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

TitleA regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels.
Publication TypeJournal Article
Year of Publication2010
AuthorsGradogna A, Babini E, Picollo A, Pusch M
JournalJ Gen Physiol
Volume136
Issue3
Pagination311-23
Date Published2010 Sep
ISSN1540-7748
KeywordsAmino Acid Sequence, Animals, Aspartic Acid, Binding Sites, Calcium, Chloride Channels, Glutamic Acid, Humans, Hydrogen-Ion Concentration, Ion Channel Gating, Kidney, Kinetics, Membrane Potentials, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Patch-Clamp Techniques, Protein Subunits, Structure-Activity Relationship, Xenopus
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.

DOI10.1085/jgp.201010455
Alternate JournalJ. Gen. Physiol.
PubMed ID20805576
PubMed Central IDPMC2931146
Grant ListGGP08064 / / Telethon / Italy