The studies described in this proposal will investigate two related hypotheses regarding hormonal regulation of membrane K+ permeability; and characterization of a recently described mechanism for inward Na+-coupled HCO3-transport in hepatocytes. These represent fundamental yet poorly understood mechanisms that directly and broadly influence specialized functions of the liver including gluconeogenesis, canalicular 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 had 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 certain hormone-receptor interactions at the cell surface to changes in membrane K+ (and other) channel activity, and the specific aims are: i) to further evaluate the mechanisms for activation of specific membrane K+ currents by cAMP analogs with particular emphasis on the role of regulatory phosphorylation of K+ channels by cAMP-dependent protein kinases (PKA); ii) to determine whether glucagon and other hormones linked to adenylate cyclase activate membrane K+ currents exclusively through cAMP or whether cAMP-independent mechanisms are also involved; and iii) to investigate the mechanisms responsible for the inhibitory effects of insulin; and the modulatory effects of Ca2+ and/or Ca2+ -dependent kinases on K+ currents. The long term objective of the second hypothesis is to determine the mechanisms responsible for membrane H+HCO3- transport a they pertain to maintenance of intracellular pH and biliary HCO3- secretion.
The specific aims are i) to determine the stoichiometry of inward Na+-coupled HCO3- transport and the regulatory influence of membrane potential difference on HCO3- influx; ii) to investigate possible allosteric regulation of this mechanism by intracellular H+, kinases, and certain bile acids; and iii) to determine the functional significance and mechanisms responsible for inhibition of membrane K+ channels by H+. Abnormalities of these mechanisms of hepatic electrolyte transport directly contribute to the cholestasis, cell swelling, and disordered transport and metabolism that accompanies most liver disease.
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