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Alkaline pH block of CLC-K kidney chloride channels mediated by a pore lysine residue.

ABSTRACT: CLC-K chloride channels are expressed in the kidney and the inner ear, where they are involved in NaCl reabsorption and endolymph production, respectively. These channels require the beta subunit barttin for proper function. Mutations in ClC-Kb and barttin, lead to Bartter's syndrome. Block of CLC-K channels by acid pH was described in a previous work, and we had identified His-497 as being responsible for the acidic block of CLC-K channels. Here, we show that ClC-K currents are blocked also by alkaline pH with an apparent pK value of ?8.7 for ClC-K1. Using noise analysis, we demonstrate that alkaline block is mediated by an allosteric reduction of the open probability. By an extensive mutagenic screen we identified K165, a highly conserved residue in the extracellular vestibule of the channel, as the major element responsible for the alkaline pH modulation. Deprotonation of K165 underlies the alkaline block. However, MTS modification of the K165C mutant demonstrated that not only the charge but also the chemical and sterical properties of lysine 165 are determinants of CLC-K gating.

SUBMITTER: Gradogna A

PROVIDER: S-EPMC3699751 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Alkaline pH block of CLC-K kidney chloride channels mediated by a pore lysine residue.

Gradogna Antonella A Pusch Michael M

Biophysical journal 20130701 1

CLC-K chloride channels are expressed in the kidney and the inner ear, where they are involved in NaCl reabsorption and endolymph production, respectively. These channels require the beta subunit barttin for proper function. Mutations in ClC-Kb and barttin, lead to Bartter's syndrome. Block of CLC-K channels by acid pH was described in a previous work, and we had identified His-497 as being responsible for the acidic block of CLC-K channels. Here, we show that ClC-K currents are blocked also by ...[more]

PMID: 23823226

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Project description:CLC-K channels belong to the CLC gene family, which comprises both Cl(-) channels and Cl(-)/H(+) antiporters. They form homodimers which additionally co-assemble with the small protein barttin. In the kidney, they are involved in NaCl reabsorption; in the inner ear they are important for endolymph production. Mutations in CLC-Kb lead to renal salt loss (Bartter's syndrome); mutations in barttin lead additionally to deafness. CLC-K channels are interesting potential drug targets. CLC-K channel blockers have potential as alternative diuretics, whereas CLC-K activators could be used for the treatment of patients with Bartter's syndrome. Several small organic acids inhibit CLC-K channels from the outside by binding to a site in the external vestibule of the ion conducting pore. Benzofuran derivatives with affinities better than 10 ?M have been discovered. Niflumic acid (NFA) exhibits a complex interaction with CLC-K channels. Below ?1?mM, NFA activates CLC-Ka, whereas at higher concentrations NFA inhibits channel activity. The co-planarity of the rings of the NFA molecule is essential for its activating action. Mutagenesis has led to the identification of potential regions of the channel that interact with NFA. CLC-K channels are also modulated by pH and [Ca(2+)](ext). The inhibition at low pH has been shown to be mediated by a His-residue at the beginning of helix Q, the penultimate transmembrane helix. Two acidic residues from opposite subunits form two symmetrically related intersubunit Ca(2+) binding sites, whose occupation increases channel activity. The relatively high affinity CLC-K blockers may already serve as leads for the development of useful drugs. On the other hand, the CLC-K potentiator NFA has a quite low affinity, and, being a non-steroidal anti-inflammatory drug, can be expected to exert significant side effects. More specific and more potent activators will be needed and it will be important to understand the molecular mechanisms that underlie NFA activation.

Project description: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.

Project description: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.

Project description:The opening and closing of chloride (Cl-) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as "fast gating" is regulated by both external and internal Cl- ions. Although the external Cl- effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl- regulation remains to be characterized. In this study, we examine the internal Cl- effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising [Cl-]i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of >1 M, a value very different from the one observed in the external Cl- regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties-the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of [Cl-]i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl- on the fast gating may also be due to the competition of Cl- with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl-. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of [Cl-]i and charges in the inner pore region. The very different nature of external and internal Cl- regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.

Dataset Information

Alkaline pH block of CLC-K kidney chloride channels mediated by a pore lysine residue.

Publications

Alkaline pH block of CLC-K kidney chloride channels mediated by a pore lysine residue.

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