A novel CLCN5 pathogenic mutation supports Dent disease with normal endosomal acidification

Area of Science:

• Molecular Biology
• Genetics
• Nephrology

Background:

• Dent disease is an X-linked renal tubular disorder caused by mutations in CLCN5, affecting the ClC-5 chloride channel.
• Previous research suggested impaired endosomal acidification due to ClC-5 dysfunction contributes to Dent disease pathogenesis.

Purpose of the Study:

• To investigate the molecular consequences of a novel pathogenic mutation (p.Glu211Gly) in the ClC-5 channel, specifically affecting the 'gating glutamate' residue.
• To explore the role of endosomal chloride concentration versus acidification in endocytosis defects associated with Dent disease 1.

Main Methods:

• Site-directed mutagenesis to create the p.Glu211Gly ClC-5 mutant.
• Expression studies in HEK293T cells and Xenopus laevis oocytes.
• Analysis of protein localization (N-glycosylation, plasma membrane, endosomes) and channel electrophysiology.
• Measurement of endosomal acidification using the pH-sensitive pHluorin2 probe.

Main Results:

• The p.Glu211Gly ClC-5 mutant showed normal localization and N-glycosylation.
• Electrophysiological studies revealed the mutant lost outward rectification and extracellular H+ sensitivity, functioning as a chloride channel.
• No significant difference in endosomal luminal pH was observed between cells expressing wild-type and p.Glu211Gly ClC-5.

Conclusions:

• The novel p.Glu211Gly mutation in ClC-5 alters its channel properties, converting it into a chloride channel.
• Impaired endosomal acidification is not the sole cause of defective endocytosis in Dent disease 1.
• Endosomal chloride concentration may play a more critical role in endocytosis than previously thought for this condition.