ClC-1 Chloride Channel: Inputs on the Structure–Function Relationship of Myotonia Congenita-Causing Mutations

Brenes, Oscar; Pusch, Michael; Morales, Fernando

doi:10.3390/biomedicines11102622

Cited by 4 publications

References 84 publications

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CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Xu,

Neelands,

Powers

et al. 2023

Preprint

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CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a ball-and-chain gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

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CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Xu,

Neelands,

Powers

et al. 2023

Preprint

View full text Add to dashboard Cite

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Case report: A CLCN1 complex variant mutation in exon 15 in a mixed-breed dog with hereditary myotonia

Eguchi,

Palumbo,

Cerri

et al. 2024

Front. Vet. Sci.

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At 4 months of age, a male dog was presented with a complaint of a stiff gait following a startle response. Neurological examination revealed no deficits, but clinical myotonia was easily induced upon requesting the patient to jump. Additionally, myotonia of the upper lip muscles was observed upon manipulation. Hereditary myotonia was suspected, and electromyography confirmed the presence of myotonic potentials. Genetic testing of the myotonic patient identified a complex of mutations, including c.[1636_1639 delins AACGGG] and c.[1644 A>T], both located in exon 15 of the CLCN1 gene leading to the formation of a premature stop codon. Genetic investigations of the mother and four littermates revealed that, except for one littermate who was wild type, all others carried a copy of the mutated gene. To the best of the authors' knowledge, these mutations have not been previously reported.

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CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism

Xu,

Neelands,

Powers

et al. 2024

eLife

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CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl−-permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl−-permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a “ball-and-chain” gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl−-permeation pathway.

show abstract

ClC-1 Chloride Channel: Inputs on the Structure–Function Relationship of Myotonia Congenita-Causing Mutations

Cited by 4 publications

References 84 publications

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Case report: A CLCN1 complex variant mutation in exon 15 in a mixed-breed dog with hereditary myotonia

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism

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