description): The goal of the proposed research is to identify the key regions within the primary sequence of the Ca2+ release channel (ryanodine receptor; RyR1) of mammalian skeletal muscle sarcoplasmic reticulum (SR) which determine (1) sensitivity to physiologic cation activators and inhibitors, and (2) the ability of the channel to sense a physiologic redox gradient. The investigators will address these problems using biochemical and molecular approaches which take advantage of their ability to express mutations of RyR1 within the environmental context of an RyR null muscle cell. This model maintains the essential protein-protein interactions present within triadic junctions of skeletal muscle, which are critical to native channel function, thereby permitting valid and direct correlation between structure and function at cellular and subcellular levels. Hypothesis I: Structural determinants of calcium activation and inactivation correlate with not only changes in binding affinity, but also free energy associated in channel gating transitions.
Specific Aim 1. Define primary sequence in RyR1 that determines sensitivity to Ca2+ and Mg2+ using site-specific mutations and RyR1/RyR3 chimera.
Specific Aim 2. Define the relative change in binding affinity and free energy associated with Ca2+-mediated channel activation for wild-type RyR1 and RyR1s possessing mutations which affect apparent sensitivity to cations and allosteric ligands.
Specific Aim 3. Define the changes in ligand binding constants and free energy associated with mutations of RyR1 both within putative Ca2+ binding motifs and outside the Ca2+ binding motifs in regions known to affect channel calcium sensitivity. Hypothesis II: Hyper-reactive sulfhydryl moieties in RyRs constitute an important element of trans-SR redox gradient sensor.
Specific Aim 1. Define the redox sensing properties of RyR1 and RyR2.
Specific Aim 2. Identify the primary sequence locations of hyper-reactive thiols in RyR1 and RyR2 essential for redox gradient sensing function.
Specific Aim 3. Mutate the hyper-reactive thiols within cytoplasmic and luminal domains of RyR1 and define their influence on redox sensing behavior.
Specific Aim 4. Define changes in susceptibility to oxidative insult in myotubes expressing mutated RyRs lacking redox sensing.
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