Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane.

For the investigation of the topology of the rabbit ileal Na+/bile-salt-cotransport system, composed of a 93-kDa integral membrane protein and a peripheral 14-kDa bile-acid-binding protein (ILBP), we have synthesized photolabile dimeric bile-salt-transport inhibitors (photoblockers), Gl-X-G2, where two bile acid moieties (GI and G2) are tethered together via a spacer, X, and where one of the two bile acid moieties carries a photoactivatable group. These photoblockers specifically interact with the ileal Na '/ bile-salt-cotransport system as demonstrated by a concentration-dependent inhibition of [iH]cholyltaurine uptake by rabbit ileal brush-border membrane vesicles and by inhibition of photolabeling of the 93-kDa and 14-kDa bile-salt-binding proteins by 7,7-azo and 3,3-azo derivatives of cholyltaurine. Ileal bile-salt uptake was specifically inhibited by the photoblockers, which were not taken up themselves by the small intestine as demonstrated by in vivo ileal perfusion.Dependent on the photoblocker used several polypeptides in the molecular-mass range of 14-130 kDa were labeled. The cytoplasmically attached 14-kDa ILBP was significantly labeled only by inhibitors that are photoactivatable in bile acid moiety GI, suggesting that during binding and translocation of a bilesalt molecule by the ileal bile-salt-transport system the steroid nucleus gets access to the cytoplasmic site of the ileal brush-border membrane first. Photoaffinity labeling in the frozen state with the transportable 3,3-azo and 7,7-azo derivatives of cholyltaurine revealed a time-dependent increase in the extent of labeling of the 14-kDa and 93-kDa proteins, suggesting a labeling of these proteins from the cytoplasmic site of the ileal brush-border membrane. By photoaffinity labeling in the frozen state with the various photoblockers time-dependent changes in the extent of photoaffinity labeling of bile-salt-binding proteins were observed, demonstrating the possibility of topological analysis of the rabbit ileal Na'hile-saltcotransport system. Keywords: bile acid ; ileal transport; topological photoaffinity labeling ; transport inhibitor; transporter protein.The enterohepatic circulation of bile salts, involving the liver, the small intestine and to a lesser extent the kidney, occurs by specific carrier proteins for bile salts in the plasma membranes and the cytosol of the respective epithelial cells, and in Abbreviurions. ILBP, ileal lipid-binding protein; 7,7-azo-TC, 2-(7,7- Enzynze. Aminopeptidase N (EC 3.4.1 1.2); y-glutamyltransferase (EC 2.3.2.2).bolic Diseases, D-65926 Frankfurt am Main, Germany blood [l]. The organotropism of bile salts for the liver, the ileum and the kidney is established by specific Na'hile-salt-cotrans~ port systems, which are located in the sinusoidal membrane of hepatocytes and the brush-border membrane of ileocytes or proximal kidney cells [2]. With photolabile derivatives of bile salts [3, 41 the putative protein components of the bile-salt carrers in blood [5, 61 and in the plasma membranes of hepatocytes [7...

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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...

The substrate specificity of the ileal and the hepatic Na ؉ /bile acid cotransporters was determined using brush border membrane vesicles and CHO cell lines permanently expressing the Na ؉ /bile acid cotransporters from rabbit ileum or rabbit liver. The hepatic transporter showed a remarkably broad specificity for interaction with cholephilic compounds in contrast to the ileal system. The anion transport inhibitor diisothiocyanostilbene disulfonate (DIDS) is a strong inhibitor of the hepatic Na ؉ /bile acid cotransporter, but does not show any affinity to its ileal counterpart. Inhibition studies and uptake measurements with about 40 different bile acid analogues differing in the number, position, and stereochemistry of the hydroxyl groups at the steroid nucleus resulted in clear structure-activity relationships for the ileal and hepatic bile acid transporters. The affinity to the ileal and hepatic Na ؉ /bile acid cotransport systems and the uptake rates by cell lines expressing those transporters as well as rabbit ileal brush border membrane vesicles is primarily determined by the substituents on the steroid nucleus. Two hydroxy groups at position 3, 7, or 12 are optimal whereas the presence of three hydroxy groups decreased affinity. Vicinal hydroxy groups at positions 6 and 7 or a shift of the 7-hydroxy group to the 6-position significantly decreased the affinity to the ileal transporter in contrast to the hepatic system. 6-Hydroxylated bile acid derivatives are preferred substrates of the hepatic Na ؉ /bile acid cotransporter. Surprisingly, the 3 ␣ -hydroxy group being present in all natural bile acids is not essential for high affinity interaction with the ileal and the hepatic bile acid transporter. The 3 ␣ -hydroxy group seems to be necessary for optimal transport of a bile acid across the hepatocyte canalicular membrane. A modification of bile acids at the 3-position therefore conserves the bile acid character thus determining the 3-position of bile acids as the ideal position for drug targeting strategies using bile acid transport pathways.