The ciliary beat in the protozoan Paramecium tetraurelia is modulated by cyclic nucleotides and Ca2+. The objective is to understand the role of protein phosphorylation in this regulation. Constitutively active forms of the cAMP dependent, cGMP-dependent, and Ca2+ dependent protein kinases previously purified from Paramecium will be prepared and either microinjected into living cells or applied to permeabilized cells. The effects of these activated enzymes upon swimming speed and direction will be compared with those elicited by cAMP, cGMP, and Ca2+, to establish that protein kinases mediate the effects of the second messengers. To determine whether ion channels are regulated by phosphorylation, voltage-clamp studies of intact cells injected with each kinase will be performed, and the effect of purified protein kinases upon the activity of single channels in excised somatic membrane patches will be studied by patch-clamping. To identify axonemal targets of regulatory phosphorylation, the effect of exogenously added protein kinases on dynein ATPase in permeabilized cells and isolated cilia will be studied. An in vitro assay system for dynein- catalyzed microtubule motility will be used to assess the effect of each protein kinase on dynein's activity. Behavioral mutant defective in membrane excitability or axonemal regulation will be assayed for each of the protein kinases, and the ability of each enzyme to repair the defect in these mutants will be tested by microinjection. The pattern of protein phosphorylation by exogenous protein kinases will be studied in permeabilized cells, isolated cilia, ciliary membranes, axonemes, and purified dyneins. The phosphorylation patterns of wild-type cells will be compared with those of behavioral mutants. These studies should clarify the role of phosphorylation in ion-channel regulation and may lead to the identification of specific membrane proteins as ion channels. The axonemal proteins that mediate regulation by cyclic nucleotides and Ca2+ will be identified, and the differential actions of cAMP and cGMP on ciliary motion may be explained. The regulation of ciliary motion is important to the normal function of ciliated epithelium in such human tissues as trachea, lungs, and oviduct, and to the motility of sperm. Ion-channel regulation is central to neural function.
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