The mechanisms by which Ca2+-store-release channels and Ca2+-entry channels are coupled to receptor activation are poorly understood. Modification of Ca2+ signals by 2-aminoethoxydiphenyl borate (2-APB), suggests the agent may target entry channels or the machinery controlling their activation. In DT40 B-cells and Jurkat T-cells, complete Ca2+ store release was induced by 2-APB (EC50 10–20 µM). At 75 µM, 2-APB emptied stores completely in both lymphocyte lines, but had no such effect on other cells. In DT40 cells, 2-APB mimicked B-cell receptor (BCR) cross-linking, but no effect was observed in mutant DT40 lines devoid of inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) or phospholipase C-γ2 (PLC-γ2). Like the BCR, 2-APB activated transfected TRPC3 (canonical transient receptor potential) channels, which acted as sensors for PLC-γ2-generated diacylglycerol in DT40 cells. The action of 2-APB on InsP3Rs and TRPC3 channels was prevented by PLC-inhibition, and required PLC-γ2 catalytic activity. However, unlike BCR activation, no increased InsP3 level could be measured in response to 2-APB. Also, calyculin A-induced cytoskeletal reorganization prevented 2-APB-induced InsP3R and TRPC3-channel activation, but not that induced by the BCR. 2-APB still activated TRPC3 channels in DT40 cells with fully depleted Ca2+ stores, indicating its action was not via Ca2+ release. Significantly, 2-APB-induced InsP3R and TRPC3 activation was prevented in DT40 knockout cells devoid of the BCR- and PLC-γ2-coupled adaptor/kinases, Syk, Lyn, Btk or BLNK. The results suggest that 2-APB activates Ca2+ signals in lymphocytes by initiating and enhancing coupling between components of the BCR–PLC-γ2 complex and both Ca2+-entry and Ca2+-release channels.
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December 2003
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Research Article|
December 15 2003
Modification of phospholipase C-γ-induced Ca2+ signal generation by 2-aminoethoxydiphenyl borate
Hong-Tao MA;
Hong-Tao MA
1
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, U.S.A.
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Kartik VENKATACHALAM;
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, U.S.A.
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Krystyna E. RYS-SIKORA;
Krystyna E. RYS-SIKORA
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, U.S.A.
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Li-Ping HE;
Li-Ping HE
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, U.S.A.
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Fei ZHENG;
Fei ZHENG
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, U.S.A.
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Donald L. GILL
Donald L. GILL
3
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, U.S.A.
3To whom correspondence should be addressed (e-mail dgill@umaryland.edu).
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Publisher: Portland Press Ltd
Received:
September 03 2003
Revision Received:
October 08 2003
Accepted:
October 15 2003
Accepted Manuscript online:
October 15 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 376 (3): 667–676.
Article history
Received:
September 03 2003
Revision Received:
October 08 2003
Accepted:
October 15 2003
Accepted Manuscript online:
October 15 2003
Citation
Hong-Tao MA, Kartik VENKATACHALAM, Krystyna E. RYS-SIKORA, Li-Ping HE, Fei ZHENG, Donald L. GILL; Modification of phospholipase C-γ-induced Ca2+ signal generation by 2-aminoethoxydiphenyl borate. Biochem J 15 December 2003; 376 (3): 667–676. doi: https://doi.org/10.1042/bj20031345
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