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The cell signaling molecules nitric oxide (NO) and Ca 2+ regulate diverse biological processes through their closely coordinated activities directed by signaling protein complexes. However, it remains unclear how dynamically the multicomponent protein assemblies behave within the signaling complexes upon the interplay between NO and Ca 2+ signals. Here we demonstrate that TRPC5 channels activated by the stimulation of G-protein-coupled ATP receptors mediate Ca 2+ influx, that triggers NO production from endothelial NO synthase (eNOS), inducing secondary activation of TRPC5 via cysteine S-nitrosylation and eNOS in vascular endothelial cells. Mutations in the caveolin-1-binding domains of TRPC5 disrupt its association with caveolin-1 and impair Ca 2+ influx and NO production, suggesting that caveolin-1 serves primarily as the scaffold for TRPC5 and eNOS to assemble into the signal complex. Interestingly, during ATP receptor activation, eNOS is dissociated from caveolin-1 and in turn directly associates with TRPC5, which accumulates at the plasma membrane dependently on Ca 2+ influx and calmodulin. This protein reassembly likely results in a relief of eNOS from the inhibitory action of caveolin-1 and an enhanced TRPC5 S-nitrosylation by eNOS localized in the proximity, thereby facilitating the secondary activation of Ca 2+ influx and NO production. In isolated rat aorta, vasodilation induced by acetylcholine was significantly suppressed by the TRPC5 inhibitor AC1903. Thus, our study provides evidence that dynamic remodeling of the protein assemblies among TRPC5, eNOS, caveolin-1, and calmodulin determines the ensemble of Ca 2+ mobilization and NO production in vascular endothelial cells., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved., Methods: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca 2+ -dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes., Results: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca 2+ -dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca 2+ elevations and a disorganized cytoskeleton., Conclusions: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca 2+ may contribute to structural damage in the podocytes., (Copyright © 2019 by the American Society of Nephrology.)

Intestinal fibrosis is an intractable complication of Crohn's disease (CD), and, when occurring excessively, causes severe intestinal obstruction that often necessitates surgical resection. The fibrosis is characterized by an imbalance in the turnover of extracellular matrix (ECM) components, where intestinal fibroblasts/myofibroblasts play active roles in ECM production, fibrogenesis and tissue remodeling, which eventually leads to the formation of stenotic lesions. There is however a great paucity of knowledge about how intestinal fibrosis initiates and progresses, which hampers the development of effective pharmacotherapies against CD. Recently, we explored the potential implications of transient receptor potential (TRP) channels in the pathogenesis of intestinal fibrosis, since they are known to act as cellular stress sensors/transducers affecting intracellular Ca 2+ homeostasis/dynamics, and are involved in a broad spectrum of cell pathophysiology including inflammation and tissue remodeling. In this review, we will place a particular emphasis on the intestinal fibroblast/myofibroblast TRPC6 channel to discuss its modulatory effects on fibrotic responses and therapeutic potential for anti-fibrotic treatment against CD-related stenosis.

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

TNF-α-NF-κB signaling plays a central role in inflammation, apoptosis, and neoplasia. One major consequence of this signaling in the gut is increased production of prostaglandin E(2) (PGE(2)) via cyclooxygenase-2 (COX-2) induction in myofibroblasts, which has been reported to be dependent on Ca(2+). In this study, we explored a potential role of canonical transient receptor potential (TRPC) proteins in this Ca(2+)-mediated signaling using a human colonic myofibroblast cell line CCD-18Co. In CCD-18Co cell, treatment with TNF-α greatly enhanced Ca(2+) influx induced by store depletion along with increased cell-surface expression of TRPC1 protein (but not of the other TRPC isoforms) and induction of a Gd(3+)-sensitive nonselective cationic conductance. Selective inhibition of TRPC1 expression by small interfering RNA (siRNA) or functionally effective TRPC1 antibody targeting the near-pore region of TRPC1 (T1E3) antagonized the enhancement of store-dependent Ca(2+) influx by TNF-α, whereas potentiated TNF-α induced PGE(2) production. Overexpression of TRPC1 in CCD-18Co produced opposite consequences. Inhibitors of NF-κB (curcumin, SN-50) attenuated TNF-α-induced enhancement of TRPC1 expression, store-dependent Ca(2+) influx, and COX-2-dependent PGE(2) production. In contrast, inhibition of calcineurin-nuclear factor of activated T-cell proteins (NFAT) signaling by FK506 or NFAT Activation Inhibitor III enhanced the PGE(2) production without affecting TRPC1 expression and the Ca(2+) influx. Finally, the suppression of store-dependent Ca(2+) influx by T1E3 antibody or siRNA knockdown significantly facilitated TNF-α-induced NF-κB nuclear translocation. In aggregate, these results strongly suggest that, in colonic myofibroblasts, NF-κB and NFAT serve as important positive and negative transcriptional regulators of TNF-α-induced COX-2-dependent PGE(2) production, respectively, at the downstream of TRPC1-associated Ca(2+) influx.

Calcium dynamics and its linked molecular interactions cause a variety of biological responses; thus, exploiting techniques for detecting both concurrently is essential. Here we describe a method for measuring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and protein-protein interactions within the same cell, using Fura-2 and superenhanced cyan and yellow fluorescence protein (seCFP and seYFP, respectively) FRET imaging techniques. Concentration-independent corrections for bleed-through of Fura-2 into FRET cubes across different time points and [Ca(2+)](i) values allowed for an effective separation of Fura-2 cross-talk signals and seCFP and seYFP cross-talk signals, permitting calculation of [Ca(2+)](i) and FRET with high fidelity. This correction approach was particularly effective at lower [Ca(2+)](i) levels, eliminating bleed-through signals that resulted in an artificial enhancement of FRET. By adopting this correction approach combined with stepwise [Ca(2+)](i) increases produced in living cells, we successfully elucidated steady-state relationships between [Ca(2+)](i) and FRET derived from the interaction of seCFP-tagged calmodulin (CaM) and the seYFP-fused CaM binding domain of myosin light chain kinase. The [Ca(2+)](i) versus FRET relationship for voltage-gated sodium, calcium, and TRPC6 channel CaM binding domains (IQ domain or CBD) revealed distinct sensitivities for [Ca(2+)](i). Moreover, the CaM binding strength at basal or subbasal [Ca(2+)](i) levels provided evidence of CaM tethering or apoCaM binding in living cells. Of the ion channel studies, apoCaM binding was weakest for the TRPC6 channel, suggesting that more global Ca(2+) and CaM changes rather than the local CaM-channel interface domain may be involved in Ca(2+)CaM-mediated regulation of this channel. This simultaneous Fura-2 and CFP- and YFP-based FRET imaging system will thus serve as a simple but powerful means of quantitatively elucidating cellular events associated with Ca(2+)-dependent functions.

Aim: The aim of the present study was to explore the mechanism for the Ca2+- dependent inactivation of the canonical transient receptor potential (TRPC) 7 channel expressed in human embryonic kidney 293 cells., Method: The whole-cell patch-clamp technique was used in the study., Results: With Ca2+-free external solution, the perfusion of 100 micromol/L carbachol to, or dialysis of the cell with 100 micromol/L guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), induced large inward currents, respectively. These currents were rapidly inhibited by the addition of 1 mmol/L Ca2+ into the bath, and recovery from this inhibition was only partial after the Ca2+ removal, unless vigorous intracellular Ca2+ buffering with 10 mmol/L 1,2 bis(2- aminophenoxy)ethane-N,N,No,No-tetraacetic acid (BAPTA) (plus 4 mmol/L Ca2+) was employed. In contrast, the current induced by a membrane-permeable analog of diacylglycerol (DAG), 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 micromol/L) did not undergo the inhibition persisting after Ca2+ removal. Interestingly, the inclusion of inositol 1,4,5 trisphosphate (IP3; 100 micromol/L) in the patch pipette rendered the OAG-induced current susceptible to the persistent Ca2+-mediated inhibition independent of the IP3 receptor in the majority of the tested cells, as evidenced by the inability of heparin and thapsigargin in reversing the effect of IP3., Conclusion: The present results suggest that Ca2+ entry via the activated TRPC7 channel plays a critical role in inactivating the channel where the cooperative actions of DAG and IP3 are essentially involved.

The mechanisms of intracellular calcium store depletion and store-related Ca(2+) dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca(2+) by BAPTA/AM (50 microM), or La(3+) (100 microM) enhanced while dantrolene (100 microM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca(2+) stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca(2+) concentrations ([Ca(2+)](i)).

It has long been known that many chemical and physical stimuli imposed on the cell from its exterior environments elicit a long-lasting Ca2+ influx through yet poorly elucidated transmembrane pathways distinct from voltage-gated and fast ligand-gated Ca2+ entry channels, thereby activating and modulating a variety of cellular functions. Recent progress in molecularly identifying these pathways, initiated from the discovery of Drosophila's visual transduction mutants transient receptor potential (TRP) proteins, has begun to reveal the presence of an enormous superfamily of non-voltage-gated Ca2+ channels. The mammalian members of TRP superfamily are (except for two members) Ca2+-permeable non-selective cation channels which are constitutively active or gated by a multitude of physicochemical stimuli such as receptor stimulation, phospholipids, oxidants, pheromones, cell volume change/shear stress, exogenous compounds affecting sensations, and changes in ambient temperature, acidity and osmolarity and cellular metabolic status. Owing to these diversities in activation and their broad distribution from brain to peripheral organs and tissues, TRP channels are now thought to be involved in divergent physiological functions including; pain and taste transductions; thermo- and mechano-sensations; regulation of mineral absorption/reabsorption; blood pressure, gut motility and airway responsiveness; cell proliferation/death, some of which seem tightly associated with specific genetic disorders. These features will render TRP channels the attractive novel molecular targets for future drug therapy. This paper briefly overviews the current knowledge available for these channels with a main interest in their possible linkage with in vivo function.

We investigated, by using the patch clamp technique, Ca2+-mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 microM)-activated TRPC6 currents (I(TRPC6)) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2+(o)) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2+(o) solely inhibited TRPC7 currents (I(TRPC7)). Vigorous buffering of intracellular Ca2+ (Ca2+(i)) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2+(o), disclosing fast potentiation (EC50: approximately 0.4 mM) and inhibition (IC50: approximately 4 mM) of I(TRPC6) and fast inhibition (IC50: approximately 0.4 mM) of I(TRPC7). This inhibition of I(TRPC6) and I(TRPC7) seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of I(TRPC6) showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of I(TRPC6) or its acceleration by Ca2+(o) probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 microM), coexpression of Ca2+-insensitive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2+(i) via CaM and conversely enhanced by IP3. In addition, the inactivation time course of I(TRPC6) was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+-CaM-dependent and -independent mechanisms.

We investigated the possible involvement of the melastatin family protein TRPM7 in Ca(2+)-mediated proliferative control of human retinoblastoma (RB) cells. The growth of RB cell was facilitated by elevating the extracellular Ca(2+) concentration with a parallel increase in the magnitude of spontaneous Ca(2+) influx. Under nystatin-perforated voltage-clamp, RB cells exhibited an outward-rectifying, spontaneous cation current (I(spont)) having Ca(2+)/Mg(2+)-inhibited but -permeating properties. Various cation channel blockers inhibiting I(spont) (Gd(3+), La(3+), LOE908, 2-APB) suppressed the spontaneous Ca(2+) influx and decelerated the growth of RB cells with similar efficacies. Excision of the RB cell membrane (inside-out) into MgATP-free solution induced a 70pS single channel activity, which was effectively inhibited by millimolar concentrations of Mg(2+) or MgATP. RT-PCR and immunocytochemical experiments revealed the expression of TRPM7 mRNA and protein in RB cells, and heterologous expression of TRPM7 in HEK293 cells reproduced the key features of I(spont). In contrast, elimination of this protein from RB cells by siRNA silencing markedly reduced I(spont) density and the magnitude of spontaneous Ca(2+) influx, which was paralleled by decreased TRPM7 immunoreactivity, decelerated cell proliferation, and retarded G(1)/S cell cycle progression. These results suggest a significant regulatory role of TRPM7 for RB cell proliferation as a spontaneously activated Ca(2+) influx pathway.

Recent investigations have revealed that mammalian homologues of transient receptor potential (TRP) protein (TRP1-7) are promising candidates for Ca2+ entry mechanisms (or channels) associated with various metabotropic G protein-coupled receptors (GPCRs) in smooth muscle, stimulation of which generates lipid second messengers and depletes internal stores. RT-PCR and immunocytochemical experiments have demonstrated that although the level of expression varies depending on tissues, the major TRP isoforms expressed in smooth muscle are TRP4, 6 and 7. In some vascular preparations, the significant expression of TRP1 mRNA and protein is also detected. Consistent with these findings, recent functional studies using TRP6- and TRP1-specific antisense oligonucleotides and antibodies have suggested that TRP6 is the essential component of alpha1-adrenoceptor activated, store depletion-independent Ca2+ entry channels, while TRP1 is partly involved in Ca2+ entry associated with store depletion or capacitative Ca2+ entry. In addition, coexpression of different TRP isoforms results in the appearance of cation channels showing novel properties reminiscent of some native GPCR-activated Ca2+-permeable non-selective cation channels. Thus, at present, TRP proteins may be the most important clues for elucidating the molecular entities of receptor- and store-operated Ca2+ entry mechanisms in smooth muscle and their roles in smooth muscle functions.

The article presents a study which investigated the effect of atrial natriuretic peptides-guanylyl cyclase-A (ANP-GC-A) signaling on transient receptor potential subfamily C(TRPC)6. C(TRPC)6 refers to a receptor-oriented calcium (Ca 2+) channel known to positively regulate prohypertrophic calcineurin-nuclear factor of activated T cells (NFAT) signaling. The researchers concluded that TRPC6 is a critical target of antihypertrophic effects. They recommend the use of TRPC6 blockade in preventing pathological cardiac remodeling.