title: Mechanism of high affinity inhibition of the human urate transporter URAT1.
abstract: Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions.
authors.affiliation: Department of Biology, Ardea Biosciences (A member of the AstraZeneca Group), 9390 Towne Centre Drive, San Diego, CA, 92121 USA.
authors.forename: Philip K
authors.initials: PK
authors.lastname: Tan
authors.affiliation: Department of Biology, Ardea Biosciences (A member of the AstraZeneca Group), 9390 Towne Centre Drive, San Diego, CA, 92121 USA.
authors.forename: Traci M
authors.initials: TM
authors.lastname: Ostertag
authors.affiliation: Department of Biology, Ardea Biosciences (A member of the AstraZeneca Group), 9390 Towne Centre Drive, San Diego, CA, 92121 USA.
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Miner
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D027321:Anion Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D027362:Organic Anion Transport Protein 1 / Q000737:chemistry; D027361:Organic Anion Transporters / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D027701:Organic Cation Transport Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism
chemical_list: D027321:Anion Transport Proteins; D027362:Organic Anion Transport Protein 1; D027361:Organic Anion Transporters; D027701:Organic Cation Transport Proteins; D011993:Recombinant Fusion Proteins; C491241:SLC22A12 protein, human; C554079:Slc22a12 protein, rat; D014527:Uric Acid