Photodynamic Activation of Cholecystokinin 1 Receptor with Different Genetically Encoded Protein Photosensitizers and from Varied Subcellular Sites

Cholecystokinin 1 receptor (CCK1R) is activated by singlet oxygen ((1)O(2)) generated in photodynamic action with sulphonated aluminum phthalocyanine (SALPC) or genetically encoded protein photosensitizer (GEPP) KillerRed or mini singlet oxygen generator (miniSOG). A large number of GEPP with varied (1)O(2) quantum yields have appeared recently; therefore, in the present work, the efficacy of different GEPP to photodynamically activate CCK1R was examined, as monitored by Fura-2 calcium imaging. KillerRed, miniSOG, miniSOG2, singlet oxygen protein photosensitizer (SOPP), flavin-binding fluorescent protein from Methylobacterium radiotolerans with point mutation C71G (Mr4511(C71G)), and flavin-binding fluorescent protein from Dinoroseobacter shibae (DsFbFP) were expressed at the plasma membrane (PM) in AR4-2J cells, which express endogenous CCK1R. Light irradiation (KillerRed: white light 85.3 mW‧cm(−2), 4’ and all others: LED 450 nm, 85 mW·cm(−2), 1.5′) of GEPP(PM)-expressing AR4-2J was found to all trigger persistent calcium oscillations, a hallmark of permanent photodynamic CCK1R activation; DsFbFP was the least effective, due to poor expression. miniSOG was targeted to PM, mitochondria (MT) or lysosomes (LS) in AR4-2J in parallel experiments; LED light irradiation was found to all induce persistent calcium oscillations. In miniSOG(PM)-AR4-2J cells, light emitting diode (LED) light irradiation-induced calcium oscillations were readily inhibited by CCK1R antagonist devazepide 2 nM; miniSOG(MT)-AR4-2J cells were less susceptible, but miniSOG(LS)-AR4-2J cells were not inhibited. In conclusion, different GEPP(PM) could all photodynamically activate CCK1R. Intracellular GEPP photodynamic action may prove particularly suited to study intracellular GPCR.