Luciferase-based bioluminescence revealed the facilitated diffusion of D-luciferin mediated by SLC17A3

SLC17A3 localized to the apical membrane of the renal proximal tubules has been implicated in the urinary excretion of drugs and endogenous/exogenous metabolites transported into the tubules by OAT1 and OAT3. Because SLC17A3 mediates the facilitated diffusion of organic anions, which requires a sensitive and rapid assay, no system has been established to evaluate its transport activity in mammalian cells. In this study, we demonstrated that the exposure of cells expressing click beetle luciferase (bLuc) and SLC17A3 to D-luciferin produces marked bioluminescence, which enables the evaluation of SLC17A3 function. The bioluminescence intensity increased under depolarized membrane potential conditions, consistent with the unique feature of SLC17A3 as a voltage-dependent organic anion transporter. SLC17A3-mediated bioluminescence was saturable with an apparent Michaelis–Menten constant (K_m) of 8.1 μM. The inhibitory effects of various compounds including OAT1/OAT3 substrates and inhibitors on bioluminescence were in good agreement with those reported in SLC17A3-expressing Xenopus oocytes using radio-labeled substrates. Interestingly, we found that sulfinpyrazone and lesinurad, uricosuric drugs that inhibit SLC22A12/URAT1, are potent SLC17A3 inhibitors, suggesting the possibility that they alter the pharmacokinetics of OAT1/OAT3-substrate drugs and urate. Taken together, the bioluminescence-based SLC17A3 functional assay is robust and reliable. This strategy enables the study of its transport activity and the identification of potential SLC17A3-mediated drug–drug interactions. This approach also provides an opportunity to elucidate the molecular mechanisms involved in the urinary excretion of organic anions.