IONIC SIGNALING MECHANISMS IN HEPATOCYTES

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This proposal focuses on the characterization of two distinct classes of ion channels that are positioned to play pivotal roles in hepatocellular responses evoked by hormones released during physiological stress as well as by growth factors linked to liver regeneration. These channels, which have been recently identified by the principal investigator, are: (a) calcium-permeable cation-selective channels and (b) potassium channels. Observations in this laboratory and by others support the following hypothesis: Cation-selective channels and potassium channels are coordinately regulated such that hormonal activation of potassium efflux through potassium channels provides favorable electrical driving forces that facilitate activation of calcium influx through cation-selective channels. The overall objectives of the proposed experiments represent a necessary first step toward testing this hypothesis. The specific aims are: (1) to determine the mechanisms by which hepatocellular cation- selective channels and potassium channels are activated, (2) to develop pharmacological tools to further elucidate the physiological roles of cation-selective channels and potassium channels in hepatocytes, and (3) to isolate functional cDNAs encoding hepatocellular cation-selective channels and potassium channels. Preliminary studies in this laboratory support the feasibility of achieving these specific aims. First, these studies have begun to reveal novel mechanisms of channel regulation by calcium and/or cyclic AMP-dependent protein kinase. Second they have identified several candidate channel blockers. Third, they have demonstrated activation of cation-selective and potassium channels by calcium or cyclic AMP-mediated signaling pathways in Xenopus oocytes that express liver mRNA. Moreover, they have led to the isolation of liver cDNAs that exhibit homology to ion channels in electrically excitable tissues and may encode hepatocellular cation-selective channels and potassium channels. The proposed experiments will employ cell physiological and molecular cloning techniques currently used in this laboratory. These technical approaches include measurements of single channel and whole cell currents using patch clamp recording techniques, measurements of intracellular cation concentrations using fluorescent dyes, cloning based on homology to known ion channel cDNAs, and ion channel expression in Xenopus oocytes. Definition of the regulation and pharmacology of hepatocellular cation-selective channels and potassium channels and isolation of their cDNAs are expected to provide a basis for new insights into control calcium entry in hepatocytes, calcium-regulated hepatocellular processes, and by extension, hormonal regulation of liver function. Just as characterization of ion channels in other cell types has led to clinical breakthroughs in diseases as diverse as hypertension and diabetes characterization of cation-selective channels and potassium channels in hepatocytes may ultimately permit the development of novel therapies for the metabolic derangements that attend liver disease.