KCNQ4, a voltage-gated potassium channel, plays an important role in maintaining cochlear ion homoeostasis and regulating hair cell membrane potential, both essential for normal auditory function. Mutations in the KCNQ4 gene lead to DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss across all frequencies. Despite recent advances in the identification of pathogenic KCNQ4 mutations, the molecular aetiology of DFNA2 remains unknown. We report here that decreased cell surface expression and impaired conductance of the KCNQ4 channel are two mechanisms underlying hearing loss in DFNA2. In HEK293T cells, a dramatic decrease in cell surface expression was detected by immunofluorescent microscopy and confirmed by Western blot for the pathogenic KCNQ4 mutants L274H, W276S, L281S, G285C, G285S, G296S and G321S, while their overall cellular levels remained normal. In addition, none of these mutations affected tetrameric assembly of KCNQ4 channels. Consistent with these results, all mutants showed strong dominant-negative effects on the wild-type (WT) channel function. Most importantly, overexpression of HSP90β, a key component of the molecular chaperone network that controls the KCNQ4 biogenesis, significantly increased cell surface expression of the KCNQ4 mutants L281S, G296S and G321S. KCNQ4 surface expression was restored or considerably improved in HEK293T cells mimicking the heterozygous condition of these mutations in DFNA2 patients. Finally, our electrophysiological studies demonstrated that these mutations directly compromise the conductance of the KCNQ4 channel, since no significant change in KCNQ4 current was observed after KCNQ4 surface expression was restored or improved.
Loss-of-function mutations in the KCNQ4 channel cause DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss. Previous studies have demonstrated that the majority of the pathogenic KCNQ4 mutations lead to trafficking deficiency and loss of KCNQ4 currents. Over the last two decades, various strategies have been developed to rescue trafficking deficiency of pathogenic mutants; the most exciting advances have been made by manipulating activities of molecular chaperones involved in the biogenesis and quality control of the target protein. However, such strategies have not been established for KCNQ4 mutants and little is known about the molecular chaperones governing the KCNQ4 biogenesis. To identify KCNQ4-associated molecular chaperones, a proteomic approach was used in this study. As a result, two major molecular chaperones, HSP70 and HSP90, were identified and then confirmed by reciprocal co-immunoprecipitation assays, suggesting that the HSP90 chaperone pathway might be involved in the KCNQ4 biogenesis. Manipulating chaperone expression further revealed that two different isoforms of HSP90, the inducible HSP90α and the constitutive HSP90β, had opposite effects on the cellular level of the KCNQ4 channel; that HSP40, HSP70, and HOP, three key components of the HSP90 chaperone pathway, were crucial in facilitating KCNQ4 biogenesis. In contrast, CHIP, a major E3 ubiquitin ligase, had an opposite effect. Collectively, our data suggest that HSP90α and HSP90β play key roles in controlling KCNQ4 homeostasis via the HSP40-HSP70-HOP-HSP90 chaperone pathway and the ubiquitin-proteasome pathway. Most importantly, we found that over-expression of HSP90β significantly improved cell surface expression of the trafficking-deficient, pathogenic KCNQ4 mutants L274H and W276S. KCNQ4 surface expression was restored by HSP90β in cells mimicking heterozygous conditions of the DFNA2 patients, even though it was not sufficient to rescue the function of KCNQ4 channels.
Background It has been observed that Slo1 knockout mice have reduced motor function, and people with certain Slo1 mutations have movement problems, but there is no answer whether the movement disorder is caused by the loss of Slo1 in the nervous system, or skeletal muscle, or both. Here, to ascertain in which tissues Slo1 functions to regulate motor function and offer deeper insight in treating related movement disorder, we generated skeletal muscle‐specific Slo1 knockout mice, studied the functional changes in Slo1‐deficient skeletal muscle and explored the underlying mechanism. Methods We used skeletal muscle‐specific Slo1 knockout mice (Myf5‐Cre; Slo1flox/flox mice, called CKO) as in vivo models to examine the role of Slo1 in muscle growth and muscle regeneration. The forelimb grip strength test was used to assess skeletal muscle function and treadmill exhaustion test was used to test whole‐body endurance. Mouse primary myoblasts derived from CKO (myoblast/CKO) mice were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Quantitative real‐time PCR, western blot and immunofluorescence approaches were used to analyse Slo1 expression during myoblast differentiation and muscle regeneration. To investigate the involvement of genes in the regulation of muscle dysfunction induced by Slo1 deletion, RNA‐seq analysis was performed in primary myoblasts. Immunoprecipitation and mass spectrometry were used to identify the protein interacting with Slo1. A dual‐luciferase reporter assay was used to identify whether Slo1 deletion affects NFAT activity. Results We found that the body weight and size of CKO mice were not significantly different from those of Slo1flox/flox mice (called WT). Deficiency of Slo1 in muscles leads to reduced endurance (~30% reduction, P < 0.05) and strength (~30% reduction, P < 0.001). Although there was no difference in the general morphology of the muscles, electron microscopy revealed a considerable reduction in the content of mitochondria in the soleus muscle (~40% reduction, P < 0.01). We found that Slo1 was expressed mainly on the cell membrane and showed higher expression in slow‐twitch fibres. Slo1 protein expression is progressively reduced during muscle postnatal development and regeneration after injury, and the expression is strongly reduced during myoblast differentiation. Slo1 deletion impaired myoblast differentiation and slow‐twitch fibre formation. Mechanistically, RNA‐seq analysis showed that Slo1 influences the expression of genes related to myogenic differentiation and slow‐twitch fibre formation. Slo1 interacts with FAK to influence myogenic differentiation, and Slo1 deletion diminishes NFAT activity. Conclusions Our data reveal that Slo1 deficiency impaired skeletal muscle regeneration and slow‐twitch fibre formation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.