A cellular and comparative approach will be used to study the excitation- contraction (EC) coupling process in single vertebrate skeletal muscle fibers. Intact fibers in their normal physiological state will be used to: (1) elucidate the conditions for detection as well as characterize the properties of Ca """"""""sparks"""""""" (spatially-localized and temporally-limited increases in myoplasmic [Ca] due to release of Ca from the sarcoplasmic reticulum); (2) characterize the time course with which the normal rise in free [Ca] spreads throughout the myoplasm; and (3) clarify the events associated with Ca binding to sites (i) on troponin that control force generation by the myofilaments, (ii) on or near the SR Ca release channels that serve to inactivate the release process, and (iii) on parvalbumin and the SR Ca pump that permit the lowering of [Ca] and fiber relaxation. Further, properties of the Ca release system will be compared in different fiber types (superfast, fast and slow twitch) of different species (frog, fish, snake and mouse). Among these fibers, there are substantial differences in the isoform composition of the SR Ca release channels (= ryanodine receptors, or RyRs). Superfast fibers of toadfish and all fiber types of snake are RyR-alpha-only; fast fibers of frog, and fast and slow fibers of fish, are RyR-alpha + RyR-beta; and fast and slow fibers of mouse are predominantly RyR1. Thus, these studies will elucidate how differences in RyR isoform composition contribute functionally to adaptations in the EC coupling mechanism in different fiber types. To carry out the proposed studies, a fluorescent calcium indicator dye such as fluo-3 or furaptra will be introduced into the myoplasm of an intact fiber, and both spatially-averaged and spatially- resolved (by means of confocal microscopy) [Ca] signals will be measured. SR Ca release will be estimated from the measured change in free [Ca] in combination with mass action calculations of Ca binding to troponin, parvalbumin and the SR Ca pump. The information obtained should contribute importantly to an understanding of how drugs and diseases alter specific events that determine muscle performance.
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