The Paramecium ciliary membrane system is a suitable model for the more complex excitable membranes of nerves and muscles that exhibit voltage-dependent Ca2+ channels, gates and probably outward-directed Ca2+ pumps. Paramecium exhibits an avoidance response to mechanical, chemical and electrical stimulation in which the organism reverses its direction of movement by reversing the motion of its cilia. Hundreds of mutant lines with altered locomotory behaviors are available. The surface membrane surrounding cilia controls ciliary activity and the defects in many Paramecium behavioral mutants involve altered surface membrane excitation. Defects in membrane excitation in some strains are known to have altered Ca2+ or K+ gating/channel mechanisms; other mutants may have defective ion pumps. The goals of this proposal are to identify structural components in the membrane that function in ion translocation, as well as to determine the structural changes that led to the loss of different function in different mutants. The methods we will use to identify, e.g., pump, channel or gate molecules, include immunochemical and affinity chromatographic techniques employing monoclonal antibodies and polyacrylamide gel isoelectric focusing and electrophoretic methods. To determine the specific function of different membrane components, we will, for example, prepare antibodies to a purified protein, determine its subcellular location and use antibodies to interfere with normal protein function. The criteria for interference with normal function will include changes in ion fluxes and locomotory behavior. The Ca2+ ATPases in the ciliary membrane will be examined for pump function utilizing sealed membrane vesicles prepared from isolated cilia. Their structures, including possible sugar and/or lipid moieties, will be studied by biochemical and immunochemical methods. By identifying structural components that regulate ion movements across the membrane, we hope to advance our understanding of the complex process of membrane excitation and the control of ciliary activity.
| Suchard, S J; Rhoads, D E; Kaneshiro, E S (1989) The inositol lipids of Paramecium tetraurelia and preliminary characterizations of phosphoinositide kinase activity in the ciliary membrane. J Protozool 36:185-90 |
| Doughty, M J; Kaneshiro, E S (1985) Divalent cation-dependent ATPase activities in ciliary membranes and other surface structures in Paramecium tetraurelia: comparative in vitro studies. Arch Biochem Biophys 238:118-28 |
