2), but not GCDCA (Fig. from that produced by native BAs, which revealed exofacial TM7 residues, thereby increasing staining. Summary Kinetic and biochemical data indicate these novel electrophilic BAs are potent and specific irreversible inhibitors of hASBT and offer new evidence about the part of TM7 in binding/translocation of bile acids. Intro The human being apical sodium-dependent bile Peptide YY(3-36), PYY, human acid transporter (hASBT; SLC10A2) is definitely a 348 amino acid protein having a molecular excess weight of 43 kDa in its fully glycosylated form (1, 2). Its physiological function as a solute symporter is definitely characterized by efficiently coupling sodium to bile acid translocation with an approximate 2:1 stoichiometry (3). hASBT is definitely a burgeoning pharmaceutical target owing to its central part in cholesterol homeostasis and is primarily indicated in the terminal ileum, kidneys and cholangiocytes (4). Despite the recent crystallization of a prokaryotic ASBT homologue (5), mechanistic understanding in the molecular level of substrate binding and translocation by mammalian ASBT is definitely hindered from the absence of high-resolution structural data. Nonetheless, recent biochemical and biophysical studies by our group on hASBT structure/function support a seven transmembrane website (TM) topology (2, 6) and reveal a critical part of amino acid residues in TM7 (7) during bile acid binding and translocation events. Substrate-like probes that interact irreversibly with proteins may provide unique mechanistic insights into substrate-transporter binding and translocation. For example, Kramer and colleagues (8, 9) synthesized photoreactive derivatives of taurocholic acid (TCA) to demonstrate the bile acid binding site of rabbit ASBT was restricted to the C-terminal portion of the protein. However, this approach relied on 7-azo derivatives which, upon activation with light, generate highly reactive carbene, that can react non-specifically with ASBT residues via nucleophilic, electrophilic, and free radical mechanisms. The present work aimed to apply electrophilic CDCA derivatives, which may interact with ASBT protein through a specific and more controlled Peptide YY(3-36), PYY, human reaction, as molecular probes to further understand hASBT function. First, we designed 3-chloro- and 7-mesyl derivatives of CDCA to assess their potential as irreversible inhibitors of hASBT. We hypothesized that an electrophilic carbon could be Peptide YY(3-36), PYY, human selectively attacked by nucleophilic amino acid residues within the binding site of hASBT, therefore forming covalent bonds that would inactivate the transporter. To the best of our knowledge, such an alkylating approach to elucidate transporter function has not been reported previously. Functional assay data, including time- and concentration-dependent kinetic studies indicate that electrophilic CDCA derivatives selectively and irreversibly inhibit hASBT. We next aimed Rabbit Polyclonal to MYLIP to employ electrophilic bile acid derivates to further examine the reported part of TM7 amino acid residues in bile acid binding and translocation events. We have previously demonstrated that exofacial residues within TM7 (Phe287-Gln297) are most sensitive to changes by methanethiosulfonate (MTS) reagents (7). Since these molecules will also be electrophilic in nature, we hypothesized that bile acids bearing electron-withdrawing substituents would display related reactivity patterns. To test this hypothesis we performed a series of biochemical studies to test whether electrophilic bile acid analogs can bind to ASBT and react with nucleophilic cysteine residues manufactured within the binding site. Results from these studies offer novel mechanistic insights concerning the part of TM7 in binding and/or translocation of bile acids via hASBT protein. MATERIALS AND METHODS Materials [3H]-Taurocholic acid (10 Ci/mmol), and [3H]-L-carnitine (66 Ci/mmol) were purchased from American Radiolabeled Chemicals, Inc, (St. Louis, MO). Taurocholic acid (TCA), glyco-chenodeoxycholic acid (GCDCA), and glyco-deoxycholic acid (GDCA) were from Sigma Aldrich (St. Louis, MO). Glyco-ursodeoxycholic acid (GUDCA) was purchased from Calbiochem (San Diego, CA). Chenodeoxycholate (CDCA) was from TCI America (Portland, OR). [2-(trimethylammonium)ethyl]-methanethio-sulfonate (MTSET) and 2-((biotinoyl)amino)-ethyl-methanethiosulfonate (MTSEA-biotin) were acquired from Toronto Study Chemicals, Inc, (North York, ON, Canada). Geneticin?, fetal bovine serum (FBS), trypsin, and DMEM were purchased from Invitrogen (Rockville, MD). All other reagents and chemicals were of the highest purity commercially available. Synthesis of electrophilic CDCA derivatives The synthesis of the electrophilic bile acids 3-chloro-7-hydroxy-5-cholan-24-oic acid (3-Cl-CDCA) and 3-hydroxy-7-mesyloxy-5-cholan-24-oic acid (7-Ms-CDCA) Peptide YY(3-36), PYY, human as explained in the Supplementary Material section (Techniques 1 and 2, respectively). Identities of electrophilic derivatives were confirmed 1D 1H NMR and 13C NMR spectra recorded having a Varian Inova 500 MHz (Varian Inc., Palo Alto, CA) (Supplemental Material, Figs. S1 and S2, panels A and B). Cell tradition and transient transfection Stably transfected hASBT-MDCK and hOCTN2-MDCK cells were cultured as previously explained (10). Briefly, cells were cultivated at 37 C, 90% relative moisture, 5% CO2.