Potent Inhibition of Ca Release-activated Ca Channels and T-lymphocyte Activation by the Pyrazole Derivative BTP2*

Ca entry through store-operated Ca release-activated Ca (CRAC) channels is essential for T-cell activation and proliferation. Recently, it has been shown that 3,5-bistrifluoromethyl pyrazole (BTP) derivatives are specific inhibitors of Ca -dependent transcriptional activity in T-cells (Trevillyan, J. M., Chiou, X. G., Chen, Y. W., Ballaron, S. J., Sheets, M. P., Smith, M. L., Wiedeman, P. E., Warrior, U., Wilkins, J., Gubbins, E. J., Gagne, G. D., Fagerland, J., Carter, G. W., Luly, J. R., Mollison, K. W., and Djuric, S. W. (2001) J. Biol. Chem. 276, 48118–48126). Whereas inhibition of Ca signals was reported for BTP2 (Ishikawa, J., Ohga, K., Yoshino, T., Takezawa, R., Ichikawa, A., Kubota, H., and Yamada, T. (2003) J. Immunol. 170, 4441–4449), it was not found for BTP3 (Chen, Y., Smith, M. L., Chiou, G. X., Ballaron, S., Sheets, M. P., Gubbins, E., Warrior, U., Wilkins, J., Surowy, C., Nakane, M., Carter, G. W., Trevillyan, J. M., Mollison, K., and Djuric, S. W. (2002) Cell. Immunol. 220, 134–142). We show that BTP2 specifically inhibits CRAC channels in T-cells with an IC50 of 10 nM. It does not interfere with other mechanisms important for Ca signals in T-cells, including Ca pumps, mitochondrial Ca signaling, endoplasmic reticulum Ca release, and K channels. BTP2 inhibits Ca signals in peripheral blood T-lymphocytes (in particular in CD4 T-cells) and in human Jurkat T-cells. Inhibition of Ca signals is independent of the stimulation method as Ca entry was blocked following stimulation with anti-CD3, which activates the T-cell receptor, and also following stimulation with thapsigargin or inositol 1,4,5-trisphosphate. BTP2 also inhibited Ca -dependent gene expression (interleukins 2 and 5 and interferon ) and proliferation of T-lymphocytes with similar IC50 values. BTP2 is the first potent and specific inhibitor of CRAC channels in primary T-lymphocytes. The inhibition of CRAC channels as well as Ca -dependent signal transduction with similar IC50 values in T-lymphocytes emphasizes the importance of CRAC channel activity during T-cell activation. Furthermore, BTP2 could prove to be a tool to finally unmask the molecular identity of CRAC channels.