title: ClC-1 mutations in myotonia congenita patients: insights into molecular gating mechanisms and genotype-phenotype correlation.
abstract: CONCLUSIONS
  Loss-of-function mutations of the skeletal muscle ClC-1 channel cause myotonia congenita with variable phenotypes. Using patch clamp we show that F484L, located in the conducting pore, probably induces mild dominant myotonia by right-shifting the slow gating of ClC-1 channel, without exerting a dominant-negative effect on the wild-type (WT) subunit. Molecular dynamics simulations suggest that F484L affects the slow gate by increasing the frequency and the stability of H-bond formation between E232 in helix F and Y578 in helix R. Three other myotonic ClC-1 mutations are shown to produce distinct effects on channel function: L198P shifts the slow gate to positive potentials, V640G reduces channel activity, while L628P displays a WT-like behaviour (electrophysiology data only). Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function.
  Myotonia congenita is an inherited disease caused by loss-of-function mutations of the skeletal muscle ClC-1 chloride channel, characterized by impaired muscle relaxation after contraction and stiffness. In the present study, we provided an in-depth characterization of F484L, a mutation previously identified in dominant myotonia, in order to define the genotype-phenotype correlation, and to elucidate the contribution of this pore residue to the mechanisms of ClC-1 gating. Patch-clamp recordings showed that F484L reduced chloride currents at every tested potential and dramatically right-shifted the voltage dependence of slow gating, thus contributing to the mild clinical phenotype of affected heterozygote carriers. Unlike dominant mutations located at the dimer interface, no dominant-negative effect was observed when F484L mutant subunits were co-expressed with wild type. Molecular dynamics simulations further revealed that F484L affected the slow gate by increasing the frequency and stability of the H-bond formation between the pore residue E232 and the R helix residue Y578. In addition, using patch-clamp electrophysiology, we characterized three other myotonic ClC-1 mutations. We proved that the dominant L198P mutation in the channel pore also right-shifted the voltage dependence of slow gating, recapitulating mild myotonia. The recessive V640G mutant drastically reduced channel function, which probably accounts for myotonia. In contrast, the recessive L628P mutant produced currents very similar to wild type, suggesting that the occurrence of the compound truncating mutation (Q812X) or other muscle-specific mechanisms accounted for the severe symptoms observed in this family. Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function.
authors.affiliation: Department of Pharmacy - Drug Sciences, University of Bari, Bari, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Imbrici
authors.affiliation: Neuroimmunology and Neuromuscular Diseases Unit, IRCCS Fondazione Istituto Neurologico 'Carlo Besta', Milano, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Maggi
authors.affiliation: Department of Pharmacy - Drug Sciences, University of Bari, Bari, Italy.
authors.forename: G F
authors.initials: GF
authors.lastname: Mangiatordi
authors.affiliation: Department of Pharmacy - Drug Sciences, University of Bari, Bari, Italy.
authors.forename: M M
authors.initials: MM
authors.lastname: Dinardo
authors.affiliation: Department of Pharmacy - Drug Sciences, University of Bari, Bari, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Altamura
authors.affiliation: Neuroimmunology and Neuromuscular Diseases Unit, IRCCS Fondazione Istituto Neurologico 'Carlo Besta', Milano, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Brugnoni
authors.affiliation: Department of Physics 'M. Merlin', INFN and TIRES, University of Bari, Bari, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Alberga
authors.affiliation: Neuroalgology and Headache Unit, IRCCS Fondazione Istituto Neurologico 'Carlo Besta', Milano, Italy.
authors.forename: G Lauria
authors.initials: GL
authors.lastname: Pinter
authors.affiliation: Department of Clinical and Experimental Medicine, Section of Neurology, University of Pisa, Pisa, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Ricci
authors.affiliation: Department of Clinical and Experimental Medicine, Section of Neurology, University of Pisa, Pisa, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Siciliano
authors.affiliation: Unit of Child Neurology and Psychiatry, Spedali Civili, Brescia, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Micheli
authors.affiliation: Regional Coordination for Rare Diseases, A. Re. S. Puglia, Bari, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Annicchiarico
authors.affiliation: Department of Physics 'M. Merlin', INFN and TIRES, University of Bari, Bari, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Lattanzi
authors.affiliation: Department of Pharmacy - Drug Sciences, University of Bari, Bari, Italy.
authors.forename: O
authors.initials: O
authors.lastname: Nicolotti
authors.affiliation: Neuroimmunology and Neuromuscular Diseases Unit, IRCCS Fondazione Istituto Neurologico 'Carlo Besta', Milano, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Morandi
authors.affiliation: Neuroimmunology and Neuromuscular Diseases Unit, IRCCS Fondazione Istituto Neurologico 'Carlo Besta', Milano, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Bernasconi
authors.affiliation: Department of Pharmacy - Drug Sciences, University of Bari, Bari, Italy.
authors.forename: J-F
authors.initials: JF
authors.lastname: Desaphy
authors.affiliation: Neuroimmunology and Neuromuscular Diseases Unit, IRCCS Fondazione Istituto Neurologico 'Carlo Besta', Milano, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Mantegazza
authors.affiliation: Department of Pharmacy - Drug Sciences, University of Bari, Bari, Italy.
authors.forename: D Conte
authors.initials: DC
authors.lastname: Camerino
mesh_terms: D000328:Adult; D000368:Aged; D002648:Child; D018118:Chloride Channels / Q000235:genetics*; D005260:Female; D056726:Genetic Association Studies / Q000379:methods; D006579:Heterozygote; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics; D008297:Male; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009224:Myotonia Congenita / Q000235:genetics*; D055815:Young Adult
chemical_list: C086321:CLC-1 channel; D018118:Chloride Channels