Abstract

Plasma membrane monoamine transporter (PMAT) is a major uptake2 monoamine transporter that shares extensive substrate and inhibitor overlap with organic cation transporters 1-3 (OCT1-3). Currently, there are no PMAT-specific inhibitors available that can be used in in vitro and in vivo studies to differentiate between PMAT and OCT activities. In this study, we showed that IDT307, a fluorescent analog of 1-methyl-4-phenylpyridinium (MPP+), is a transportable substrate for PMAT; and that IDT307-based fluorescence assay can be used to rapidly identify and characterize PMAT inhibitors. Using the fluorescent substrate-based assays, we analyzed the interactions of eight HIV protease inhibitors (PIs) with human PMAT and OCT1-3 in HEK293 cells stably transfected with individual transporters. Our data revealed that PMAT and OCTs exhibit distinct sensitivity and inhibition patterns towards HIV PIs. PMAT is most sensitive to PI inhibition whereas OCT2 and OCT3 are resistant. OCT1 showed an intermediate sensitivity and a distinct inhibition profile from PMAT. Importantly, lopinavir is a potent PMAT inhibitor and exhibited >120 fold selectivity towards PMAT (IC₅₀ = 1.4 ± 0.2 μM) over OCT1 (IC₅₀ = 174 ± 40 μM). Lopinavir has no inhibitory effect on OCT2 and OCT3 at maximal tested concentrations. Lopinavir also exhibited no or much weaker interactions with uptake1 monoamine transporters. Together, our results revealed that PMAT and OCTs have distinct specificity exemplified by their differential interaction with HIV PIs. Further, we demonstrate that lopinavir can be used as a selective PMAT inhibitor to differentiate PMAT-mediated monoamine and organic cation transport from those mediated by OCT1-3.