During the past four years we have made major progress in the development of several model systems for studying the regulation of muscle contraction. These include reconstituted thin filaments, TnC depleted reconstituted skinned fibers and intact muscle fibers. These systems combined with the techniques of fluorescence spectroscopy, flash photolysis of caged compounds, time resolved X-ray diffraction and proteolytic fragments of the regulatory proteins have allowed us to discover many new features about muscle activation/relaxation. In the present application, we propose to pursue several major questions related to these processes and have divided the application into two major areas of study as follows: I. The use of skinned and intact muscle fibers to study muscle activation and relaxation processes. The following specific aims will be pursued: I-1. What are the temporal relationships between: a) the binding of Ca2+ to the regulatory sites of TnC (measured by incorporated fluorescent TnC derivatives) and the activation of force development and stiffness of skinned fiber systems from rabbit, frog and barnacle muscle following rapid step changes in free Ca2+ (photolysis of caged Ca2+) which are not limited by diffusion processes and: b) the dissociation of Ca2+ from the regulatory sites of TnC and force relaxation and stiffness following rapid step decreases in free Ca2+ (photolysis of the caged Ca2+ chelator, diazo-2)? I-2. What are the effects of different crossbridge states (e.g. increment[ATP], increment [ADP]/[ATP], increment[Pi], rigor (-ATP), weakly attached (N- phenylmaleimide treated) and numbers (e.g. different sarcomere lengths, length perturbations) on these processes? I-3. What is the time course for Ca2+ binding to TnC in intact single muscle fibers, particularly barnacle, when activated electrically? I-4. During relaxation, what is the relation between free Ca2+ and the attached crossbridge states? I-5. Can the rates of force development and force relaxation observed in electrically excited intact frog, barnacle and scallop (myosin regulated) fibers be increased by laser flash photolysis of caged calcium or caged calcium chelator injected directly into the cell? I-6. What is the Ca2+ affinity of rabbit light chain-2 of myosin in situ and can it play a role in muscle regulation? II. The use of proteolytic fragments of Tn and Tm to study muscle regulation. The following specific aims will be pursued: II-1. What are the roles the different regions of TnC in the regulation of contraction? II-2. What are the roles of the head-to-tail interaction of Tm and the NH2-terminal region of TnT in the cooperative activation of muscle contraction? II-3. What are the interactions of the different regions of TnI with the other thin filament proteins and how are these related to the activation and relaxation of muscle contraction? Taken together these unique approaches should yield a much clearer view of the temporal and molecular events involved in muscle activation and relaxation.
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