Acid-activated Cl- channels in endocytic function in kidney and beyond

• Applicant: Professor Dr. Thomas J. Jentsch
• Subject Area: Anatomy and Physiology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 527664055

Luminal ion homeostasis is crucial for vesicular trafficking and function. Important ions are not only H+ (pH), but also luminal Cl-. This is evident from pathologies resulting from disruption of each of the five endolysosomal CLC 2Cl-/H+-exchangers, which we have studied extensively previously. For instance, disruption of ClC-7 leads to osteopetrosis, loss of ClC-3 to severe neurodegeneration, and inactivation of ClC-5 to kidney stones in Dent’s disease. ClC-5 is important for the acidification and Cl- accumulation of apical proximal tubular (PT) endosomes. Without ClC-5, apical endocytosis and recycling of membrane proteins is severely impaired, resulting in proteinuria and secondary changes in Ca2+ metabolism. The same pathology is observed when ClC-5 is converted into an uncoupled Cl conductance in Clcn5 unc mice and in humans carrying similar point mutations. We recently identified Tmem206 as the hitherto enigmatic, broadly expressed acid-activated Cl- channel ASOR. Its role in acidotoxicity is consistent with its partial presence at the plasma-membrane. However, recent work suggests that it is mainly expressed on endosomes, which are normally acidified, and plays a role in endocytic trafficking. We found that ASOR is required for the shrinkage of macropinosomes. More recently we detected abundant ASOR expression in the proximal tubule, where it co-localizes with ClC-5 on apical endosomes. Using our Tmem206 and Clcn5 mouse models, together with acute injection of labelled proteins, or hormones known to induce apical protein recycling, we propose to study endocytic processes in vivo, thus avoiding problems associated with cell culture studies. Exploiting chimeric gene deletion in the same tubule we can compare WT and KO cells side by side under exactly the same conditions. Functional interactions with ClC-5 will be studied in (mostly chimeric) double KO mice comparing all possible cellular genotypes. Further studies will investigate functional interactions with the linear, pH-insensitive endosomal Cl conductance in Clcn5 unc mice. Preliminary results suggest that ASOR KO partially rescues impaired PT endocytosis of Clcn5- mice, suggesting that Tmem206 might be a modifier gene for Dent’s disease. Based on these findings, we will ask whether ASOR disruption alleviates the neurodegeneration associated with Clcn3 disruption. We anticipate that these projects will lead to important insights not only for kidney and CNS physiology and disease, but for endocytosis in general.

• DFG Programme: Research Grants