A novel fluorescent cell membrane-permeable caged cyclic ADP-ribose analogue.
Cyclic adenosine diphosphate ribose is an endogenous Ca(2+) mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca(2+) 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 Ca(2+) release from endoplasmic reticulum, with concomitant Ca(2+) influx in Jurkat cells. Ca(2+) release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca(2+) influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca(2+) 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 Ca(2+) release from endoplasmic reticulum via RyRs and triggers Ca(2+) influx via TRPM2.
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
Pei-Lin
PL
Yu
Zhe-Hao
ZH
Zhang
Bai-Xia
BX
Hao
Yong-Juan
YJ
Zhao
Li-He
LH
Zhang
Hon-Cheung
HC
Lee
Liangren
L
Zhang
Jianbo
J
Yue
D000475:Alkenes / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D036563:Cyclic ADP-Ribose / Q000031:analogs & derivatives* / Q000378:metabolism; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism; D000071737:Stromal Interaction Molecule 1; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism
D000475:Alkenes; D003374:Coumarins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; C092958:caged cyclic ADP-ribose; D036563:Cyclic ADP-Ribose; C030123:coumarin; C013658:propylene; D002118:Calcium