Coexpression of complementary fragments of ClC-5 and restoration of chloride channel function in a Dent's disease mutation.
The human hereditary disorder Dent's disease is linked to loss-of-function mutations of the chloride channel ClC-5. Many of these mutations involve insertion of premature stop codons, resulting in truncation of the protein. We determined whether the functional activity of ClC-5 could be restored by coexpression of the truncated protein (containing the NH2-terminal region) with its complementary "missing" COOH-terminal region. Split channel constructs for ClC-5, consisting of complementary N and C protein regions, were created at an arbitrary site in the COOH-terminal region (V655) and at four Dent's disease mutation sites (R347, Y617, R648, and R704). Coexpression of complementary fragments for the split channel at V655 produced currents with anion and pH sensitivity similar to those of wild-type ClC-5. Channel activity was similarly restored when complementary split channel constructs made for Dent's mutation R648 were coexpressed, but no ClC-5 currents were found when split channels for mutations R347, Y617, or R704 were coexpressed. Immunoblot and immunofluorescence studies of COS-7 cells revealed that N or C protein fragments could be transiently expressed and detected in the plasma membrane, even in split channels that failed to show functional activity. The results suggest that ClC-5 channel activity can be restored for specific Dent's mutations by expression of the missing portion of the ClC-5 molecule.
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005455:Fluorescent Antibody Technique; D006801:Humans; D007669:Kidney Calculi / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009865:Oocytes; D010446:Peptide Fragments / Q000378:metabolism; D051437:Renal Insufficiency / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution; D014981:Xenopus
C097503:CLC-5 chloride channel; D018118:Chloride Channels; D010446:Peptide Fragments