Kirchberger Tanja, Moreau Christelle, Wagner Gerd K, Fliegert Ralf, Siebrands Cornelia C, Nebel Merle, Schmid Frederike, Harneit Angelika, Odoardi Francesca, Flügel Alexander, Potter Barry V L, Guse Andreas H
The Calcium Signalling Group, University Medical Center Hamburg-Eppendorf, Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Hamburg, Germany.
Biochem J. 2009 Jul 29;422(1):139-49. doi: 10.1042/BJ20082308.
cADPR (cyclic ADP-ribose) is a universal Ca(2+) mobilizing second messenger. In T-cells cADPR is involved in sustained Ca(2+) release and also in Ca(2+) entry. Potential mechanisms for the latter include either capacitative Ca(2+) entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N(1)-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N(1)-cIDPR evoked Ca(2+) signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca(2+) signalling induced by 8-Br-N(1)-cIDPR consisted of Ca(2+) release and Ca(2+) entry. Whereas Ca(2+) release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca(2+) entry was inhibited by the Ca(2+) entry blockers Gd(3+) (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca(2+) entry evoked by 8-Br-N(1)-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H(2)O(2) was enhanced dramatically in those cells, Ca(2+) signalling induced by 8-Br-N(1)-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N(1)-cIDPR. In summary, the sensitivity to the Ca(2+) entry blockers Gd(3+) and SKF-96365 is in favour of the concept of capacitative Ca(2+) entry, secondary to store depletion by 8-Br-N(1)-cIDPR. Taken together, 8-Br-N(1)-cIDPR appears to be the first cADPR agonist affecting Ca(2+) release and secondary Ca(2+) entry, but without effect on TRPM2.
环ADP核糖（cADPR）是一种通用的可动员钙离子的第二信使。在T细胞中，cADPR参与持续性钙离子释放以及钙离子内流。后者的潜在机制包括cADPR导致储存库耗竭后的容量性钙离子内流，或非选择性阳离子通道瞬时受体电位阳离子通道亚家族M成员2（TRPM2）的直接激活。在此，我们对新描述的可透过细胞膜的cADPR激动剂8-溴-N(1)-环异鸟苷二磷酸核糖（8-Br-N(1)-cIDPR）的分子靶点进行了表征。8-Br-N(1)-cIDPR可在人T淋巴瘤细胞系Jurkat和原代大鼠T淋巴细胞中引发钙离子信号。8-Br-N(1)-cIDPR诱导的钙离子信号包括钙离子释放和钙离子内流。钙离子释放对兰尼碱受体（RyR）阻断剂钌红（RuRed）和cADPR拮抗剂8-溴-cADPR（8-Br-cADPR）均敏感，而钙离子内流则受到钙离子内流阻断剂钆离子（Gd(3+)）、SKF-96365以及8-Br-cADPR的抑制。为了阐明TRPM2在8-Br-N(1)-cIDPR诱发的持续性钙离子内流中的潜在作用，我们在人胚肾（HEK）-293细胞中过表达了TRPM2。然而，尽管这些细胞中过氧化氢（H(2)O(2)）诱导的激活显著增强，但8-Br-N(1)-cIDPR诱导的钙离子信号几乎未受影响。同样，对TRPM2电流的直接分析也未揭示8-Br-N(1)-cIDPR对TRPM2的激活或协同激活作用。总之，对钙离子内流阻断剂Gd(3+)和SKF-96365的敏感性支持了8-Br-N(1)-cIDPR导致储存库耗竭后引发容量性钙离子内流的概念。综上所述，8-Br-N(1)-cIDPR似乎是首个影响钙离子释放和继发性钙离子内流但对TRPM2无作用的cADPR激动剂。
