The proposed projects are designed to (1) clarify the roles of the intramembranous charge components, Qbeta, Qgamma and Qdelta, in triggering Ca release from the sarcoplasmic reticulum, (2) elucidate the mechanisms underlying Qbeta, Qgamma and Qdelta, and (3) study how these charge components and Ca release are related to dihydropyridine receptors and ryanodine receptors. Charge movement and Ca-dye signal will be measured simultaneously i frog cut twitch fibers voltage-clamped by the double Vaseline-gap technique to correlate the kinetics of Ibeta, Igamma Idelta and the voltage distributions of the magnitudes of Qbeta, Qgamma and Qdelta with those of Ca-dye signal. Ca release will be blocked with TMB-8 or ruthenium red, or Ca2+ in the SR will be depleted to test the hypotheses that Igamma is not, but Idelta is, caused by the release. Varying concentrations of EGTA will be applied to the internal solution to test the effect of chelating free Ca++ on Ibeta, Igamma, Idelta and Ca-dye signal. Ibeta, Igamma Idelta and Ca-dye signal will be recorded in a fiber partially paralyzed, or partially revived, in the presence of D600 to correlate Qbeta, Qgamma and Qdelta with the Ca-dye signal in each state. Either Ibeta or Igamma will be perturbed and its effect on Ca-dye signal will be studied to test the hypotheses that Qbeta is not, and Qgamma is, responsible for Ca release. Some of the above experiments on cut fibers will be repeated on intact twitch fibers, voltage-clamped by the 3-microelectrode technique, to confirm the findings from cut fibers or to contrast the physiological states of the two preparations. Ibeta, Igamma, Idelta and Ca-dye signal will also be measured in intact slow fibers to compare the mechanism underlying contractile activation in the two fiber types. Ibeta, Igamma and potentiometric dye signal will be measured simultaneously in cut twitch fibers to probe the electric field associated with Qbeta and Qgamma and to test the hypothesis that Qbeta and Qgamma operate in different locations in the tubular membrane. The effect of ryanodine ester derivatives on Ibeta, Igamma, Idelta and Ca-dye signal will be studied in cut twitch fibers to test their agonism or antagonism on Ca release channels an to test if there is any feedback from the ryanodine receptors to the voltage sensors. The proposed experiments are part of 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 the causes of some diseases in human muscles. To reach the latter goal, experiments will be started to measure charge movement and Ca-dye signal in cut fibers or myotubes from normal mammals. This will pave the way for future studies of these signals in muscle cells from dysgenic or transgenic animals.
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