This project addresses the regulation of a cyclic AMP-dependent Na+ current, one of a class widely distributed among tissues and phyla. In olfactory epithelium the current is mediated by direct nucleotide binding, not phosphorylation. Evidence suggests that the current in molluskan neurons, like that of vertebrate olfactory epithelium, is mediated by direct binding. Despite the lack of kinase regulation, the current is richly modulated by multiple factors: both extracellular and intracellular a Ca2+, intracellular pH and a factor sensitive to calmodulin blockers. We have developed a model for regulation of the current at the channel level by interactions of Ca and cyclic AMP based on observations of the whole cell current. The character of the model and the kinetics of the current have allowed formulation of a relatively non-invasive and quantitative electrophysiological assay for adenylate cyclase and phosphodiesterase activities, and for levels of cyclic AMP in vivo. We propose 1) to test the independence of I(Na)(cAMP) from phosphorylation by single channel recording; 2) to assay the effects of intracellular and extracellular Ca2+ in regulating cyclic AMP activation of I(Na)(cAMP) and the conductance in single channels; 3) to compare the regulatory mechanisms of I(Na)(cAMP)in neurons where its voltage dependence differs; 4) to complete development of the in vivo assay and test it against radioimmunoassay; 5) to assess the role of cyclic AMP phosphodiesterase in mediating some effects of Ca 2+, pH(i), and calmodulin blockers. The likely broad distribution of the type of current under study suggests that the mechanisms regulating it are also widely distributed and participate in diverse cell functions and pathological states. The assay method under development may be also broadly applicable.
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