The physiologic activity of many cells, including muscle, neurons and several secretory cells, is strongly influenced by cytoplasmic Ca. To regulate Ca, cells possess intracellular organelles that sequester and release Ca, and Ca transport proteins in the cell membrane, that extrude Ca or allow Ca to enter. This grant has three aims. The first is to explore the mechanism of Ca release from internal stores of a striated muscle (the myotomal lamellae of the chordate Amphioxus) and a secretory cell (the rat peritoneal mast cell). Cytoplasmic Ca will be monitored by a fluorescent Ca indicator, using fluorescence microscopy, microfluorimetry and patch voltage clamp. The preparations were chosen because they offer uniquely favorable conditions for the experiments proposed; the insights gained are almost certain to apply also to mammalian muscle and other secretory cells.
The second aim i s to characterize Ca channels, which regulate Ca entry into muscle, Ca channels will be studied in adult and embryonic skeletal muscle, and in oocytes that have inserted muscle-type Ca channels into their cell membranes under direction of previously injected mRNA. Special attention will be given to (a) the mechanism of ion selectivity, which is of crucial significance for a transport protein with the task of selectively transporting an ion present at low concentration, and (b) the interaction of Ca channels with dihydropyridines, a clinically used Ca antagonist that is regarded as a specific, high-affinity marker for Ca channels and has become fundamental for biochemical studies of Ca channels.
A third aim i s to test whether Ca-influx (e.g. through an antigen-activated Ca channel) contributes to the increase in cytoplasmic Ca in mast cells. If an influx-mediated component is found, attempts will be made to record antigen-activated Ca currents.
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