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J Gen Physiol. 2012 Dec;140(6):681-96. doi: 10.1085/jgp.201210878. Epub 2012 Nov 12.

Dissecting a regulatory calcium-binding site of CLC-K kidney chloride channels.

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1
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genoa, Italy.

Abstract

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.

PMID:
23148261
PMCID:
PMC3514729
DOI:
10.1085/jgp.201210878
[Indexed for MEDLINE]
Free PMC Article
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