Excitation-contraction coupling, control of activator calcium release, and sequestration and redistribution of intracellular calcium, are all vital functions of cardiac SR. Defects in these functions would impair cardiac contractility and is thought to contribute to heart failure. Calsequestrin is the major SR Ca2+- binding protein in heart and is highly localized to junctional SR lumens. Despite decades of speculation concerning its role in physiological release of Ca2+, the exact function of calsequestrin remains unknown. It is known to bind to junctional face proteins junction and triadan, which are thought to couple Ca2+ binding by calsequestrin to release of activator Ca2+ through the ryanodine receptor. In addition, calsequestrin undergoes a highly specific phosphorylation within intracellular membrane compartments that persists in the mature protein; a reaction that occurs for only a select set of resident SR and ER proteins. We propose to carry out biochemical, physiological, and immunocytochemical experiments to determine calsequestrin function in heart cells, and to test the hypothesis that calsequestrin phosphorylation is part of is physiological function or its cell biological processing and targeting.
Aim 1 will investigate mechanisms of calsequestrin phosphorylation and dephosphorylation, and transduction of this signal in heart cells. These experiments will be directed at identifying protein targets of phospho-calsequestrin relative to the dephosphorylated protein using affinity-chromatographic techniques, and by localization of the cellular site of calsequestrin phosphorylation using subcellular fractionation following adenovirus infections with calsequestrin or phosphorylation-site mutants.
Aim 2 will investigate the effects of wild-type and non-phosphorylatable calsequestrin on heart cell physiology by examining effects of these protein forms on voltage and caffeine- induced Ca2+ transients, on the rate and extent of tension development, on kinetics of Ca2+ accumulation by SR vesicles, and effects of twitch on the extent of calsequestrin phosphorylation.
Aim 3 will use immunocytochemistry and electron microscopy to compare the subcellular localization of calsequestrin or phosphorylation state mutants following overexpression in heart cells. The experiments outlined in these Specific Aims investigate new areas of SR biology by investigating cell biological features that are specific to calsequestrin, reliant upon its phosphorylation, and thereby unique to the cardiac isoform and cardiac SR function.
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