Depolarization of a skeletal muscle fiber causes the release of Ca2+ from the SR. The release of Ca2+ occurs via ryanodine receptor Ca2+ channels located in the SR, gated by voltage sensor in adjacent t-tubular membranes. The mechanism by which the voltage sensors control the activation of ryanodine receptors remains a central question.
The aim of the present proposal is to study the activation of individual ryanodine receptor channels at the level of single triads in voltage-clamped frog skeletal muscle fibers with intact control of SR Ca2+ release by voltage sensors. The proposed experiments will be carried out using confocal line-scan imaging of the fluorescence of Ca2+-sensitive indicator dyes inside the fiber. This approach has been used by the investigator, as well as by another lab to measure the Ca2+ arising from single SR Ca2+ release channels or from small clusters of interacting channels. The proposed experiments have five Specific Aims.
In Aim 1, the investigator will study the average amplitude and the spatio-temporal waveforms of local Ca2+ release events in single, voltage-clamped frog skeletal muscle fibers and relate these characteristics to the gating properties of the SR Ca2+ release channels.
The second aim addresses the voltage dependence of local Ca2+ release events. Ca2+ release events will be recorded after small depolarizations and under conditions that affect DHPR inactivation so that large depolarizations will produce relative few individual Ca2+ release events. These studies will also investigate the frequency of occurrence and the properties of Ca2+ release events that occur spontaneously under conditions that should inactivate the voltage sensors.
The third aim will investigate the effects of agents on both the voltage-activated and the spontaneous Ca2+ release events. These will include, caffeine, procaine, cADPR, diminished Mg2+ levels, and the ability of Ca2+ to induce further Ca2+ release (CICR).
Aim 4 will investigate if single or unitary Ca2+ release events and multiple amplitude release events are related in a causal way or if they are independent.
