Structural and functional analysis of the ryanodine receptor: Multiple conductance states regulated by allosteric cation interactions and the identification of hyperreactive sulfhydryls

摘要

Ryanodine receptors (RyRs) are large (∼2.3 MDa) homotetrameric Ca 2+ release channels of endo/sarcoplasmic reticulum (ER/SR), expressed throughout the body as three isoforms (RyR1--3). RyR mediated increases in cytosolic Ca2+ are a critical component of many intracellular signaling cascades. Thus, cellular homeostasis requires tight, mulit-faceted regulation of RyRs. The focus of this dissertation is a functional analysis of RyR regulation by Ca2+ and Mg2+ using RyR3/RyR1 chimeras and a structural identification of redox-sensing, hyperreactive sulfhydryls.;Ca2+ and Mg2+ are important endogenous modulators of the RyR. Ca2+ activation and inhibition likely occur through multiple coordinated sites: muM activation through high affinity (H) sites and mM inhibition through low affinity (L) sites. Mg2+ inhibition is thought to occur through competition with Ca2+ for H sites and binding at L sites. In this dissertation, biochemical and biophysical analyses of RyR3/RyR1 chimeras reveal previously unreported allosteric interactions between cation activation and inhibition sites, which regulate multiple conductance states.;RyR1 has long been known to undergo redox modulation, with initial studies focusing on oxidative damage evolving to include subtle and generally reversible redox modulations associated with intracellular signaling. Recent reports indicate the RyR undergoes covalent adduction by nitric oxide (NO), redox-induced shifts in cation regulation, and noncovalent interactions driven by the transmembrane redox potential that enable redox sensing. These discrete, usually reversible RyR modulations are thought to be mediated by 6--8 hyperreactive sulfhydryls. Only cys-3635, considered the exclusive NO binding site, has previously been identified. This work outlines the development and successful application of a mass spectrometric methodology to identify 7 hyperreactive redox-sensing sulfhydryls of RyR1.;The structural and functional studies presented here advance the understanding of RyR regulation by Ca2+, Mg2+ and provide the primary sequence location of redox-sensitive cysteines. The chimeric analysis introduces a novel allosteric and multi-state model of RyR regulation by cations. The identification of redox-sensing sulfhydryls introduces a mass spectrometric assay for reactive cysteines and provides the locations for point mutational analysis of redox regulation. The new RyR allosteric model and cysteine identities established here serve as a foundation for future investigations of RyR regulation.