Ca2+ channels of intracellular Ca2+ stores, such as ryanodine and inositol 1, 4, 5 trisphosphate (IP3) receptors, are ultimately responsible for the changes in cytosolic Ca2+ observed in cells stimulated by voltage, neurotransmitters, and hormones. The kinetics of activation and inactivation of intracellular Ca2+ channels is likely also to be responsible for the ubiquitous intracellular Ca2+ release mechanism known as Ca2+- induced Ca2+ release (CICR). The proposal focuses on the molecular properties of ryanodine receptors and IP3 receptors of cardiac and skeletal muscle cells with the interest in understanding the contribution of these channels to excitation-contraction (EC) coupling. The objectives of the proposal are to study 1) the kinetics of the Ca2+- dependent activation and possibly, inactivation of ryanodine receptors. This will establish the ionic conditions under which ryanodine receptors may mediate CICR in the cell; 2) the defects in Ca2+ binding sites of ryanodine receptors in the porcine model of Malignant Hyperthermia which is a genetic disorder affecting ryanodine receptors. This is a promising model that may help us to understand the structural and kinetic basis of Ca2+-dependent activation and inactivation; 3) the size of the Ca2+ pools controlled by, and rates of Ca2+ release mediated by, ryanodine and IP3 receptors in cardiac and skeletal muscle. This is important given that both types of channels may serve to increase the Ca2+ permeability of the sarcoplasmic reticulum; and 4) the block or activation of ryanodine receptors by scorpion toxins. This will establish if toxins could be used to dissect the contribution of ryanodine receptors to EC coupling. The four goals are drawn from the experience gained in the previous funding period on the functional reconstitution of Ca2+ channels, of intracellular and surface origin, using a combination of 45Ca2+ fluxes, protein purification, and planar bilayer recording. The strength of the proposal resides in the resolving power of these techniques when used within a quantitative framework. The experiments are essential for understanding the functional properties of intracellular Ca2+ channels and the kinetic restrictions imposed on these channels by the intracellular environment. The proposal should provide new information regarding the mechanisms of Ca2+ release from intracellular stores operative in striated muscle cells and provide rigorous tests of novel hypothesis concerning Ca2+ release triggered by Ca2+ and IP3.
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