Functional and Transport Analysis of CLCN5 Mutations Found in Dent Disease Patients

Abstract

Dent disease, an X-linked kidney disease caused by mutations in the CLCN5 gene, is characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets, and progressive renal failure. In published Dent patients, S244L is the most frequently found mutation. Q629X and R345W are two novel mutations diagnosed by the Mayo Clinic RKSC. T657S was reported as pathogenic in a Dent disease patient and high minor-allele frequency (0.391%) in the African-American population without any pathophysiology data. The transport properties of CLC-5 were characterized by electrophysiology experiments showing rectified outward currents at positive holding voltages in the presence of 104 mM Cl- while removal of chloride abolished the currents. Currents of WT and ClC-5 mutants were inhibited by extracellular pH at 5.5 but not pH8.5. The T657S and R345W showed same anion selectivity sequence as WT (SCN->NO3-∼Cl->Br->I-). However, the S244L and Q629X lost the anion conductance sequence. A HA tag, shown not to interfere CLC-5 transport function, was added to the extracellular loop of the CLC-5 protein enabling chemiluminescence method to quantify ClC-5 surface expression. Oocytes expressing T657S, R345W, and Q629X mutations showed positive correlations between transport function and cell membrane protein expression indicating protein trafficking problem. In contrast, S244L function was significantly lower than WT CLC-5 whereas protein expression did not significantly differ from the HA tagged CLC-5 WT suggesting defected transport function. The function and trafficking of T657S was equivalent to the CLC-5 WT and seems to be a benign mutation. DK101405, AHA-SDG2640146