The Calsequestrin Mutation CASQ2D307H Does Not Affect Protein Stability and Targeting to the Junctional Sarcoplasmic Reticulum but Compromises Its Dynamic Regulation of Calcium Buffering*

  1. Muthu Periasamy‡,1
  1. From the ‡Department of Physiology and Cell Biology, The Ohio State University College of Medicine, Columbus, Ohio 43210,
  2. the §Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and
  3. the Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, Tennessee 37232
  1. 1 To whom correspondence should be addressed: 304 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Fax: 614-292-4888; E-mail: periasamy.1{at}osu.edu.

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 CASQ2D307H 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 Ca2+ 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 Ca2+. These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca2+ activation.

Footnotes

  • ↵* This work was supported, in whole or in part, by National Institutes of Health Grant R0-1 HL64014 (to M. P.) and an American Heart Association pre-doctoral fellowship (to A. K.).

  • ↵3 A. Kalyanasundaram, N. C. Bal, C. Franzini Armstrong, B. C. Knollmann, and M. Periasamy, unpublished data.

  • ↵2 The abbreviations used are:

    CASQ
    calsequestrin
    CPVT
    catecholaminergic polymorphic ventricular tachycardia
    GdnHCl
    guanidine hydrochloride
    TPCK
    l-1-tosylamido-2-phenylethyl chloromethyl ketone
    CD
    circular dichroism
    SR
    sarcoplasmic reticulum
    jSR
    junctional sarcoplasmic reticulum
    WT
    wild type
    ER
    endoplasmic reticulum.

    • Received August 7, 2009.
    • Revision received November 12, 2009.
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  1. First Published on November 17, 2009 doi: 10.1074/jbc.M109.053892 The Journal of Biological Chemistry 285, 3076-3083.
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