Modification of phospholipase C-gamma-induced Ca2+ signal generation by 2-aminoethoxydiphenyl borate.
The mechanisms by which Ca(2+)-store-release channels and Ca(2+)-entry channels are coupled to receptor activation are poorly understood. Modification of Ca(2+) 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 Ca(2+) store release was induced by 2-APB (EC(50) 10-20 microM). At 75 microM, 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 (InsP(3)) receptors (InsP(3)Rs) or phospholipase C-gamma2 (PLC-gamma2). Like the BCR, 2-APB activated transfected TRPC3 (canonical transient receptor potential) channels, which acted as sensors for PLC-gamma2-generated diacylglycerol in DT40 cells. The action of 2-APB on InsP(3)Rs and TRPC3 channels was prevented by PLC-inhibition, and required PLC-gamma2 catalytic activity. However, unlike BCR activation, no increased InsP(3) level could be measured in response to 2-APB. Also, calyculin A-induced cytoskeletal reorganization prevented 2-APB-induced InsP(3)R and TRPC3-channel activation, but not that induced by the BCR. 2-APB still activated TRPC3 channels in DT40 cells with fully depleted Ca(2+) stores, indicating its action was not via Ca(2+) release. Significantly, 2-APB-induced InsP(3)R and TRPC3 activation was prevented in DT40 knockout cells devoid of the BCR- and PLC-gamma2-coupled adaptor/kinases, Syk, Lyn, Btk or BLNK. The results suggest that 2-APB activates Ca(2+) signals in lymphocytes by initiating and enhancing coupling between components of the BCR-PLC-gamma2 complex and both Ca(2+)-entry and Ca(2+)-release channels.
Ma
Hong-Tao
HT
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, USA.
Venkatachalam
Kartik
K
Rys-Sikora
Krystyna E
KE
He
Li-Ping
LP
Zheng
Fei
F
Gill
Donald L
DL
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D001896:Boron Compounds / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism* / Q000502:physiology; D020013:Calcium Signaling; D002460:Cell Line; D002645:Chickens; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels / Q000378:metabolism; D017136:Ion Transport; D019169:Jurkat Cells; D008214:Lymphocytes / Q000378:metabolism*; D008954:Models, Biological; D051966:Phospholipase C gamma; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000502:physiology; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D050052:TRPC Cation Channels; D010738:Type C Phospholipases / Q000378:metabolism* / Q000502:physiology
D001896:Boron Compounds; D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels; D011947:Receptors, Antigen, B-Cell; D018160:Receptors, Cytoplasmic and Nuclear; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; C109986:2-aminoethoxydiphenyl borate; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium