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The kidney and inner ear CLC-K chloride channels, which are involved in salt absorption and endolymph production, are regulated by extracellular Ca(2+) in the millimolar concentration range. Recently, Gradogna et al. (2010. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201010455) identified a pair of acidic residues (E261 and D278) located in the loop between helices I and J as forming a putative intersubunit Ca(2+)-binding site in hClC-Ka. In this study, we sought to explore the properties of the binding site in more detail. First, we verified that the site is conserved in hClC-Kb and rClC-K1. In addition, we could confer Ca(2+) sensitivity to the Torpedo marmorata ClC-0 channel by exchanging its I-J loop with that from ClC-Ka, demonstrating a direct role of the loop in Ca(2+) binding. Based on a structure of a bacterial CLC and a new sequence alignment, we built homology models of ClC-Ka. The models suggested additional amino acids involved in Ca(2+) binding. Testing mutants of these residues, we could restrict the range of plausible models and positively identify two more residues (E259 and E281) involved in Ca(2+) coordination. To investigate cation specificity, we applied extracellular Zn(2+), Mg(2+), Ba(2+), Sr(2+), and Mn(2+). Zn(2+) blocks ClC-Ka as well as its Ca(2+)-insensitive mutant, suggesting that Zn(2+) binds to a different site. Mg(2+) does not activate CLC-Ks, but the channels are activated by Ba(2+), Sr(2+), and Mn(2+) with a rank order of potency of Ca(2+) > Ba(2+) > Sr(2+) = Mn(2+) for the human CLC-Ks. Dose-response analysis indicates that the less potent Ba(2+) has a lower affinity rather than a lower efficacy. Interestingly, rClC-K1 shows an altered rank order (Ca(2+) > Sr(2+) >> Ba(2+)), but homology models suggest that residues outside the I-J loop are responsible for this difference. Our detailed characterization of the regulatory Ca(2+)-binding site provides a solid basis for the understanding of the physiological modulation of CLC-K channel function in the kidney and inner ear.
Pubmed ID: 23148261
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A multiple sequence alignment server which can align Protein, DNA and RNA sequences.
View all literature mentionsSoftware tool as Program for Comparative Protein Structure Modelling by Satisfaction of Spatial Restraints. Used for homology or comparative modeling of protein three dimensional structures. User provides alignment of sequence to be modeled with known related structures and MODELLER automatically calculates model containing all non hydrogen atoms.
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