A key feature of the enterohepatic circulation for bile acids is their rapid intrahepatic vectorial transport from the sinusoidal to the canalicular pole of the hepatocyte. We previously demonstrated critical interactions of bile acids with specific cytosolic bile acid binding proteins that influence trans-hepatic bile acid transport. We have characterized the biochemical and bile acid binding properties of the human and rat bile acid binders and recently cloned their cDNAs. Using these cDNAs, we recently generated a family of recombinant chimeric and site specific mutated proteins to evaluate the structure/function domains of these bile acid binding proteins. In this proposal, we will examine the tissue distribution, gene regulation in human hepatoma cell line and begin to determine the physiological function of a novel human bile acid binding (HBAB) protein. Using the-Xenopus laevis oocyte as a model system for bile acid transport, we will explore the concept that changes in the free intracellular bile acid concentration by cytosolic binding proteins alter the functional capacity of co-expressed plasma membrane bile acid transporters. Specific goals include: 1) Determination of HBAB tissue distribution by RNA analysis, 2) Determining bile acid dissociation constants for a family of recombinant chimeric and site specific mutated proteins, 3) Characterization of the modulation of bile acid transport in the Xenopus laevis oocyte by co-expressing different cytosolic bile acid binding proteins with transmembrane bile acid transporters, and 4) Determination of HBAB gene expression in a Hep G2 cell line. These studies will characterize the expression and the physiologic function of a novel human bile acid binding protein.
