Canonical transient receptor potential (TRPC) channels control influxes of Ca 2؉ and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca 2؉ influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptorinduced Ca 2؉ response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca 2؉ -dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.Ca 2ϩ signaling ͉ pyrazole compounds ͉ TRPC channels ͉ TRPC3

Flubendiamide is a benzenedicarboxamide derivative that shows selective insecticidal activity against lepidopterous insects. The specific modulatory effects of flubendiamide on ryanodine binding in insect muscle microsomal membranes suggest that the ryanodine receptor (RyR) Ca(2+) release channel is a primary target of flubendiamide. However, the molecular mechanisms underlying the species-specific action of flubendiamide are unclear. We have cloned cDNA encoding a novel RyR from the lepidopterous silkworm RyR (sRyR) and tested the sensitivity to flubendiamide of the recombinant sRyR in HEK293 cells. Confocal localization studies and Ca(2+) imaging techniques revealed that sRyRs form Ca(2+) release channels in the endoplasmic reticulum. Importantly, flubendiamide induced release of Ca(2+) through the sRyR, but not through the rabbit RyR isoforms. Photoaffinity labeling of sRyR deletion mutants using a photoreactive derivative revealed that flubendiamide is mainly incorporated into the transmembrane domain (amino acids 4111-5084) of the sRyR. The rabbit cardiac muscle isoform RyR2 (rRyR2) and the RyR mutant carrying a replacement of the transmembrane domain (residues 4084-5084) with its counterpart sequence from rRyR2 (residues 3936-4968) were not labeled by the photoreactive compound. This replacement in the sRyR significantly impaired the responses to flubendiamide but only marginally reduced the sensitivity to caffeine, a general RyR activator. Furthermore, deletion of the N-terminal sequence (residues 183-290) abolished the responses of the sRyR to flubendiamide but not the sensitivity to caffeine. Our results suggest that the transmembrane domain plays an important role in the formation of an action site for flubendiamide, while the N-terminus is a structural requirement for flubendiamide-induced activation of the sRyR.

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca 2؉ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca 2؉ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca 2؉

Pim kinases are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Here in this study, we investigated the preclinical profile of novel pan-Pim kinase inhibitors with imidazopyridazine and thiazolidinedione structure. Imidazopyridazine-thiazolidinediones inhibited activities of Pim kinases with IC50 values of tens to hundreds nanomolar. With YPC-21440 and/or YPC-21817, which exhibited especially high inhibitory activities against Pim kinases, we investigated in vitro and in vivo activities of imidazopyridazine-thiazolidinediones. In silico analysis of binding mode of YPC-21440 and Pim kinases revealed that it directly bound to ATP-binding pockets of Pim kinases. In the kinase panel tested, YPC-21440 and YPC-21817 were highly specific to Pim kinases. These compounds exerted antiproliferative activities against various cancer cell lines derived from hematological malignancies and solid carcinomas. Furthermore, they suppressed phosphorylation of Pim kinase substrates, arrested cell cycle at the G1 phase, and induced apoptosis in cultured cancer cells. In tumor xenograft models, YPC-21440 methanesulfonate and YPC-21817 methanesulfonate exerted antitumor activities. Furthermore, pharmacodynamic analysis with a xenograft model suggested that YPC-21817 methanesulfonate inhibited Pim kinases in tumors. In conclusion, our data revealed that imidazopyridazine-thiazolidinediones are novel Pim kinases inhibitors, effective on various types of cancer cell lines both in vitro and in vivo.