Functional and transport analyses of CLCN5 genetic changes identified in Dent disease patients.
Dent disease type 1, an X-linked inherited kidney disease is caused by mutations in electrogenic Cl(-)/H(+) exchanger, ClC-5. We functionally studied the most frequent mutation (S244L) and two mutations recently identified in RKSC patients, Q629X and R345W. We also studied T657S, which has a high minor-allele frequency (0.23%) in the African-American population, was published previously as pathogenic to cause Dent disease. The transport properties of CLC-5 were electrophysiologically characterized. WT and ClC-5 mutant currents were inhibited by pH 5.5, but not affected by an alkaline extracellular solution (pH 8.5). The T657S and R345W mutations showed the same anion selectivity sequence as WT ClC-5 (SCN(-)>NO3(-)≈Cl(-)>Br(-)>I(-)). However, the S244L and Q629X mutations abolished this anion conductance sequence. Cell surface CLC-5 expression was quantified using extracellular HA-tagged CLC-5 and a chemiluminescent immunoassay. Cellular localization of eGFP-tagged CLC-5 proteins was also examined in HEK293 cells with organelle-specific fluorescent probes. Functional defects of R345W and Q629X mutations were caused by the trafficking of the protein to the plasma membrane since proteins were mostly retained in the endoplasmic reticulum, and mutations showed positive correlations between surface expression and transport function. In contrast, although the S244L transport function was significantly lower than WT, cell surface, early endosome, and endoplasmic reticulum expression was equal to that of WT CLC-5. Function and trafficking of T657S was equivalent to the WT CLC-5 suggesting this is a benign variant rather than pathogenic. These studies demonstrate the useful information that can be gained by detailed functional studies of mutations predicted to be pathogenic.
O'Brien Urology Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota Division of Nephrology, Shanghai Changzheng Hospital Second Military Medical University, Shanghai, China.
Xiaojing
X
Tang
O'Brien Urology Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota Physiology & Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota Wayne State University, Detroit, Michigan.
Matthew R
MR
Brown
Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota.
Andrea G
AG
Cogal
O'Brien Urology Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota.
Daniel
D
Gauvin
Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota.
Peter C
PC
Harris
O'Brien Urology Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, Minnesota.
John C
JC
Lieske
O'Brien Urology Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota Physiology & Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota.
Michael F
MF
Romero
O'Brien Urology Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota Physiology & Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota chang.minhwang@mayo.edu.
Min-Hwang
MH
Chang
D000595:Amino Acid Sequence; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D057973:Dent Disease / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D021381:Protein Transport / Q000235:genetics; D012042:Registries; D014162:Transfection
C097503:CLC-5 chloride channel; D018118:Chloride Channels
Anion selectivity; pH dependence; protein trafficking; voltage clamp