In vertebrate twitch muscle fibers, contraction is normally activated by a depolarization of the membranes of the transverse tubular system. This leads to a movement of Ca from inside the sarcoplasmic reticulum (SR) into myoplasm where it can bind to the Ca-regulatory sites on troponin so that contraction can occur. The long-term objectives of the research in this application are to understand the factors that control SR CA release. One of these factors is the voltage across the tubular membranes, which is monitored by a voltage sensor (the dihydropyrydine receptor) hat is located in the tubular membranes. Structural rearrangements of movements of this sensor give rise to intramembranous charge movement currents, which can be detected with the voltage-clamp method. Another factor that controls SR Ca release is the value of free [Ca], either in the bulk myoplasmic solution or near the myoplasmic surface of the SR release sites. Ca-induced Ca release may play a role in the activation of some of the SR Ca channels and Ca inactivation of Ca release may play a role in shutting off SR Ca channels once sufficient Ca has been released to bind most of the Ca-regulatory sites on troponin. Understanding these processes is fundamental to understanding how skeletal muscle is activated. The proposed experiments will be carried out on frog cut twitch fibers mounted in a double Vaseline-gap chamber. SR Ca release will be elicited by either action-potential or voltage-clamp stimulation. In most experiments, the amount and time course of release will be measured with the EGTA-phenol red method. Currents from intramembranous change movement will be measured with the voltage-clamp method. Ca release from single SR CA channels will be measured with fluo-3 fluorescence in a laser scan confocal microscope, as """"""""calcium sparks."""""""" Some of the questions that the experiments should answer are: A. Does Ca inactivation of Ca release markedly reduce the peak rate of SR Ca release during a positive voltage step? B. Is Ca inactivation of Ca release reduced or eliminated when the Ca content of the SR is reduced? C. How does intramembranous change movement regulate the turning on and turning off of Ca release (in the absence of effects of Ca inactivation of Ca release)? D. Does Ca inactivation of Ca release contribute to the turning off of Ca release during repolarization after an action potential or voltage pulse in a fiber with normal SR Ca content? E. Are reprimed SR Ca channels able to develop Ca inactivation of Ca release? If so, is a single reprimed channel, shielded by EGTA from increases in free [Ca] from neighboring open channels, able to develop Ca inactivation of Ca release? F. Can calcium sparks be recorded from single SR Ca channels in skeletal muscle? If so, what is their spatial distribution, how does voltage affect their frequency, and how long do they last?
