Identification of Gln422 in transmembrane domain 9 as a key amino acid residue for the selective transport of ginsenoside Re by organic anion transporting polypeptide 1B3.

Organic anion transporting polypeptide (OATP) 1B1 and 1B3 are the 2 highly homologous liver-specific uptake transporters. Although they share many common substrates, OATP1B3 exhibits selective transport for some protopanaxatriol-type ginsenosides such as ginsenoside Re. In this study, we successfully identified Gln422 (Q422) in transmembrane domain 9 as a key residue for OATP1B3's selectivity toward 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 the Q422Y mutation might cause steric hindrance for ginsenoside Re binding/translocation. Q422 plays a significant role in the transport of protopanaxatriol-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. SIGNIFICANCE STATEMENT: Protopanaxatriol-type ginsenosides are primarily eliminated via hepatobiliary excretion. They are transported by organic anion transporting polypeptide (OATP) 1B3 but not by OATP1B1. This study successfully identified that Q422 in transmembrane domain 9 plays a pivotal role in OATP1B3's uptake for ginsenosides Re, Rg1, and notoginsenoside R1. The naturally occurring Q422H variant may largely compromise OATP1B3's function and thus impair the hepatobiliary excretion of these ginsenosides. These findings would advance our knowledge of the molecular mechanism for OATP1B3's selectivity toward protopanaxatriol-type ginsenosides.