A Pyrazole Derivative Potently Inhibits Lymphocyte Ca Influx and Cytokine Production by Facilitating Transient Receptor Potential Melastatin 4 Channel Activity

3,5-Bis(trifluoromethyl)pyrazole derivative (BTP2) or N-[4–3, 5-bis(trifluromethyl)pyrazol-1-yl]-4-methyl-1,2,3-thiadiazole-5carboxamide (YM-58483) is an immunosuppressive compound that potently inhibits both Ca influx and interleukin-2 (IL-2) production in lymphocytes. We report here that BTP2 dosedependently enhances transient receptor potential melastatin 4 (TRPM4), a Ca -activated nonselective (CAN) cation channel that decreases Ca influx by depolarizing lymphocytes. The effect of BTP2 on TRPM4 occurs at low nanomolar concentrations and is highly specific, because other ion channels in T lymphocytes are not significantly affected, and the major Ca influx pathway in lymphocytes, ICRAC, is blocked only at 100fold higher concentrations. The efficacy of BTP2 in blocking IL-2 production is reduced approximately 100-fold when preventing TRPM4-mediated membrane depolarization, suggesting that the BTP2-mediated facilitation of TRPM4 channels represents the major mechanism for its immunosuppressive effect. Our results demonstrate that TRPM4 channels represent a previously unrecognized key element in lymphocyte Ca signaling and that their facilitation by BTP2 supports cell membrane depolarization, which reduces the driving force for Ca entry and ultimately causes the potent suppression of cytokine release. In many cells, receptor activation evokes an increase in cytosolic Ca concentration ([Ca ]i) composed of release from intracellular stores followed by store-operated Ca entry via calcium release-activated calcium (CRAC) channels (Penner et al., 1993; Parekh and Penner, 1997; Putney et al., 2001). These channels are functionally best characterized in mast cells (Hoth and Penner, 1992, 1993) and T lymphocytes (Zweifach and Lewis, 1993; Premack et al., 1994) and are considered key elements for the activation of immune cells. The study of CRAC has been hampered by the lack of potent and specific pharmacological tools. At present, the most widely used organic blockers of store-operated calcium entry pathways are imidazole antimycotics, such as econazole, the closely related compound SKF-96365, and 2-aminoethoxydiphenyl borate (Mason et al., 1993; Chung et al., 1994; Franzius et al., 1994; Christian et al., 1996; Hermosura et al., 2002). However, none of these compounds is very potent or