Identification of Gln422 in transmembrane domain 9 as a key amino acid residue for the selective transport of ginsenoside Re by OATP1B3

Organic anion transporting polypeptide 1B1 (OATP1B1) and 1B3 are two highly homologous liver-specific uptake transporters. While they share many common substrates, OATP1B3 exhibits selective transport for some protopanaxatriol-type (PPT-type) ginsenosides such as ginsenoside Re. In this study, we successfully identified Q422 in transmembrane domain 9 (TM9) as a key residue for OATP1B3’s selectivity towards ginsenoside Re. Substitution of Q422 with bulky residues tyrosine and phenylalanine will almost totally destroy OATP1B3’s transport for ginsenoside Re, which is not due to the reduction of transporter’s surface expression but due to the reduction of its transport activity per se . Kinetic studies showed that the decreased transport activity of Q422Y was mainly due to its reduced turnover number. Molecular docking indicated that Q422Y mutation might cause steric hindrance for ginsenoside Re binding/translocation. Q422 also plays a significant role in the transport of PPT-type ginsenoside Rg1 and notoginsenoside R1. However, it only has a trivial effect on the transport of estradiol-17β-glucuronide, estrone-3-sulfate, and rosuvastatin. Taken together, Q422 is crucial for ginsenoside transport by OATP1B3, but its role is substrate-dependent.