Comparative analysis of the single-molecule transport kinetics of OATP1B1 genetic variants

Several genetic variants of OATP1B1, a hepatic uptake transporter, increase the blood concentrations of substrate drugs, e.g., ∗15 carriers exhibit higher blood substrate concentrations than ∗1b carriers. It remains unclear whether these differences are due to changes in expression or intrinsic activity (transport activity per OATP1B1 molecule). This study compared the intrinsic activity of four OATP1B1 variants, ∗1a, ∗1b, ∗5, and ∗15, using HEK293 cell lines that co-expressed large-conductance Ca²⁺-activated K⁺ (BK) channels and one of the OATP1B1 variants. To estimate the kinetic parameters K_m and V_max, 2', 7'-dichlorofluorescein uptake was evaluated. The number of OATP molecules per cell (Q_T) was calculated from BK channel-mediated whole-cell conductance and the OATP1B1/BK channel expression ratio (ρ) (determined by LC-MS/MS). V_max,int (maximum intrinsic transport velocity) was obtained by dividing V_max by Q_T, and intrinsic clearance (CL_int) was calculated as V_max,int/K_m. The K_m values of ∗1a, ∗1b, ∗5, and ∗15 were 12.5, 9.19, 7.53, and 10.4 μM, and their V_max,int values were 3.0, 7.0, 1.5, and 1.2 × 10^-21 mol/OATP molecule/min, respectively. Accordingly, the CL_int value for OATP1B1∗15 was 15 % lower than that for OATP1B1∗1b, suggesting that the increased blood substrate concentrations observed in OATP1B1∗15 carriers may be due to the decreased intrinsic activity of OATP1B1∗15.