A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels.
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.
Gradogna
Antonella
A
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genoa, Italy.
Babini
Elena
E
Picollo
Alessandra
A
Pusch
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D013329:Structure-Activity Relationship; D014981:Xenopus
C489932:CLCNKA protein, human; C412733:CLCNKB protein, human; D018118:Chloride Channels; D021122:Protein Subunits; D001224:Aspartic Acid; D018698:Glutamic Acid; D002118:Calcium
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