Communication between neighboring ryanodine receptor channels in skeletal muscle

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The goal of this project is to understand the coupled function of skeletal muscle ryanodine receptor channels (RyRs). The RyRs are intracellular calcium release channels located in the membranes of intracellular calcium stores, where they form orderly arrays. After cell stimuli, groups of RyRs briefly open and rapidly release to the cytosol the bulk of calcium required for the twitch muscle contraction. The characteristics of these calcium release events strongly indicate that the participating RyRs work in synchrony and they all activate and deactivate together within few milliseconds. It is still unknown how RyRVs communicate for synchronizing their gating in cells. Consequently, it is difficult to understand the genesis of anomalous excitation-contraction coupling as found in various muscle diseases, including those (Malignant Hyperthermia, Central Core Disease) where mutations in the genes encoding RyR1 have been identified. As RyRs are intracellular channels, electrophysiological studies require cell subfractionation, channel isolation and reconstitution into artificial lipid bilayers. Nearly all reports from these bilayers studies describe a single (individual) RyR1 or independent behavior of multiple RyRs. Only Dr. A. Marks's laboratory (Marx et al, 1998; 2000) described neighboring RyRs (2-4 channels) that gated with synchronous coordination if FK506 binding protein (FKBP) molecules were associated with the channels. This process, new for the field of ion channels, was named "coupled gating". Despite its importance to understand calcium release, little else is known about this inter-RyR1 communication. During the last years, the PI has developed methodologies and accumulated preliminary data that clearly evidence the feasibility of studying channels coupling with his bilayer system. Consequently, the goal of this proposal is to define the mechanisms of communication between RyR channels for coupled gating. The specific aims of this proposal is: Specific Aim #1: Define the mechanisms of communication between RyR1 channels. This aim is subdivided in three sub-Aims: A) Define the role of ATP/Mg2+ in RyR1 coordination; B) Define the role of Ca2+ in coordinated gating; and C) Define the role of FKBP12 (Calstabilinl) and other ancillary proteins.