The mechanisms involved in the """"""""active"""""""" (uphill) extrusion of Ca from muscle fibers, and in the regulation of steady Ca balance, are incompletely understood. Many types of cells, including invertebrate muscle and vertebrate cardiac muscle, employ a Ca transport mechanism in which Na ions exchange for Ca (""""""""Na-Ca exchange""""""""). Thus, the Na electrochemical gradient may provide at least some of the energy for Ca extrusion. ATP also plays a role in part via an effect on Na-Ca exchange kinetics, and perhaps in part as fuel for a parallel Ca-ATPase. Internally perfused single giant barnacle fibers are employed to study Ca transport. This preparation is especially useful because internal as well as external solute composition are controlled, and unidirectional ion fluxes are measured. A voltage-clamp is used to determine current flow associated with transport. Preliminary data suggest that Na-Ca exchange is the predominant mode of Ca extrusion in barnacle muscle. We will use Na and Ca flux measurements and voltage clamp data to determine the stoichiometry of Na-Ca exchange. The effects of ATP, calmodulin and cyclic AMP on Ca extrusion will be investigated, as will the effects of various drugs. We will determine how these substances influence the Na-Ca exchange kinetics, and whether there is a parallel Ca-ATPase transport system. In addition, we will use ion-specific intracellular micro-electrodes to measure the ionized concentrations of free Na+ and Ca++ in intact muscle fibers; the effects of changes in the Na electrochemical gradient on the intracellular Ca++ concentration will be determined. The data should enable us to refine current models of Ca extrusion and Ca regulation in barnacle muscle. The results should also be applicable to many vetebrate cells that use similar, if not identical, mechanisms for Ca extrusion. The perfused giant barnacle muscle fiber preparation should also prove useful for studying many other transport mechanisms such as those that mediate amino acid transport.
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