The objective of this proposal is to determine the molecular properties of a Ca2+ release channel in sarcoplasmic reticulum of muscle. The Ca2+ release channel in isolated sarcoplasmic reticulm vesicles is capable of mediating Ca2+ fluxes with a physiological rate suggesting that it plays an important role in the process of excitation-contraction coupling in muscle. Sedimentation and equilibrium centrifugation techniques will be used to isolate the following subfractions from rabbit skeletal muscle: """"""""heavy"""""""" sarcoplasmic reticulum vesicles containing the CA2+ release channel, """"""""light"""""""" sarcoplasmic reticulum vesicles lacking the Ca2+ release channel, surface membranes (T-system and plasmalemma), and triads (sarcoplasmic reticulum/T-system junctional complexes). Monoclonal antibodies to enriched triad fractions and sarcoplasmic reticulum vesicles containing the Ca2+ release channel will be generated in order to localize and establish the function of specific membrane components in excitation-contraction coupling. The mechanism of regulation of the Ca2+ release channel by Ca2+, Mg2+ adenine nucleotides, calmodulin, and other factors will be determined. The C2+ release channel of sarcoplasmic reticulum will be incorporated into planar lipid bilayers so that single channel conductance, ion selectivity and voltage-dependence, as well as the kinetics of channel opening and closing can be investigated. Ca2+ efflux from sarcoplasmic reticulum vesicles will be measured, using rapid quench techniques. T-system depolarization-induced Ca2+ release from the sarcoplasmic reticulum compartment will be measured using isolated triads in order to determine how a muscle action potential at the cell surface induces release of Ca2+, and thereby muscle contraction. In addition, affinity labeling techniques and/or monoclonal antibodies will be used to identify, purify and reconstitute the channel protein(s).

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
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Physiology Study Section (PHY)
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Xu, Le; Mowrey, David D; Chirasani, Venkat R et al. (2018) G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to cytosolic and luminal Ca2. J Biol Chem 293:2015-2028
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Manno, Carlo; Figueroa, Lourdes; Royer, Leandro et al. (2013) Altered Ca2+ concentration, permeability and buffering in the myofibre Ca2+ store of a mouse model of malignant hyperthermia. J Physiol 591:4439-57
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