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Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix.

ABSTRACT: Myotonia is a state of hyperexcitability of skeletal-muscle fibres. Mutations in the ClC-1 Cl- channel cause recessive and dominant forms of this disease. Mutations have been described throughout the protein-coding region, including three sequence variations (A885P, R894X and P932L) in a distal C-terminal stretch of residues [CTD (C-terminal domain) region] that are not conserved between CLC proteins. We show that surface expression of these mutants is reduced in Xenopus oocytes compared with wild-type ClC-1. Functional, biochemical and NMR spectroscopy studies revealed that the CTD region encompasses a segment conserved in most voltage-dependent CLC channels that folds with a secondary structure containing a short type II poly-proline helix. We found that the myotonia-causing mutation A885P disturbs this structure by extending the poly-proline helix. We hypothesize that this structural modification results in the observed alteration of the common gate that acts on both pores of the channel. We provide the first experimental investigation of structural changes resulting from myotonia-causing mutations.

SUBMITTER: Macias MJ

PROVIDER: S-EPMC1828897 | biostudies-other | 2007 Apr

REPOSITORIES: biostudies-other

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Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix.

Macías María J MJ Teijido Oscar O Zifarelli Giovanni G Martin Pau P Ramirez-Espain Ximena X Zorzano Antonio A Palacín Manuel M Pusch Michael M Estévez Raúl R

The Biochemical journal 20070401 1

Myotonia is a state of hyperexcitability of skeletal-muscle fibres. Mutations in the ClC-1 Cl- channel cause recessive and dominant forms of this disease. Mutations have been described throughout the protein-coding region, including three sequence variations (A885P, R894X and P932L) in a distal C-terminal stretch of residues [CTD (C-terminal domain) region] that are not conserved between CLC proteins. We show that surface expression of these mutants is reduced in Xenopus oocytes compared with wi ...[more]

PMID: 17107341

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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:The voltage-dependent ClC-1 chloride channel, whose open probability increases with membrane potential depolarization, belongs to the superfamily of CLC channels/transporters. ClC-1 is almost exclusively expressed in skeletal muscles and is essential for stabilizing the excitability of muscle membranes. Elucidation of the molecular structures of human ClC-1 and several CLC homologs provides important insight to the gating and ion permeation mechanisms of this chloride channel. Mutations in the human CLCN1 gene, which encodes the ClC-1 channel, are associated with a hereditary skeletal muscle disease, myotonia congenita. Most disease-causing CLCN1 mutations lead to loss-of-function phenotypes in the ClC-1 channel and thus increase membrane excitability in skeletal muscles, consequently manifesting as delayed relaxations following voluntary muscle contractions in myotonic subjects. The inheritance pattern of myotonia congenita can be autosomal dominant (Thomsen type) or recessive (Becker type). To date over 200 myotonia-associated ClC-1 mutations have been identified, which are scattered throughout the entire protein sequence. The dominant inheritance pattern of some myotonia mutations may be explained by a dominant-negative effect on ClC-1 channel gating. For many other myotonia mutations, however, no clear relationship can be established between the inheritance pattern and the location of the mutation in the ClC-1 protein. Emerging evidence indicates that the effects of some mutations may entail impaired ClC-1 protein homeostasis (proteostasis). Proteostasis of membrane proteins comprises of biogenesis at the endoplasmic reticulum (ER), trafficking to the surface membrane, and protein turn-over at the plasma membrane. Maintenance of proteostasis requires the coordination of a wide variety of different molecular chaperones and protein quality control factors. A number of regulatory molecules have recently been shown to contribute to post-translational modifications of ClC-1 and play critical roles in the ER quality control, membrane trafficking, and peripheral quality control of this chloride channel. Further illumination of the mechanisms of ClC-1 proteostasis network will enhance our understanding of the molecular pathophysiology of myotonia congenita, and may also bring to light novel therapeutic targets for skeletal muscle dysfunction caused by myotonia and other pathological conditions.

Dataset Information

Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix.

Publications

Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix.

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