Permanent Photodynamic Activation of the Cholecystokinin 2 Receptor.

Wen Zhu Tang,Zong Jie Cui

doi:10.3390/biom10020236

Abstract

The cholecystokinin 2 receptor (CCK2R) is expressed in the central nervous system and peripheral tissues, playing an important role in higher nervous and gastrointestinal functions, pain sensation, and cancer growth. CCK2R is reversibly activated by cholecystokinin or gastrin, but whether it can be activated permanently is not known. In this work, we found that CCK2R expressed ectopically in CHO-K1 cells was permanently activated in the dark by sulfonated aluminum phthalocyanine (SALPC/AlPcS4, 10–1000 nM), as monitored by Fura-2 fluorescent calcium imaging. Permanent CCK2R activation was also observed with AlPcS2, but not PcS4. CCK2R previously exposed to SALPC (3 and 10 nM) was sensitized by subsequent light irradiation (>580 nm, 31.5 mW·cm−2). After the genetically encoded protein photosensitizer mini singlet oxygen generator (miniSOG) was fused to the N-terminus of CCK2R and expressed in CHO-K1 cells, light irradiation (450 nm, 85 mW·cm−2) activated in-frame CCK2R (miniSOG-CCK2R), permanently triggering persistent calcium oscillations blocked by the CCK2R antagonist YM 022 (30 nM). From these data, it is concluded that SALPC is a long-lasting CCK2R agonist in the dark, and CCK2R is photogenetically activated permanently with miniSOG as photosensitizer. These properties of SALPC and CCK2R could be used to study CCK2R physiology and possibly for pain and cancer therapies.

Highlights

The G protein-coupled cholecystokinin type 2 receptor (CCK2R) is widely distributed in the central nervous system (CNS) [1,2]
On the basis of the above reports, in the present work we investigated the photodynamic modulation of CCK2R, with both SALPC and mini singlet oxygen generator (miniSOG) as photosensitizers
CCK2R was transiently expressed in Chinese hamster ovary K1 (CHO-K1) cells, immunocytochemistry 48 h after transfection revealed plasma membrane expression of CCK2R (Figure 1A)

Summary

Introduction

The G protein-coupled cholecystokinin type 2 receptor (CCK2R) is widely distributed in the central nervous system (CNS) [1,2]. Evidence for the involvement of CCK2R in pain sensitization/perception is abundant. CCK2R has been confirmed as being expressed in dorsal root ganglion neurons to mediate the inhibition of a Kv current (IA) to increase pain sensation [16]. The CCK2R antagonist is known to reduce the need for opioid administration in relieving pain in a mouse model of burn-induced pain [17], likely by antagonizing CCK2R–μ-opioid receptor (MOR) heterodimerization, which inhibits MOR signaling [18]. CCK infusion into amygdala in rats has been found to activate a descending CCK2R-mediated pathway and to inhibit spinal neuron discharge, leading to hindpaw analgesia [19]. CCK2R knockout is known to relieve pain in mouse models of chronic constrictive injury [20]

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Discussion

Conclusion