Paul A. Dawson, PhD
Bile acids, cholesterol metabolism, molecular cloning, gene expression and regulation, molecular genetics
Molecular Genetics of Ileal Bile Acid Transporter. My lab identified and cloned the human ileal bile acid transporter cDNA and gene. These probes are being used to identify dysfunctional mutations in patients with bile acid malabsorption. Various classes of dysfunctional mutations in the ileal bile acid transporter gene have been identified. In addition to null mutations (i.e., splicing defects), we have also identified missense mutations that interfere with bile acid transporter processing and mechanism of action. The Class 2 mutations cause misfolding and ER retention of the transporter. More interesting are the Class 3 and 4 mutations that block bile acid transport at the substrate binding and solute translocation steps. The actions of these mutations are being studied to gain insight into the molecular mechanism of sodium-coupled solute transport. The association of these mutations with other gastrointestinal and lipid metabolism disorders including gallstone disease, irritable bowel syndrome, hypocholesterolemia, and hypertriglyceridemia is currently being investigated.
Ileal Bile Acid Transporter Biology. In addition to the ileum, the ileal bile acid transporter is expressed in the proximal tubules of the kidney and in the cholangiocytes lining the bile ducts. The role of the transporter in the ileum and kidney is the quantitative reclamation of bile acids. However, its role in bile duct epithelium is unknown. We are generating an ileal bile acid transporter knockout mouse as a model to further understand the physiological role of the transporter in different tissues.
Intracellular Lipid Transport. The transcellular transport of bile acids is a defined process that involves uptake across the apical membrane, trafficking to the basolateral membrane and secretion across the basolateral membrane. Currently, we have identified and cloned cDNAs for the apical membrane transporter responsible for the initial uptake of bile acids, and the cytoplasmic binding protein thought to be responsible for the transcellular transport. We are devising expression-cloning strategies to identify additional bile acid transporters as well as the basolateral membrane transporter. Using the cloned transporters in polarized cells, the entire pathway can be reconstituted and analyzed. This model system should yield insight into more complex pathways for intracellular lipid trafficking.
FIGURE LEGEND: Disruption of the Ileal Bile Acid Transporter Gene in Mice. A) This figure shows the structure of the mouse ileal bile acid transporter gene (slc10a2), the targeting vector, as well as the predicted disrupted gene. Exons are numbered and indicated by the boxes. TK, viral thymidine kinase gene; NEO, neomycin resistance gene. B) The lower panel shows the results of PCR analysis of the ileal bile acid transporter gene in wild type mice (+/+) or mice that are heterozygous (+/-) or homozygous (-/-) for disruption of slc10a2. The results are shown for a Neo-specific probe (Neo gene), the wild type Slc10a2 gene, or lecithin cholesterol acyl-transferase (LCAT) as control.
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