Leukoencephalopathy‐causing CLCN2 mutations are associated with impaired Cl⁻ channel function and trafficking

Abstract

Mutations in CLCN2 have been recently identified in patients suffering from a type of leukoencephalopathy involving intramyelinic oedema. Here, we characterised most of these mutations that reduce the function of the chloride channel ClC-2 and impair its plasma membrane (PM) expression. Detailed biochemical and electrophysiological analyses of the Ala500Val mutation revealed that defective gating and increased cellular and PM turnover contributed to defective A500V-ClC-2 functional expression. Co-expression of the adhesion molecule GlialCAM, which forms a tertiary complex with ClC-2 and megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1), rescued the functional expression of the mutant by modifying its gating properties. GlialCAM also restored the PM levels of the channel by impeding its turnover at the PM. This rescue required ClC-2 localisation to cell-cell junctions, since a GlialCAM mutant with compromised junctional localisation failed to rescue the impaired stability of mutant ClC-2 at the PM. Wild-type, but not mutant, ClC-2 was also stabilised by MLC1 overexpression. We suggest that leukodystrophy-causing CLCN2 mutations reduce the functional expression of ClC-2, which is partly counteracted by GlialCAM/MLC1-mediated increase in the gating and stability of the channel.

CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

“…In primary aldosteronism, gain-of-function mutations in the CLCN2 gene lead to enhanced Cl − efflux and therefore membrane depolarization in aldosterone-producing adrenal glomerulosa cells, manifesting as constitutive aldosterone secretion, hypertension, and hypokalemia [ 11 , 12 , 13 , 14 , 15 ]. On the other hand, loss-of-function mutations in the CLCN2 gene have been linked to a type of leukodystrophy (white matter disorder), CLCN2 -related leukoencephalopathy, characterized by intramyelinic edema in the brain [ 16 , 17 , 18 ], which is reminiscent of the myelin vacuolation found in ClC-2 knockout mice [ 10 ]. Moreover, another type of leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts, is associated with genetic mutations in the ClC-2-binding proteins GlialCAM and megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1), whose disease-related defects significantly disrupt the subcellular localization of ClC-2 channels in astrocytes and oligodendrocytes, as well as reducing ClC-2 protein stability at the plasma membrane [ 19 , 20 , 21 ].…”

“…The mechanism underlying the enhanced cell surface Cl − conductance in aldosteronism can be attributed to altered voltage-dependent gating properties that increase the current amplitude of mutant ClC-2 channels [ 12 , 13 , 14 ]. In contrast, leukodystrophy-associated mutations result in altered voltage-dependent gating properties that reduce the current amplitude of mutant ClC-2 channels [ 18 ]. Importantly, leukodystrophy-associated mutations also lead to reduced ClC-2 protein levels that may involve defective protein stability and impaired membrane trafficking [ 16 , 18 ].…”

“…In contrast, leukodystrophy-associated mutations result in altered voltage-dependent gating properties that reduce the current amplitude of mutant ClC-2 channels [ 18 ]. Importantly, leukodystrophy-associated mutations also lead to reduced ClC-2 protein levels that may involve defective protein stability and impaired membrane trafficking [ 16 , 18 ]. It remains unclear whether aldosteronism-causing mutations may also affect the biochemical property of ClC-2 channels by, for example, promoting ClC-2 protein expression.…”

Evaluation of Gene-Based Family-Based Methods to Detect Novel Genes Associated With Familial Late Onset Alzheimer Disease

“…CLCN2 , chloride voltage-gated channel 2, has several functions including the regulation of cell volume: membrane potential stabilization, signal transduction and transepithelial transport. It has been associated with different epilepsy modes (Saint-Martin et al, 2009 ; Cukier et al, 2014 ) and leukoencephalopathy (Gaitán-Peñas et al, 2017 ). CHRD and CLCN2 show co-expression which could be due to their close proximity, both belong to a gene cluster at 3q27.…”

Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice

“…Xenopus oocytes injection and voltage-clamp recordings. All procedures for plasmids construction, Xenopus oocyte preparation, injection, and voltage-clamp recordings were performed as described in detail previously (41,58). Briefly, fluorescently tagged plasmids were linearized by NotI digestion prior to in vitro transcription using the mMessage mMachine SP6 kit (Ambion).…”