Abstract: SA-PO1174
Electrogenic Transport Function of Low Temperature Rescued CLC-5: R345W Mutation and Towards Dent Disease Novel Therapies
Session Information
- NIDDK KUH Summer Undergraduate Research Program Posters
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1002 Genetic Diseases of the Kidneys: Non-Cystic
Author
Group or Team Name
- Meihui Zhang. Mayo Clinic, Rochester, MN.
Background
Dent Disease type 1 (DD1) is an X-linked disorder characterized by low-molecularweight proteinuria, hypercalciuria, nephrocalcinosis, and kidney stones. DD1 has no effective therapy or treatment, although it is known to be caused by mutations in the CLCN5 gene encoding ClC-5, a voltage-gated nCl-/H+ exchanger found at the apical membrane of the proximal tubule and within endosomal membranes. Our previous studies revealed that functional defects of two novel CLCN5 mutations (R345W, Q629X) were due to abnormal ClC-5 trafficking to the plasma membrane. Interestingly, R345W surface expression in cells could be rescued by lowering the incubation temperature from 37 to 30°C after transfection. However, the transport function of the rescued R345W has not been investigated.
Methods
Thus, we used whole cell patch-clamp electrophysiology to measure transport functions of transiently transfected renal proximal tubule cells (LLC-PK1) expressing wild type (WT) or R345W mutated ClC-5 incubated at 37 or 27°C.
Results
Cells expressing WT exhibited strongly outward rectifying current at positive holding voltages whereas R345W expressing cells incubated at 37°C demonstrated reduced current. Intriguingly, R345W expressing cells incubated at 27°C demonstrated significantly increased transport function compared to R345W expressing cells incubated at 37°C. We have also successfully established LLC-PK1 cell lines that stably express eGFP/HA double-tagged WT and mutant ClC-5. Using Western blot analysis, we documented that ClC-5 expression levels were sufficient to use these cells to screen candidate chaperones.
Conclusion
These results establish a rationale and the tools to explore the effect of pharmacological chaperones to facilitate proper protein folding and maturation of DD1 mutations, including R345W. Results could suggest new therapies for some populations of DD1 patients who currently have no specific therapy available.