Inhibition of TRPC4/5 Channel Is Effective in Protecting against Histamine-Induced Hyperpermeability by Blocking Ca2+ Influx in Lung Microvascular Endothelial Cells.

Dysfunction of lung microvascular endothelium is a major feature in the pathobiology of pulmonary edema and hypoxic respiratory failure. Histamine induces lung microvascular endothelial barrier disruption and hyperpermeability upon evoking intracellular Ca2+ ([Ca2+]i) dynamics via binding to its receptors. Transient receptor potential canonical (TRPC) channels are Ca2+-permeable channel and stimulated by the agonists of G-protein-coupled receptors (GPCR). Here, we assessed histamine induced [Ca2+]i increases in human lung microvascular endothelial cells (HLMVEC) by using live cell Ca2+ imaging. We found that histamine increased [Ca2+]i was maintained at a static elevated level after a transient peak. The elevated Ca2+ plateau was vanished when extracellular Ca2+ was removed, indicating Ca2+ influx from extracellular mediated the histamine-induced Ca2+ plateau. TRPC4/5 channels antagonists, ML204 (10 µM) and HC070 (1 µM), significantly inhibited the Ca2+ plateaus, which was not influenced by Pyr3 or larixyl, the antagonists of TRPC3/6. Furthermore, ML204 or HC070 effectively suppressed the permeability response to histamine in HLMVEC. Our results indicated that histamine-induced Ca2+ influx may be mediated by TRPC4/5 channels and the antagonist of the channel significantly improved histamine-induced HLMVEC dysfunction.

Calcium Channels and Calcium-Regulated Channels in Human Red Blood Cells.

Free Calcium (Ca2+) is an important and universal signalling entity in all cells, red blood cells included. Although mature mammalian red blood cells are believed to not contain organelles as Ca2+ stores such as the endoplasmic reticulum or mitochondria, a 20,000-fold gradient based on a intracellular Ca2+ concentration of approximately 60 nM vs. an extracellular concentration of 1.2 mM makes Ca2+-permeable channels a major signalling tool of red blood cells. However, the internal Ca2+ concentration is tightly controlled, regulated and maintained primarily by the Ca2+ pumps PMCA1 and PMCA4. Within the last two decades it became evident that an increased intracellular Ca2+ is associated with red blood cell clearance in the spleen and promotes red blood cell aggregability and clot formation. In contrast to this rather uncontrolled deadly Ca2+ signals only recently it became evident, that a temporal increase in intracellular Ca2+ can also have positive effects such as the modulation of the red blood cells O2 binding properties or even be vital for brief transient cellular volume adaptation when passing constrictions like small capillaries or slits in the spleen. Here we give an overview of Ca2+ channels and Ca2+-regulated channels in red blood cells, namely the Gárdos channel, the non-selective voltage dependent cation channel, Piezo1, the NMDA receptor, VDAC, TRPC channels, CaV2.1, a Ca2+-inhibited channel novel to red blood cells and i.a. relate these channels to the molecular unknown sickle cell disease conductance Psickle. Particular attention is given to correlation of functional measurements with molecular entities as well as the physiological and pathophysiological function of these channels. This view is in constant progress and in particular the understanding of the interaction of several ion channels in a physiological context just started. This includes on the one hand channelopathies, where a mutation of the ion channel is the direct cause of the disease, like Hereditary Xerocytosis and the Gárdos Channelopathy. On the other hand it applies to red blood cell related diseases where an altered channel activity is a secondary effect like in sickle cell disease or thalassemia. Also these secondary effects should receive medical and pharmacologic attention because they can be crucial when it comes to the life-threatening symptoms of the disease.

Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes.

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 Ca2+-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 Ca2+-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 Ca2+ 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 Ca2+ may contribute to structural damage in the podocytes.

TRPC1 as a negative regulator for TRPC4 and TRPC5 channels.

Transient receptor potential canonical (TRPC) channels are calcium permeable, non-selective cation channels with wide tissue-specific distribution. Among 7 TRPC channels, TRPC 1/4/5 and TRPC3/6/7 are subdivided based on amino acid sequence homology. TRPC4 and TRPC5 channels exhibit cationic current with homotetrameric form, but they also form heterotetrameric channel such as TRPC1/4 or TRPC1/5 once TRPC1 is incorporated. The expression of TRPC1 is ubiquitous whereas the expressions of TRPC4 and TRPC5 are rather focused in nervous system. With the help of conditional knock-out of TPRC1, 4 and/or 5 genes, TRPC channels made of these constituents are reported to be involved in various pathophysiological functions such as seizure, anxiety-like behaviour, fear, Huntington's disease, Parkinson's disease and many others. In heterologous expression system, many issues such as activation mechanism, stoichiometry and relative cation permeabilites of homomeric or heteromeric channels have been addressed. In this review, we discussed the role of TRPC1 channel per se in plasma membrane, role of TRPC1 in heterotetrameric conformation (TRPC1/4 or TRPC1/5) and relationship between TRPC1/4/5 channels, calcium influx and voltage-gated calcium channels.

Switching between persistent firing and depolarization block in individual rat CA1 pyramidal neurons.

The hippocampal formation plays a role in mnemonic tasks and epileptic discharges in vivo. In vitro, these functions and malfunctions may relate to persistent firing (PF) and depolarization block (DB), respectively. Pyramidal neurons of the CA1 field have previously been reported to engage in either PF or DB during cholinergic stimulation. However, it is unknown whether these cells constitute disparate populations of neurons. Furthermore, it is unclear which cell-specific peculiarities may mediate their diverse response properties. However, it has not been shown whether individual CA1 pyramidal neurons can switch between PF and DB states. Here, we used whole cell patch clamp in the current clamp mode on in vitro CA1 pyramidal neurons from acutely sliced rat tissue to test various intrinsic properties which may provoke individual cells to switch between PF and DB. We found that individual cells could switch from PF to DB, in a cholinergic agonist concentration dependent manner and depending on the parameters of stimulation. We also demonstrate involvement of TRPC and potassium channels in this switching. Finally, we report that the probability for DB was more pronounced in the proximal than in the distal half of CA1. These findings offer a potential mechanism for the stronger spatial modulation in proximal, compared to distal CA1, as place field formation was shown to be affected by DB. Taken together, our results suggest that PF and DB are not mutually exclusive response properties of individual neurons. Rather, a cell's response mode depends on a variety of intrinsic properties, and modulation of these properties enables switching between PF and DB.

Pretreatment-Etidronate Alleviates CoCl2 Induced-SH-SY5Y Cell Apoptosis via Decreased HIF-1α and TRPC5 Channel Proteins.

Chronic hypoxic damage is one of the most common pathogenic factors that can cause neurodegenerative disorder in most cases. Etidronate (Eti) is one of the best-known earlier-generations of bisphosphonate derivatives for the treatment of bone-loss related diseases. Building on the preceding study of our laboratory, we found that Eti showed neuroprotective effects against 2-vessel occlusion induced vascular dementia (VD) in rats. Therefore, in this study, we attempted to elucidate the mechanism of action, which Eti protected cells from chronic hypoxic damage caused by CoCl2 in SH-SY5Y cells in vitro. Our data showed that the pretreatment with 100 µM Eti partially improved hypoxic damage in cell viability and reduced the hypoxia-inducible factor-1α (HIF-1α) expression, which indicated chronic hypoxic level. Furthermore, the protein expression of TRPC5 channel and its mediated intracellular calcium ion concentration ([Ca2+]i) were decreased. In addition, the apoptosis-related proteins caspase-9, and caspase-3 as well as calcium/calmodulin-dependent protein kinase II (CaMK-II) were down-regulated after treatment with Eti. In conclusion, Eti shows neuroprotective effects on SH-SY5Y cells injured by CoCl2 through resisting apoptosis caused by calcium influx, which may be related to the inhibition of HIF-1α protein and the decreased TRPC5 channel protein.

Muscarinic receptors in adrenal chromaffin cells: physiological role and regulation of ion channels.

Adrenal medullary chromaffin cells in mammals are innervated by sympathetic preganglionic nerve fibers, as are sympathetic ganglion neurons. Acetylcholine in the ganglion neurons is well established as mediating fast and slow excitatory postsynaptic potentials through nicotinic and muscarinic acetylcholine receptors (AChRs), respectively. The role of muscarinic AChRs during neuronal transmission in chromaffin cells varies among different mammals. Furthermore, the ion channel mechanisms associated with the muscarinic AChR-mediated increase in excitability of chromaffin cells are complicated and different from the excitation of ganglion neurons, which has been ascribed to the inhibition of M-type K+ channels. In this review, we focus on muscarinic receptor-mediated excitation in rodent and guinea pig chromaffin cells, in particular, on the role of muscarinic receptors in neuronal transmission, the muscarinic receptor subtypes involved in excitation and secretion, and the muscarinic regulation of ion channels including TWIK-related acid-sensitive K+ channels. Finally, we discuss prospectively the future of muscarinic receptor research in adrenal chromaffin cells.

5-HT promotes pulmonary arterial smooth muscle cell proliferation through the TRPC channel.

Pulmonary arterial hypertension is caused by an imbalance of pulmonary vasoconstriction and vasodilation. Pulmonary arteriolar remodeling is a primary pathological change and proliferation of pulmonary arterial smooth muscle cells (PASMC) is an important pathological basis for pulmonary arteriolar remodeling. Vasoactive substances, such as 5-HT, may play a role in proliferation of PASMC via unknown mechanisms. In vitro experiments with PASMC showed that the TRPC channel inhibitor SKF96365 inhibited the effects 5-HT and DOI on PASMC proliferation and G2M percentage increase, and decreased expression of TRPC1, TRPC6 and calcineurin A/NFATc3 induced by 5-HT and DOI. SKF96365 inhibited binding of NFATc3 and DNA promoted by 5-HT and DOI. Therefore, 5-HT may affect the TRPC channel to promote proliferation of PASMC; upregulate expression of TRPC1, TRPC6, and calcineurin A/NFATc3; and therefore promote NFATc3 nuclear translocation. There may be crosstalk between 5-HT and TRPC, which may contribute to the pathogeneis of pulmonary arterial hypertension and this may be a novel therapeutic target for treating pulmonary arterial hypertension.

Electrophysiological Mechanism of Peripheral Hormones and Nutrients Regulating Energy Homeostasis.

In organism, energy homeostasis is a biological process that involves the coordinated homeostatic regulation of energy intake (food intake) and energy expenditure. The human brain, particularly the hypothalamic proopiomelanocortin (POMC)- and agouti-related protein/neuropeptide Y (AgRP/NPY)-expressing neurons in the arcuate nucleus, plays an essential role in regulating energy homeostasis. The regulation process is mainly dependent upon peripheral hormones such as leptin and insulin, as well as nutrients such as glucose, amino acids, and fatty acids. Although many studies have attempted to illustrate the exact mechanisms of glucose and hormones action on these neurons, we still cannot clearly see the full picture of this regulation action. Therefore, in this review we will mainly discuss those established theories and recent progresses in this area, demonstrating the possible physiological mechanism by which glucose, leptin, and insulin affect neuronal excitability of POMC and AgRP neurons. In addition, we will also focus on some important ion channels which are expressed by POMC and AgRP neurons, such as KATP channels and TRPC channels, and explain how these channels are regulated by peripheral hormones and nutrients and thus regulate energy homeostasis.

The interaction domains of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heteromultimeric channels.

Transient receptor potential canonical (TRPC) family contains a non-selective cation channel, and four TRPC subunits form a functional tetrameric channel. TRPC4/5 channels form not only the homotetrameric channel but also a heterotetrameric channel with TRPC1. We investigated the interaction domain required for TRPC1/4 or TRPC1/5 heteromultimeric channels using FRET and the patch-clamp technique. TRPC1 only localized at the plasma membrane (PM) when it was coexpressed with TRPC4 or TRPC5. The TRPC1/4 or TRPC1/5 heteromultimeric showed the typical outward rectifying I/V curve. When TRPC1 and TRPC4 form a heteromeric channel, the N-terminal coiled-coil domain (CCD) and C-terminal 725-745 region of TRPC1 interact with the N-terminal CCD and C-terminal 700-728 region of TRPC4. However, when TRPC1 and TRPC5 form a heteromeric channel, the N-terminal CCD and C-terminal 673-725 region of TRPC1 interact with the N-terminal CCD and C-terminal 707-735 region of TRPC5. In conclusion, the N-terminal CCD of TRPC channels is essential for the heteromultimeric structure of TRPC channels, whereas specific C-terminal regions are required for unique heteromerization between subgroups of TRPC channels.

Dystrophin complex functions as a scaffold for signalling proteins.

Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and ß-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Periphery Pre-S1 and S1 helix nexus for PIP2 at TRPC3 channel.

Transient receptor potential canonical 3 (TRPC3) is a calcium-permeable, non-selective cation channel known to be regulated by components of the phospholipase C (PLC)-mediated signaling pathway, such as Ca2+, diacylglycerol (DAG) and phosphatidylinositol 4,5-biphosphate (PI(4,5)P2). However, the molecular gating mechanism by these regulators is not yet fully understood, especially its regulation by PI(4,5)P2, despite the importance of this channel in cardiovascular pathophysiology. Recently, Clarke et al. (2024) have reported that PI(4,5)P2 is a positive modulator for TRPC3 using molecular dynamics simulations and patch-clamp techniques. They have demonstrated a multistep gating mechanism of TRPC3 with the binding of PI(4,5)P2 to the lipid binding site located at the pre-S1/S1 nexus, and the propagation of PI(4,5)P2 sensing to the pore domain via a salt bridge between the TRP helix and the S4-S5 linker.

Transient receptor potential canonical 3 inhibitor Pyr3 improves outcomes and attenuates astrogliosis after intracerebral hemorrhage in mice.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) stems from the rupture of blood vessels in the brain, with the subsequent accumulation of blood in the parenchyma. Increasing evidence suggests that blood-derived factors induce excessive inflammatory responses that are involved in the progression of ICH-induced brain injury. Thrombin, a major blood-derived factor, leaks into the brain parenchyma on blood-brain barrier disruption and induces brain injury and astrogliosis. Furthermore, thrombin dynamically upregulates transient receptor potential canonical 3 channel, which contributes to pathological astrogliosis through a feed-forward upregulation of its own expression. The present study investigated whether Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), a specific transient receptor potential canonical 3 inhibitor, can improve functional outcomes and attenuate astrogliosis after ICH in mice. METHODS: Male C57BL6 mice received an intracerebral infusion of collagenase or autologous blood to induce ICH. Pyr3 was given both intracerebroventricularly and intraperitoneally after ICH induction. ICH-induced brain injury was evaluated by quantitative assessment of neurological deficits, brain swelling, and injury volume after ICH. Astrocyte activation was evaluated by immunohistochemical assessment of changes in S100 protein expression. RESULTS: Neurological deficits, neuronal injury, brain edema, and astrocyte activation were all significantly improved by administration of Pyr3. Moreover, delayed administration of Pyr3 at 6 hours or 1 day after blood or collagenase infusion, respectively, also improved the symptoms. CONCLUSIONS: Pyr3, a specific inhibitor of transient receptor potential canonical 3, reduced the perihematomal accumulation of astrocytes and ameliorated ICH-induced brain injury. Therefore, transient receptor potential canonical 3 provides a new therapeutic target for the treatment of hemorrhagic brain injury.

Enhancement of muscarinic receptor-mediated excitation in spontaneously hypertensive rat adrenal medullary chromaffin cells.

One of the mechanisms for hypertension is an increase in blood catecholamines due to increased secretion from sympathetic nerve terminals and adrenal medullary chromaffin (AMC) cells. Spontaneously hypertensive rats (SHRs) are used as an animal model of hypertension. Catecholamine secretion in AMC cells occurs in response to humoral factors and neuronal inputs from the sympathetic nerve fibres. Acetylcholine (ACh) released from the nerve terminals activates nicotinic as well as muscarinic ACh receptors. The present experiment aimed to elucidate whether muscarinic receptor-mediated excitation is altered in SHR AMC cells and, if it is, how. Compared with normotensive rat AMC cells, muscarinic stimulation induced greater catecholamine secretion and larger depolarising inward currents in SHR AMC cells. In contrast to normotensive rat AMC cells, the muscarine-induced current consisted of quinine-sensitive and quinine-insensitive components. The former and the latter are possibly ascribed to nonselective cation channel activation and TWIK-related acid-sensitive K+ (TASK) channel inhibition, as noted in guinea pig AMC cells. In fact, immunoreactive material for TASK1 and several isoforms of transient receptor potential canonical (TRPC) channels was detected in SHR AMC cells. Stromal interaction molecule 1 (STIM1), which plays an essential role for heteromeric TRPC1-TRPC4 channel formation and is not expressed in normotensive rat AMC cells, was detected in the cytoplasm and co-localised with TRPC1. The expression of muscarinic M1 receptors was enhanced in SHR AMC cells compared with normotensive rats. The results indicate that muscarinic excitation is enhanced in SHR AMC cells, probably through facilitation of TRPC channel signalling.

Reduction of TRPC1/TRPC3 mediated Ca2+-signaling protects oxidative stress-induced COPD.

Oxidative stress is a predisposing factor in Chronic Obstructive Pulmonary Disease (COPD). Specifically, pulmonary epithelial (PE) cells reduce antioxidant capacity during COPD because of the continuous production of reactive oxygen species (ROS). However, the molecular pathogenesis that governs such ROS activity is unclear. Here we show that the dysregulation of intracellular calcium concentration ([Ca2+]i) in PE cells from COPD patients, compared to the healthy PE cells, is associated with the robust functional expressions of Transient Receptor Potential Canonical (TRPC)1 and TRPC3 channels, and Ca2+ entry (SOCE) components, Stromal Interaction Molecule 1 (STIM1) and ORAI1 channels. Additionally, the elevated expression levels of fibrotic, inflammatory, oxidative, and apoptotic markers in cells from COPD patients suggest detrimental pathway activation, thereby reducing the ability of lung remodeling. To further delineate the mechanism, we used human lung epithelial cell line, A549, since the behavior of SOCE and the expression patterns of TRPC1/C3, STIM1, and ORAI1 were much like PE cells. Notably, the knockdown of TRPC1/C3 in A549 cells substantially reduced the SOCE-induced [Ca2+]i rise, and reversed the ROS-mediated oxidative, fibrotic, inflammatory, and apoptotic responses, thus confirming the role of TRPC1/C3 in SOCE driven COPD-like condition. Higher TRPC1/C3, STIM1, and ORAI1 expressions, along with a greater Ca2+ entry, via SOCE in ROS-induced A549 cells, led to the rise in oxidative, fibrotic, inflammatory, and apoptotic gene expression, specifically through the extracellular signal-regulated kinase (ERK) pathway. Abatement of TRPC1 and/or TRPC3 reduced the mobilization of [Ca2+]i and reversed apoptotic gene expression and ERK activation, signifying the involvement of TRPC1/C3. Together these data suggest that TRPC1/C3 and SOCE facilitate the COPD condition through ROS-mediated cell death, thus implicating their likely roles as potential therapeutic targets for COPD. SUMMARY: Alterations in Ca2+ signaling modalities in normal pulmonary epithelial cells exhibit COPD through oxidative stress and cellular injury, compromising repair, which was alleviated through inhibition of store-operated calcium entry. SUBJECT AREA: Calcium, ROS, Cellular signaling, lung disease.

Structural mechanism of human TRPC3 and TRPC6 channel regulation by their intracellular calcium-binding sites.

TRPC3 and TRPC6 channels are calcium-permeable non-selective cation channels that are involved in many physiological processes. The gain-of-function (GOF) mutations of TRPC6 lead to familial focal segmental glomerulosclerosis (FSGS) in humans, but their pathogenic mechanism remains elusive. Here, we report the cryo-EM structures of human TRPC3 in both high-calcium and low-calcium conditions. Based on these structures and accompanying electrophysiological studies, we identified both inhibitory and activating calcium-binding sites in TRPC3 that couple intracellular calcium concentrations to the basal channel activity. These calcium sensors are also structurally and functionally conserved in TRPC6. We uncovered that the GOF mutations of TRPC6 activate the channel by allosterically abolishing the inhibitory effects of intracellular calcium. Furthermore, structures of human TRPC6 in complex with two chemically distinct inhibitors bound at different ligand-binding pockets reveal different conformations of the transmembrane domain, providing templates for further structure-based drug design targeting TRPC6-related diseases such as FSGS.

TRPC1/5-Ca V 3 Complex Mediates Leptin-Induced Excitability in Hypothalamic Neurons.

Leptin regulates hypothalamic POMC+ (pro-opiomelanocortin) neurons by inducing TRPC (Transient Receptor Potential Cation) channel-mediate membrane depolarization. The role of TRPC channels in POMC neuron excitability is clearly established; however, it remains unknown whether their activity alone is sufficient to trigger excitability. Here we show that the right-shift voltage induced by the leptin-induced TRPC channel-mediated depolarization of the resting membrane potential brings T-type channels into the active window current range, resulting in an increase of the steady state T-type calcium current from 40 to 70% resulting in increased intrinsic excitability of POMC neurons. We assessed the role and timing of T-type channels on excitability and leptin-induced depolarization in vitro in cultured mouse POMC neurons. The involvement of TRPC channels in the leptin-induced excitability of POMC neurons was corroborated by using the TRPC channel inhibitor 2APB, which precluded the effect of leptin. We demonstrate T-type currents are indispensable for both processes, as treatment with NNC-55-0396 prevented the membrane depolarization and rheobase changes induced by leptin. Furthermore, co-immunoprecipitation experiments suggest that TRPC1/5 channels and Ca V 3.1 and Ca V 3.2 channels co-exist in complex. The functional relevance of this complex was corroborated using intracellular Ca2+ chelators; intracellular BAPTA (but not EGTA) application was sufficient to preclude POMC neuron excitability. However, leptin-induced depolarization still occurred in the presence of either BAPTA or EGTA suggesting that the calcium entry necessary to self-activate the TRPC1/5 complex is not blocked by the presence of BAPTA in hypothalamic neurons. Our study establishes T-type channels as integral part of the signaling cascade induced by leptin, modulating POMC neuron excitability. Leptin activation of TRPC channels existing in a macromolecular complex with T-type channels recruits the latter by locally induced membrane depolarization, further depolarizing POMC neurons, triggering action potentials and excitability.

TRPC Channels in Cardiac Plasticity.

The heart flexibly changes its structure in response to changing environments and oxygen/nutrition demands of the body. Increased and decreased mechanical loading induces hypertrophy and atrophy of cardiomyocytes, respectively. In physiological conditions, these structural changes of the heart are reversible. However, chronic stresses such as hypertension or cancer cachexia cause irreversible remodeling of the heart, leading to heart failure. Accumulating evidence indicates that calcium dyshomeostasis and aberrant reactive oxygen species production cause pathological heart remodeling. Canonical transient receptor potential (TRPC) is a nonselective cation channel subfamily whose multimodal activation or modulation of channel activity play important roles in a plethora of cellular physiology. Roles of TRPC channels in cardiac physiology have been reported in pathological cardiac remodeling. In this review, we summarize recent findings regarding the importance of TRPC channels in flexible cardiac remodeling (i.e., cardiac plasticity) in response to environmental stresses and discuss questions that should be addressed in the near future.

TRPC Channels: Dysregulation and Ca2+ Mishandling in Ischemic Heart Disease.

Transient receptor potential canonical (TRPC) channels are ubiquitously expressed in excitable and non-excitable cardiac cells where they sense and respond to a wide variety of physical and chemical stimuli. As other TRP channels, TRPC channels may form homo or heterotetrameric ion channels, and they can associate with other membrane receptors and ion channels to regulate intracellular calcium concentration. Dysfunctions of TRPC channels are involved in many types of cardiovascular diseases. Significant increase in the expression of different TRPC isoforms was observed in different animal models of heart infarcts and in vitro experimental models of ischemia and reperfusion. TRPC channel-mediated increase of the intracellular Ca2+ concentration seems to be required for the activation of the signaling pathway that plays minor roles in the healthy heart, but they are more relevant for cardiac responses to ischemia, such as the activation of different factors of transcription and cardiac hypertrophy, fibrosis, and angiogenesis. In this review, we highlight the current knowledge regarding TRPC implication in different cellular processes related to ischemia and reperfusion and to heart infarction.

Mechanisms for pituitary adenylate cyclase-activating polypeptide-induced increase in excitability in guinea-pig and mouse adrenal medullary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on adrenal medullary (AM) cells as a neurotransmitter of the sympathetic preganglionic nerve. In guinea-pig AM cells, PACAP induces little catecholamine secretion, but enhances secretion evoked by stimulants, whereas in other animals, such as mouse, PACAP itself induces depolarization and/or catecholamine secretion. The present studies aim to explore the physiological implication of these species differences in PACAP actions, the ion channel mechanism for PACAP-induced depolarization, and the mechanism for facilitation of muscarinic receptor-mediated cation currents in mouse and guinea-pig AM cells. The perforated patch clamp technique was used to record the whole-cell current in isolated AM cells. The amplitudes of 3 nM PACAP-induced inward currents were significantly larger in mouse AM cells than guinea-pig, whereas 1 µM muscarine-induced currents were larger in guinea-pig AM cells than mouse. Exposure to PACAP consistently resulted in enhancement of muscarine-induced currents in guinea-pig AM cells and facilitation of cell membrane insertion of heteromeric TRPC1-TRPC4 channels in response to muscarine in PC12 cells. The PACAP-induced current was inhibited by 30 µM 9-phenanthrol, a specific TRPM4 channel inhibitor, and abolished by replacement of external Na+ with N-methyl D-glucamine. TRPM4-like immunoreactivity was located at the cell periphery in AM cells. The present results indicate that PACAP and muscarinic receptors are major metabotropic receptors mediating generation of depolarizing inward currents in mouse and guinea-pig AM cells, respectively. We conclude that PACAP activates TRPM4-like channels and enhance the muscarinic current through facilitating the membrane insertion of TRPC1-TRPC4 channels in AM cells.