Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.

Ssu-Ju Fu,Tsung-Yu Chen,Chung-Jiuan Jeng,An-Ting Cheng,Meng-Chun Hu,Cheng-Tsung Hsiao,Chih-Yung Tang

doi:10.3390/ijms22115859

Abstract

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90β (Hsp90β) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90β-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

Highlights

By employing heterologous expression in the human embryonic kidney (HEK) 293T cells, we explored the therapeutic potential of correcting leukodystrophy-associated anomalous human ClC-2 proteostasis by modulating chaperone/co-chaperone activity
Previous biochemical evidence supports the interaction between the ClC-2 Cl− channel and the molecular chaperone heat shock protein 90 (Hsp90) in the mouse brain [35]
We began by asking whether this well-known molecular chaperone, as well as its associated molecular chaperone and co-chaperones, may interact with endogenous ClC-2 protein profusely expressed in mouse testes (Figure 1A)

Summary

Introduction

The ubiquitously expressed ClC-2 chloride (Cl− ) channel is activated by membrane hyperpolarization and osmotic cell swelling, and plays an essential role in the regulation of Cl− homeostasis in a wide variety of different tissues [1,2,3,4,5,6,7]. Clcn gene, which encodes the voltage-gated ClC-2 channel, results in neuronal fluid accumulation and myelin vacuolation in the brain, as well as substantial degeneration of the retina and the testis [8,9,10]. Along with other studies in different types of Clcn2-deficient mice [15,16], these findings support the notion that the ClC-2 channel is important for extracellular ion homeostasis in the brain and the testis

Objectives

Results

Conclusion