title: The calsequestrin mutation CASQ2D307H does not affect protein stability and targeting to the junctional sarcoplasmic reticulum but compromises its dynamic regulation of calcium buffering.
abstract: Mutations in cardiac ryanodine receptor (RYR2) and cardiac calsequestrin (CASQ2) genes are linked to catecholaminergic polymorphic ventricular tachycardia, a life-threatening genetic disease. They predispose young individuals to cardiac arrhythmia in the absence of structural abnormalities. One such mutation that changes an aspartic residue to histidine at position 307 in CASQ2 has been linked to catecholaminergic polymorphic ventricular tachycardia. In this study we made a transgenic mouse model expressing the mutant CASQ2(D307H) protein in a CASQ2 null background and investigated if the disease is caused by accelerated degradation of the mutant protein. Our data suggest that the mutant protein can be expressed, is relatively stable, and targets appropriately to the junctional sarcoplasmic reticulum. Moreover, it partially normalizes the ultrastructure of the sarcoplasmic reticulum, which was altered in the CASQ2 null background. In addition, overexpression of the mutant protein does not cause any pathology and/or structural changes in the myocardium. We further demonstrate, using purified protein, that the mutant protein is very stable under chemical and thermal denaturation but shows abnormal Ca(2+) buffering characteristics at high calcium concentrations. In addition, trypsin digestion studies reveal that the mutant protein is more susceptible to protease activity only in the presence of high Ca(2+). These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca(2+) activation.
authors.affiliation: Department of Physiology and Cell Biology, The Ohio State University College of Medicine, Columbus, Ohio 43210, USA.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Kalyanasundaram
authors.forename: Naresh C
authors.initials: NC
authors.lastname: Bal
authors.forename: Clara
authors.initials: C
authors.lastname: Franzini-Armstrong
authors.forename: Björn C
authors.initials: BC
authors.lastname: Knollmann
authors.forename: Muthu
authors.initials: M
authors.lastname: Periasamy
mesh_terms: D000818:Animals; D002021:Buffers; D002118:Calcium / Q000737:chemistry*; D002155:Calsequestrin / Q000235:genetics* / Q000502:physiology; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal / Q000379:methods; D009154:Mutation; D009206:Myocardium / Q000473:pathology; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D017180:Tachycardia, Ventricular / Q000378:metabolism
chemical_list: D002021:Buffers; D002155:Calsequestrin; D011506:Proteins; D002118:Calcium