Intramembranous charge movements observed in frog twitch and slow muscle fibers are believed to play a role in contractile activation. In both fiber types, charge movement consists of two components, the main Qbeta and a secondary Qgamma. Both Qbeta and Qgamma disappear when a twitch fiber is paralyzed by D600, and reappear when the fiber is revived. Three-microelectrode voltage clamp experiments will be performed to measure Qbeta and Qgamma in a partially paralyzed or a revived twitch fiber in order to correlate these charge components with the contractile state of the fiber. Experiments using D890, TMB8 and other Ca++ blockers will also be performed to probe the site-of-action of the paralyzing agents and to see whether an entry of Ca++ is involved in paralysis. Results from these experiments might give clues concerning the physiological role of Qbeta or Qgamma and, hopefully, help us understand how charge movement triggers calcium release. Injection of metallochromic calcium-indicators into twitch fibers have enabled muscle physiologists to monitor the rise in myoplasmic calcium level in the fibers during an action potential or a voltage clamp pulse. Experiments are planned to apply this optical technique to voltage-clamped slow fibers. Intensities of light beams at four different wavelengths and two states of polarization will be monitored simultaneously to give information about dye concentration, movement artifact and calcium transient. A direct comparison of the characteristics of calcium transient in slow fibers with that in twitch fibers might give additional support to the hypothesis that Qgamma is responsible for triggering calcium release and, more importantly, help us to understand the comparative contractile behavior of the two types of fibers. Simultaneous measurements of charge movement and calcium signal will be performed on twitch fibers when they are either chronically depolarized, fatigued, partially paralyzed or revived. Pharmacological experiments will be done to investigate how Qbeta, Qgamma and calcium signal simultaneously respond to the application of dantrolene sodium, tetracaine, dibucaine, intracellular EGTA, caffeine, perchlorate, formamide or hypertonicity. A few of these experiments will also be tried on slow fibers. The results will hopefully provide a direct link between charge movement and calcium signal, which is just the first step in my long term project designed to elucidate the complete sequence of events underlying excitation-contraction coupling in normal muscle so that ultimately we can understand what causes the diseases in striated muscles.
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