Calsequestrin 1 (CASQ1) is the main Ca2+ binding protein of the sarcoplasmic reticulum (SR) of skeletal muscle fibers. We identified in patients with a vacuolar myopathy characterized by distinctive inclusions in muscle fibers a mutation in the CASQ1 gene, which results in a substitution of a conserved aspartic acid in position 244 to a glycine (D244G). This mutation represents the first mutation in CASQ1 linked to an inherited human muscle disease. The affected aspartic acid is located in a high-affinity Ca2+ binding site, close to a region of the protein proposed to be relevant for interactions between adjacent monomers. Analysis of the predicted three-dimensional structure of the mutated CASQ1 revealed a conformational change in the affected Ca2+ binding site. Analysis of muscle biopsies from patients revealed, beside the presence of inclusions composed of SR proteins aggregates, altered Ca2+ release kinetics. Expression of the mutated protein in COS7 cells showed a reduced ability of CASQ1 in forming rod-shape polymers, whereas in cultured myotubes induced the formation of protein aggregates positive for CASQ1. In-vivo electroporation of mouse muscle fibers of the mutated CASQ1 resulted in formation of inclusions resembling those observed in biopsies from patients. In addition, transfection of COS7 cells with the mutated CASQ1 revealed a reduction of Ca2+ binding ability when compared with cells transfected with the wild type CASQ1 protein. Altogether, these data describe molecular and functional abnormalities of the mutated protein, which support the view that this mutation is responsible for the clinical phenotype observed in patients.
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