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Published on: December 27, 2016
Potent inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2
Christof Zitt1, Bettina Strauss, Eva C Schwarz
1Department of Biochemistry (RDR/B2), ALTANA Pharma AG, 78467 Konstanz, Germany.
Abstract:
Ca2+ entry through store-operated Ca2+release-activated Ca2+ (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 Ca2+-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 Ca2+ 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 IC(50) of approximately 10 nm. It does not interfere with other mechanisms important for Ca2+ signals in T-cells, including Ca2+ pumps, mitochondrial Ca2+ signaling, endoplasmic reticulum Ca2+ release, and K+ channels. BTP2 inhibits Ca2+ signals in peripheral blood T-lymphocytes (in particular in CD4+ T-cells) and in human Jurkat T-cells. Inhibition of Ca2+ signals is independent of the stimulation method as Ca2+ 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 Ca2+-dependent gene expression (interleukins 2 and 5 and interferon gamma) and proliferation of T-lymphocytes with similar IC(50) values. BTP2 is the first potent and specific inhibitor of CRAC channels in primary T-lymphocytes. The inhibition of CRAC channels as well as Ca2+-dependent signal transduction with similar IC(50) 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.
Insights
BTP2 is a potent and specific inhibitor of calcium release-activated calcium (CRAC) channels in T-cells. This compound blocks calcium entry, gene expression, and proliferation, highlighting CRAC channels
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Calcium (Ca2+) entry via store-operated Ca2+ release-activated Ca2+ (CRAC) channels is crucial for T-cell activation and proliferation.
- 3,5-bistrifluoromethyl pyrazole (BTP) derivatives have been identified as inhibitors of Ca2+-dependent transcriptional activity in T-cells.
- Previous studies showed conflicting results regarding the inhibitory effects of BTP2 and BTP3 on Ca2+ signals in T-cells.
Purpose of the Study:
- To investigate the specificity and potency of BTP2 as an inhibitor of CRAC channels in T-lymphocytes.
- To determine if BTP2 interferes with other Ca2+ signaling mechanisms in T-cells.
- To evaluate the effect of BTP2 on T-cell activation, gene expression, and proliferation.
Main Methods:
- Measurement of Ca2+ influx through CRAC channels in primary T-lymphocytes and Jurkat T-cells using BTP2.
- Assessment of BTP2's effect on Ca2+ pumps, mitochondrial Ca2+ signaling, endoplasmic reticulum Ca2+ release, and K+ channels.
- Evaluation of BTP2's impact on anti-CD3, thapsigargin, and inositol 1,4,5-trisphosphate-induced Ca2+ signals.
- Analysis of BTP2's inhibition of Ca2+-dependent gene expression (IL-2, IL-5, IFN-γ) and T-lymphocyte proliferation.
Main Results:
- BTP2 specifically inhibits CRAC channels in T-cells with an IC50 of approximately 10 nM.
- BTP2 does not affect other critical Ca2+ signaling pathways in T-cells.
- BTP2 effectively inhibits Ca2+ signals, gene expression, and proliferation in both primary T-lymphocytes and Jurkat T-cells, irrespective of stimulation method.
Conclusions:
- BTP2 is the first potent and specific inhibitor of CRAC channels identified in primary T-lymphocytes.
- The findings underscore the critical role of CRAC channel activity in T-cell activation.
- BTP2 represents a valuable pharmacological tool for further research into the molecular identity and function of CRAC channels.
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