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Canonical transient receptor potential (TRPC) channels control influxes of Ca[sup2+] 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 TRPC5 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[sup2+] influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptorinduced Ca[sup2+] response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca[sup2+]-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 TRPC3selective inhibitor Pyr3 isa 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. [ABSTRACT FROM AUTHOR]

Depletion of intracellular calcium (Ca2+) stores induces store-operated Ca2+ (SOC) entry across the plasma membrane (PM). STIM1, a putative Ca2+ sensor in the endoplasmic reticulum (ER), has been recently shown to be necessary for SOC channel activation. Here we show that STIM1 dynamically moves in tubulovesicular shape on the ER and its subcompartment in resting living cells, whereas, upon Ca2+ store depletion, it is rapidly redistributed into discrete puncta that are located underneath, but not inserted into the PM. Normal constitutive movement of STIM1 is mediated through the coiled-coil and Ser/Thr-rich C-terminal domains in the cytoplasmic region of STIM1, whereas subsequent inducible puncta formation further requires the sterile α motif domain protruding into the ER lumen. Each of these three domains (coiled-coil, Ser/Thr-rich, and sterile α motif) was essential for activating SOC channels. Hence, our findings based on structure-function experiments suggest that constitutive dynamic movement of STIM1 in the ER and its subcompartment is obligatory for subsequent depletion-dependent redistribution of STIM1 into puncta underneath the PM and activation of SOC channels. [ABSTRACT FROM AUTHOR]

The tom2-1 mutation of Arabidopsis thaliana reduces the efficiency of intracellular multiplication of toba-moviruses. The tom2-1 mutant was derived from fast-neutron-irradiated seeds, and the original mutant tine also carries ttm1, a dominant modifier that increases tobamovirus multiplication efficiency in a tobamo- virus-strain-specific manner in the tom2-1 genetic background. Here, we show that the tom2-1 mutation involved a deletion of ∼20 kb in the nuclear genome. The deleted region included two genes named TOM2A and TOM2B that were both associated with the tom2-1 phenotype, whereas ttm1 corresponded to the translocation of part of the deleted region that included intact TOM2B but not TOM2A. TOM2A encodes a 280 amino acid putative four-pass transmembrane protein with a C-terminal farnesylation signal, white TOM2B encodes a 122 amino acid basic protein. The split-ubiquitin assay demonstrated an interaction of TOM2A both with itself and with TOM1, an integral membrane protein of A.thaliana presumed to be an essential constituent of tobamovirus replication complex. The data presented here suggest that TOM2A is also an integral part of the tobamovirus replication complex. [ABSTRACT FROM AUTHOR]

Ca2+ signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca2+ entry activated by Ins(1,4,5)P3-induced Ca2+ release from endoplasmic reticulum, the importance of DAG-activated Ca2+ entry remains elusive. Here, we describe the physiological role of DAG-activated Ca2+ entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cβ (PKCβ) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCβ that maintained localization of PKCβ at the PM. Thus, TRPC3 functions as both a Ca2+-permeable channel and a protein scaffold at the PM for downstream PKCβ activation in B cells. [ABSTRACT FROM AUTHOR]