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Conserved positively charged amino acid residues in the putative binding pocket are important for OATP1B1 function
Author(s) -
Miao Yi,
Hagenbuch Bruno
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a196-d
Subject(s) - alanine , mutant , amino acid , wild type , biochemistry , alanine scanning , mutagenesis , site directed mutagenesis , chemistry , binding site , biology , conserved sequence , peptide sequence , gene
OATP1B1 and 1B3 are closely related transport proteins that mediate translocation of amphipathic substrates across the sinusoidal membrane of hepatocytes. Using comparative modeling based on structures of major facilitator superfamily members, a putative structural model of OATP1B3 with a “Positive Binding Pocket” has been predicted (Meier‐Abt et al., J. Membr. Biol . 208 :, 2005). Therefore we tested the hypothesis that positive amino acids, conserved in the OATP1B family and predicted to be in this positive binding pocket, are important for OATP1B1 function. We performed site‐directed mutagenesis and expressed the resulting constructs using the vaccinia virus T7 system in HeLa cells for functional analysis. Alanine substitution of Arg181 (R181A) reduced estradiol‐17β‐glucuronide uptake rates to about 30% compared to wild type OATP1B1. Mutant R181K did not transport at all, whereas R181H exhibited 20% of wild‐type transport activity. Mutants R580A, R580K and R580H similarly demonstrated reduced uptake rates. Kinetic experiments revealed that R181A has a lower affinity while R580A has a lower maximal transport rate than wild‐type OATP1B1. Surface biotinylation experiments confirmed that R580A is hardly expressed in the plasma membrane. In conclusion, conserved positively charged amino acid residues in the putative binding pocket affect both transport function and surface expression of OATP1B1.