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The peripheral sensory nerve must be maintained to perceive environmental changes. Daily physiological mechanical stimulations, like gravity, floor reaction force, and occlusal force, influence the nerve homeostasis directly or indirectly. Although the direct axonal membrane stretch enhances axon outgrowth via mechanosensitive channel activation, the indirect mechanisms remain to be elucidated. In this study, we identified the indirect pathways where Wnt5a was a molecular cue released by mechanically stimulated rat periodontal ligament (rPDL) cells. qRT-PCR and ELISA showed that mechanically stimulated rPDL cells enhanced Wnt5a expression level and Wnt5a protein in a Ca2+-dependent manner. The inhibitors of PI3K (LY294002) and MEK1/2 (U0126) suppressed the Akt/PKB and ERK1/2 phosphorylation, respectively, in Western blotting analysis and consequently abolished the increase in Wnt5a expression. Similarly, PF573228, a focal adhesion kinase inhibitor, attenuated Akt- and ERK1/2-phosphorylation and Wnt5a expression. Importantly, the culture medium of stretched PDL cells enhanced neurite elongation, sprouting, and branching in trigeminal ganglion neurons that project to PDL. Moreover, treatment with an anti-Wnt5a antibody (to neutralize Wnt5a activity), AP7677a (anti-Ryk antibody, to block Ryk receptor activity), or strictinin (Ror1 inhibitor) suppressed the morphological changes. These findings reveal the indirect mechanisms that Wnt5a, released from the connective tissues in response to mechanical stimulation, enhances the outgrowth of the peripheral nerves. Our study suggests that the peripheral connective tissues regulate peripheral nerve homeostasis and that Wnt5a signaling could be targeted for the treatment of peripheral nerve disorders.

Myoepithelial cells (MECs) are responsible for receiving stimuli from the central nervous system and translating their responses into the form of secretion into glandular tissue, including salivary glands (SG), sweet glands, and mammary glands. SG MECs cause the secretion of serous saliva by contracting of acini/ductal cells with acetylcholine (Ach) from parasympathetic nerves via muscarinic receptors. To response the parasympathetic physiological stimulation, SG epithelial cell-derived MECs are supposed to be induced and placed adjacent to parasympathetic system nerve ends in SGs by forming a neuro-myoepithelial junction. For salivary secretion to function under parasympathetic control, therefore, specific regions of salivary gland epithelial cells must be mapped and the epithelium near the nerve must differentiate into MECs in order to form a nerve-myoepithelial junction during organogenesis. We hypothesized that the epithelium near the parasympathetic nerves is induced the differentiation into MECs by which the neurotransmitter acetylcholine via muscarinic receptors. qPCR and whole-mount immunohistochemical analysis in ex vivo organ culture system revealed that SG epithelial cells near a parasympathetic nerve were found to be induced to differentiate into MECs via the cholinergic receptor muscarinic 1 by carbachol (CCh), an acetylcholine agonist. In addition, CCh stimulated ERK and Akt signaling for the induction of MEC differentiation in rat submandibular gland epithelial cells. These findings indicate that muscarinic action is required for the induction of MECs and formation of a neuro-myoepithelial junction in developing SGs. This study proposes a novel concept for tissue architecture to form a neuro-myoepithelial junction during neurofunctional organogenesis including SGs.

The transient receptor potential vanilloid 1 (TRPV1) channel is a polymodal receptor in sensory nerves and involved in pain sensation. TRPV1 has at least three distinct activation modes that are selectively induced by different stimuli capsaicin, noxious heat, and protons. Although many mode-selective TRPV1 antagonists have been developed for their anticipated analgesic effects, there have been few successful reports because of adverse effects due to burn injuries and hyperthermia. Eugenol is a vanilloid that has been used as an analgesic in the dental treatment, and its TRPV1 activation ability has been reported. However, our knowledge about the underlying mechanisms of the antagonistic effects of eugenol on TRPV1 activation induced by three different modes is limited. Here, we show that eugenol dose-dependently inhibited the capsaicin-activated inward currents of mouse TRPV1 expressed in human embryonic kidney 293 (HEK293) cells. Under low pH conditions, low concentrations of eugenol only enhanced the proton-induced TRPV1 currents, whereas high eugenol concentrations initially potentiated but then immediately abrogated TRPV1 currents. Finally, eugenol had no modulatory effects on heat-activated TRPV1 in electrophysiological and Fura-2-based Ca2+ imaging experiments. Our results demonstrate that eugenol is a mode-selective antagonist of TRPV1 and can be evaluated as a lead compound of analgesics targeting TRPV1 without serious side effects.

Excessive triglyceride accumulation in lipid-metabolizing tissues is associated with an increased risk of a variety of metabolic diseases. Kamishoyosan (KSS) is a Kampo composed of 10 constituent herbs, and contains moutan cortex (MC) and paeonol (PN) as the major ingredient of MC. Here, we demonstrate the molecular mechanism underlying the effect of KSS on the differentiation of mouse preadipocytes (3T3-L1 cells). KSS inhibited the accumulation of triglycerides in a dose-dependent manner in 3T3-L1 cells that were induced to differentiate into adipocytes. We also found that MC and PN were responsible for the anti-adipogenetic effect of KSS and significantly suppressed the expression of CCAAT/enhancer-binding proteins-&delta, (C/EBP-&delta, ) mRNA 3 days after the induction of differentiation. Thus, PN may contribute to the anti-adipogenetic property of MC in 3T3-L1 cells. In addition, PN inhibited dexamethasone (Dex)-induced glucocorticoid receptor (GR) promoter activity. Taken together, these results suggest that PN suppresses C/EBP-&delta, expression by inhibiting Dex-induced GR promoter activity at the early stage of differentiation and, consequently, delays differentiation into mature adipocytes. Our results suggest that the habitual intake of Kampo-containing PN contributes to the prevention of the onset of metabolic diseases by decreasing the excessive accumulation of triglycerides in lipid-metabolizing tissues.

Background and purpose The development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Nav 1.1-Nav 1.7. Experimental approach Each VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings. Key results The inhibitory effects of TTX on Nav 1.1-Nav 1.7 were observed in accordance with those reported in the literature; however, the 5-deoxy-10,7-lactone-type analogues and 4,9-anhydro-type analogues did not cause inhibition. Chiriquitoxin showed less binding to Nav 1.7 compared to the other TTX-sensitive subtypes. Two amino acid residues in the TTX binding site of Nav 1.7, Thr1425 and Ile1426 were mutated to Met and Asp, respectively, because these residues were found at the same positions in other subtypes. The two mutants, Nav 1.7 T1425M and Nav 1.7 I1426D, had a 16-fold and 5-fold increase in binding affinity for chiriquitoxin, respectively. Conclusions and implications The reduced binding of chiriquitoxin to Nav 1.7 was attributed to its C11-OH and/or C12-NH2 , based on reported models for the TTX-VSSC complex. Chiriquitoxin is a useful tool for probing the configuration of the TTX binding site until a crystal structure for the mammalian VSSC is solved.

T-type voltage-gated Ca 2+ channels (T-VGCCs) function in the pathophysiology of epilepsy, pain and sleep. However, their role in cognitive function remains unclear. We previously reported that the cognitive enhancer ST101, which stimulates T-VGCCs in rat cortical slices, was a potential Alzheimer's disease therapeutic. Here, we introduce a more potent T-VGCC enhancer, SAK3 (ethyl 8′-methyl-2′,4-dioxo-2-(piperidin-1-yl)-2′H-spiro[cyclopentane-1,3′-imidazo [1,2-a]pyridin]-2-ene-3-carboxylate), and characterize its pharmacological properties in brain. Based on whole cell patch-clamp analysis, SAK3 (0.01–10 nM) significantly enhanced Cav3.1 currents in neuro2A cells ectopically expressing Cav3.1. SAK3 (0.1–10 nM nM) also enhanced Cav3.3 but not Cav3.2 currents in the transfected cells. Notably, Cav3.1 and Cav3.3 T-VGCCs were localized in cholinergic neurve systems in hippocampus and in the medial septum. Indeed, acute oral administration of SAK3 (0.5 mg/kg, p.o.), but not ST101 (0.5 mg/kg, p.o.) significantly enhanced acetylcholine (ACh) release in the hippocampal CA1 region of naive mice. Moreover, acute SAK3 (0.5 mg/kg, p.o.) administration significantly enhanced hippocampal ACh levels in olfactory-bulbectomized (OBX) mice, rescuing impaired memory-related behaviors. Treatment of OBX mice with the T-VGCC-specific blocker NNC 55-0396 (12.5 mg/kg, i.p.) antagonized both enhanced ACh release and memory improvements elicited by SAK3 administration. We also observed that SAK3-induced ACh releases were significantly blocked in the hippocampus from Cav3.1 knockout (KO) mice. These findings suggest overall that T-VGCCs play a key role in cognition by enhancing hippocampal ACh release and that the cognitive enhancer SAK3 could be a candidate therapeutic in Alzheimer's disease.

Crambescin B carboxylic acid, a synthetic analog of crambescin B, was recently found to inhibit the voltage-sensitive sodium channels (VSSC) in a cell-based assay using neuroblastoma Neuro 2A cells. In the present study, whole-cell patch-clamp recordings were conducted with three heterologously expressed VSSC subtypes, Na v 1.2, Na v 1.6 and Na v 1.7, in a human embryonic kidney cell line HEK293T to further characterize the inhibition of VSSC by crambescin B carboxylic acid. Contrary to the previous observation, crambescin B carboxylic acid did not inhibit peak current evoked by depolarization from the holding potential of −100 mV to the test potential of −10 mV in the absence or presence of veratridine (VTD). In the presence of VTD, however, crambescin B carboxylic acid diminished VTD-induced sustained and tail currents through the three VSSC subtypes in a dose-dependent manner, whereas TTX inhibited both the peak current and the VTD-induced sustained and tail currents through all subtypes of VSSC tested. We thus concluded that crambescin B carboxylic acid does not block VSSC in a similar manner to TTX but modulate the action of VTD, thereby causing an apparent block of VSSC in the cell-based assay.

Activation of transient receptor potential melastatin 2 (TRPM2), an oxidative stress-sensitive Ca2+-permeable channel, contributes to the aggravation of cerebral ischemia-reperfusion (CIR) injury. Recent studies indicated that treatment with the antidepressant duloxetine for 24 hours (long term) attenuates TRPM2 activation in response to oxidative stress in neuronal cells. To examine the direct effects of antidepressants on TRPM2 activation, we examined their short-term (0-30 minutes) treatment effects on H2O2-induced TRPM2 activation in TRPM2-expressing human embryonic kidney 293 cells using the Ca2+ indicator fura-2. Duloxetine exerted the strongest inhibitory effects on TRPM2 activation among the seven antidepressants tested. These inhibitory effects appeared to be due to the inhibition of H2O2-induced TRPM2 activation via an open-channel blocking-like mechanism, because duloxetine reduced the sustained phase but not the initial phase of increases in intracellular Ca2+ concentrations. In a whole-cell patch-clamp study, duloxetine reduced the TRPM2-mediated inward current during the channel opening state. We also examined the effects of duloxetine in a mouse model of CIR injury. The administration of duloxetine to wild-type mice attenuated CIR injury, similar to that in Trpm2 knockout (KO) mice. The administration of duloxetine did not reduce CIR injury further in Trpm2 KO mice, suggesting that it exerts neuroprotective effects against CIR injury by inhibiting TRPM2 activation. Regarding drug repositioning, duloxetine may be a useful drug in reperfusion therapy for ischemic stroke because it has already been used clinically in therapeutics for several disorders, including depression.

Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Cav2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail. To gain insight into the functional role of the AS in vivo and whether modulating the splice patterns at this locus can be a potential therapeutic strategy for SCA6, here we created knockin mice that exclusively express MPc by inserting the splice-site mutation. The resultant Cacna1aCtmKO/CtmKO mice developed non-progressive neurological phenotypes, featuring early-onset ataxia and absence seizure without significant alterations in the basic properties of the channel. Interactions of Cav2.1 with Cavβ4 and Rimbp2 were significantly reduced while those with GABAB2 were enhanced in the cerebellum of Cacna1aCtmKO/CtmKO mice. Treatment with the GABAB antagonist CGP35348 partially rescued the motor impairments seen in Cacna1aCtmKO/CtmKO mice. These results suggest that the carboxyl-terminal domain of Cav2.1 is not essential for maintaining the basic properties of the channel in the cerebellar Purkinje neurons but is involved in multiple interactions of Cav2.1 with other proteins, and plays an essential role in preventing a complex neurological disease.

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel that controls Ca(2+) signalling. The activation of Janus kinase 2 (Jak2) by oxidative stress is implicated in the production of inflammatory mediators. We found that AG490, a Jak2 inhibitor, had an inhibitory effect on H2O2-induced TRPM2 activation. The purpose of this study was to examine the underlying mechanisms of the inhibitory effects of AG490. Activation of TRPM2 in TRPM2-expressing human embryonic kidney 293 (TRPM2/HEK) cells or the human monocytic cell line U937 was monitored by fluorescence-based Ca(2+) imaging and patch-clamp techniques. Treatment with AG490 almost completely blocked H2O2-induced increase in intracellular Ca(2+) in TRPM2/HEK and U937 cells. In the patch-clamp study, AG490 inhibited the H2O2-evoked inward current but not the ADP-ribose-induced inward current in TRPM2/HEK cells. In contrast, Jak inhibitor 1 (pyridone 6) and staurosporine, both of which inhibit Jak2, had no effect on H2O2-induced increase in intracellular Ca(2+). Moreover, AG490 decreased intracellular reactive oxygen species level, which was measured by using a hydroperoxide-sensitive fluorescent dye, on incubation with H2O2. In the cell-free assay system, AG490 scavenged hydroxyl radicals but not H2O2. These findings indicate that AG490 significantly reduces H2O2-induced TRPM2 activation, presumably by scavenging hydroxyl radicals rather than Jak2-dependent mechanisms. Although transient receptor potential ankyrin 1 (TRPA1) channel is also activated by H2O2, the H2O2-induced Ca(2+) entry through TRPA1 was only slightly delayed by AG490. This validates the potential use of AG490, as one of the materials for characterizing the role of TRPM2 channels in pathological models.

We synthesized novel environment-sensitive fluorophores using a unique fluorogenic core quinolyl-pyrrole (QP, 2-(1H-pyrrol-2-yl)quinoline). QP was obtained from isatins and 2-acetylpyrrole by Pfitzinger quinoline synthesis. Derivatization at the quinoline and pyrrole rings was carried out using the Sonogashira–Hagiwara cross coupling and Vilsmeier–Haack reactions, respectively. The obtained dyes showed strong fluorescence solvatochromism, but their absorption spectra remained nearly unchanged. The introduction of a dimethylaminophenylethynyl moiety at the quinoline side of the QP resulted in a change in the fluorescence color from blue to red within a narrow relative permittivity range of roughly 2–10, demonstrating that the substitution strengthened the sensitivity of the dye to the low polarity environment. The dye also reflected voltage changes across liposomal membranes, showing a 3.7-fold enhanced fluorescence intensity when tested under the same conditions as di-4-ANEPPS, a popular voltage sensitive dye, which only changed its fluorescence intensity by a factor of 1.2. The voltage sensitivity of the dye was calculated to be 2.3%/mV, much larger than that of di-4-ANEPPS (0.13 – 0.18%/mV). These dyes may be utilized for the studies on the activities of neurons or chloroplasts.

T-type voltage-gated Ca

Lavender oil (LO) is a commonly used essential oil in aromatherapy as non-traditional medicine. With an aim to demonstrate LO effects on the body, we have recently established an animal model investigating the influence of orally administered LO in rat tissues, genome-wide. In this brief, we investigate the effect of LO ingestion in the blood of rat. Rats were administered LO at usual therapeutic dose (5 mg/kg) in humans, and following collection of the venous blood from the heart and extraction of total RNA, the differentially expressed genes were screened using a 4 × 44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA) in conjunction with a two-color dye-swap approach. A total of 834 differentially expressed genes in the blood were identified: 362 up-regulated and 472 down-regulated. These genes were functionally categorized using bioinformatics tools. The gene expression inventory of rat blood transcriptome under LO, a first report, has been deposited into the Gene Expression Omnibus (GEO): GSE67499. The data will be a valuable resource in examining the effects of natural products, and which could also serve as a human model for further functional analysis and investigation.

Assembly of voltage-dependent Ca(2+) channels (VDCCs) with their associated proteins regulates the coupling of VDCCs with upstream and downstream cellular events. Among the four isoforms of the Rab3-interacting molecule (RIM1 to -4), we have previously reported that VDCC beta-subunits physically interact with the long alpha isoform of the presynaptic active zone scaffolding protein RIM1 (RIM1alpha) via its C terminus containing the C(2)B domain. This interaction cooperates with RIM1alpha-Rab3 interaction to support neurotransmitter exocytosis by anchoring vesicles in the vicinity of VDCCs and by maintaining depolarization-triggered Ca(2+) influx as a result of marked inhibition of voltage-dependent inactivation of VDCCs. However, physiological functions have not yet been elucidated for RIM3 and RIM4, which exist only as short gamma isoforms (gamma-RIMs), carrying the C-terminal C(2)B domain common to RIMs but not the Rab3-binding region and other structural motifs present in the alpha-RIMs, including RIM1alpha. Here, we demonstrate that gamma-RIMs also exert prominent suppression of VDCC inactivation via direct binding to beta-subunits. In the pheochromocytoma PC12 cells, this common functional feature allows native RIMs to enhance acetylcholine secretion, whereas gamma-RIMs are uniquely different from alpha-RIMs in blocking localization of neurotransmitter-containing vesicles near the plasma membrane. Gamma-RIMs as well as alpha-RIMs show wide distribution in central neurons, but knockdown of gamma-RIMs attenuated glutamate release to a lesser extent than that of alpha-RIMs in cultured cerebellar neurons. The results suggest that sustained Ca(2+) influx through suppression of VDCC inactivation by RIMs is a ubiquitous property of neurons, whereas the extent of vesicle anchoring to VDCCs at the plasma membrane may depend on the competition of alpha-RIMs with gamma-RIMs for VDCC beta-subunits.

Membrane excitability is mainly regulated by ion channels, including both voltage-gated and ligand-gated channels. In addition to these conventional types of ion channel, a new class of ionic channel is also involved in regulation of the membrane potential. Transient receptor potential (TRP) channels are expressed not only in excitable cells, such as sensory organs, neurons, and cardiac myocytes, but also in non-excitable cells such as endothelial cells, B lymphocytes, and rat basophilic leukemia mast cells. Most TRP channels are Ca 2+ -permeable, and some are regulated by intracellular Ca 2+ . The aim of this review is to provide an overview of the TRP channel superfamily, and describe the electrophysiological properties and physiological roles of the neuronal Ca 2+ -sensitive non-selective cation channel TRPC5.

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 Ca2+ 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 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 Ca2+ influx that may lead to dysregulated cell growth in ADPKD. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc., Publisher's version/PDF may be used after 12 months embargo

Spinocerebellar ataxia type 6 (SCA6) is caused by polyglutamine expansion in P/Q-type Ca2+ channels (Ca(v)2.1) and is characterized by predominant degeneration of cerebellar Purkinje cells. To characterize the Ca(v)2.1 channel with an SCA6 mutation in cerebellar Purkinje cells, we have generated knock-in mouse models that express human Ca(v)2.1 with 28 polyglutamine repeats (disease range) and with 13 polyglutamine repeats (normal range). Patch-clamp recordings of the Purkinje cells from homozygous control or SCA6 knock-in mice revealed a non-inactivating current that is highly sensitive to a spider toxin omega-Agatoxin IVA, indicating that the human Ca(v)2.1 expressed in Purkinje cells exhibits typical P-type properties in contrast to the previous data showing Q-type properties, when it was expressed in cultured cell lines. Furthermore, the voltage dependence of activation and inactivation and current density were not different between SCA6 and control, though these properties were altered in previous reports using non-neuronal cells as expression systems. Therefore, our results do not support the notion that the alteration of the channel properties may underlie the pathogenic mechanism of SCA6.

The rocker mice are hereditary ataxic mutants that carry a point mutation in the gene encoding the CaV2.1 (P/Q-type) Ca2+ channel alpha1 subunit, and show the mildest symptoms among the reported CaV2.1 mutant mice. We studied the basic characteristics of the rocker mutant Ca2+ channel and their impacts on excitatory synaptic transmission in cerebellar Purkinje cells (PCs). In acutely dissociated PC somas, the rocker mutant channel showed a moderate reduction in Ca2+ channel current density, whereas its kinetics and voltage dependency of gating remained nearly normal. Despite the small changes in channel function, synaptic transmission in the parallel fiber (PF)-PC synapses was severely impaired. The climbing fiber inputs onto PCs showed a moderate impairment but could elicit normal complex spikes. Presynaptic function of the PF-PC synapses, however, was unexpectedly almost normal in terms of paired-pulse facilitation, sensitivity to extracellular Ca2+ concentration and glutamate concentration in synaptic clefts. Electron microscopic analyses including freeze-fracture replica labeling revealed that both the number and density of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors substantially decreased without gross structural changes of the PF-PC synapses. We also observed an abnormal arborization of PC dendrites in young adult rocker mice (approximately 1 month old). These lines of evidence suggest that even a moderate dysfunction of CaV2.1 Ca2+ channel can cause substantial changes in postsynaptic molecular composition of the PF-PC synapses and dendritic structure of PCs.

Vertebrate sensory cells such as vomeronasal neurons and Drosophila photoreceptor cells use TRP channels to respond to exogenous stimuli. In mammalian taste cells, bitter and sweet substances as well as some amino acids are received by G protein-coupled receptors (T2Rs or T1Rs). As a result of activation of G protein and phospholipase Cbeta2, the TRPM5 channel is activated. Intracellular Ca(2+) is known to be a TRPM5 activator, but the participation of lipid activators remains unreported. To clarify the effect of arachidonic acid on TRPM5 in taste cells, we investigated the expression profile of a series of enzymes involved in controlling the intracellular free arachidonic acid level, with the result that in a subset of taste bud cells, monoglyceride lipase (MGL) and cyclooxygenase-2 (COX-2) are expressed as well as the previously reported group IIA phospholipase A(2) (PLA(2)-IIA). Double-labeling analysis revealed that MGL, COX-2 and PLA(2)-IIA are co-expressed in some cells that express TRPM5. We then investigated whether arachidonic acid activates TRPM5 via a heterologous expression system in HEK293 cells, and found that its activation occurred at 10 microM arachidonic acid. These results strongly suggest the possibility that arachidonic acid acts as a modulator of TRPM5 in taste signaling pathways.

Ca 2+ channels play critical roles in the regulation of synaptic activity. In contrast to the well established function of voltage-activated Ca 2+ channels in the presynaptic membrane for neurotransmitter release, some studies are just beginning to elucidate the functions of the Ca 2+ channels in the postsynaptic membrane. In this study, we demonstrated the functional association of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with the neuronal Ca 2+ channels. A series of biochemical studies showed the specific association of Ca v 2.1 (α 1A -class) and Ca v 2.2 (α 1B -class) with AMPA receptors in the postsynaptic membrane. Our electrophysiological and Ca 2+ imaging analyses of recombinant Ca v 2.1 and AMPA receptors also showed functional coupling of the two channels. Considering the critical roles of postsynaptic intracellular concentration of Ca 2+ ([Ca 2+ ] i ) increase and AMPA receptor trafficking for long-term potentiation (LTP) and long-term depression (LTD), the functional association of Ca 2+ channels with the AMPA receptors may provide new insights into the mechanism of synaptic plasticity.

Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca2+ influx in vertebrate cells. We previously reported the involvement of intracellular Ca2+ in the receptor-mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca2+, and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca2+ entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, was abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca2+–calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique was inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch was unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca2+–calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.

The electrophysiological properties of nicotinic ACh receptors (nAChR) were investigated in acutely dissociated area postrema (AP) neurons of the immature rat brain using the whole-cell patch-clamp recording method. ACh induced a transient inward current exhibiting a strong inward rectification. The ACh response was mimicked by nicotine and cytisine, and was inhibited by nAChR antagonists, but not by 10(-7) M atropine. Muscarinic AChR agonists did not induce any current. We confirmed the Ca2+ permeability of nAChR. These results indicate the presence of nAChR on AP neurons, and suggest that the activation of nAChR play important roles in cardiovascular functions in rats.

Hereditary ataxic mice, tottering (tg) and rolling Nagoya (tg(rol)), carry mutations in the P/Q-type Ca(2+) channel alpha(1A) subunit gene. The positions of the mutations and the neurological phenotypes are known, but the mechanisms of how the mutations cause the symptoms and how the different mutations lead to various onset and severity have remained unsolved. Here we compared fundamental properties of excitatory synaptic transmission in the cerebellum and roles of Ca(2+) channel subtypes therein among wild-type control, tg, and tg(rol) mice. The amplitude of EPSC of the parallel fiber-Purkinje cell (PF-PC) synapses was considerably reduced in ataxic tg(rol). Although the amplitude of the parallel fiber-mediated EPSC was only mildly decreased in young non-ataxic tg mice, it was drastically diminished in adult ataxic tg mice of postnatal day 28-35, showing a good correlation between the impairment of the PF-PC synaptic transmission and manifestation of ataxia. In contrast, the EPSC amplitude of the climbing fiber-Purkinje cell (CF-PC) synapses was preserved in tg, and it was even increased in tg(rol), which was associated with altered properties of the postsynaptic glutamate receptors. The climbing fiber-mediated EPSC was more dependent on other Ca(2+) channel subtypes in mutant mice, suggesting that such compensatory mechanisms contribute to maintaining the CF-PC synaptic transmission virtually intact. The results indicate that different mutations of the P/Q-type Ca(2+) channel not only cause the primary effect of different severity but also lead to diverse additional secondary effects, resulting in disruption of well balanced neural networks.

We investigated differences in electrophysiological characteristics between peripheral and central N-type Ca(2+) channels, containing alpha(1B-a) and a(1B-c), respectively. In addition, we examined the inhibitory effects of cilnidipine, a dihydropyridine (DHP) derivative, on both channels. Both alpha(1B) subunits were transiently expressed in BHK cells, and then analyzed using the whole-cell patch-clamp technique. The current-voltage relationship showed that alpha(1B-c) currents were activated at more negative potentials than alpha(1B-a) currents. The voltage-dependent steady-state inactivation and activation showed that the V(1/2) values for inactivation and activation of alpha(1B-c) (-88.5+/-1.3 and -33.2+/-1.3 mV) were both significantly more negative than those for alpha(1B-a) (-83.3+/-1.3 and -27.9+/-2.3 mV). Despite the different electrophysiological characteristics of these two N-type channels, cilnidipine blocked both with similar potency within the range 0.1 to 10 microM. Furthermore, cilnidipine had no effect on the I-V relationships or the steady-state inactivation curves. Our data indicate that the spliced positions of alpha(1B-a) and a(1B-c) may affect not only their voltage-sensing abilities but also the kinetics of channel activation and inactivation. The data also suggest that cilnidipine binds to sites independent of those controlling voltage-sensing and channel kinetics in these alpha(1B) subunits.

Using a confocal laser-scanning microscope, we measured the changes in the cytosolic Ca(2+) concentration ([Ca]i) of chromaffin cells on adrenal slice preparations of mouse. The spontaneous fluctuations of [Ca]i were often observed in situ, as reported in isolated rat and bovine cells. Intriguingly, the spontaneous [Ca]i changes in one cell were often transmitted to one or two adjacent cells, and the synchronized [Ca]i changes were often observed in two adjacent cells, both of which failed to respond to ATP. The synchronized [Ca]i changes between two cells were much less frequently observed in rat than in mouse adrenals. The spontaneous [Ca]i changes were also synchronized between closely touching mouse chromaffin cells in culture. These results suggest that the spread of Ca(2+) signaling is mediated by low-conductance intercellular coupling.

Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.

Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Ca2+-permeable cation channel, LTRPC2, activated by micromolar levels of H2O2 and agents that produce reactive oxygen/nitrogen species. This sensitivity of LTRPC2 to redox state modifiers was attributable to an agonistic binding of nicotinamide adenine dinucleotide (beta-NAD+) to the MutT motif. Arachidonic acid and Ca2+ were important positive regulators for LTRPC2. Heterologous LTRPC2 expression conferred susceptibility to death on HEK cells. Antisense oligonucleotide experiments revealed physiological involvement of "native" LTRPC2 in H2O2- and TNFalpha-induced Ca2+ influx and cell death. Thus, LTRPC2 represents an important intrinsic mechanism that mediates Ca2+ and Na+ overload in response to disturbance of redox state in cell death.

Cholinergically induced network activity is a useful analogue of theta rhythms involved in memory processing or epileptiform activity in the hippocampus, providing a powerful tool to elucidate the mechanisms of synchrony in neuronal networks. In absence epilepsy, although its association with cognitive impairments has been reported, the mechanisms underlying hippocampal synchrony remain poorly investigated. Here we simultaneously recorded electrical activities from 64 sites in hippocampal slices of CaV2.1 Ca(2+) channel mutant tottering (tg) mice, a well-established mouse model of spontaneous absence epilepsy, to analyze the spatiotemporal pattern of cholinergically induced hippocampal network activity. The cholinergic agonist carbachol induced oscillatory discharges originating from the CA3 region. In tg/tg mice, this hippocampal network activity was characterized by enhanced occupancy of discharges of relatively high frequency (6-10 Hz) compared to the wild type. Pharmacological analyses of slices, patch clamp electrophysiological characterization of isolated neurons, and altered patterns of hippocampal GABAA receptor subunit and Cl(-) transporter messenger RNA (mRNA) transcript levels revealed that this abnormality is attributable to a developmental retardation of GABAergic inhibition caused by immature intracellular Cl(-) regulation. These results suggest that the inherited CaV2.1 Ca(2+) channel mutation leads to developmental abnormalities in Cl(-) transporter expression and GABAA receptor compositions in hippocampal neurons and that compromised maturation of GABAergic inhibition contributes to the abnormal synchrony in the hippocampus of tg absence epileptic mice.

The role of N-type Ca(2+) channels in nociceptive transmission was examined in genetically engineered mice lacking the alpha(1B) subunit of N-type channels and in their heterozygote and wild-type littermates. In alpha(1B)-deficient mice, N-type channel activities in dorsal root ganglion neurons and spinal synaptoneurosomes were eliminated without compensation by other types of voltage-dependent Ca(2+) channels. The alpha(1B)-deficient mice showed a diminution in the phase 2 nociceptive responses more extensively than in the phase 1 nociceptive responses of the formalin test. The alpha(1B)-deficient mice exhibited significantly increased thermal nociceptive thresholds in the hot plate test, but failed to increase mechanical nociceptive thresholds in the tail pinch test. These results suggest a crucial role of N-type channels in nociceptive transmission, especially for persistent pain like phase 2 of the formalin test and for nociception induced by thermal stimuli.

Recent genetic analyses have revealed an important association of the gene encoding the P/Q-type voltage-dependent Ca2+channel α1Asubunit with hereditary neurological disorders. We have identified the ataxic mouse mutation,rolling Nagoya(tgrol), in the α1Agene that leads to a charge-neutralizing arginine-to-glycine substitution at position 1262 in the voltage sensor-forming segment S4 in repeat III. Ca2+channel currents in acutely dissociated Purkinje cells, where P-type is the dominant type, showed a marked decrease in slope and a depolarizing shift by 8 mV of the conductance–voltage curve and reduction in current density intgrolmouse cerebella, compared with those in wild-type. Compatible functional change was induced by thetgrolmutation in the recombinant α1Achannel, indicating that a defect in voltage sensor of P/Q-type Ca2+channels is the direct consequence of thetgrolmutation. Furthermore, somatic whole-cell recording of mutant Purkinje cells displayed only abortive Na+burst activity and hardly exhibited Ca2+spike activity in cerebellar slices. Thus, intgrolmice, reduced voltage sensitivity, which may derive from a gating charge defect, and diminished activity of the P-type α1ACa2+channel significantly impair integrative properties of Purkinje neurons, presumably resulting in locomotor deficits.

1. We systematically examined the biophysical properties of omega-conotoxin GVIA-sensitive neuronal N-type channels composed of various combinations of the alpha1B, alpha2/delta and beta1b subunits in Xenopus oocytes. 2. Whole-cell recordings demonstrated that coexpression of the beta1b subunit decelerated inactivation, whereas the alpha2/delta accelerated both activation and inactivation, and cancelled the kinetic effects of the beta1b. The alpha2/delta and the beta1b controlled voltage dependence of activation differently: the beta1b significantly shifted the current-voltage relationship towards the hyperpolarizing direction; however, the alpha2/delta shifted the relationship only slightly in the depolarizing direction. The extent of voltage-dependent inactivation was modified solely by the beta1b. 3. Unitary currents measured using a cell-attached patch showed stable patterns of opening that were markedly different among subunit combinations in their kinetic parameters. The alpha2/delta and the beta1b subunits also acted antagonistically in regulating gating patterns of unitary N-type channels. Open time was shortened by the alpha2/delta, while the fraction of long opening was enhanced by the beta1b. The alpha2/delta decreased opening probability (Po), while the beta1b increased Po. alpha1Balpha2/deltabeta1b produced unitary activity with an open time distribution value in between those of alpha1Balpha2/delta and alpha1Bbeta1b. However, both the alpha2/delta and the beta1b subunits reduced the number of null traces. 4. These results suggest that the auxiliary subunits alone and in combination contribute differently in forming gating apparatuses in the N-type channel, raising the possibility that subunit interaction contributes to the generation of functional diversity of N-type channels in native neuronal preparations also.

Recent genetic and molecular biological analyses have revealed many forms of inherited channelopathies. Homozygous ataxic mice, tottering (tg) and leaner (tgla) mice, have mutations in the P/Q-type Ca2+ channel alpha1A 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 tgla mice, and compared them with the properties of the wild-type and mutant alpha1A channels recombinantly expressed with the alpha2 and beta 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 approximately 60 and approximately 40% of control in tg and tgla 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 the tgla mice showed a more distinct change in voltage dependence of activation and inactivation, being shifted in the depolarizing direction by approximately 10 mV, with a broader voltage dependence of inactivation. In the recombinant expression system, the tg channel with a missense mutation (P601L) and one form of the two possible tgla aberrant splicing products, tgla (short) channel, showed a significant reduction in current density, while the other form of the tgla channels, tgla (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 tgla (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.

Interactions of G-protein α (Gα) and βγ subunits (Gβγ) with N- (α1B) and P/Q-type (α1A) Ca2+ channels were investigated using the Xenopus oocyte expression system. Gi3α was found to inhibit both N- and P/Q-type channels by receptor agonists, whereas Gβ1γ2 was responsible for prepulse facilitation of N-type channels. L-type channels (α1C) were not regulated by Gα or Gβγ. For N-type, prepulse facilitation mediated via Gβγ was impaired when the cytoplasmic I-II loop (loop 1) was deleted or replaced with the α1C loop 1. Gα-mediated inhibitions were also impaired by substitution of the α1C loop 1, but only when the C terminus was deleted. For P/Q-type, by contrast, deletion of the C terminus alone diminished Gα-mediated inhibition. Moreover, a chimera of L-type with the α1B loop 1 gained Gβγ-dependent facilitation, whereas an L-type chimera with the N- or P/Q-type C terminus gained Gα-mediated inhibition. These findings provide evidence that loop 1 of N-type channels is a regulatory site for Gβγ and the C termini of P/Q- and N-types for Gα.

Voltage-dependent Ca2+ channels serve as the only link to transduce membrane depolarization into cellular Ca(2+)-dependent reactions. A wide variety of chemical substances that have the ability to modulate Ca2+ channels have been demonstrated both for their clinic utility and for importance in elucidating the molecular basis of various biological responses. Recently, introduction of molecular biology to pharmacology has brought a great deal of information about the molecular basis of drug action in Ca2+ channels. In this review, we attempt to overview recent progress in understanding the interactions between Ca2+ channels and their blockers, namely Ca2+ antagonists, from a molecular and structural point of view.

Fat is one of the three major nutrients but it is not a sapid substance which activates one of the five basic tastes, sweet, bitter, sour, salt, and umami. However, we have experience that fat changes the taste of dishes largely. Transient receptor potential M5 (TRPM5) channel is not a receptor of the five basic tastes but it is gated after stimulation of the gustducin-coupled receptors for sweet, bitter, and umami. TRPM5 channel is a cation channel and depolarizes the taste cell, indicating that TRPM5 channel relates to the formation of tastes. The activities of some TRP channels (TRPV2, TRPV4, TRPM2, and TRPA1 channels) are regulated by lipids. I will review the TRP channels and taste transduction.

A unique structural variant of the cardiac L-type voltage-dependent calcium channel alpha 1 subunit cDNA was isolated from libraries derived from normal human heart mRNA. The deduced amino acid sequence shows significant homology to other calcium channel alpha 1 subunits. However, differences from the rabbit heart alpha 1 include a shortened N-terminus, a unique C-terminal insertion, and both forms of an alternatively spliced motif IV S3 region. The shortened N-terminus provides optimal access to consensus sequences thought to facilitate translation. Northern blot analysis revealed a single hybridizing mRNA species of 9.4 kb. The gene for the human heart alpha 1 subunit was localized specifically to the distal region of chromosome 12p13. The cloned alpha 1 subunit was expressed in Xenopus oocytes and single-channel analyses revealed native-like pharmacology and channel properties.

Although febrile seizures (FSs) are the most common convulsive syndrome in infants and childhood, the etiology of FSs has remained unclarified. Several missense mutations of the Nav1.1 channel (SCN1A), which alter channel properties, have been reported in a familial syndrome of GEFS+ (generalized epilepsy with febrile seizures plus). Here, we generatedScn1a-targeted rats carrying a missense mutation (N1417H) in the third pore region of the sodium channel by gene-driven ENU (N-ethyl-N-nitrosourea) mutagenesis. Despite their normal appearance under ordinary circumstances,Scn1amutant rats exhibited remarkably high susceptibility to hyperthermia-induced seizures, which involve generalized clonic and/or tonic–clonic convulsions with paroxysmal epileptiform discharges. Whole-cell patch-clamp recordings from HEK cells expressing N1417H mutant channels and from hippocampal GABAergic interneurons of N1417H mutant rats revealed a significant shift of the inactivation curve in the hyperpolarizing direction. In addition, clamp recordings clearly showed the reduction in action potential amplitude in the hippocampal interneurons of these rats. These findings suggest that a missense mutation (N1417H) of the Nav1.1 channel confers susceptibility to FS and the impaired biophysical properties of inhibitory GABAergic neurons underlie one of the mechanisms of FS.

Mechanotransduction is a fundamental process converting mechanical force into electrical and chemical responses, in which mechanosensitive channels are thought to play crucial roles. They are expressed in a variety of cells, including hair cells, baroreceptors, muscle spindle, and bone cells. Recent molecular biological analyses have revealed that several members of transient receptor potential (TRP) channels may play important roles in detection of mechanical stimuli. Twenty-seven trp-related genes in human have been identified to date. TRP proteins can be classified into six subfamilies: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPP (Polycystin), TRPML (Mucolipin), and TRPA (Ankyrin). Among the TRP superfamily TRPC1, TRPC6, TRPV1, TRPV2, TRPP1/TRPP2, TRPM4, TRPM7, and TRPA1 channels mediate mechanosensation. TRPC1, TRPC5, TRPC6, TRPV2, TRPV4, TRPM3, and TRPM7 channels are involved in osmosensation. I will review mechanosensitive channels and discuss their activation mechanism.

The effect of a new type of organic Ca2+ channel blocker, NC-1100 [(+/-)-1-(3,4-dimethoxyphenyl)-2-(4-diphenylmethylpiperazinyl)etha nol dihydrochloride], on both low- and high-threshold Ca2+ currents was studied in the whole-cell mode of the pyramidal neurons freshly dissociated from rat hippocampal CA1 region under voltage-clamp condition. The NC-1100 reversibly reduced the high-threshold Ca2+ current (HVA ICa) in a concentration-dependent manner without affecting the current-voltage relationship. The values of half-inhibition (IC50) were 1.3 x 10(-5) and 9.1 x 10(-6) M in external solution containing 10 and 2.5 mM Ca2+, respectively. The NC-1100 also decreased the low-threshold Ca2+ current (LVA ICa) in a concentration-dependent manner. The inhibitory potency was augmented by increasing the stimulation frequency and/or decreasing the extracellular Ca2+ concentration to a physiological range (2.5 mM). The IC50 value decreased to 7.7 x 10(-7) M in external solution containing 2.5 mM Ca2+ at a stimulation frequency of 1 Hz. The NC-1100 delayed the reactivation of LVA Ca2+ channel and enhanced voltage-dependently the steady-state inactivation, suggesting that this drug bound not only the resting LVA Ca2+ channel but also the inactivated one.

The CACNA1A gene encodes the poreforming, voltage-sensitive subunit of the voltage-dependent Cav2.1 calcium channel. Mutations in this gene have been linked to several human disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. In mice, mutations of the homolog Cacna1a cause recessively inherited phenotypes in tottering, rolling Nagoya, rocker, and leaner mice. Here we describe two knockdown mice with 28.4 ± 3.4% and 13.8 ± 3.3% of the wild-type Cav2.1 quantity. 28.4 ± 3.4% level mutants displayed ataxia, absence-like seizures and progressive cerebellar atrophy, although they had a normal life span. Mutants with 13.8 ± 3.3% level exhibited ataxia severer than the 28.4 ± 3.4% level mutants, absence-like seizures and additionally paroxysmal dyskinesia, and died premature around 3 weeks of age. These results indicate that knock down of Cav2.1 quantity to 13.8 ± 3.3% of the wild-type level are sufficient to induce the all neurological disorders observed in natural occurring Cacna1a mutants. These knockdown animals with Cav2.1 calcium channels intact can contribute to functional studies of the molecule in the disease.

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.