Bile acids play a critical role in numerous biological processes such as a) digestion, b) formation of bile, which functions as an excretory vehicle for cholesterol and metabolites of drugs and carcinogens, c) the regulation of cholesterol metabolism and d) the modulation of hepatocyte signaling pathways. Defects in the transport of these molecules leads to cholestasis, resulting in numerous pathological conditions such as liver injury and failure. Previous studies have demonstrated that microsomal epoxide hydrolase (mEH), which plays a central role in carcinogen metabolism, is also one of the membrane carrier proteins capable of mediating sodium- dependent transport of bile acids across the hepatocyte blood sinusoidal plasma membrane. This protein exists in two distinct topological orientations in the endoplasmic reticulum, one of which is targeted to the plasma membrane. Recent studies have identified several patients with extremely elevated serum bile acid levels (hypercholanemic) that appear to have a greatly reduced capacity to transport bile acids. One of these subjects has been shown to also express greatly reduced levels of mEH and mEH mRNA and the absence of exon 1 in the mEH gene (EPHX1). The long term goals of these studies are to 1) characterize the structure, function and mechanism of action of mEH as a bile acid transporter, and 2) characterize alterations in mEH expression or structure leading to defects in transport.
The specific aims of this proposal are to characterize a) the membrane architecture of mEH, b) the effects of expression levels, mediated by development, pregnancy and drugs, on the targeting and functional expression of mEH at the plasma membrane and c) mutations in the mEH gene (EPHX1) in subjects with hypercholanemia to establish the molecular basis for the observed transport defects. Membrane architecture and functional expression of mEH on the hepatocyte cell surface will be investigated using antibody and biotinylation reagents, confocal fluorescence microscopy, glycosylation site insertion technology, co- immunoprecipitation, proteolysis protection analysis, membrane protein crosslinking procedures, and cell cytometry, in conjunction with transport assays. The effect of mutations derived from the sequencing of EPHX1 from hypercholanemic patients will be studied in regard to their effect on promoter activity, or synthesis of mEH with altered transport and/or targeting characteristics. These studies should substantially increase out knowledge of this important transport system which plays a critical role in numerous physiological processes and in the etiology of several major diseases.
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