Depletion of intracellular calcium (Ca 2؉ ) stores induces storeoperated Ca 2؉ (SOC) entry across the plasma membrane (PM). STIM1, a putative Ca 2؉ 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 Ca 2؉ 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 Ser͞Thr-rich C-terminal domains in the cytoplasmic region of STIM1, whereas subsequent inducible puncta formation further requires the sterile ␣ motif domain protruding into the ER lumen. Each of these three domains (coiled-coil, Ser͞Thr-rich, and sterile ␣ 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.B cell receptor ͉ calcium signaling ͉ DT40 ͉ store-operated calcium C ytosolic Ca 2ϩ signals are a key to the regulation of various physiological events (1, 2). Two stages of calcium mobilization have been distinguished in lymphocytes and other nonexcitable cells (3-5). The first stage involves activation of phospholipase C by trimeric G protein-or tyrosine kinase-coupled receptors. This enzyme hydrolyzes phosphatidylinositol bisphosphate to release the second messenger inositol-1,4,5-trisphosphate, which binds to its receptor in the endoplasmic reticulum (ER) membrane, thereby causing rapid but transient release of Ca 2ϩ from ER stores. The second stage involves a sustained influx of extracellular Ca 2ϩ across the plasma membrane (PM) in a process termed store-operated Ca 2ϩ (SOC) entry. In this process, depletion of Ca 2ϩ within the ER lumen serves as the primary trigger to open SOC channels residing in the PM.STIM1 has recently emerged to play a critical role in coupling the first and second stages of calcium mobilization (6, 7). The STIM1 protein is thought to function primarily as a sensor of Ca 2ϩ within the ER stores, because a single N-terminal EF-hand Ca 2ϩ binding motif is located within the ER lumen (7,8). The activation mechanism of STIM1, however, has remained elusive. For instance, Zhang and colleagues (9, 10) proposed that insertion of STIM1 from the ER to the PM, presumably through vesicular transport, would be a prerequisite for subsequent SOC channel activation. Furthermore, STIM1 in the PM has been reported to play a role for SOC activation (11). But others have shown that STIM1 redistributed into puncta near the PM without inserting into the PM and proposed that this aggregated STIM1 might activate SOC channels (7, 12).To elucidate the mechanisms by which STIM1 activates SOC channels, we have constructed STIM1 mutants and ...

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

Summary Ca2+ signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca 2+ entry activated by Ins(1,4,5)P 3 -induced Ca 2+ release from endoplasmic reticulum, the importance of DAG-activated Ca 2+ entry remains elusive. Here, we describe the physiological role of DAG-activated Ca 2+ 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 C (PKC) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKC that maintained localization of PKC at the PM. Thus, TRPC3 functions as both a Ca 2+ -permeable channel and a protein scaffold at the PM for downstream PKC activation in B cells.