RESUMO
Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca(2+) from internal stores to initiate muscle contraction. Defects in excitation-contraction coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the excitation-contraction coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca(2+) imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 is the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fissura Palatina/genética , Fissura Palatina/fisiopatologia , Acoplamento Excitação-Contração , Hipertermia Maligna/genética , Hipertermia Maligna/fisiopatologia , Mutação/genética , Miotonia Congênita/genética , Miotonia Congênita/fisiopatologia , Proteínas do Tecido Nervoso/genética , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Embrião não Mamífero/metabolismo , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Miofibrilas/metabolismo , Miofibrilas/ultraestrutura , Miotonia Congênita/patologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Natação , Tato , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
Autosomal dominant sensory ataxia is a rare genetic condition that results in a progressive ataxia that is caused by degeneration of the posterior columns of the spinal cord. To date only two families have been clinically ascertained with this condition, both from Maritime Canada. We previously mapped both families to chromosome 8p12-8q12 and have now screened the majority of annotated protein-coding genes in the shared haplotype region by direct DNA sequencing. We have identified a putative pathogenic mutation in the gene encoding ring-finger protein RNF170, a potential ubiquitin ligase. This mutation is a rare non-synonymous change in a well-conserved residue and is predicted to be pathogenic by SIFT, PolyPhen, PANTHER and Align-GVD. Microinjection of wild-type or mutant orthologous messenger RNAs into zebrafish (Danio rerio) embryos confirmed that the mutation dominantly disrupts normal embryonic development. Together these results suggest that the mutation in RNF170 is causal for the sensory ataxia in these families.
Assuntos
Ataxia/genética , Mutação de Sentido Incorreto , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Animais , Ataxia/metabolismo , Morte Celular/efeitos dos fármacos , Variações do Número de Cópias de DNA , Técnicas de Silenciamento de Genes/métodos , Humanos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Peixe-ZebraRESUMO
When wild-type zebrafish embryos are touched at 24 hours post-fertilization (hpf), they typically perform two rapid alternating coils of the tail. By contrast, accordion (acc) mutants fail to coil their tails normally but contract the bilateral trunk muscles simultaneously to shorten the trunk, resulting in a pronounced dorsal bend. Electrophysiological recordings from muscles showed that the output from the central nervous system is normal in mutants, suggesting a defect in muscles is responsible. In fact, relaxation in acc muscle is significantly slower than normal. In vivo imaging of muscle Ca2+ transients revealed that cytosolic Ca2+ decay was significantly slower in acc muscle. Thus, it appears that the mutant behavior is caused by a muscle relaxation defect due to the impairment of Ca2+ re-uptake. Indeed, acc mutants carry a mutation in atp2a1 gene that encodes the sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1), a Ca2+ pump found in the muscle sarcoplasmic reticulum (SR) that is responsible for pumping Ca2+ from the cytosol back to the SR. As SERCA1 mutations in humans lead to Brody disease, an exercise-induced muscle relaxation disorder, zebrafish accordion mutants could be a useful animal model for this condition.