Structural and functional characterization of the human apical sodium-dependent bile acid transporter
AdvisorSwaan, Peter W.
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AbstractThe human apical sodium-dependent bile acid transporter (hASBT), an essential component of the enterohepatic circulation (EHC), is responsible for bile acid reabsorption from the lumen of the distal ileum and plays a critical role in bile acid and cholesterol homeostasis. Lack of a crystal structure for ASBT, limits our understanding of the structural and functional determinants of transport. The work in this dissertation was carried out to characterize the structural components and determine their overall role in ASBT function. In particular, the work described here was aimed to (1) elucidate and further understand the topological framework of hASBT by epitope insertion, (2) evaluate the role of N-glycosylation and the N-terminal domain in ASBT function by alanine scanning mutagenesis, (3) probe the role of the endogenous cysteines using thiol modifiers and various bile acid conjugates, (4) and to determine the sodium, and bile acid translocation pathway comprising residues of transmembrane (TM) domain seven and extracellular loop (EL) three using substituted cysteine accessibility method. Due to conflicting experimental evidence, the membrane topography of ASBT, predicted to comprise 7 to 9 putative TM domains, remains unresolved. Our results from epitope insertion clearly, support a 7TM model. The work that followed was aimed at characterizing some of the functionally critical domains of this transporter. Alanine scanning mutagenesis showed that the N-terminal region was vital for function and mutation of the N-glycosylation site was responsible for reduced uptake activity but did not impact trafficking of the protein to the plasma membrane. Evaluation of the endogenous cysteine residues revealed that multiple Cys residues are essential for hASBT function and C270A in combination with methanethiosulfonate (NITS) reagents and bile acid-NITS conjugates can aid in defining the putative ligand binding region(s). Cysteine mutants of EL3 and TM7 were generated using C270A, and in conjunction with thiol-modification it was shown that EL3 forms the primary sodium interaction site while TM7 lines the substrate translocation pathway. Furthermore, critical residues involved in the process were also identified. Overall, the work carried out in this dissertation will aid in the advancement of drug design and improve our understanding of the features that are essential for recognition of effective inhibitors for this transporter.
DescriptionUniversity of Maryland, Baltimore. Pharmaceutical Sciences. Ph.D. 2005
Health Sciences, Pharmacology
human apical sodium-dependent bile acid transporter
Bile Acids and Salts