Taste cells respond to gustatory stimuli by depolarizing their membrane potential as denouement of yet incompletely characterized transduction mechanisms. This depolarization relies on a diverse repertoire of ion channels, constituently integrated into intracellular signal transduction cascades, that ultimately results in neurotransmitter release. Prior research focused on characterizing these basic conductances, which were monitored by whole cell patch clamp recordings on dissociated rat posterior test cells. This proposal seeks to examine how cAMP intervenes between the application of bitter and, to a lesser extent, sweet stimuli and consequent changes on ionic currents. Bitter stimuli have been hypothesized to use both cAMP and IP3 transduction cascades. For cAMP, bitter stimuli have been proposed to increase or decrease its levels with and without the utilization of G-proteins and/or phosphorylation. Using an agonist driven assay that monitors how application of a tastant (e.g. quinine) reversibly affects an isolated current (e.g. K+ currents), the role of G proteins in these proposed cascades will be studied using guanine nucleotide analogues, pertussis toxin, and G-protein alpha-specific antibodies. Roles for gustducin and transducin in particular taste stimuli will be sought. Experiments are planned to continue study of cAMP by examining the kinetics of cAMP inhibition on potassium and sodium currents using cAMP, cAMP analogues, and photolysis of caged cAMP and to study the requirement of phosphorylation and phosphatases in tastant responses. In addition a cAMP-suppressible cation conductance, proposed as key to one bitter transduction theory, will be sought. Finally, preliminary data suggests that NE and 5HT affect potassium and chloride currents in taste cells. These data also suggest that NE uses a beta-receptor, which strongly links it to cAMP production. The 5HT mechanism is not yet known. These neurotransmitter actions will be further characterized for receptor class and signal transduction mechanism. They may represent another modulatory influence over cAMP in these cells. In summary these experiments promise to explore in detail how external stimuli affect cAMP production in posterior taste receptor cells and to explore its subsequent electrophysiological actions.
