ILEAL BILE ACID TRANSPORTER METABOLISM AND REGULATION

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The identification and cloning of the ileal apical Na/bile acid cotransporter (ASBT) cDNA has provided new information regarding its primary structure, tissue expression, regulation, transport mechanism, and role in human disease. However, little is known about the assembly and structure of the functional ASBT, the regulation of its plasma membrane expression and turnover, or bile acid transcellular transport and efflux. The proposed research will focus on the molecular mechanism for enterocyte bile acid uptake. The long-range goal of this work is to understand the mechanism and regulation of intestinal bile acid transport as it relates to diet lipid metabolism in normal and disease states. The following questions will be addressed: (1) What are the general structure and subunit stoichiometry of the ASBT? What are the steps in the ASBT biogenesis? In contrast to its primary structure, the subunit stoichiometry and assembly of the function transporter complex are poorly understood. Preliminary studies suggest that intra-subunit and possibly inter subunit disulfide bonding are prerequisite steps for ASBT folding and transporter complex formation. In proposed studies, the assembly and subunit stoichiometry of the ASBT will be determined in transfect MDCK cells and normal rat cholangiocytes. The identity of associated subunits will be determined using combination of chemical cross-linking, coimmunoprecipitation, and a yeast two-hybrid screening strategy. (2) How is the plasma membrane expression of the ASBT regulated? ASBT expression is regulated by substrate, hormones such as glucocorticoids and secretin, and by cAMP. Whereas part of this regulation is transcriptionally, post-translational regulation of ASBT protein or plasma membrane activity appears to be an important component. In the proposed studies, the mechanism for ASBT exocytosis/endocytosis and degradation will be determined. (3) What is the role of the intestinal Oatp3 in bile acid absorption? Bile acids are absorbed by active sodium dependent transport and passive facilitative transport. A candidate transporter for the facilitative transporter Oatp3, has been isolated. The Oatp3's cellular expression and role in intestinal bile acid transport will be determined.