Cellular Localization and Cl−/H+ Coupling Properties of wild type ClC‐5 and patient‐specific mutants
Authors: Chang Minhwang , Brown Matthew , Liu Yiran , Gainullin Vladimir G , Harris Peter C , Romero Michael F , Lieske John C 
Published: November 2023

Dent disease 1, an X‐linked inherited kidney disease caused by mutations in the CLCN5 gene, is characterized by low molecular weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets, and progressive renal failure. CLCN5 encodes the CLC‐5 protein found at the apical membrane in the kidney proximal tubule as well as endosome membranes of other tubule segments. Approximately 80 human mutations of the CLCN5 gene have been identified consisting of nonsense, missense, splice site, insertion and deletion mutations. However, the genotype and phenotype correlation are poor, and the mechanisms of CLC‐5 dysfunction that cause disease are still unclear. Moreover, there has been a lack of systematic study of CLC‐5 mutational effects on Cl−/H+ exchanger activity. To address this need we used ion selective microelectrodes to monitor intracellular pH (pHi) and intracellular Cl− concentration ([Cli]) of Xenopus oocytes expressing WT and mutant CLC‐5 while clamping at +40 mV. Common and novel mutations were identified in Dent patients by collaborators in the Rare Kidney Stone Consortium. The apparent H+:Cl− coupling ratio through the CLC‐5 protein was calculated using the Gibbs‐free energy equation. Cellular localization and protein trafficking of WT and mutant CLC‐5 in renal epithelial cells were also examined by immunofluorescence microscopy using eGFP/HA double‐tagged CLC‐5 and organelle‐specific markers and quantified by flow cytometry.In the presence of 104 mM Cl−(ND96), the pHi and [Cl−]i of the CLC‐5 WT increased 340 ± 59 ×10−4 pH unit/min and 2.8 ± 0.5 mM/min, respectively. T657S increased at a similar rate as WT, while there was a significant decrease in the rate of pHi and [Cl−]i change for S244L, R345W, and Q629X compared to WT. Decreasing extracellular chloride to 5 mM decreased WT ClC‐5 pHi and [Cl−]i by reversing transport: −7 ± 65 ×10−4 pH unit/min and −0.2 ± 0.2 mM/min, respectively. Using these measurements, the stoichiometry of WT CLC‐5 was calculated to be 1.80 ± 0.02 (nCl−:H+) in ND96, similar to the stoichiometry estimated for T657S. However, the stoichiometry was significantly altered for S244L, R345W, and Q629X.WT ClC‐5 acts as Cl−/H+ exchanger at +40mV with 104 mM Cl− in the extracellular solution. Lowering extracellular Cl− reverses the transport direction, and the ClC‐5 exchanger functions at a significantly slower rate. Although S244L has normal surface expression, S244L and R345W mutations have significantly decreased pH and Cl− exchange rates in response to solution manipulations, suggesting these mutations have an altered ion exchange coupling ratio (stoichiometry). Interestingly, while R345W was retained in the ER suggesting defective protein maturation, R345W surface localization was rescued by lowering the incubation temperature from 37 to 30°C. Q629X was retained in the ER, not localized in early endosomes, and also has abolished Cl−/H+ exchange activity, suggesting that Q629X affects protein maturation and trafficking while T657S is a benign, non‐disease causing mutation. These data suggest studying CLC‐5 mutational effects on both transport function and cellular localization is essential to better understand divergent phenotypes in Dent disease.Support or Funding InformationNIH DK101405, U54KD083908, U54DK100227, Mayo O'Brien Urology Research Center, Mayo Clinic Rare Kidney Stone Consortium