The overall objective of this work is to establish how a high-conductance, ligand-gated Ca2+ release channel in sarcoplasmic reticulum (SR) regulates the intracellular Ca2+ ion concentration of skeletal muscle. The SR Ca2+ release channel has been recently purified, reconstituted into lipid bilayers, and shown to be identical with the large protein bridges (""""""""feet"""""""") that span the transverse (T-) tubule-sarcoplasmic reticulum junction. The proposed research is directed toward the further characterization of the structure and function of the SR Ca2+ release channel. T-tubule/SR junctional complexes (triads) and T-tubule detached (heavy) SR vesicles will be isolated to analyze T-tubule depolarization-induced and ligand- regulated SR Ca2+ release. Regulation of the T-tubule linked, T-tubule detached and purified Ca2+ release channel will be determined, on a time scale of milliseconds to minutes, in vesicles and/or at the single channel level. Effectors to be studied include the endogenous ligands Ca2+, Mg2+, H+, adenine nucleotides an calmodulin, T-tubule membrane potentials, membrane phosphorylation and drugs such as ryanodine and methylxanthines. These studies will test the hypothesis that T-tubule linked and T-tubule detached SR CA2+ release channels respond differently under identical physiological and pharmacological conditions. The domain structure of the SR CA2+ release channel will be determined with the use of proteases and site-directed antibodies. The effects of controlled proteolysis and antibodies will be assessed by immunoblot analysis, radiolabeling of regulatory ligand binding sites and peptide sequencing, sedimentation analysis of soluble channel complexes, and vesicle-ion flux and single channel measurements. Ca2+ release channel complementary DNAs will be expressed to localize the channel domain and/or regulatory site domains. Expressed proteins will be characterized as outlined above using cellular, microsomal and purified channel protein preparations.
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