Dataset Information

Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule.

ABSTRACT: ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.

SUBMITTER: Engh AM

PROVIDER: S-EPMC2234078 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule.

Engh Anita M AM Maduke Merritt M

The Journal of general physiology 20050516 6

ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis ...[more]

PMID: 15897295

Similar Datasets

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-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.

Dataset Information

Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule.

Publications

Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets