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Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita

Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential a...

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Autores principales: Altamura, Concetta, Conte, Elena, Campanale, Carmen, Laghetti, Paola, Saltarella, Ilaria, Camerino, Giulia Maria, Imbrici, Paola, Desaphy, Jean-François
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9403836/
https://www.ncbi.nlm.nih.gov/pubmed/36034862
http://dx.doi.org/10.3389/fphar.2022.958196
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author Altamura, Concetta
Conte, Elena
Campanale, Carmen
Laghetti, Paola
Saltarella, Ilaria
Camerino, Giulia Maria
Imbrici, Paola
Desaphy, Jean-François
author_facet Altamura, Concetta
Conte, Elena
Campanale, Carmen
Laghetti, Paola
Saltarella, Ilaria
Camerino, Giulia Maria
Imbrici, Paola
Desaphy, Jean-François
author_sort Altamura, Concetta
collection PubMed
description Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in “gating-defective” or “expression-defective” mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 µM NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations.
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spelling pubmed-94038362022-08-26 Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita Altamura, Concetta Conte, Elena Campanale, Carmen Laghetti, Paola Saltarella, Ilaria Camerino, Giulia Maria Imbrici, Paola Desaphy, Jean-François Front Pharmacol Pharmacology Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in “gating-defective” or “expression-defective” mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 µM NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations. Frontiers Media S.A. 2022-08-11 /pmc/articles/PMC9403836/ /pubmed/36034862 http://dx.doi.org/10.3389/fphar.2022.958196 Text en Copyright © 2022 Altamura, Conte, Campanale, Laghetti, Saltarella, Camerino, Imbrici and Desaphy. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Pharmacology
Altamura, Concetta
Conte, Elena
Campanale, Carmen
Laghetti, Paola
Saltarella, Ilaria
Camerino, Giulia Maria
Imbrici, Paola
Desaphy, Jean-François
Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita
title Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita
title_full Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita
title_fullStr Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita
title_full_unstemmed Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita
title_short Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita
title_sort chaperone activity of niflumic acid on clc-1 chloride channel mutants causing myotonia congenita
topic Pharmacology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9403836/
https://www.ncbi.nlm.nih.gov/pubmed/36034862
http://dx.doi.org/10.3389/fphar.2022.958196
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