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 longterm objectives of the research proposed in this application are to understand (A) how Ca movement from the SR into myoplasm is regulated by the potential difference across the membranes of the transverse tubular system and (B) what happens to the Ca after it is released into the myoplasm. The experiments will be carried out on frog cut twitch fibers mounted in a double Vaseline-gap chamber. Changes in myoplasmic Ca will be measured with Ca indicators after either action-potential stimulation or voltage-clamp depolarization. Currents from intramembranous charge movement, which are thought to play a role in the regulation of SR Ca release by the tubular membranes, will be measured with the voltage-clamp method.
The specific aims are: 1. To measure Ca release from the SR of cut muscle fibers in which free [Ca] is buffered to a nearly constant level by a large concentration of a high affinity Ca indicator. 2. To measure the reuptake of Ca by the SR, after its release, in cut muscle fibers in which free [Ca] is buffered to a nearly constant level by a large concentration of a high affinity Ca indicator. 3. To measure charge movement in cut muscle fibers in which free [Ca] is buffered to a nearly constant level by a large concentration of a high affinity Ca indicator or buffer. 4. To simultaneously measure and compare charge movement, or some component of it, and SR Ca release in cut muscle fibers in which free [Ca] is buffered to a nearly constant level by a large concentration of a high affinity Ca indicator. 5. To simultaneously measure and compare charge movement, or some component of it, and SR Ca release in cut muscle fibers under more normal physiological conditions in which free [Ca] is not heavily buffered by extrinsic Ca buffers.
