Electrical depolarization of a skeletal muscle fiber triggers a relatively large and rapid release of Ca2+ ions from the sarcoplasmic reticulum (SR), the intracellular membrane enclosed Ca2+-sequestering compartment in muscle, to the myofilament space. The released Ca2+ ions bind to thin filament troponin C molecules, thereby producing mechanical activation by removing the inhibition of contractile filament interaction. The membrane depolarization that triggers release is carried rapidly into the fiber by the transverse tubule (TT) system, a lattice of tubular invaginations of the external membrane into the fiber occurring once per sarcomere at the level of the z line in frog muscle. The SR constitutes a second organized membrane system in the cell, surrounding each myofibril and coming into close apposition with a TT lattice at each end of the sarcomere. Voltage sensors (dihydropyridine receptors) for initiating SR calcium release are located in the TT membrane but control calcium release channels (ryanodine receptors) located in the apposed junctional SR membrane, possibly via the large cytosolic domain of the ryanodine receptor which may contact the TT voltage sensor. In previous studies we have identified two components of SR calcium release during step depolarization, an inactivatable (probably calcium-induced) component giving a sharp early peak of release and a non- inactivatable component giving the steady level, possibly corresponding to indirect and direct components of activation by the voltage sensors. Our first aim will be to investigate the mechanisms whereby the TT voltage sensor and other putative physiological activators (Ca2+ itself in positive feedback, ATP, IP3) and inhibitors (Ca2+ in negative feedback, Mg2+) interact to control the SR release channel in functioning muscle fibers. The effect of each modulator on the two components of release will be specifically investigated for the first time in these studies. Our second aim is to characterize the spatial and temporal distribution of [Ca2+] within the sarcomere before, during and after calcium release using either a masked recording and illumination fluorescence system or digital video fluorescence microscopy. All studies will be carried out on voltage clamped fibers, allowing electrical measurements of voltage sensor charge movements. For the studies of control and modulation of release average myoplasmic [Ca2+] will be monitored simultaneously with two Ca2+ indicators: the relatively high affinity but somewhat slowly equilibrating fluorescent indicator fura-2 and the lower affinity faster absorbance indicator antipyrylazo III. The calcium localization studies will be carried out using relatively high concentrations of the low affinity fluorescent Ca2+ indicator furaptra, which has negligible delay in its equilibration with [Ca2+]. The rate of release of calcium from the SR will be calculated from each measured [Ca2+] transient after characterizing the calcium removal properties of the fiber under study. Our results should provide new insights regarding the factors controlling calcium release and how they influence the spatial and temporal integration of the release process.