Role of Calsequestrin in Ca2+ Storage & Cardiac Disease

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DESCRIPTION (provided by applicant): The studies I propose in this grant will dissect the role of the C-terminus of CSQ in Ca2+ buffering. CSQ is a Ca2+ storage protein in the junctional sarcoplasmic reticulum (jSR) that plays an important in Ca2+ handling during excitation-contraction coupling. The specific structural elements that mediate Ca2+ buffering are unknown. For specific purposes of this research proposal, my first specific aim is to establish whether the clusters of aspartate residues present in the C-terminal region have an active role in Ca2+ buffering capacity of CSQ. To accomplish this, mutant CSQ where its aspartate clusters present in the C-terminus will be mutated to alanine and expressed in cultured rat cardiac myocytes by adenoviral gene transfer. After expression of these mutants, I will assess the impact they have on the SR Ca2+ stores by measuring Ca2+ transients and SR Ca2+ load. Second, I want to determine how a naturally occurring mutation found in CSQ affects Ca2+ buffering in vitro and in vivo. The mutation is a D307->H amino acid change and was correlated with Chatecolaminergic Polymorphic Ventricular Tachycardia (CPVT). To determine whether the mutation affect Ca2+ buffering, I will generate the D307->H mutation and express it in cultured rat cardiac myocytes by adenoviral gene transfer. The effect on Ca2+ buffering will be measured in vivo by measuring Ca2+ transients and SR Ca2+ load, which will help to determine if the mutant CSQ has indeed a lower Ca2+ buffering capacity. In vitro, the Ca2+ buffering capacity of the D307->H CSQ mutant will be determined using the previously established equilibrium dialysis technique. I believe that a detailed study of Ca2+ buffering will provide new and useful insights in Ca2+ handling and the role of CSQ in excitation-contraction coupling.