The amount of force produced by a muscle cell is a function of the intracellular calcium ion concentration. An intracellular organelle called the sarcoplasmic reticulum sequesters calcium from the cytoplasm to cause muscle relaxation and releases it to initiate contraction. Although it is known that a change in the electrical voltage across the cell surface membrane can stimulate calcium release from the SR, the cellular mechanisms that control and regulate the release process are poorly understood. This project is designed to gain a greater understanding of these mechanisms by studying the way in which a variety of drugs and ions modulate calcium release from the sarcoplasmic reticulum. Single amphibian muscle cells will be used. The surface membranes of these cells are removed by microdissection so that the conditions surrounding the SR can be rigorously controlled. This technique causes only minor disruption of normal cellular structure. A quantitative measure of the rate of calcium release from the sarcoplasmic reticulum in these isolated cells will be obtained using an optical technique employing the calcium sensitive dye Arsenazo III. In other experiments the permeability of the SR membrane to potassium ion will be monitored using radioactive tracer techniques. Specific experiments are designed to determine the mechanism of action of calcium channel blocking agents in decoupling the spread of activation from the transverse tubular membranes to the sarcoplasmic reticulum. In addition, the importance of counterion movement in modulating the magnitude of calcium efflux will be investigated by using potassium channel blocking agents. The results of these experiments will add to our knowledge of the cellular processes involved in the activation of contraction in skeletal muscle. A better understanding of the mechanisms underlying this process in healthy cells will improve our understanding of the changes which occur in the activation of cells under a variety of pathological states.
