The Importance of Val386 in Transmembrane Domain 8 for the Activation of OATP1B3 by Epigallocatechin Gallate

Abstract

Estrone-3-sulfate (E3S) uptake mediated by organic anion transporting polypeptide 1B3 (OATP1B3) can be activated by epigallocatechin gallate (EGCG). In this study, by using chimeric transporters and site-directed mutagenesis, we found that Val386 in transmembrane domain 8 (TM8) is essential for OATP1B3's activation by EGCG. Kinetic studies showed that the loss of activation of 1B3-TM8 and 1B3-V386F in the presence of EGCG is due to their decreased substrate binding affinity and reduced maximal transport rate. The overall transport efficiencies of OATP1B3, 1B3-TM8, and 1B3-V386F in the absence and presence of EGCG are 8.6 ± 0.7 vs 15.9 ± 1.4 (p < 0.05), 11.2 ± 2.1 vs 2.7 ± 0.3 (p < 0.05), and 10.2 ± 1.0 vs 2.5 ± 0.3 (p < 0.05), respectively. While 1B3-V386F cannot be activated by EGCG, its transport activity for EGCG is also diminished. OATP1B3's activation by EGCG is substrate-dependent as EGCG inhibits OATP1B3-mediated pravastatin uptake. Furthermore, the activation of OATP1B3-mediated E3S uptake by quercetin 3-O-α-L-arabinopyranosyl(1 → 2)-α-L-rhamnopyranoside is not affected by TM8 and V386F. Taken together, the activation of OATP1B3 by small molecules is substrate-and modulator-dependent, and V386 in TM8 plays a critical role in the activation of OATP1B3-mediated E3S uptake by EGCG.

G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17βG Uptake by Clotrimazole

“…Amino acid sequence alignment showed that in both, TM1 and TM8, 5 amino acid residues were different between OATP1B3 and 1B1. , To determine which amino acid residues in TM1 and TM8 are important for the activation of OATP1B3 by clotrimazole, we constructed 5 single mutants for each of TM1 and TM8, by replacing each residue in OATP1B3 with its corresponding residue in OATP1B1. As shown in Figure A, all these 10 mutants had comparable surface expression levels as wild-type OATP1B3.…”

“…Antibodies for detecting the six-His tag and the Na + /K + -ATPase α subunit were purchased from Proteintech (66005–1-Ig) and Abcam (ab76020), respectively. Other reagents were obtained from the same companies as previously indicated …”

“…Accordingly, we have investigated structural domains and amino acid residues that are crucial for the activation of OATP1B3-mediated E3S uptake by EGCG in previous studies. 11,12 By functional analysis of a series of chimeras between OATP1B3 and 1B1, we successfully identified that TMs 1, 8, and 10 are critical for the activation of OATP1B3 by EGCG. 11,12 With site-directed mutagenesis, we further pinpointed that G45, V386 and F555 are the most important amino acid residues in TMs 1, 8, and 10, respectively, for the activation of OATP1B3.…”

Importance of N-Glycosylation for the Expression and Function of Human Organic Anion Transporting Polypeptide 2B1

“…Twenty-four hours post-transfection, cell surface biotinylation experiment was carried out. The procedures for surface biotinylation and immunoblot analysis were the same as previously described . In brief, plasma membrane proteins were labeled with sulfo- N -hydroxysuccinimide-SS-biotin and isolated with streptavidin–agarose beads after cell lysis.…”

“…The procedures for surface biotinylation and immunoblot analysis were the same as previously described. 44 In brief, plasma membrane proteins were labeled with sulfo-N-hydroxysuccinimide-SS-biotin and isolated with streptavidin−agarose beads after cell lysis. Total proteins in cell lysate and isolated membrane proteins were then subjected to immunoblot analysis.…”