Abstract

Inside-out–oriented membrane vesicles are useful tools to investigate whether a compound can be an inhibitor of efflux transporters such as multidrug resistance–associated protein 2 (MRP2). However, because of technical limitations of substrate diffusion and low dynamic uptake windows for interacting drugs used in the clinic, estradiol-17β-glucuronide (E17βG) remains the probe substrate that is frequently used in MRP2 inhibition assays. Here we recapitulated the sigmoidal kinetics of MRP2-mediated transport of E17βG, with apparent Michaelis-Menten constant (K_m) and V_max values of 170 ±17 µM and 1447 ± 137 pmol/mg protein/min, respectively. The Hill coefficient (2.05 ± 0.1) suggests multiple substrate binding sites for E17βG transport with cooperative interactions. Using E17βG as a probe substrate, 51 of 97 compounds tested (53%) showed up to 6-fold stimulatory effects. Here, we demonstrate for the first time that coproporphyrin-I (CP-I) is a MRP2 substrate in membrane vesicles. The uptake of CP-I followed a hyperbolic relationship, adequately described by the standard Michaelis-Menten equation (apparent K_m and V_max values were 7.7 ± 0.7 µM and 48 ± 11 pmol/mg protein/min, respectively), suggesting the involvement of a single binding site. Of the 47 compounds tested, 30 compounds were inhibitors of human MRP2 and 8 compounds (17%) stimulated MRP2-mediated CP-I transport. The stimulators were found to share the basic backbone structure of the physiologic steroids, which suggests a potential in vivo relevance of in vitro stimulation of MRP2 transport. We concluded that CP-I could be an alternative in vitro probe substrate replacing E17βG for appreciating MRP2 interactions while minimizing potential false-negative results for MRP2 inhibition due to stimulatory effects.