9410756 Hennessey Paramecium is an excellent system for studying sensory transduction mechanisms in eukaryotic cells because behavioral mutants can be selected to genetically dissect a sensory transduction pathway. Also, swimming behavior can be used as a convenient bioassay to estimate the effects of drugs, antibodies, and mutations on the electrophysiological properties of the cell. In Paramecium, attractants generally cause hyperpolarization and fast forward swimming while repellents depolarize and can cause backward swimming. External GTP is a repellent at concentrations as low as 0.01 micromolar. It elicits novel repetitive somatic (body) depolarizations which cause bouts of backward swimming. Adaptation occurs within 1 minute, causing a return to normal swimming. This research is designed to test the hypothesis that external GTP is recognized by a body plasma membrane purinergic receptor to activate a novel somatic calcium conductance. This triggers repetitive somatic plateau depolarizations. if receptor-operated channels are involved, there may be second messenger involvement in either their activation, deactivation, or adaptation. The objectives are to: 1) Identify and characterize the ion channels or transporters responsible for GTP-induce somatic depolarizations and describe the ion dependence, pharmacology, kinetics, and possible second messenger involvements. Established intracellular current injection and voltage clamp procedures will be used. The second messengers cAMP, cGMP and calcium will be assayed during exposure to GTP; 2) Use behavioral bioassays to screen drugs and mutations for effects on responses to external GTP. New behavioral mutants will be selected by behavioral selection techniques and characterized; and 3) Use microinjection "curing" of behavioral mutants to identify the altered gene products of mutants. The goal is to understand the biochemical and electrophysiological mechanisms involved in GTP-induced responses and sensory a daptation in Paramecium. This will contribute to a better understanding of purinergic responses and chemosensory transduction mechanisms in eukaryotic cells. %%% Living cells can sense and respond to chemicals in their environment. The first indication of such a cellular response is usually a change in the electrical properties of the cell membrane. The goal of this research is to understand how single cells sense chemicals and respond to them. The free-swimming, single-celled organism, Paramecium provides an excellent opportunity to study the molecular bases of these responses. Paramecium can be grown in large numbers in solutions resembling pond water; the electrical properties of its membrane can be studied using techniques of electrophysiology; and its genetics are well understood and mutants can be generated that affect the organism's swimming behavior. Swimming behavior can be used as a convenient assay for changes in its chemical sensing machinery. One chemical that Paramecium can sense very well at very low concentrations is an organic molecule, GTP. External GTP is a powerful repellent for these cells. They swim away from it at concentrations as low as 10.0 nanomolar. Many types of behavioral mutants will be generated that do not respond to GTP, and the details of their biochemical and physiological defects will be characterized. In this way the important components of the cellular GTP sensation pathway will be identified. The results of this research will provide important insights into how cells sense chemicals in their environment and respond to them. ***
