Functional and Structural Characterization of ClC-1 and Nav1.4 Channels Resulting from CLCN1 and SCN4A Mutations Identified Alone and Coexisting in Myotonic Patients.

Oscar Brenes,Rebeca Vindas-Smith,Melissa Vásquez,Gerardo Del Valle,Luis Bermúdez-Guzmán,Fernando Morales,Jeffrey Roig,Michael Pusch,Sara Bertelli,Raffaella Barbieri,Adarli Romero

doi:10.3390/cells10020374

Abstract

Non-dystrophic myotonias have been linked to loss-of-function mutations in the ClC-1 chloride channel or gain-of-function mutations in the Nav1.4 sodium channel. Here, we describe a family with members diagnosed with Thomsen’s disease. One novel mutation (p.W322*) in CLCN1 and one undescribed mutation (p.R1463H) in SCN4A are segregating in this family. The CLCN1-p.W322* was also found in an unrelated family, in compound heterozygosity with the known CLCN1-p.G355R mutation. One reported mutation, SCN4A-p.T1313M, was found in a third family. Both CLCN1 mutations exhibited loss-of-function: CLCN1-p.W322* probably leads to a non-viable truncated protein; for CLCN1-p.G355R, we predict structural damage, triggering important steric clashes. The SCN4A-p.R1463H produced a positive shift in the steady-state inactivation increasing window currents and a faster recovery from inactivation. These gain-of-function effects are probably due to a disruption of interaction R1463-D1356, which destabilizes the voltage sensor domain (VSD) IV and increases the flexibility of the S4-S5 linker. Finally, modelling suggested that the p.T1313M induces a strong decrease in protein flexibility on the III-IV linker. This study demonstrates that CLCN1-p.W322* and SCN4A-p.R1463H mutations can act alone or in combination as inducers of myotonia. Their co-segregation highlights the necessity for carrying out deep genetic analysis to provide accurate genetic counseling and management of patients.

Highlights

Non-dystrophic myotonias (NDM) are a group of hereditary muscle diseases characterized by myotonia, muscle stiffness and a non-dystrophic phenotype [1]
All family members clinically evaluated at that time (NDM1, NDM2, NDM4-10 and NDM15) were affected with DMC (Figure 1) [17]
It has been widely recognized that NDMs are not easy to distinguish, clinically or genetically, mainly due to the phenotypic overlap among these diseases and the great number of mutations with different effects, inheritance patterns and associated phenotypes [3]

Summary

Introduction

Non-dystrophic myotonias (NDM) are a group of hereditary muscle diseases characterized by myotonia, muscle stiffness and a non-dystrophic phenotype [1]. Mutations in the CLCN1 and SCN4A voltage-gated ion channel genes, encoding the skeletal muscle chloride (ClC-1) and sodium α-subunit (Nav1.4) channels, respectively, are responsible for these diseases [6,7]. A particular advantage of Xenopus oocytes compared to transfection-based approaches is that the single cell injection allows to control the stoichiometric amount of RNA delivered. This allows for a straightforward quantification of effects of mutants on current densities, comparing WT and mutant in the same batch of oocytes. This is even more important for multimeric channel complexes, for example, for dimeric ClC channels and transporters. Heterozygosity of mutant/WT can be quantitatively reproduced and possible dominant effects of mutants in the dimeric complex can be investigated [9,12,13]

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Results

Conclusion