The muscle specific chloride channel ClC-1 and myotonia congenita in Northern Finland

Abstract Functional defects in the muscle specific chloride channel ClC-1 result in reduced chloride conductance and electrical hyperexcitability, which in turn impairs muscle relaxation and leads to myotonia. The gene CLCN 1 codes for ClC-1 in humans, and mutations in CLCN 1 cause the disease known as myotonia congenita. Worldwide over 80 mutations in CLCN1 have been described, but only three were found in patients in Northern Finland. These included two missense mutations and a nonsense mutation. The behavior and localization of the normal and mutated ClC-1 mRNA and protein were analyzed in muscle cell cultures. In intact muscle the ClC-1 protein was seen in the sarcolemma, but after myofiber isolation the protein was located intracellularly. Sarcolemmal localization was restored when myofibers were electrically stimulated or treated with a protein kinase C inhibitor. When mutated ClC-1 proteins were examined in a myofiber cell culture system, retardation in the ER was observed with the two missense mutations. The nonsense mutation did not have an effect on the transport from the ER to the Golgi elements, but the mutated ClC-1 was degraded more rapidly than the wild type ClC-1, at least in myotubes. Both retardation and degradation of the mutated ClC-1 are likely to result in too few channels present at the plasma membrane of the muscle cell to maintain normal physiological function. A very strict quality control in muscle cells was observed. The behavior and survival of multinuclear skeletal muscle cells is dependent on innervation and muscle activity, and the balance between the phosphorylation and dephosphorylation pathways modulates the function of muscle chloride channels.Tiivistelmä Lihasspesifisen kloridikanavan ClC-1:n toiminnalliset virheet johtavat alentuneeseen kloridin johtumiseen solukalvon läpi ja lihassolun ylieksitoitumiseen. Tämän seurauksena lihaksen rentoutuminen vaikeutuu ja havaitaan myotoniaa, lihasjäykkyyttä. Pohjoissuomalaisesta potilasmateriaalista tautiin johtavia geenimutaatioita löytyi kolme erilaista. Poikkeuksellista havainnoissa on erilaisten mutaatioiden vähyys, mikä on tyypillistä suomalaiselle tautiperinnölle. Yhteensä tämän kloridikanavan mutaatioita on julkaistu yli 80 erilaista. Tutkiessamme normaalin ja mutatoidun ClC-1 lRNA:n ja proteiinin käyttäytymistä ja sijaintia lihassoluviljelmissä. Havaitsimme eron lihasleikkeiden ja eristettyjen myofiibereiden välillä. Lihasleikkeissä ClC-1 paikantui solun pinnalle sarkolemmalle, mutta eristetyissä myofiibereissä lähinnä solun sisälle. Stimuloimalla eristettyjä myofiibereitä sähkövirralla tai käsittelemällä proteiini kinaasi C inhibiittorilla, saimme kloridikanava-proteiinin siirtymään takaisin solun pinnalle. Proteiinitasolla kuljetuksessa on havaittavissa eroja. Aminohappomuutokseen johtavat pistemutaatiot aiheuttivat proteiinin jäämisen endoplasmiseen kalvostoon, kun taas ennenaikaisen stop-kodonin johdosta lyhentynyt proteiini kuljetetaan eteenpäin Golgin laitteeseen. Myotuubeissa tämä lyhentynyt proteiini kuitenkin hajotettiin nopeammin kuin normaali kloridikanavaproteiini. Sekä kuljetuksen hidastuminen että nopeampi hajotus johtavat tilanteeseen, jossa lihassolun solukalvolla on liian vähän kloridikanavia ylläpitämään lihaksen normaalia fysiologista toimintaa. Monitumaisten lihassolujen laaduntarkkailu havaittiin vielä monitahoisemmaksi kuin yksitumaisilla. Monitumainen lihassolu on riippuvainen hermoärsytyksestä ja lihasaktiivisuudesta. Lisäksi fosforylaatioon liittyvä signalointi on tärkeää ClC-1 proteiinin oikealle paikantumiselle lihassolussa

Functional complementation of truncated human skeletal-muscle chloride channel (hCIC-1) using carboxyl tail fragments

Copyright © 2006 The Biochemical SocietyCrystal structures of bacterial CLC (voltage-gated chloride channel family) proteins suggest the arrangement of permeation pores and possible gates in the transmembrane region of eukaryotic CLC channels. For the extensive cytoplasmic tails of eukaryotic CLC family members, however, there are no equivalent structural predictions. Truncations of cytoplasmic tails in different places or point mutations result in loss of function or altered gating of several members of the CLC family, suggesting functional importance. In the present study, we show that deletion of the terminal 100 amino acids (N889X) in human ClC-1 (skeletal-muscle chloride channel) has minor consequences, whereas truncation by 110 or more amino acids (from Q879X) destroys channel function. Use of the split channel strategy, co-injecting mRNAs and expressing various complementary constructs in Xenopus oocytes, confirms the importance of the Gln879–Arg888 sequence. A split between the two CBS (cystathionine b-synthase) domains (CBS1 and CBS2) gives normal function (e.g. G721X plus its complement), whereas a partial complementation, eliminating the CBS1 domain, eliminates function. Surprisingly, function is retained even when the region Gly721–Ala862 (between CBS1 and CBS2, and including most of the CBS2 domain) is omitted from the complementation. Furthermore, even shorter peptides from the CBS2-immediate post-CBS2 region are sufficient for functional complementation. We have found that just 26 amino acids from Leu863 to Arg888 are necessary since channel function is restored by co-expressing this peptide with the otherwise inactive truncation, G721X.Weiping Wu, Grigori Y. Rychkov, Bernard P. Hughes and Allan H. Breta

In tandem analysis of CLCN1 and SCN4A greatly enhances mutation detection in families with non-dystrophic myotonia.

Contains fulltext : 69798.pdf (publisher's version ) (Closed access)Non-dystrophic myotonias (NDMs) are caused by mutations in CLCN1 or SCN4A. The purpose of the present study was to optimize the genetic characterization of NDM in The Netherlands by analysing CLCN1 and SCN4A in tandem. All Dutch consultant neurologists and the Dutch Patient Association for Neuromuscular Diseases (Vereniging Spierziekten Nederland) were requested to refer patients with an initial diagnosis of NDM for clinical assessment and subsequent genetic analysis over a full year. Based on clinical criteria, sequencing of either CLCN1 or SCN4A was performed. When previously described mutations or novel mutations were identified in the first gene under study, the second gene was not sequenced. If no mutations were detected in the first gene, the second gene was subsequently also analysed. Underlying NDM mutations were explored in 54 families. In total, 20% (8 of 40) of our probands with suspected chloride channel myotonia showed no CLCN1 mutations but subsequent SCN4A screening revealed mutations in all of them. All 14 probands in whom SCN4A was primarily sequenced showed a mutation. In total, CLCN1 mutations were identified in 32 families (59%) and SCN4A in 22 (41%), resulting in a diagnostic yield of 100%. The yield of mutation detection was 93% with three recessive and three sporadic cases not yielding a second mutation. Among these mutations, 13 in CLCN1 and 3 in SCN4A were novel. In conclusion, the current results show that in tandem analysis of CLCN1 and SCN4A affords high-level mutation ascertainment in families with NDM