title: A cytoplasmic domain mutation in ClC-Kb affects long-distance communication across the membrane.
abstract: BACKGROUND
  ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix.
  
  
  RESULTS
  Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface.
  
  
  CONCLUSIONS
  The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change--ranging from the cytoplasmic side of the protein to the extracellular side of the protein--is involved in the Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure.
authors.affiliation: Department of Molecular and Cellular Physiology and Program in Biophysics, Stanford University. Stanford, California, United States of America.
authors.forename: Gilbert Q
authors.initials: GQ
authors.lastname: Martinez
authors.forename: Merritt
authors.initials: M
authors.lastname: Maduke
mesh_terms: D000818:Animals; D001477:Bartter Syndrome / Q000175:diagnosis / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000235:genetics* / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism*; D007758:Ear, Inner / Q000378:metabolism*; D004594:Electrophysiology; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D014981:Xenopus
chemical_list: C489932:CLCNKA protein, human; C412733:CLCNKB protein, human; D018118:Chloride Channels; D002118:Calcium