The studies described in this proposal will investigate two related hypotheses regarding hormonal regulation of membrane ion permeability, and characterization of a recently described mechanism for Na+/HCO3- cotransport in hepatocytes. These represent fundamental yet poorly understood mechanisms that directly and broadly influence many specialized functions of the liver including gluconeogenesis, bile formation, and membrane solute transport. The proposed studies will utilize a variety of methods but emphasize direct measurement of whole cell and single channel currents with patch clamp recording techniques which have little prior application to these or other aspects of hepatocellular function. The long term objective of the first hypothesis is to define the mechanisms which link hormone-receptor interactions to regulated changes in membrane K+ and other ion channel activity, and the Specific Aims are: i) to evaluate the changes in membrane potential difference and ion permeability which characterize activation of receptors coupled to Ca2+ mobilization and cAMP generation; ii) to identify individual K+ channels responsible for cAMP- and glucagon- dependent increases in K+ efflux; assess the relative roles of cAM- dependent protein kinases and GTP-binding proteins in channel activation, and investigate the mechanisms responsible for the inhibitory effects of insulin; iii) to evaluate stimulatory and inhibitory interactions between Ca2+ and protein kinase C in regulation of Ca2+ permeable cation channels; and iv) to evaluate the complement of hepatic receptor(s) activated by extracellular ATP. The long term objective of the second hypothesis to assess the role of Na+/HCO3-cotransport in regulation of membrane acid/base transport, and the Specific Aims are i) to determine the stoichiometry of Na+/HCO3-cotransport, ii) assess the roles of membrane potential difference and of intracellular H, Ca2+, and kinases as potential regulatory factors; and iii) to evaluate the mechanisms which contribute to pHi-dependent modulation of membrane potential difference through measurement of the effects of [H+] on opening of K+ and other channels. Abnormalities of these mechanisms contribute directly and importantly to the cholestasis, cell swelling, and disordered transport and metabolism that accompany most liver diseases.
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