title: Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3.
abstract: OBJECTIVE
  Organic anion transporting polypeptide 1B3 (OATP1B3) (SLCO1B3) mediates the uptake of endogenous substrates (e.g. estrone-3-sulphate) and drugs (e.g. pravastatin) from blood into hepatocytes. Structure-based modelling of OATP1B3 suggested that a pore with a positive electrostatic potential contributes to the transport mechanism. Therefore, we investigated the role of conserved positively charged amino acids for OATP1B3-mediated uptake of sulphobromophthalein (BSP) and pravastatin.
  
  
  METHODS
  Residues Lys28, Lys41 and Arg580 in OATP1B3 were substituted by alanine, arginine, glutamine, glycine or lysine. Using immunofluorescence, immunoblot analysis and cellular uptake assays, the effect of these mutations on protein expression and transport activity was investigated.
  
  
  RESULTS
  Immunofluorescence revealed that all mutants were localized in the plasma membrane with partial intracellular retention of the Arg580>Ala and Arg580>Lys mutants. Lys41>Ala, Lys41>Gln, Lys41>Gly, Arg580>Gly and Arg580>Lys showed significantly reduced transport for BSP and pravastatin. Kinetic analyses of BSP transport revealed a significant reduction of V(max) normalized to cell surface protein expression for Lys41>Ala (wild type: 190 +/- 8, Lys41>Ala:16 +/- 4 pmol (mg protein)(-1) min(-1), P < 0.001), whereas V(max) of Lys41>Arg and Arg580>Lys (103 +/- 8 and 123 +/- 14 pmol (mg protein)(-1) min(-1), P > 0.05) did not change significantly. This suggests that the positive charges at positions 41 and 580 are important for transport activity of BSP. Structural modelling indicated that the positively charged side chain of Lys41 is flexible within the pore. The orientation of Arg580 is defined by adjacent residues Glu74 and Asn77, which was confirmed by kinetic analysis of Glu74>Ala.
  
  
  CONCLUSIONS
  We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany. hartmut.glaeser@pharmakologie.med.uni-erlangen.de
authors.forename: H
authors.initials: H
authors.lastname: Glaeser
authors.forename: K
authors.initials: K
authors.lastname: Mandery
authors.forename: H
authors.initials: H
authors.lastname: Sticht
authors.forename: M F
authors.initials: MF
authors.lastname: Fromm
authors.forename: J
authors.initials: J
authors.lastname: König
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D022082:Cellular Structures / Q000378:metabolism; D004970:Estrone / Q000031:analogs & derivatives; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D027361:Organic Anion Transporters / Q000737:chemistry / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism; D017035:Pravastatin / Q000378:metabolism; D017433:Protein Structure, Secondary; D013448:Sulfobromophthalein / Q000378:metabolism
chemical_list: D027361:Organic Anion Transporters; D004364:Pharmaceutical Preparations; D013448:Sulfobromophthalein; D004970:Estrone; D001120:Arginine; D008239:Lysine; D017035:Pravastatin; D000409:Alanine; C017296:estrone sulfate; D005998:Glycine