The goal of this research is to understand, at the molecular level, how Ca is accumulated and released by subspecialized regions of sarcoplasmic reticulum (SR) in cardiac muscle. Five key cardiac SR proteins regulating intracellular Ca flux will be studied: phospholamban, a free SR protein regulating the Ca pump and recently shown to have Ca channel activity; the ryanodine receptor, the primary Ca release channel of junctional SR; calsequestrin, the intraluminal Ca binding protein localized to junctional SR; the 26-kDa calsequestrin-binding protein, a protein which may anchor calsequestrin to the junctional SR membrane; and 100-kDa and 90-kDa proteins, putative Ca storage proteins localized to free SR. Studies on phospholamban will address membrane protein topology and basic mechanisms of action. We will determine whether phospholamban regulates the Ca pump directly (by a protein:protein interaction) or indirectly (by altering membrane surface charge), and whether the Ca channel activity of phospholamban is necessary for Ca pump modulation. These studies will be facilitated by expressing native and mutated phospholamban in cultured atrial tumor cells. Three important regulatory sites of the cardiac ryanodine receptor will be sequenced and characterized functionally: a unique phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase; the calmodulin-binding site(s); and calpain II cleavage sites. All three classes of sites modulate channel activity, and localization and characterization of these sites will give new insight into topological domains required for channel function. In a collaborative study, cardiac calsequestrin will be crystallized and the three dimensional structure determined to localize the Ca binding sites, the Ca-regulated hydrophobic domain, and the unique C-terminal tail. The 26-kDa calsequestrin-binding protein will be purified and sequenced, in order to determine whether this protein anchors calsequestrin to the junctional face membrane. Finally, the 100-kDa and 90-kDa proteins, which may be intraluminal proteins adapted for Ca handling inside free SR, will be purified and sequenced. Comparison of these latter proteins to calsequestrin should yield new information on the significance and function of intraluminal Ca storage proteins which are localized to discrete SR regions. Completion of the studies described will increase our understanding of the role of the SR in basic mechanisms of excitation- contraction coupling in cardiac muscle.
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