Characterization of Two Human Skeletal Calsequestrin Mutants Implicated in Malignant Hyperthermia and Vaculolar Aggreagtes Myopathy

Abstract

Calsequestrin 1 (Casq1) is the principal Ca2+ storage protein of the sarcoplasmic reticulum of skeletal muscle. Its inheritable D244G mutation causes a myopathy with vacuolar aggregates and muscle weakness (Rossi et al 2014), while its M87T “variant” (Kraeva et al 2013) is weakly associated with malignant hyperthermia (MH). We characterized the consequences of these mutations with studies of the human proteins in vitro. In the WT, studied by equilibrium dialysis, Ca2+ binding features a second stage of high capacity, which occurs at high [Ca2+]. For both mutants, but especially D244G, this stage was smaller in capacity and required higher [Ca2+]. Turbidity measurements showed that D244G aggregates abruptly and abnormally as [Ca2+] increases, while M87T aggregates very weakly. This property fully explains the protein inclusions that characterize the disease phenotype of D244G and suggests that polymerization of M87G is defective. Crystals of D244G grown in low Ca2+ concentrations lack two Ca2+ ions normally present in WT. This weakens the hydrophobic core of homology Domain II in the folded molecule. D244G crystallized in high Ca2+ concentrations regains its missing ions and Domain II order, but shows a dimeric interaction not present in WT. The crystals of M87T show a major shift of the α-helix bearing the mutated residue. If a similar shift occurred in vivo, it would significantly weaken the back-to-back interface essential for tetramerization-the second stage of polymerization. D244G exhibited the more severe structural and biophysical property changes, which matches the higher pathophysiological impact of this mutation. Supported by NIH grant GM111254 to ER and CHK.