Functional and structural analysis of ClC-K chloride channels involved in renal disease.
ClC-K channels belong to the CLC family of chloride channels and are predominantly expressed in the kidney. Genetic evidence suggests their involvement in transepithelial transport of chloride in distal nephron segments; ClC-K1 gene deletion leads to nephrogenic diabetes insipidus in mice, and mutations of the hClC-Kb gene cause Bartter's syndrome type III in humans. Expression of rClC-K1 in Xenopus oocytes yielded voltage-independent currents that were pH-sensitive, had a Br(-) > NO(3)(-) = Cl(-) > I(-) conductance sequence, and were activated by extracellular calcium. A glutamate for valine exchange at amino acid position 166 induced strong voltage dependence and altered the conductance sequence of ClC-K1. This demonstrates that rClC-K1 indeed functions as an anion channel. By contrast, we did not detect currents upon hClC-Kb expression in Xenopus oocytes. Using a chimeric approach, we defined a protein domain that, when replaced by that of rClC-K1, allowed the functional expression of a chimera consisting predominantly of hClC-Kb. Its currents were linear and were inhibited by extracellular acidification. Contrasting with rClC-K1, they displayed a Cl(-) > Br(-)> I(-) > NO(3)(-) conductance sequence and were not augmented by extracellular calcium. Insertion of point mutations associated with Bartter's syndrome type III destroyed channel activity. We conclude that ClC-K proteins form constitutively open chloride channels with distinct physiological characteristics.
Waldegger
S
S
Zentrum für Molekulare Neurobiologie, University of Hamburg, Martinistr. 85, D-20246 Hamburg, Germany. siegfried.waldegger@zmnh.uni-hamburg.de
Jentsch
T J
TJ
D019943:Amino Acid Substitution; D000818:Animals; D027321:Anion Transport Proteins; D001477:Bartter Syndrome / Q000235:genetics; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017353:Gene Deletion; D006801:Humans; D007674:Kidney Diseases / Q000235:genetics*; D008564:Membrane Potentials; D008565:Membrane Proteins; D051379:Mice; D008958:Models, Molecular; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D029867:Xenopus Proteins; D014982:Xenopus laevis
D027321:Anion Transport Proteins; C489932:CLCNKA protein, human; C412733:CLCNKB protein, human; D018118:Chloride Channels; C489933:Clcnka protein, mouse; C486573:Clcnkb protein, mouse; C486572:Clcnkb protein, rat; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C489934:clcnka protein, Xenopus