Excitation-contraction coupling (ECC) will be studied in intact vertebrate skeletal muscle fibers by cellular and comparative methods. A major goal is to elucidate the function of ryanodine receptors (RYRs), the Ca release channels of the sarcoplasmic reticulum (SR), in intact fibers as they function in their native environment. Experiments will be carried out on adult twitch fibers of frogs, mouse and fish. Individual fibers will be micro-injected with a Ca indicator (e.g., fluo-3) and studied on a confocal microscope having excellent spatial resolution. Signals to be measured include: (i) """"""""Ca sparks,"""""""" which are brief, localized increases in myoplasmic Ca caused by the opening of one, or a small number of, RYRs; and (ii) large-scale Ca release events (e.g., triggered by action potentials), which reflect the synchronous activation of many RYRs. Results will be interpreted with the aid of computer models that quantify the amplitude and time course of Ca release, the binding and diffusion of Ca and its buffers within the myoplasm, and the re-sequestration of Ca within the SR. The approaches will yield new knowledge regarding: (1) the number of active RYRs that contribute to sparks under physiological conditions; (2) the mechanism(s) that control the activation and termination (de-activation and inactivation) of ryanodine receptor openings; (3) the reaction kinetics between Ca and the quantitatively important Ca buffers (e.g., troponin C); (4) the mechanisms by which pharmacological agents such as caffeine affect RYRs. The comparative studies (frog vs. mouse vs. fish) will permit an assessment of the role of RYR isoform variation in the Ca release process. Fast and slow fibers of mouse and """"""""superfast"""""""" fibers of toadfish all have one major RYR isoform (denoted RYR1), whereas fast fibers of frog and fish have two isoforms (RyR1 and RyR3; found in similar proportion), and slow fibers of fish have a unique variant of RyR1 (termed RyR1slow). Functional comparisons among fibers having different isoform composition should yield important information regarding control and molecular adaptation of RYRs. Overall, the knowledge obtained will contribute importantly to understanding the mechanisms of intracellular Ca signaling and the mechanisms by which specific drugs and diseases affect muscle performance.
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