Abstract
In contrast to reversible activation by agonist, cholecystokinin 1 receptor (CCK1R) is permanently activated by singlet oxygen generated in photodynamic action, with sulphonated aluminium phthalocyanine or genetically encoded mini singlet oxygen generator (miniSOG) as photosensitizer. In these works, a halogen light source was used to power photodynamic action. For possible in vivo application of photodynamic CCK1R physiology, bearing a cumbersome light-delivery device connected to an external light source by experimental animals might interfere with their behavior. Therefore, in the present work, the possibility of bioluminescence-driven miniSOG photodynamic CCK1R activation was examined, as monitored by Fura-2 calcium imaging. In parallel experiments, it was found that, after plasma membrane (PM)-localized expression of miniSOG(PM) in AR4-2J cells, light irradiation with blue light-emitting diode (LED) (450 nm, 85 mW·cm(-2), 1.5 min) induced persistent calcium oscillations that were blocked by CCK1R antagonist devazepide 2 nM. NanoLuc was expressed bicistronically with miniSOG(PM) via an internal ribosome entry site (IRES) sequence (pminiSOG(PM)-IRES-NanoLuc). The resultant miniSOG(PM)-IRES-NanoLuc-AR4-2J cells were found to generate strong bioluminescence upon addition of NanoLuc substrate coelenterazine. Strikingly, coelenterazine 5 microM was found to trigger long-lasting calcium oscillations (a hallmark for permanent CCK1R activation) in perifused miniSOG(PM)-IRES-NanoLuc-AR4-2J cells. These data indicate that NanoLuc bioluminescence can drive miniSOG(PM) photodynamic CCK1R activation, laying the foundation for its future in vivo applications.