A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels (Articolo in rivista)

Type
Label
  • A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels (Articolo in rivista) (literal)
Anno
  • 2010-01-01T00:00:00+01:00 (literal)
Alternative label
  • Gradogna A, Babini E, Picollo A, Pusch M. (2010)
    A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels
    (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#autori
  • Gradogna A, Babini E, Picollo A, Pusch M. (literal)
Pagina inizio
  • 311 (literal)
Pagina fine
  • 323 (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#numeroVolume
  • 136 (literal)
Note
  • ISI Web of Science (WOS) (literal)
Titolo
  • A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels (literal)
Abstract
  • 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. (literal)
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