In nonexcitable cells, Ca2+ entry is mediated predominantly through the store depletion-dependent Ca2+ channels called store-operated Ca2+ (SOC) or Ca2+ release-activated Ca2+ channels. YM-58483, a pyrazole derivative, inhibited an anti-CD3 mAb-induced sustained Ca2+ influx in acute T cell leukemia, Jurkat cells. But it did not affect an anti-CD3 mAb-induced transient intracellular Ca2+ increase in Ca2+-free medium, nor anti-CD3 mAb-induced phosphorylation of phospholipase Cγ1. It was suggested that YM-58483 inhibited Ca2+ influx through SOC channels without affecting the TCR signal transduction cascade. Furthermore, YM-58483 inhibited thapsigargin-induced sustained Ca2+ influx with an IC50 value of 100 nM without affecting membrane potential. YM-58483 inhibited by 30-fold the Ca2+ influx through SOC channels compared with voltage-operated Ca2+ channels, while econazole inhibited both SOC channels and voltage-operated Ca2+ channels with an equivalent range of IC50 values. YM-58483 potently inhibited IL-2 production and NF-AT-driven promoter activity, but not AP-1-driven promoter activity in Jurkat cells. Moreover, this compound inhibited delayed-type hypersensitivity in mice with an ED50 of 1.1 mg/kg. Therefore, we concluded that YM-58483 was a novel store-operated Ca2+ entry blocker and a potent immunomodulator, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 would be a candidate for the study of capacitative Ca2+ entry mechanisms through SOC/CRAC channels and for identification of putative Ca2+ channel genes.

TRPM7 is a ubiquitously expressed and constitutively active divalent cation-selective ion channel, whose basal activity is regulated by intracellular levels of Mg 2؉ and Mg⅐ATP. We have investigated receptor-mediated mechanisms that may actively regulate TRPM7 activity. We here report that TRPM7 currents are suppressed by intracellular GTP␥S, suggesting the involvement of heterotrimeric G proteins. TRPM7 currents are also inhibited by stimulating endogenous muscarinic receptors, which is mediated by Gi because the inhibitory effect is blunted by pertussis toxin. Conversely, stimulation of endogenous Gs-coupled ␤-adrenergic receptors potentiates TRPM7 currents, whereas Gq-coupled thrombin receptors have little effect. Consistent with the involvement of Gs͞Gi in controlling adenylyl cyclase activity, elevations of intracellular cAMP levels enhance TRPM7 activity and prevent receptor-mediated modulation of TRPM7 activity by muscarinic and adrenergic agonists. This cAMP-dependent effect requires the functional integrity of both protein kinase A (PKA) and the endogenous kinase domain of TRPM7 because cAMP-mediated effects are abolished when treating cells with the PKA inhibitors H89 or KT5720 as well as in cells expressing phosphotransferase-deficient TRPM7 constructs. These mutant channels are also much less susceptible to GTP␥S-mediated inhibition, suggesting that the main regulatory effect occurs through Gi-and Gs-mediated changes in cAMP. Taken together, our results demonstrate that TRPM7 activity is up-and down-regulated through its endogenous kinase in a cAMP-and PKA-dependent manner.B ased on sequence similarities, the mammalian transient receptor potential (TRP) channel family is divided into three subfamilies [TRP classical (formerly short TRP channel)], TRP vanilloid [(formerly osm TRP channel), and TRP melastatin (formerly long TRP channel)] (1-4). TRPM7 (long TRP channel 7, TRP-phospholipase C interacting kinase and channel kinase 1), a Ca 2ϩ -and Mg 2ϩ -permeable divalent cation channel of the TRP melastatin ion channel subfamily, is required for cellular viability and noted for its ubiquitous distribution profile (5, 6). We have previously demonstrated that TRPM7 is regulated by millimolar levels of intracellular Mg 2ϩ and Mg⅐ATP and its activity appears to be linked to cellular energy metabolism (5, 7). In resting cells, physiological levels of these molecules strongly suppress the activity of TRPM7 channels and only a small constitutive activity remains, sufficient to maintain basal divalent cation fluxes. In whole-cell patch-clamp experiments, intracellular solutions that lack added Mg⅐ATP lead to activation of TRPM7-mediated currents (Fig.