title: Putative irreversible inhibitors of the human sodium-dependent bile acid transporter (hASBT; SLC10A2) support the role of transmembrane domain 7 in substrate binding/translocation.
abstract: OBJECTIVE
  To explore the involvement of transmembrane domain (TM) 7 of the human apical sodium-dependent bile acid transporter (hASBT) on bile acid (BA) binding/translocation, using two electrophilic BA derivatives as molecular probes.
  
  
  METHODS
  Two electrophilic derivatives of chenodeoxycholic acid (CDCA) were designed, synthesized and evaluated for their ability to inactivate hASBT, and the human organic cation/carnitine transporter (hOCTN2) as a control (i.e. a non-BA transporting model). The ability of electrophilic derivatives to interact with hASBT was evaluated by 2-aminoethyl-methanethiosulfonate (MTSEA)-biotin labeling of thiol groups in TM7 cysteine mutants.
  
  
  RESULTS
  Unlike native BAs, the electrophilic CDCA derivatives specifically inactivated hASBT, but not hOCTN2, and inhibited hASBT in a time- and concentration-dependent fashion. Preincubation of hASBT Cys-mutants in the exofacial half of TM7 with reactive electrophilic probes blocked transporter biotinylation by MTSEA-biotin, similar to 2-(trimethylammonium)ethyl-methanethiosulfonate (MTSET) blocking. This blocking pattern differed from that produced by native BAs, which exposed exofacial TM7 residues, thereby increasing staining.
  
  
  CONCLUSIONS
  Kinetic and biochemical data indicate these novel electrophilic BAs are potent and specific irreversible inhibitors of hASBT and offer new evidence about the role of TM7 in binding/translocation of bile acids.
authors.affiliation: Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile, Santiago, Chile. pmgonzal@uc.cl
authors.forename: Pablo M
authors.initials: PM
authors.lastname: González
authors.forename: Naissan
authors.initials: N
authors.lastname: Hussainzada
authors.forename: Peter W
authors.initials: PW
authors.lastname: Swaan
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Mackerell
authors.forename: James E
authors.initials: JE
authors.lastname: Polli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001647:Bile Acids and Salts / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002635:Chenodeoxycholic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D015335:Molecular Probes / Q000737:chemistry* / Q000494:pharmacology*; D008969:Molecular Sequence Data; D029363:Organic Anion Transporters, Sodium-Dependent / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027701:Organic Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D000074058:Solute Carrier Family 22 Member 5; D027981:Symporters / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001647:Bile Acids and Salts; D015335:Molecular Probes; D029363:Organic Anion Transporters, Sodium-Dependent; D027701:Organic Cation Transport Proteins; C492020:SLC22A5 protein, human; D000074058:Solute Carrier Family 22 Member 5; D027981:Symporters; D002635:Chenodeoxycholic Acid; C071908:sodium-bile acid cotransporter