Long-term objectives are to gain a better understanding of the regulation of both intracellular free calcium (Ca2+)i and free protons (H+)i and the interdependence between these two ion concentrations in nerve cells.
Specific aims are to study, both in isolated axoplasm and in intact axons of the Myxicola giant axon, (Ca2+)i regulatory mechanisms at four loci: (a) the plasma membrane, (b) the mitochondrion, (c) organelles such as the endoplasmic reticulum that sequester Ca by ATP hydrolysis, and (d) the cell constituents that bind Ca by purely physio-chemical processes. The approach will be to physiologically distinguish classes of Ca binding sites in situ by studying succinate-dependent binding (mitochondria), ATP-dependent binding, and energy-independent binding. A description of these Ca buffering sites in isolated axoplasm will be made from three types of measurements: (a) The Ca content of the axoplasm will be determined as a function of the free Ca with which it is in equilibrium. (b) Isotopic self-exchange between Ca40 of isolated axoplasm (held in a polyethelene tube) and Ca45 of an adjacent solution containing the same free Ca will be measured. The profile of Ca45 that has migrated into the axoplasm will be determined by freezing the sample, slicing the cylindrical plug of axoplasm in 25 um thick disks, and assaying the radioactivity. Results will be analyzed by a method which yields the Ca diffusion coefficient and properties of the Ca binding reactions. (c) Net diffusion of Ca into axoplasm will be monitored with ion-sensitive microelectrodes and the behavior analyzed using the diffusion-reaction equation. In intact axons, the kinetics of recovery from injected Ca loads will be examined as a function of the location of the load with respect to the plasma membrane. Comparison of these results with results in isolated axoplasm will provide a measure for the role of the plasmalemma in Ca regulation. In other experiments, the effect of pHi on Na-Ca exchange will be studied. While pHi is varied, pCai will be held constant through the use of an exogenous pH-independent Ca buffering system injected into the axon. The inhibition of Na-o-dependent Ca efflux and Ca-o-dependent Na efflux by H+i will be examined kinetically to determine whether the interaction between H+ and Ca2+ or between H+ and Na+ is competitive, uncompetitive, or mixed.
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