Abstract
Voltage-gated ClC-2 channels are essential for chloride homeostasis. Complete knockout of mouse ClC-2 leads to testicular degeneration and neuronal myelin vacuolation. Gain-of-function and loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the genetic diseases aldosteronism and leukodystrophy, respectively. The protein homeostasis (proteostasis) mechanism of ClC-2 is currently unclear. Here, we aimed to identify the molecular mechanism of endoplasmic reticulum-associated degradation of ClC-2, and to explore the pathophysiological significance of disease-associated anomalous ClC-2 proteostasis. In both heterologous expression system and native neuronal and testicular cells, ClC-2 is subject to significant regulation by cullin-RING E3 ligase-mediated polyubiquitination and proteasomal degradation. The cullin 4 (CUL4)-damage-specific DNA binding protein 1 (DDB1)-cereblon (CRBN) E3 ubiquitin ligase co-exists in the same complex with and promotes the degradation of ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide and the cullin E3 ligase inhibitor MLN4924 promotes and attenuates, respectively, proteasomal degradation of ClC-2. Analyses of disease-related ClC-2 mutants reveal that aldosteronism and leukodystrophy are associated with opposite alterations in ClC-2 proteostasis. Modifying CUL4 E3 ligase activity with lenalidomide and MLN4924 ameliorates disease-associated ClC-2 proteostasis abnormality. Our results highlight the significant role and therapeutic potential of CUL4 E3 ubiquitin ligase in regulating ClC-2 proteostasis.
Highlights
Voltage-gated ClC-2 chloride (Cl− ) channels are broadly expressed in virtually all tissues, with abundant expression levels in neurons, glial cells, and epithelia cells [1,2,3,4,5]
Proteasomal Degradation of ClC-2 is Mediated by Cullin 4 E3 Ubiquitin Ligase
We began by investigating whether proteasomal degradation contributes to the proteolytic mechanism of mouse ClC-2 proteins overexpressed in HEK293T cells
Summary
Voltage-gated ClC-2 chloride (Cl− ) channels are broadly expressed in virtually all tissues, with abundant expression levels in neurons, glial cells, and epithelia cells [1,2,3,4,5]. Complete knockout of ClC-2 in mice leads to severe retinal and testicular degeneration [8,9], as well as prominent fluid accumulation and myelin vacuolation in central neurons [10]. Together, these observations suggest that ClC-2 channels may regulate ion homeostasis in narrow extracellular spaces, such as those in blood–retinal, blood–testis, and blood–brain barriers. Gain-of-function mutations in the CLCN2 gene lead to enhanced Cl− efflux and membrane depolarization in aldosterone-producing adrenal glomerulosa cells, manifesting as constitutive aldosterone secretion, hypertension, and hypokalemia [11,12,13,14,15]. 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]
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