Depolarization of a skeletal muscle fiber causes the release of calcium ions (Ca2+) from the intracellular sarcoplasmic reticulum (SR). The elevation of myoplasmic Ca2+ results in the activation of contraction of the muscle fiber. The release of Ca2+ occurs via ryanodine receptor Ca2+ channels located in the SR membrane, however, the mechanism by which voltage sensors control the activation of ryanodine receptors remains a central question of skeletal muscle biology. The long-term goal of this project is to elucidate the mechanisms controlling Ca2+ release from the SR.
The aim of the present proposal is to study the properties of discrete, local elevations of Ca2+, termed Ca2+ sparks, which reflect the underlying activation of Ca2+ release channels. The experiments will investigate Ca2+ sparks in a preparation in which the activation of SR Ca2+ release is close to the native state of the fiber. Confocal line-scan imaging of the fluorescence of a Ca2+-indicator dye loaded into the myoplasm of voltage-clamped, and saponin-permeabilized frog skeletal muscle fibers will be used to characterize the Ca2+ sparks. The voltage dependence of Ca2+ spark waveform will be examined using a two-pulse protocol to determine the mechanism of termination of Ca2+ sparks by voltage sensor deactivation and Ca2+-induced inactivation. The relative contribution of Ca2+ sparks to the early peak, and later steady level of release will be determined. The consequences of Ca2+ release and elevation of myoplasmic Ca2+ will be examined on Ca2+ spark waveform and frequency in permeabilized fiber preparation which allows control of the composition of solutions bathing the myoplasmic compartment of the fiber. The results of these experiments will provide new information about basic mechanisms of skeletal muscle activation and will help lead to an understanding of genetic defects of molecules associated with Ca2+ homeostasis in diseases of skeletal muscle.
