Mapping ligand binding pockets in chloride ClC‐1 channels through an integrated <i>in silico</i> and experimental approach using anthracene‐9‐carboxylic acid and niflumic acid

Altamura, Concetta; Mangiatordi, Giuseppe Felice; Nicolotti, Orazio; Sahbani, Dalila; Farinato, Alessandro; Leonetti, Francesco; Mr, Carratù; Conte, Diana; Desaphy, Jean-François; Imbrici, Paola

doi:10.1111/bph.14192

Cited by 15 publications

(15 citation statements)

References 56 publications

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“…The pore aperture of the extracellular vestibule is constricted by a hydrophobic barrier with Met485 (Met427 in ClC-K), but in contrast to ClC-K, the gate opening is also controlled by Lys231 (of αE–F) and Arg421 (of αL) (Fig 2B–2F) that may orchestrate Cl − permeation to or from the extracellular environment [25–27]. This difference can be attributed to αE–F, with its Glu GATE and Lys231 adopting a more CLC-transporter–like configuration because this loop is considerably shorter than in ClC-K, alongside a side-chain reorientation of Arg421 (Fig 2C–2F).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, the different gating profiles of ClC-1 and ClC-2 likely do not necessitate major structural differences. These residues are generally surface exposed and localized to the extracellular half, including the vestibule and the pore-constricting residues Lys231 and Arg421 (Fig 4B) [26, 27, 32–35]. In contrast, many dominant mutations exert a “shift” of the common gate to open probability to positive voltages, leading to significant reduction of g Cl at the physiological membrane potential [36].…”

Section: Resultsmentioning

confidence: 99%

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Structure of the human ClC-1 chloride channel

et al. 2019

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ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue (“fast gate”) known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-β-synthase (CBS) domains and the intracellular vestibule (“slow gating”). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1–related diseases.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structure of the human ClC-1 chloride channel

et al. 2019

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“…The mutations of ClC-1 associated with myotonia congenita can be correlated to anomalous gating-permeation or reduced ClC-1 protein abundance at the plasma membrane (Lee et al, 2013;Jeng et al, 2020). This distinction, together with the new and important advance in knowledge of the mechanisms behind proteostasis (Peng et al, 2016), biogenesis (Chen et al, 2015) and those governing trafficking to the surface membrane (Peng et al, 2018), has opened the doors to the novel pharmacological approach for increasing ClC-1 in plasma membrane (Altamura et al, 2018;Jeng et al, 2020). By studying the biophysical mechanism to induce an increase of ClC-1 in plasma membrane, certain inhibitors of ligase E3 (Lee et al, 2013) or blockers of ubiquitin inhibitors (Chen et al, 2015) are emerging.…”

Section: Discussionmentioning

confidence: 99%

Changes in Expression and Cellular Localization of Rat Skeletal Muscle ClC-1 Chloride Channel in Relation to Age, Myofiber Phenotype and PKC Modulation

Conte¹,

Fonzino²,

Cibelli³

et al. 2020

Front. Pharmacol.

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The ClC-1 chloride channel 1 is important for muscle function as it stabilizes resting membrane potential and helps to repolarize the membrane after action potentials. We investigated the contribution of ClC-1 to adaptation of skeletal muscles to needs induced by the different stages of life. We analyzed the ClC-1 gene and protein expression as well as mRNA levels of protein kinase C (PKC) alpha and theta involved in ClC-1 modulation, in soleus (SOL) and extensor digitorum longus (EDL) muscles of rats in all stage of life. The cellular localization of ClC-1 in relation to age was also investigated. Our data show that during muscle development ClC-1 expression differs according to phenotype. In fasttwitch EDL muscles ClC-1 expression increased 10-fold starting at 7 days up to 8 months of life. Conversely, in slow-twitch SOL muscles ClC-1 expression remained constant until 33 days of life and subsequently increased fivefold to reach the adult value. Aging induced a downregulation of gene and protein ClC-1 expression in both muscle types analyzed. The mRNA of PKC-theta revealed the same trend as ClC-1 except in old age, whereas the mRNA of PKC-alpha increased only after 2 months of age. Also, we found that the ClC-1 is localized in both membrane and cytoplasm, in fibers of 12-day-old rats, becoming perfectly localized on the membrane in 2-month-old rats. This study could represent a point of comparison helpful for the identification of accurate pharmacological strategies for all the pathological situations in which ClC-1 protein is altered.

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“…For pharmacological experiments, transfected HEK293T cells were incubated with the proteasome inhibitor MG132 (MG-132 Ready Made Solution, Sigma-Aldrich, M7449) at the final concentration of 20 μM for 16 h and then used for patch-clamp recordings. The reducing agent dithiothreitol (DTT, Pierce, Rockford, IL, USA) was added at the final concentration of 1 or 2 mM in the culture medium for cell incubation or in the extracellular patch-clamp solution for acute gravity-driven diffusion around the patched cell ( 13 ).…”

Section: Methodsmentioning

confidence: 99%

Pathomechanisms of a CLCN1 Mutation Found in a Russian Family Suffering From Becker's Myotonia

et al. 2020

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Objective: Myotonia congenita (MC) is a rare muscle disease characterized by sarcolemma over-excitability inducing skeletal muscle stiffness. It can be inherited either as an autosomal dominant (Thomsen's disease) or an autosomal recessive (Becker's disease) trait. Both types are caused by loss-of-function mutations in the CLCN1 gene, encoding for ClC-1 chloride channel. We found a ClC-1 mutation, p.G411C, identified in Russian patients who suffered from a severe form of Becker's disease. The purpose of this study was to provide a solid correlation between G411C dysfunction and clinical symptoms in the affected patient. Methods: We provide clinical and genetic information of the proband kindred. Functional studies include patch-clamp electrophysiology, biotinylation assay, western blot analysis, and confocal imaging of G411C and wild-type ClC-1 channels expressed in HEK293T cells. Results: The G411C mutation dramatically abolished chloride currents in transfected HEK cells. Biochemical experiments revealed that the majority of G411C mutant channels did not reach the plasma membrane but remained trapped in the cytoplasm. Treatment with the proteasome inhibitor MG132 reduced the degradation rate of G411C mutant channels, leading to their expression at the plasma membrane. However, despite an increase in cell surface expression, no significant chloride current was recorded in the G411C-transfected cell treated with MG132, suggesting that this mutation produces non-functional ClC-1 chloride channels. Conclusion: These results suggest that the molecular pathophysiology of G411C is linked to a reduced plasma membrane expression and biophysical dysfunction of mutant channels, likely due to a misfolding defect. Chloride current abolition confirms that the mutation is responsible for the clinical phenotype.

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Mapping ligand binding pockets in chloride ClC‐1 channels through an integrated in silico and experimental approach using anthracene‐9‐carboxylic acid and niflumic acid

Abstract: This study represents the first effort to delineate the binding sites of ClC-1. This information is fundamental to discover compounds useful in the treatment of ClC-1-associated dysfunctions and might represent a starting point for specifically targeting other CLC proteins.

Cited by 15 publications

References 56 publications

Structure of the human ClC-1 chloride channel

Structure of the human ClC-1 chloride channel

Changes in Expression and Cellular Localization of Rat Skeletal Muscle ClC-1 Chloride Channel in Relation to Age, Myofiber Phenotype and PKC Modulation

Pathomechanisms of a CLCN1 Mutation Found in a Russian Family Suffering From Becker's Myotonia

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