A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

J Gen Physiol. 2010 Sep;136(3):311-23. doi: 10.1085/jgp.201010455.


The two human CLC Cl(-) channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel beta subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca(2+) and H(+) on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca(2+)](ext) without full saturation up to 50 mM. However, in the absence of Ca(2+), ClC-Ka currents were still 20% of currents in 10 mM [Ca(2+)](ext), demonstrating that Ca(2+) is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H(+)](ext) with a practically complete block at pH 6. Ca(2+) and H(+) act as gating modifiers without changing the single-channel conductance. Dose-response analysis suggested that two protons are necessary to induce block with an apparent pK of approximately 7.1. A simple four-state allosteric model described the modulation by Ca(2+) assuming a 13-fold higher Ca(2+) affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca(2+) and H(+). A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca(2+)-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca(2+). Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H(+) block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H(+) -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing conformational changes underlying the complex gating of CLC-K channels.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aspartic Acid
  • Binding Sites
  • Calcium / metabolism*
  • Chloride Channels / chemistry
  • Chloride Channels / genetics
  • Chloride Channels / metabolism*
  • Glutamic Acid
  • Humans
  • Hydrogen-Ion Concentration
  • Ion Channel Gating*
  • Kidney / metabolism*
  • Kinetics
  • Membrane Potentials
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Patch-Clamp Techniques
  • Protein Subunits
  • Structure-Activity Relationship
  • Xenopus


  • CLCNKA protein, human
  • CLCNKB protein, human
  • Chloride Channels
  • Protein Subunits
  • Aspartic Acid
  • Glutamic Acid
  • Calcium