Several Conserved Positively Charged Amino Acids in OATP1B1 are Involved in Binding or Translocation of Different Substrates

Several Conserved Positively Charged Amino Acids in OATP1B1 are Involved in Binding or... OATP1B1 and 1B3 are related transporters mediating uptake of numerous compounds into hepatocytes. A putative model of OATP1B3 with a “positive binding pocket” containing conserved positively charged amino acids was predicted (Meier-Abt et al. J Membr Biol 208:213–227, 2005). Based on this model, we tested the hypothesis that these positive amino acids are important for OATP1B1 function. We made mutants and measured surface expression and uptake of estradiol-17β-glucuronide, estrone-3-sulfate and bromosulfophthalein in HEK293 cells. Two of the mutants had low surface expression levels: R181K at 10% and R580A at 30% of wild-type OATP1B1. A lysine at position 580 (R580K) rescued the expression of R580A. Mutations of several amino acids resulted in substrate-dependent effects. The largest changes were seen for estradiol-17β-glucuronide, while estrone-3-sulfate and bromosulfophthalein transport were less affected. The wild-type OATP1B1 K m value for estradiol-17β-glucuronide of 5.35 ± 0.54 μM was increased by R57A to 30.5 ± 3.64 μM and decreased by R580K to 0.52 ± 0.18 μM. For estrone-3-sulfate the wild-type high-affinity K m value of 0.55 ± 0.12 μM was increased by K361R to 1.8 ± 0.47 μM and decreased by R580K to 0.1 ± 0.04 μM. In addition, R580K reduced the V max values for all three substrates to <25% of wild-type OATP1B1. Mutations at intracellular K90, H92 and R93 mainly affected V max values for estradiol-17β-glucuronide uptake. In conclusion, the conserved amino acids R57, K361 and R580 seem to be part of the substrate binding sites and/or translocation pathways in OATP1B1. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Several Conserved Positively Charged Amino Acids in OATP1B1 are Involved in Binding or Translocation of Different Substrates

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Publisher
Springer-Verlag
Copyright
Copyright © 2010 by Springer Science+Business Media, LLC
Subject
Life Sciences; Human Physiology ; Biochemistry, general
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-010-9300-3
Publisher site
See Article on Publisher Site

Abstract

OATP1B1 and 1B3 are related transporters mediating uptake of numerous compounds into hepatocytes. A putative model of OATP1B3 with a “positive binding pocket” containing conserved positively charged amino acids was predicted (Meier-Abt et al. J Membr Biol 208:213–227, 2005). Based on this model, we tested the hypothesis that these positive amino acids are important for OATP1B1 function. We made mutants and measured surface expression and uptake of estradiol-17β-glucuronide, estrone-3-sulfate and bromosulfophthalein in HEK293 cells. Two of the mutants had low surface expression levels: R181K at 10% and R580A at 30% of wild-type OATP1B1. A lysine at position 580 (R580K) rescued the expression of R580A. Mutations of several amino acids resulted in substrate-dependent effects. The largest changes were seen for estradiol-17β-glucuronide, while estrone-3-sulfate and bromosulfophthalein transport were less affected. The wild-type OATP1B1 K m value for estradiol-17β-glucuronide of 5.35 ± 0.54 μM was increased by R57A to 30.5 ± 3.64 μM and decreased by R580K to 0.52 ± 0.18 μM. For estrone-3-sulfate the wild-type high-affinity K m value of 0.55 ± 0.12 μM was increased by K361R to 1.8 ± 0.47 μM and decreased by R580K to 0.1 ± 0.04 μM. In addition, R580K reduced the V max values for all three substrates to <25% of wild-type OATP1B1. Mutations at intracellular K90, H92 and R93 mainly affected V max values for estradiol-17β-glucuronide uptake. In conclusion, the conserved amino acids R57, K361 and R580 seem to be part of the substrate binding sites and/or translocation pathways in OATP1B1.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Sep 4, 2010

References

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