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Abstract

The kidney and inner ear CLC-K chloride channels, which are involved in salt absorption and endolymph production, are regulated by extracellular Ca²⁺ 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 Ca²⁺-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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ coordination. To investigate cation specificity, we applied extracellular Zn²⁺, Mg²⁺, Ba²⁺, Sr²⁺, and Mn²⁺. Zn²⁺ blocks ClC-Ka as well as its Ca²⁺-insensitive mutant, suggesting that Zn²⁺ binds to a different site. Mg²⁺ does not activate CLC-Ks, but the channels are activated by Ba²⁺, Sr²⁺, and Mn²⁺ with a rank order of potency of Ca²⁺ > Ba²⁺ > Sr²⁺ = Mn²⁺ for the human CLC-Ks. Dose–response analysis indicates that the less potent Ba²⁺ has a lower affinity rather than a lower efficacy. Interestingly, rClC-K1 shows an altered rank order (Ca²⁺ > Sr²⁺ >> Ba²⁺), but homology models suggest that residues outside the I–J loop are responsible for this difference. Our detailed characterization of the regulatory Ca²⁺-binding site provides a solid basis for the understanding of the physiological modulation of CLC-K channel function in the kidney and inner ear.