INTERACTIONS BETWEEN H+.1 AND CA2+1 IN MYXICOLA AXON

长期目标是获得更好的理解

Long-term objectives are to gain a better understanding of the regulation of both intracellular free calcium (Ca2+) i, and free protons, (H+), and the interdependence between these two ion concentrations. These ions are now known or suspected to regulate or influence numberous cell functions, including muscle contraction, neuro-secretion, ion permeability, intercellular electrical coupling, cell division and differentiation, and metabolism. Calcuium regulatory mechanisms will be examined at four loci: (a) endoplasmic reticulum, (b) plasma membrane, (c) intracellular Ca-binding proteins, and (d) mitochondria. Calcium uptake and release from the endoplasmic reticulum of neural cytoplasm iwll be characterized using a new Ca electrode that detects free Ca in the nM concentration range. ATP-dependent Ca uptake will be studied in the presence of mitochondrial inhibitors as a function of ATP, pH, and (cA2+). The effects of caffeine and inositol phosphates on Ca release from organelles close to the plasma membrane will be compared to organelles near the center of the axon. Intracellular Ca-binding proteins will be studied with special emphasis on neurofilaments. The 150,000- 160,000 molecular-weight neurofilament proteins will be isolated and the binding of Ca to these proteins will be quantitiatively examined in solutions of different pH, ionic strength, and temperature. Using limited, specific proteolytic cleavage and autoradiographic analysis, the portions of the neurofilament molecule containg the calcium-binding site will be examined. The influence of pH on mitochondria will be studied. Using tetraphenol phosphonium (TPP+), a TPP+-microelectrode, and samples of axoplasm containing mitochondria, the effect of pH on the mitochondrial membrane potential will be examined to see if this explains a previus observation concerning pH and Ca uptake. Using pH microelectrodes and isolated samples of cytoplasm, the "effective" hydrogen ion diffusion coefficient in neural cytoplasm will be determined, and the importance of fixed and mobile pH buffers in dissipating intracellular pH gradients will be evaluated.