Asunto(s)
Conexinas/genética , Desequilibrio de Ligamiento/genética , Epilepsia Mioclónica Juvenil/genética , Estudios de Casos y Controles , Citosina/metabolismo , Elementos de Facilitación Genéticos/genética , Exones/genética , Pruebas Genéticas/métodos , Genotipo , Haplotipos/genética , Humanos , Conformación de Ácido Nucleico , Polimorfismo de Nucleótido Simple/genética , Empalme del ARN/genética , ARN Mensajero/química , ARN Mensajero/genética , Timina/metabolismo , Proteína delta-6 de Union ComunicanteRESUMEN
Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.
Asunto(s)
Magnesio/farmacología , Hipertermia Maligna/veterinaria , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/genética , Enfermedades de los Porcinos/metabolismo , Animales , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Cafeína/farmacología , Calcio/farmacología , Halotano/farmacología , Humanos , Técnicas In Vitro , Cinética , Hipertermia Maligna/genética , Hipertermia Maligna/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Concentración Osmolar , Mutación Puntual , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Porcinos , Enfermedades de los Porcinos/genéticaRESUMEN
The inhibitory effect of myoplasmic Mg2+ on Ca2+ release from the sarcoplasmic reticulum (SR) was examined in mechanically skinned skeletal muscle fibers from pigs of different ryanodine-receptor (RyR) genotypes. In fibers from pigs homozygous for the normal RyR allele, the free Mg2+ concentration ([Mg2+]) had to be lowered from the normal resting level of 1 to approximately 0.1 mM to induce Ca2+ release and a force response. Fibers from pigs heterozygous or homozygous for the RyR allele associated with malignant hyperthermia (MH) needed only a smaller reduction in free [Mg2+] to induce Ca2+ release (reduction to 0.1-0.2 and > or = 0.2 mM, respectively). Dantrolene (20 microM) counteracted the effect of this reduced Mg2+ inhibition in MH muscle. The response of muscle fiber bundles to the caffeine-halothane contracture test in the three genotypes correlated well with the responsiveness of single fibers to reduced [Mg2+]. Thus the abnormal responsiveness of MH muscle to various stimuli may largely result from the reduced ability of myoplasmic Mg2+ to inhibit Ca2+ release from the SR.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Magnesio/farmacología , Hipertermia Maligna/genética , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Animales , Cafeína/farmacología , Dantroleno/farmacología , Predisposición Genética a la Enfermedad , Genotipo , Halotano/farmacología , Contracción Muscular , Fibras Musculares Esqueléticas/fisiología , Concentración Osmolar , Porcinos/genéticaRESUMEN
The molecular defect predisposing to the majority of malignant hyperthermia (MH) cases is unknown, although various point mutations in the ryanodine receptor gene (RYR1) have been associated with susceptibility in a small proportion of cases. We report here that one of these, the Arg163Cys substitution, does not cosegregate with MH susceptibility. Comparison of cDNA sequences encoding the skeletal muscle specific components of the dihydropyridine receptor alpha 1 subunit between MH susceptible (MHS) and MH non-susceptible (MHN) patients was made in subjects without the reported MH linked RYR1 mutations. There were no differences within the sequence encoding the II-III loop or the IS3/IS3-IS4 segment, excluding defects in these functional segments of the alpha 1 subunit as frequent causes of MH.
Asunto(s)
Canales de Calcio/genética , Hipertermia Maligna/genética , Proteínas Musculares/genética , Mutación Puntual , Secuencia de Bases , Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio Tipo L , ADN Complementario/genética , Femenino , Heterogeneidad Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático/metabolismoRESUMEN
We have cloned and sequenced the cDNA encoding triadin, a junctional terminal cisternae protein from human skeletal muscle. The cDNA, 2941 base pairs in length, encodes a protein of 729 amino acids with a predicted molecular mass of 81,545 Da. Hydropathy analysis indicates that triadin of human skeletal muscle has the same topology in the myoplasmic, transmembrane and sarcoplasmic reticulum luminal domains as that of triadin from rabbit skeletal muscle. The number and relative position of potential modulation sites are also conserved between the human and rabbit proteins. The cDNA sequence of the predicted sarcoplasmic reticulum luminal domain of human triadin diverged from that of rabbit, with an observed similarity of 82%, translating to an identity of 77% in amino acid sequence. Two insertions of 9 and 12 residues in the amino acid sequence were observed in the predicted luminal domain of triadin, although the structural and functional consequences of such insertions are expected to be minimal. Using fluorescence in situ hybridisation, we have assigned the gene encoding human triadin to the long arm of chromosome 6 in the region 6q22-6q23. Our structural analysis of human triadin supports a central role for this protein in the mechanism of skeletal muscle excitation/contraction coupling.
