsy. Presumably, valosin-containing protein (VCP)/p97 extracts misfolded subunits from the endoplasmic reticulum membrane to the cytosolic proteasome for degradation. Here we showed that inhibiting VCP using Eeyarestatin I reduces the endoplasmic reticulum-associated degradation of the α1(A322D) subunit without an apparent effect on its dynamin-1 dependent endocytosis and that this treatment enhances its trafficking. Furthermore, coapplication of Eeyarestatin I and suberanilohydroxamic acid, a known small molecule that promotes chaperone-assisted folding, yields an additive restoration of surface expression of α1(A322D) subunits in HEK293 cells and neuronal SH-SY5Y cells. Consequently, this combination significantly increases GABA-induced chloride currents in whole-cell patch clamping experiments than either chemical compound alone in HEK293 cells. Our findings suggest that VCP inhibition without stress induction, together with folding enhancement, represents a new strategy to restore proteostasis of misfolding-prone GABAA receptors and, therefore, a potential remedy for idiopathic epilepsy.
Highlights
The ␣1 subunit harboring the A322D mutation is subject to excessive endoplasmic reticulum-associated degradation (ERAD)
Our findings suggest that valosin-containing protein (VCP) inhibition without stress induction, together with folding enhancement, represents a new strategy to restore proteostasis of misfolding-prone GABAA receptors and, a potential remedy for idiopathic epilepsy
VCP Interacts Stronger with Misfolding-prone ␣1(A322D) Subunits Than with WT ␣1 Subunits—It has been reported that the A322D mutation in the ␣1 subunit resulted in rapid degradation of the ␣1(A322D) subunit mainly by ERAD [29]
Summary
Results: VCP inhibition using Eeyarestatin I reduces the ERAD of ␣1(A322D) subunits, and coapplication of SAHA additively enhances their proteostasis. Coapplication of Eeyarestatin I and suberanilohydroxamic acid, a known small molecule that promotes chaperone-assisted folding, yields an additive restoration of surface expression of ␣1(A322D) subunits in HEK293 cells and neuronal SH-SY5Y cells. This combination significantly increases GABA-induced chloride currents in whole-cell patch clamping experiments than either chemical compound alone in HEK293 cells. Our findings suggest that VCP inhibition without stress induction, together with folding enhancement, represents a new strategy to restore proteostasis of misfolding-prone GABAA receptors and, a potential remedy for idiopathic epilepsy. Proteostasis deficiency in ion channels leads to a variety of ion channel diseases called channelopathies, which are often
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