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

Yuan Li,Zong Jie Cui

doi:10.3390/biom10101423

Abstract

Cholecystokinin 1 receptor (CCK1R) is activated by singlet oxygen (1O2) 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 1O2 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 (Mr4511C71G), 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 GEPPPM-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 miniSOGPM-AR4-2J cells, light emitting diode (LED) light irradiation-induced calcium oscillations were readily inhibited by CCK1R antagonist devazepide 2 nM; miniSOGMT-AR4-2J cells were less susceptible, but miniSOGLS-AR4-2J cells were not inhibited. In conclusion, different GEPPPM could all photodynamically activate CCK1R. Intracellular GEPP photodynamic action may prove particularly suited to study intracellular GPCR.

Highlights

CCK1R is unique among A class G protein-coupled receptors (GPCR) in that it is permanently activated ligand-independently by the lowest lying excited state molecular oxygen, the delta singlet oxygen (∆1O2 or 1O2), usually generated in type II photodynamic action with chemical photosensitizer sulphonated aluminum phthalocyanine (SALPC) after a brief cellular incubation [29,30] in freshly isolated rat pancreatic acini or with genetically encoded protein photosensitizer (GEPP) KillerRed or mini singlet oxygen generator target-expressed at the plasma membrane (PM) in rat pancreatic acinar tumor cell AR4-2J [31,32]
Other than targeting mini singlet oxygen generator (miniSOG) to the plasma membrane (PM) like previously reported [31,32], we examined the miniSOG photodynamic action from the mitochondria (MT) or lysosomes (LS)
It was found that PM-expressed KillerRed, miniSOG, miniSOG2, singlet oxygen protein photosensitizer (SOPP), Mr4511C71G, and DsFbFP after light irradiation all photodynamically activated CCK1R to induce persistent cytosolic calcium oscillations in AR4-2J cells, but the photodynamic effect of DsFbFP was much reduced in comparison, likely due to poor protein expression

Summary

Introduction

Cholecystokinin 1 receptor (CCK1R) is expressed prominently in highly restricted brain regions, such as the basal ganglia, hippocampus, thalamus, hypothalamus, medulla oblongata [1,2,3,4,5,6,7,8,9], in the dorsal horn of the spinal cord [7,8], and in the peripheral ganglion and enteric neurons [10,11,12,13,14,15,16]. CCK1R is unique among A class G protein-coupled receptors (GPCR) in that it is permanently activated ligand-independently by the lowest lying excited state molecular oxygen, the delta singlet oxygen (∆1O2 or 1O2), usually generated in type II photodynamic action with chemical photosensitizer sulphonated aluminum phthalocyanine (SALPC) after a brief cellular incubation [29,30] in freshly isolated rat pancreatic acini or with genetically encoded protein photosensitizer (GEPP) KillerRed or mini singlet oxygen generator (miniSOG) target-expressed at the plasma membrane (PM) in rat pancreatic acinar tumor cell AR4-2J [31,32]. In contrast to chemical photosensitizers such as porphyrins and phthalocyanines, genetic manipulations will ensure that GEPP could be targeted to specified cell types or subcellular organelles with high precision

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Discussion

Conclusion