title: Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity.
abstract: OBJECTIVE
  The uptake carrier organic anion-transporting polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes, and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms and to analyze their functional relevance.
  
  
  METHODS
  We used DNA of ethnically diverse individuals for polymerase chain reaction, and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis. We then created variant OATP1B3 expression plasmids by site-directed mutagenesis, which were transiently expressed and functionally characterized in human cervical carcinoma (HeLa) cells using radiolabeled substrates.
  
  
  RESULTS
  We identified six nonsynonymous polymorphisms including novel variants such as 439A>G (Thr147Ala), 767G>C (Gly256Ala), 1559A>C (His520Pro), and 1679T>C (Val560Ala). Allelic frequencies occurred to be ethnicity-dependent, with the latter observed only in African-Americans (3.6%). After expression in HeLa cells, His520Pro, Val560Ala, and Met233Ile or Met233Ile_Ser112Ala haplotype variants showed decreased uptake activity compared with wild type for cholecystokinin-8 and rosuvastatin, but not for atorvastatin. Kinetic cholecystokinin-8 analysis showed reduced Vmax without altering Km. His520Pro and Val560Ala exhibited decreased total and plasma membrane protein expressions. Val560 mapped onto a structural model of OATP1B3 showed that this is a key region for substrate-transporter interaction. His520 resides in a predicted extracellular region thought to be critical to the pH-dependent component of OATP1B3 activity. Loss of activity at pH 7.4 and 8.0 relative to pH 6.5 was significantly greater for the Pro520 variant.
  
  
  CONCLUSIONS
  OATP1B3 polymorphisms that result in altered expression, substrate specificity, and pH-dependent activity may be of potential relevance to hepatic clearance of substrate drugs in vivo.
authors.affiliation: Division of Clinical Pharmacology, Department of Medicine, University of Western Ontario, Canada.
authors.forename: Ute I
authors.initials: UI
authors.lastname: Schwarz
authors.forename: Henriette E
authors.initials: HE
authors.lastname: Meyer zu Schwabedissen
authors.forename: Rommel G
authors.initials: RG
authors.lastname: Tirona
authors.forename: Atsuko
authors.initials: A
authors.lastname: Suzuki
authors.forename: Brenda F
authors.initials: BF
authors.lastname: Leake
authors.forename: Younes
authors.initials: Y
authors.lastname: Mokrab
authors.forename: Kenji
authors.initials: K
authors.lastname: Mizuguchi
authors.forename: Richard H
authors.initials: RH
authors.lastname: Ho
authors.forename: Richard B
authors.initials: RB
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000069059:Atorvastatin; D005464:Fluorobenzenes / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006538:Heptanoic Acids / Q000378:metabolism; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D029362:Organic Anion Transporters, Sodium-Independent / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D011743:Pyrimidines / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism; D000068718:Rosuvastatin Calcium; D012844:Sincalide / Q000378:metabolism; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D013449:Sulfonamides / Q000378:metabolism
chemical_list: D005464:Fluorobenzenes; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D029362:Organic Anion Transporters, Sodium-Independent; D011743:Pyrimidines; D011758:Pyrroles; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D013449:Sulfonamides; D000068718:Rosuvastatin Calcium; D000069059:Atorvastatin; D012844:Sincalide