Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3

Abstract

Background and purpose: 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. Experimental approach: 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. Key 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 Vmax normalized to cell surface protein expression for Lys41>Ala (wild type: 190 Ϯ 8, Lys41>Ala:16 Ϯ 4 pmol (mg protein) -1 min -1 , P < 0.001), whereas Vmax of Lys41>Arg and Arg580>Lys (103 Ϯ 8 and 123 Ϯ 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 and implications:We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.

Influence of the flavonoids apigenin, kaempferol, and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1

“…Kaempferol showed the highest amounts in leek and endive with values of 30 and 46 mg / kg of fresh edible part, respectively. A further analyses of different beverages revealed that quercetin can 19 be found in red wine (highest concentration measured: 16 mg/l), apple juice, tomato juice, and different tea brands [2]. Taken the high and regular consumption of these vegetables, fruits, and beverages into consideration, it seems comprehensible that the concomitant intake of such foods with drugs such as fexofenadine or atorvastatin may lead to a reduced absorption of these drugs.…”

“…The uptake experiments were performed as previously described [19][20]. Briefly, HEK293-OATP1A2, HEK293-OATP2B1, and the respective HEK293-VC cells were seeded in poly-D-lysine coated 12-well plates at an initial density of 7.5x10 5 cells / well.…”

“…After geneticin (G-418; 500 µg/ml) treatment, single colonies were selected and characterized for OATP1A2 mRNA and protein expression using real-time PCR, immunofluorescence, and immunoblot analysis as previously described [19][20]. The polyclonal rabbit anti human …”

“…The uptake experiments were performed as previously described [19][20]. Briefly, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 12 the inhibitory constants (K i ) of flavonoids for OATP1A2 and OATP2B1 were performed using Dixon-Plot analyses [24].…”

“…Human embryonic kidney cells (HEK293) were transfected with the plasmid pQCXIN-OATP1A2 using a retroviral gene transfer and expression kit (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France). After geneticin (G-418; 500 µg/ml) treatment, single colonies were selected and characterized for OATP1A2 mRNA and protein expression using real-time PCR, immunofluorescence, and immunoblot analysis as previously described [19][20]. The polyclonal rabbit anti human …”