Identification of a Ligand-binding Site in the Na+/Bile Acid Cotransporting Protein from Rabbit Ileum

Abstract

Reabsorption of bile acids occurs in the terminal ileum by a Na؉ -dependent transport system composed of several subunits of the ileal bile acid transporter (IBAT) and the ileal lipid-binding protein. To identify the bile acid-binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7-azi-derivative of cholyltaurine followed by enrichment of IBAT protein by preparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypsin yielded IBAT peptide fragments in the molecular range of 20.4 -4 kDa. With epitope-specific antibodies generated against the C terminus a peptide of molecular mass of 6.6 -7 kDa was identified as the smallest peptide fragment carrying both the C terminus and the covalently attached radiolabeled bile acid derivative. This clearly indicates that the ileal Na ؉ /bile acid cotransporting protein IBAT contains a bile acid-binding site within the C-terminal 56 -67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the seventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site is localized to the ninth transmembrane domain and the C terminus.Enterohepatic circulation of bile acids with a specific and highly efficient extraction of bile acids from the intestinal lumen, portal blood, and the primary filtrate in the kidney occurs by Na ϩ -dependent transport systems localized in the apical membrane of ileocytes and renal proximal tubule cells and the basolateral membrane of hepatocytes (1, 2). The functional ileal and hepatic Na ϩ /bile acid cotransport systems are protein complexes composed of several transporter protein subunits (3, 4). The ileal Na ϩ /bile acid cotransport system with a molecular mass of 451 Ϯ 35 kDa contains additionally several subunits of the cytoplasmically attached ileal lipid-binding protein (ILBP) 1 (3, 5, 6). The mammalian Na ϩ /bile acid cotransport proteins have 347-362 amino acids with 35-37% identity and 46 -48% similarity between the ileal and the hepatic transporter (Refs. 7-12 and GenBank TM accession numbers 254357 and AJ131361). Originally a topology model with seven transmembrane domains has been suggested (2), but novel investigations with in vitro translation approaches favor a topology model with nine transmembrane domains (13). Investigations of the substrate specificity of the ileal and hepatic Na ϩ /bile acid cotransporters using recombinant cell lines and brush border membrane vesicles (14) enabled us to generate the first predictive three-dimensional quantitative structure activity relationships pharmacophore model for mammalian Na ϩ /bile acid cotransporting proteins (15). As a next step to the understanding of intestinal bile acid absorption we describe in the present manuscript the identification of a bile acid-binding site of the rabbit ileal Na ϩ /cotransporter localized to the C-term...

Energetics by NMR: Site-specific binding in a positively cooperative system

“…A determination of the structural correlates of the cooperativity must await a careful comparison of the three-dimensional structures of the doubly ligated and unligated proteins. Curiously, solution-state NMR structures have been reported for the singly ligated forms of the porcine and human I-BABP (18,19). Because of the cooperativity, the population of the singly ligated protein should not exceed a few percent.…”

The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships

“…Proteins were grouped according to their phylogenetic relationship (see below, "Phylogenetic relationships and evolutionary origin of the SLC10 family"). Amino acid sequences were taken from the following GenBank accession numbers: NTCP/NP_003040, P4/NP_689892, ASBT/NP_000443, SOAT/NP_932069, P3/NP_062822, and P5/NP_001010893 predominant bile acid binder in the cytosol of ileal enterocytes (Stengelin et al 1996;Kramer et al 1997Kramer et al , 2001a. In the intestine ASBT is thought to be the major route for active bile acid uptake as emphasised by loss-offunction mutations in the human ASBT gene that cause primary bile acid malabsorption, an interruption of the enterohepatic circulation of bile acids, and reduced plasma LDL cholesterol levels Oelkers et al 1997).…”

Apical Sodium Dependent Bile Acid Transporter (ASBT, SLC10A2): A Potential Prodrug Target

“…There is a general lack of information about the spatial requirements for substrate binding to and transport by hASBT 40,41,66 . Most studies were conducted using ex-vivo tissue or organ preparations, or uptake studies using non-polarized cell culture models 29,33,67 .…”