The objective of this proposal is to determine the functional properties of a ligand-gated """"""""Ca2+ release"""""""" channel in cardiac sarcoplasmic reticulum (SR). In isolated sarcoplasmic reticulum membrane fractions, this channel mediates Ca2+ fluxes with rates sufficiently rapid to suggest a central role in the process of excitation-contraction coupling. Sedimentation and equilibrium centrifugation techniques will be used to isolate """"""""Ca2+ release"""""""" vesicles containing the cardiac SR Ca2+ release channel, """"""""control"""""""" SR vesicles lacking the Ca2+ release channel, as well as junctional complexes composed of SR and surface membranes. Regulation of the cardiac SR Ca2+ release channel by Ca2+, Mg2+, H+, adenine nucleotides, calmodulin, and other factors will be determined on a time scale of milliseconds to minutes, using radioisotope flux -rapid quench and filtration techniques. In addition, cardiac SR Ca2+ release channels will be incorporated into planar lipid bilayers so that single channel conductance, ion selectivity and voltage-dependence, as well as the kinetics of channel activation and inactivation can be investigated. Surface membrane depolarization-induced Ca2+ release will be studied by measuring, in the presence and absence of transient membrane potentials, Ca+2 efflux from the SR compartment of surface membrane/SR junctional complexes. The effects of drugs will be tested to suggest ways to identifying and purifying the Ca2+ release channel components as well as modifying Ca2+ release in vivo. The Ca2+ release properties of membrane fractions isolated from ischemic hearts will be analyzed in order to detect a possible defect in SR Ca2+ release that may be related to or may potentiate loss of function during heart failure.
