The project aims to understand the mechanisms that regulate Ca2+ inside neural cells. Ca2+ fulfills an essential role in neural cells, controlling key excitatory responses including release of neurotransmitters, axonal membrane conductance, generation of action potentials, electrical coupling between neurons, and fast axonal transport. The proposed studies involve a combination of cell biological and biochemical approaches to analyze the molecular basis of mechanisms which control Ca2+ release into the neuronal cytoplasm and transfer Ca2+ inside neural cells. The project centers on Ca2+ transport mechanisms activated either by inositol phosphates or quanine nucleotides. The studies have three objectives: (1) Elucidation of the identity and organization of intracellular Ca2+ pools: studies will assess the function of Ca2+ regulatory organelles in clonal neuroblastoma cell cultures and isolated synaptosomes, identify at the subcellular level those organelles responsive to inositol 1,4,5-trisphophate (InsP3) and GTP, and use electron microscopy and electron probe microanalysis to localize intracellular Ca2+ pools within the neural systems. (2) Determination of the mechanisms of intracellular Ca2+ transport activated by InsP3 and GTP; measurements will be made on the nature and specificity of ion transport activated by InsP3, and on the physical and biochemical basis of the process by which a GTP-regulated mechanism mediates the translocation of Ca2+ between distinct Ca2+-regulatory compartments within neuronal cells. (3) Molecular characterization of the Ca2+ translocation mechanisms; studies are designed to identify the proteins involved in InsP3- and GTP-activated Ca2+ translocation, and to apply immunochemical approaches to the determinations of the existence of known Ca2+ - regulatory proteins within discrete membrane subfractions of neural cells. The project utilizes a combination of isotopic transport measurements, electron microscopic structural analyses, biophysical measurements on the interactions between membranes, protein analytical and immunochemical approaches, aa directed at ascertaining how Ca2+ translocation occurs within neural cells and what regulates it. In view of the fundamental role of Ca2+ in controlling neural functional, identification and characterization of the mechanisms that mediate and regulate Ca2+ signals in neural cells will permit understanding of the generation of neural excitability, and the development of methods to control the deleterious effects of diseases that alter neuronal conduction and excitability.
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