title: Functional analysis of nonsynonymous single nucleotide polymorphisms of multidrug resistance-associated protein 2 (ABCC2).
abstract: BACKGROUND
  Multidrug resistance-associated protein 2 (MRP2; ABCC2) mediates the biliary excretion of glutathione, glucuronide, and sulfate conjugates of endobiotics and xenobiotics. Single nucleotide polymorphisms (SNPs) of MRP2 contribute to interindividual variability in drug disposition and ultimately in drug response.
  
  
  OBJECTIVE
  To characterize the transport function of human wild-type (WT) MRP2 and four SNP variants, S789F, A1450T, V417I, and T1477M.
  
  
  METHODS
  The four SNP variants were expressed in Sf9 cells using recombinant baculovirus infection. The kinetic parameters [Km, (μmol/l); V(max), (pmol/mg/min); the Hill coefficient] of ATP-dependent transport of leukotriene C(4) (LTC(4)), estradiol-3-glucuronide (E(2)3G), estradiol-17β-glucuronide (E(2)17G), and tauroursodeoxycholic acid (TUDC) were determined in Sf9-derived plasma membrane vesicles. Transport activity was normalized for expression level.
  
  
  RESULTS
  The V(max) for transport activity was decreased for all substrates for S789F, and for all substrates except E(2)17G for A1450T. V417I showed decreased apparent affinity for LTC(4), E(2)3G, and E(2)17G, whereas transport was similar between wild-type (WT) and T1477M, except for a modest increase in TUDC transport. Examination of substrate-stimulated MRP2-dependent ATPase activity of S789F and A1450T, SNPs located in MRP2 nucleotide-binding domains (NBDs), demonstrated significantly decreased ATPase activity and only modestly decreased affinity for ATP compared with WT.
  
  
  CONCLUSIONS
  SNPs in the NBDs (S789F in the D-loop of NBD1, or A1450T near the ABC signature motif of NBD2) variably decreased the transport of all substrates. V417I in membrane spanning domain 1 selectively decreased the apparent affinity for the glutathione and glucuronide conjugated substrates, whereas the T1477M SNP in the carboxyl terminus altered only TUDC transport.
authors.affiliation: Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536-0305, USA.
authors.forename: Vandana
authors.initials: V
authors.lastname: Megaraj
authors.forename: Tianyong
authors.initials: T
authors.lastname: Zhao
authors.forename: Christian M
authors.initials: CM
authors.lastname: Paumi
authors.forename: Phillip M
authors.initials: PM
authors.lastname: Gerk
authors.forename: Richard B
authors.initials: RB
authors.lastname: Kim
authors.forename: Mary
authors.initials: M
authors.lastname: Vore
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D016367:Baculoviridae; D054316:Biomarkers, Pharmacological; D018432:Drug Resistance, Multiple / Q000235:genetics*; D004958:Estradiol / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D005822:Genetic Vectors; D020719:Glucuronides / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D017997:Leukotriene C4 / Q000493:pharmacokinetics; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D013655:Taurochenodeoxycholic Acid / Q000493:pharmacokinetics
chemical_list: D000251:Adenosine Triphosphatases; D054316:Biomarkers, Pharmacological; D004958:Estradiol; D020719:Glucuronides; D005978:Glutathione; D017997:Leukotriene C4; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D013655:Taurochenodeoxycholic Acid; C000715087:ABCC2 protein, human; C025482:estradiol-3-glucuronide; C025483:estradiol-17 beta-glucuronide; C031655:ursodoxicoltaurine