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Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels.

ABSTRACT: The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO42-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 A from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 A, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.

SUBMITTER: Chen MF

PROVIDER: S-EPMC2229543 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels.

Chen Mei-Fang MF Chen Tsung-Yu TY

The Journal of general physiology 20030801 2

The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence ...[more]

PMID: 12885875

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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.

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:CLC-0, a prototype Cl- channel in the CLC family, employs two gating mechanisms that control its ion-permeation pore: fast gating and slow gating. The negatively-charged sidechain of a pore glutamate residue, E166, is known to be the fast gate, and the swinging of this sidechain opens or closes the pore of CLC-0 on the millisecond time scale. The other gating mechanism, slow gating, operates with much slower kinetics in the range of seconds to tens or even hundreds of seconds, and it is thought to involve still-unknown conformational rearrangements. Here, we find that low intracellular pH (pHi) facilitates the closure of the CLC-0's slow gate, thus generating current inhibition. The rate of low pHi-induced current inhibition increases with intracellular H+ concentration ([H+]i)-the time constants of current inhibition by low pHi = 4.5, 5.5 and 6 are roughly 0.1, 1 and 10 sec, respectively, at room temperature. In comparison, the time constant of the slow gate closure at pHi = 7.4 at room temperature is hundreds of seconds. The inhibition by low pHi is significantly less prominent in mutants favoring the slow-gate open state (such as C212S and Y512A), further supporting the fact that intracellular H+ enhances the slow-gate closure in CLC-0. A fast inhibition by low pHi causes an apparent inverted voltage-dependent activation in the wild-type CLC-0, a behavior similar to those in some channel mutants such as V490W in which only membrane hyperpolarization can open the channel. Interestingly, when V490W mutation is constructed in the background of C212S or Y512A mutation, the inverted voltage-dependent activation disappears. We propose that the slow kinetics of CLC-0's slow-gate closure may be due to low [H+]i rather than due to the proposed large conformational change of the channel protein. Our results also suggest that the inverted voltage-dependent opening observed in some mutant channels may result from fast closure of the slow gate by the mutations.

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.

Dataset Information

Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels.

Publications

Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels.

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