화학공학소재연구정보센터
Biochemical and Biophysical Research Communications, Vol.495, No.3, 2152-2157, 2018
Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on (D)-luciferin luciferase reaction in living cells by facilitating the intracellular accumulation of D-luciferin
Bioluminescence (BL) imaging based on (D)-luciferin ((D)-luc) luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the (D)-luc luciferase reaction, an approach to increase intracellular levels of (D)-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a (D)-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of (D)-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of (D)-luc was higher than that in control cells, and the specific (D)-luc uptake mediated by OAT1 was saturable with a Michaelis constant (K-m) of 0.23 M. The interaction between OAT1 and (D)-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that (D)-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent Km of 0.36 mu M. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OATI-mediated transport of (D)-luc could be a rate-limiting step in the (D)-luc luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of (D)-luc. Our findings suggest that because OAT1 is capable of transporting (D)-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells. (C) 2017 Elsevier Inc. All rights reserved.