A Novel Fluorescent Cell Membrane-permeable Caged Cyclic ADP-ribose Analogue

• Published in: The Journal of biological chemistry 2012-07, Vol.287 (29), p.24774-24783
• Main Authors: Yu, Pei-Lin, Zhang, Zhe-Hao, Hao, Bai-Xia, Zhao, Yong-Juan, Zhang, Li-He, Lee, Hon-Cheung, Zhang, Liangren, Yue, Jianbo
• Format: Article
• Language: English
• Subjects: Alkenes - metabolism; Blotting, Western; Calcium; Calcium Signaling; Cell Line; Cell Membrane - metabolism; Coumarins - metabolism; Cyclic ADP-ribose; Cyclic ADP-Ribose - analogs & derivatives; Cyclic ADP-Ribose - metabolism; Fluorescence; HEK293 Cells; Humans; Jurkat Cells; Membrane Proteins - genetics; Membrane Proteins - metabolism; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Real-Time Polymerase Chain Reaction; Ryanodine Receptor; Ryanodine Receptor Calcium Release Channel - genetics; Ryanodine Receptor Calcium Release Channel - metabolism; Signal Transduction; SOCE; Stromal Interaction Molecule 1; TRP Channels; TRPM Cation Channels - genetics; TRPM Cation Channels - metabolism; TRPM2
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.329854

Cyclic adenosine diphosphate ribose is an endogenous Ca2+ mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca2+ increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca2+ release from endoplasmic reticulum, with concomitant Ca2+ influx in Jurkat cells. Ca2+ release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca2+ influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca2+ release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca2+ release from endoplasmic reticulum via RyRs and triggers Ca2+ influx via TRPM2. Background: The available agonists for cADPR, an endogenous Ca2+-mobilizing nucleotide, are either weak or not cell-permeant. Results: We synthesized a coumarin-caged isopropylidene-protected cIDPRE (Co-i-cIDPRE), which is a potent and cell-permeant cADPR agonist. Conclusion: Uncaging of Co-i-cIDPRE activates RyRs for Ca2+ mobilization and triggers Ca2+ influx via TRPM2. Significance: Co-i-cIDPRE should provide a valuable tool to study cADPR/Ca2+ signaling.