Functional Reconstitution of Human OATP1B1 and OATP1B3 in Proteoliposomes: Effect of Bicarbonate Gradients and Substrate-specific Stimulation on Transport

The hepatic organic anion transporting polypeptides OATP1B1 and OATP1B3 are critical for the clearance of widely prescribed drugs, hepatotoxins, and endogenous metabolites. However, the thermodynamic driving force governing their transport has remained a subject of debate due to the complexity of cellular models. Here, we report a defined, reconstituted proteoliposome system to study the transport kinetics of isolated human OATP1B1 and OATP1B3. Through direct functional assays, real-time counter-ion tracking, and a novel ABCG2-OATP1B1 functional co-reconstitution, we identify an outwardly directed bicarbonate gradient as a thermodynamic driving force for concentrative substrate uptake. In contrast we find no evidence for the proposed role of reduced glutathione efflux as a possible driving force. Targeted mutagenesis experiments revealed that the conserved TM3 residue His115 is needed to couple the bicarbonate gradient to concentrative substrate uptake, mechanistically linking substrate antiport to local pH-sensitive bicarbonate processing. We also investigated the substrate-specific stimulatory effect of the green tea catechin epigallocatechin gallate (EGCG) on OATP1B3 transport, confirming that this effect is intrinsic to OATP1B3, substrate specific, and does not need the involvement of other cellular proteins. Our findings define the energetic basis of OATP1B1 and OATP1B3 hepatic xenobiotic clearance.