Abstract
Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.
Highlights
In skeletal muscles, the voltage-gated CLC-1 chloride (Cl−) channel may contribute up to 70%-80% of the resting membrane conductance and plays a critical role in controlling the membrane excitability[1,2,3]
We propose that the cullin 4A (CUL4A)/B E3 ubiquitin ligase mediates the protein degradation of CLC-1 channels
We evaluated the effect of DN-CUL4A/B co-expression on CLC-1 protein stability by using the cycloheximide chase assay
Summary
The voltage-gated CLC-1 chloride (Cl−) channel may contribute up to 70%-80% of the resting membrane conductance and plays a critical role in controlling the membrane excitability[1,2,3]. The pathophysiological mechanisms of a significant number of myotonia-related mutations can be attributed to the disruption of the gating functions of CLC-18–11. Several disease-associated CLCN1 mutations, have been shown to yield functional CLC-1 channels with gating properties either only slightly different or virtually indistinguishable from those of wild-type (WT) channels[6,12]. Emerging evidence suggests that the effects of myotonia-related CLC-1 mutations may entail mechanisms other than defective channel gating. The molecular basis of ER-associated degradation (ERAD) for CLC-1 protein is virtually unknown. One important step to addressing the molecular pathophysiology of myotonia congenita is to elucidate the protein ubiquitination mechanism of CLC-1 channels. We propose that the CUL4A/B E3 ubiquitin ligase mediates the protein degradation of CLC-1 channels
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