The proposed studies will elucidate the structure and function of cardiac, skeletal, and smooth muscle ligand-gated calcium release channels (LGCRC) in isolate membranes and purified receptor preparations. Particular emphasis is placed on defining the mechanisms by which pertinent classes of pharmacological agents and environmental toxicants alter normal channel function in vitro and assesses their toxicological relevance in vivo. Analysis of (3H)ryanodine receptor binding with concomitant spectrophotometric assays of Ca2+ uptake and release from junctional vesicles with the metallochromic Ca2+ indicator antipyrylazo III allows direct inspection of receptor and Ca2+ channel function. Fluorescent and photoaffinity probes will test the major premise that four distinct effector domains modulate the gating behavior of LGCRC. The principal hypotheses tested are: 1. (3H)ryanodine specifically binds to the Ca2+-induced open state of LGCRC, and hence can directly assess modulation of LGCRC by physiologically-relevant ligands or xenobiotics. Photoaffinity labelling of the ryanoid-binding site reveals its position within junctional foot oligomer. 2. The Ca2+ regulatory domain is primarily responsible for gating LGCRC and unmasking the (3H)ryanodine-binding site. Lanthanides compete for the Ca2+-binding sites while thiol-reactive heavy metals and aryldisulfides specifically interact with critical thiols associated with this domain and serve as functional and structural probe. Fluorescent sulfhydryl reagents clarify the role and position of this domain within the native LGCRC oligomer and discriminate major differences among muscle types. 3. The xanthine domain allosterically influences the sensitivity of LGCRC and the (3H)ryanodine binding site to activation by Ca2+- . Antineoplastic anthracyclines specifically bind to this domain causing potent sensitization to activation by Ca2+ which can be antagonized by caffeine. 4. The adenine nucleotide domain enhances (3H)ryanodine receptor occupancy and the intensity of the LGCRC response to Ca2+ and functionally overlaps the xanthine domain. The combined use of physiological, biochemical, and toxicological endpoints will yield important new information about LGCRC structure and function and assess its involvement as a target for pharmacological and toxicological agents and characterize the mechanisms involved.
