The ciliated protozoan Paramecium specifically detects extracellular GTP at submicromolar concentrations and responds with slow oscillations (depolarizations) in membrane potential (Vm), corresponding to episodes of backward swimming, which are known to result from elevated intraciliary Ca2+. The long-term goal is to understand in molecular detail each step in the sensory pathways by which the cell (1) detects GTP, (2) alters its ciliary motility , then (3) adapts to the signal and ceases to respond. These studies will exploit the special advantages of this unicellular organism: motile behavior that is easily observed and quantified, availability of many behavioral mutants, easy selection of new mutants with defective swimming behavior, and well-established background information on the electrophysiology and biochemistry of ciliary motiiity. The ionic basis of the oscillation in Vm will be investigated by voltage-clamp electrophysiology, with wild-type cells and with existing mutants with known defects in specific ion conductances. Fluorescent probes for intracellular free [Ca2+] will be used with confocal microscopy to measure oscillations in intracellular (Ca2+] and to determine whether and where the Ca2+ transients are localized within the cytosol. The source of the Ca2+ entering the cytosol (extracellular or intracellular) will be determined by depleting internal pools with drugs that inhibit Ca2+-sequestering pumps, omitting Ca2+ from the surrounding medium, and measuring 45Ca fluxes across the plasma and alveolar membranes. The possible role of cAMP-dependent phosphorylation in adaptation to GTP will be investigated by measuring [cAMP] after exposure to GTP, by studying the patterns of protein phosphorylation induced by GTP and by benzoyl-cAMP a permeant cAMP analog,that makes Paramecium nonresponsive to GTP, and by determining the effect of phosphoprotein phosphatase inhibitors on the re-acquisition of GTP- sensitivity. The role of cGMP-dependent protein kinase (PKG) and cGMP in initiating and sustaining Ca2+ transients will be explored with permeable c0MP analogs. by microinjecting constitutively active PKO or peptide inhibitors of PKG or antibodies against PKG, and by overexpressing the cloned PKG gene. Mutants defective in either the motile response to GTP or adaptation to GTP will be selected and compared with wild-type cells to define steps in the transduction process. These studies may provide fundamental understanding of the transduction and desensitization mechanisms for purinoceptors, the molecular events in the triggering and maintenance of Ca2+ oscillations, and the regulation of ciliary motion by extracellular nucleotides, all of which processes occur in a wide variety of mammalian cells and tissues, and are essential to human health.
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