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 receptor-induced 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.

Depletion of intracellular calcium (Ca ²⁺ ) stores induces store-operated 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 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.

Characterization of mammalian homologues of Drosophila TRP proteins, which induce light-activated Ca²⁺ conductance in photoreceptors, has been an important clue to understand molecular mechanisms underlying receptor-activated Ca²⁺ influx in vertebrate cells. We have here isolated cDNA that encodes a novel TRP homologue, TRP5, predominantly expressed in the brain. Recombinant expression of the TRP5 cDNA in human embryonic kidney cells dramatically potentiated extracellular Ca²⁺-dependent rises of intracellular Ca²⁺ concentration ([Ca²⁺]_i) evoked by ATP. These [Ca²⁺]_i transients were inhibited by SK&F96365, a blocker of receptor-activated Ca²⁺ entry, and by La³⁺. Expression of the TRP5 cDNA, however, did not significantly affect [Ca²⁺]_i transients induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺-ATPases. ATP stimulation of TRP5-transfected cells pretreated with thapsigargin to deplete internal Ca²⁺stores caused intact extracellular Ca²⁺-dependent [Ca²⁺]_itransients, whereas ATP suppressed [Ca²⁺]_i in thapsigargin-pretreated control cells. Furthermore, in ATP-stimulated, TRP5-expressing cells, there was no significant correlation between Ca²⁺ release from the internal Ca²⁺ store and influx of extracellular Ca²⁺. Whole-cell mode of patch-clamp recording from TRP5-expressing cells demonstrated that ATP application induced a large inward current in the presence of extracellular Ca²⁺. Omission of Ca²⁺ from intrapipette solution abolished the current in TRP5-expressing cells, whereas 10 nm intrapipette Ca²⁺ was sufficient to support TRP5 activity triggered by ATP receptor stimulation. Permeability ratios estimated from the zero-current potentials of this current wereP _Ca:P _Na:P _Cs= 14.3:1.5:1. Our findings suggest that TRP5 directs the formation of a Ca²⁺-selective ion channel activated by receptor stimulation through a pathway that involves Ca²⁺ but not depletion of Ca²⁺ store in mammalian cells.

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca²⁺ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca²⁺ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC β-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The β construct beta-AID dominant negative, which disrupts the RIM1-β association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with β in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca²⁺ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

Recent genetic and molecular biological analyses have revealed many forms of inherited channelopathies. Homozygous ataxic mice, tottering(tg) and leaner (tg ^la) mice, have mutations in the P/Q-type Ca²⁺ channel α_1A subunit gene. Although their clinical phenotypes, histological changes, and locations of gene mutations are known, it remains unclear what phenotypes the mutant Ca²⁺ channels manifest, or whether the altered channel properties are the primary consequence of the mutations. To address these questions, we have characterized the electrophysiological properties of Ca²⁺channels in cerebellar Purkinje cells, where the P-type is the dominant Ca²⁺ channel, dissociated from the normal, tg,and tg ^la mice, and compared them with the properties of the wild-type and mutant α_1A channels recombinantly expressed with the α₂ and β subunits in baby hamster kidney cells. The most striking feature of Ca²⁺ channel currents of mutant Purkinje cells was a marked reduction in current density, being reduced to ∼60 and ∼40% of control in tg and tg ^la mice, respectively, without changes of cell size. The Ca²⁺channel currents in the tg Purkinje cells showed a relative increase in non-inactivating component in voltage-dependent inactivation. Besides the same change, those of thetg ^la mice showed a more distinct change in voltage dependence of activation and inactivation, being shifted in the depolarizing direction by ∼10 mV, with a broader voltage dependence of inactivation. In the recombinant expression system, thetg channel with a missense mutation (P601L) and one form of the two possible tg ^la aberrant splicing products, tg ^la (short) channel, showed a significant reduction in current density, while the other form of thetg ^la channels, tg ^la(long), had a current density comparable to the normal control. On the other hand, the shift in voltage dependence of activation and inactivation was observed only for the tg ^la(long) channel. Comparison of properties of the native and recombinant mutant channels suggests that single tottering mutations are directly responsible for the neuropathic phenotypes of reduction in current density and deviations in gating behavior, which lead to neuronal death and cerebellar atrophy.