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).
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