To Transient Receptor Potential Research Articles

Interaction between TRP channels and anoctamins

Anoctamin 1 (ANO1) binds to transient receptor potential (TRP) channels (protein-protein interaction) and then is activated by TRP channels (functional interaction). TRP channels are non-selective cation channels that are expressed throughout the body and play roles in multiple physiological functions. Studies on TRP channels increased after the identification of TRP vanilloid 1 (TRPV1) in 1997. Calcium-activated chloride channel anoctamin 1 (ANO1, also called TMEM16A and DOG1) was identified in 2008. ANO1 plays a major role in TRP channel-mediated functions, as first shown in 2014 with the demonstration of a protein-protein interaction between TRPV4 and ANO1. In cells that co-express TRP channels and ANO1, calcium entering cells through activated TRP channels causes ANO1 activation. Therefore, in many tissues, the physiological functions related to TRP channels are modulated through chloride flux associated with ANO1 activation. In this review, we summarize the latest understanding of TRP-ANO1 interactions, particularly interaction of ANO1 with TRPV4, TRP canonical 6 (TRPC6), TRPV3, TRPV1, and TRPC2 in the salivary glands, blood vessels, skin keratinocytes, primary sensory neurons, and vomeronasal organs, respectively.

Progress on role of ion channels of cardiac fibroblasts in fibrosis.

Cardiac fibrosis is defined as excessive deposition of extracellular matrix (ECM) in pathological conditions. Cardiac fibroblasts (CFs) activated by injury or inflammation differentiate into myofibroblasts (MFs) with secretory and contractile functions. In the fibrotic heart, MFs produce ECM which is composed mainly of collagen and is initially involved in maintaining tissue integrity. However, persistent fibrosis disrupts the coordination of excitatory contractile coupling, leading to systolic and diastolic dysfunction, and ultimately heart failure. Numerous studies have demonstrated that both voltage- and non-voltage-gated ion channels alter intracellular ion levels and cellular activity, contributing to myofibroblast proliferation, contraction, and secretory function. However, an effective treatment strategy for myocardial fibrosis has not been established. Therefore, this review describes the progress made in research related to transient receptor potential (TRP) channels, Piezo1, Ca2+ release-activated Ca2+ (CRAC) channels, voltage-gated Ca2+ channels (VGCCs), sodium channels, and potassium channels in myocardial fibroblasts with the aim of providing new ideas for treating myocardial fibrosis.

Development of enhanced drug delivery vehicles for three cannabis-based terpenes using poly(lactic-co-glycolic acid) based nanoparticles

Terpenes derived from different varieties of Cannabis sativa have shown potentially useful pharmacological activities, including the ability to bind to transient receptor potential (TRP) channels. Beta-myrcene (MC), beta-caryophyllene (CPh), and nerolidol (NL) are among the most abundant and studied terpenes found in cannabis. Despite the potential pharmacological effectiveness of these terpenes, their volatile and hydrophobic nature, and associated poor solubility and low bioavailability limit their suitability as pharmaceutical agents. To address these limitations, the current study sought to design poly(DL-lactide-co-glycolide) (PLGA) based nanoparticles (NPs) that encapsulate MC, CPh, and NL. First, several preparation methods and formulations were tested to develop MC-loaded PLGA NPs. The NPs produced had a mean diameter between 200–350 nm, a narrow size distribution, and a negative zeta potential in the range of -25 to -30 mV. Optimal NPs were produced using emulsion-solvent evaporation, purified and collected by ultra-filtration, and freeze-dried in the presence of trehalose 5% (w/w), achieving the highest encapsulation efficiency (EE%: ∼8%). Utilizing the optimized formulation and preparation method, PEG-PLGA NPs encapsulating MC, CPh, or NL were also fabricated and characterized. The encapsulation capacities of CPh (EE%: 64.9 %) and NL (EE%: 55.7 %) PEG-PLGA NPs were approximately 3-fold that of MC NPs (EE%: 18.1 %). In conclusion, these three terpenes were successfully encapsulated in PLGA-based NPs. The nanosystems developed herein can be proposed as promising therapeutic tools for the enhanced delivery of cannabis-derived terpenes.

Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4.

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators—calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein–protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

TRPA1 and TRPV1 channels participate in atmospheric-pressure plasma-induced [Ca2+]i response

Despite successful clinical application of non-equilibrium atmospheric pressure plasma (APP), the details of the molecular mechanisms underlying APP-inducible biological responses remain ill-defined. We previously reported that exposure of 3T3L1 cells to APP-irradiated buffer raised the cytoplasmic free Ca2+ ([Ca2+]i) concentration by eliciting Ca2+ influx in a manner sensitive to transient receptor potential (TRP) channel inhibitors. However, the precise identity of the APP-responsive channel molecule(s) remains unclear. In the present study, we aimed to clarify channel molecule(s) responsible for indirect APP-responsive [Ca2+]i rises. siRNA-mediated silencing experiments revealed that TRPA1 and TRPV1 serve as the major APP-responsive Ca2+ channels in 3T3L1 cells. Conversely, ectopic expression of either TRPA1 or TRPV1 in APP-unresponsive C2C12 cells actually triggered [Ca2+]i elevation in response to indirect APP exposure. Desensitization experiments using 3T3L1 cells revealed APP responsiveness to be markedly suppressed after pretreatment with allyl isothiocyanate or capsaicin, TRPA1 and TRPV1 agonists, respectively. APP exposure also desensitized the cells to these chemical agonists, indicating the existence of a bi-directional heterologous desensitization property of APP-responsive [Ca2+]i transients mediated through these TRP channels. Mutational analyses of key cysteine residues in TRPA1 (Cys421, Cys621, Cys641, and Cys665) and in TRPV1 (Cys258, Cys363, and Cys742) have suggested that multiple reactive oxygen and nitrogen species are intricately involved in activation of the channels via a broad range of modifications involving these cysteine residues. Taken together, these observations allow us to conclude that both TRPA1 and TRPV1 channels play a pivotal role in evoking indirect APP-dependent [Ca2+]i responses.

Mitochondrial dysfunction increases bronchopulmonary C‐fiber excitability via PKC alpha signaling

Airway sensory nerve excitability is a key determinant of respiratory disease‐associated reflexes and sensations such as cough and dyspnea. One notable feature of peripheral terminals of sensory nerves is that they are packed with mitochondria. Mitochondrial dysfunction and subsequent oxidative stress has been reported for a variety of cell types in inflammatory diseases. We have previously shown that reactive oxygen species (ROS) produced by Antimycin A (a mitochondrial complex III Qi site inhibitor) increase excitability in bronchopulmonary nociceptive C‐fibers. This increased excitability decreases the mechanical threshold for activation and increases action potential firing in response to a P2X2/3 agonist. This Antimycin A–induced hyperexcitability is dependent on mitochondrial ROS and is blocked by intracellular antioxidants.Given that ROS are known to activate protein kinase C (PKC), we hypothesized that this increased excitability was due to PKC activation. We used dissociated vagal ganglion neurons to study activation of the PKC isoforms: alpha, beta1, beta2, delta and epsilon in response to Antimycin‐A. Cells were stained with antibodies against the specific isoforms to determine their cellular location. Of these isoforms, only PKC alpha translocated to the region of the cell membrane in response to Antimycin A treatment.We also examined the effects of Antimycin A induced ROS production on sensory nerve terminals. We recorded action potential firing from the terminals of individual bronchopulmonary C‐fibers using a mouse ex vivo lung‐vagal ganglia preparation. C‐fibers were characterized as nociceptors or non‐nociceptors based upon conduction velocity and response to transient receptor potential (TRP) channel agonists. Antimycin A–induced hyperexcitability was inhibited by the PKC inhibitor bisindolylmaleimide (BIM) I, but not by its inactive analog BIM V. Further, this BIM I inhibition occurred at a concentration that specifically blocks activity of PKC alpha over other isoforms. In conclusion, ROS evoked by mitochondrial dysfunction caused nociceptor hyperexcitability via the translocation and activation of PKC alpha.Support or Funding InformationThis work was supported by the National Heart Lung and Blood Institute in Bethesda, USA (R01‐HL119802)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Closely Associated Phospholipase C Regulates Cation Channel Function through Phosphoinositide Hydrolysis.

In the hemaphroditic sea snail, Aplysia californica, reproduction is initiated when the bag cell neurons secrete egg-laying hormone during a protracted afterdischarge. A source of depolarization for the afterdischarge is a voltage-gated, nonselective cation channel, similar to transient receptor potential (TRP) channels. Once the afterdischarge is triggered, phospholipase C (PLC) is activated to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3). We previously reported that a DAG analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), activates a prominent, inward whole-cell cationic current that is enhanced by IP3 To examine the underlying mechanism, we investigated the effect of exogenous OAG and IP3, as well as PLC activation, on cation channel activity and voltage dependence in excised, inside-out patches from cultured bag cell neurons. OAG transiently elevated channel open probability (PO) when applied to excised patches; however, coapplication of IP3 prolonged the OAG-induced response. In patches exposed to OAG and IP3, channel voltage dependence was left-shifted; this was also observed with OAG, but not to the same extent. Introducing the PLC activator, m-3M3FBS, to patches increased channel PO, suggesting PLC may be physically linked to the channels. Accordingly, blocking PLC with U-73122 ablated the m-3M3FBS-induced elevation in PO Treatment with m-3M3FBS left-shifted cation channel voltage dependence to a greater extent than exogenous OAG and IP3 Finally, OAG and IP3 potentiated the stimulatory effect of PKC, which is also associated with the channel. Thus, the PLC-PKC signaling system is physically localized such that PIP2 breakdown products liberated during the afterdischarge modulate the cation channel and temporally influence neuronal activity.SIGNIFICANCE STATEMENT Using excised patches from Aplysia bag cell neurons, we present the first evidence of a nonselective cation channel physically associating with phospholipase C (PLC) at the single-channel level. PLC-mediated breakdown of phospholipids generates diacylglycerol and inositol trisphosphate, which activate the cation channel. This is mimicked by exogenous lipids; furthermore, these second messengers left-shift channel voltage dependence and enhance the response of the channel to protein kinase C. PLC-mediated lipid signaling controls single-channel currents to ensure depolarization is maintained for an extended period of firing, termed the afterdischarge, when the bag cell neurons secrete egg-laying hormone to trigger reproduction.

Why individual thermo sensation and pain perception varies? Clue of disruptive mutations in TRPVs from 2504 human genome data

ABSTRACTEvery individual varies in character and so do their sensory functions and perceptions. The molecular mechanism and the molecular candidates involved in these processes are assumed to be similar if not same. So far several molecular factors have been identified which are fairly conserved across the phylogenetic tree and are involved in these complex sensory functions. Among all, members belonging to Transient Receptor Potential (TRP) channels have been widely characterized for their involvement in thermo-sensation. These include TRPV1 to TRPV4 channels which reveal complex thermo-gating behavior in response to changes in temperature. The molecular evolution of these channels is highly correlative with the thermal response of different species. However, recent 2504 human genome data suggest that these thermo-sensitive TRPV channels are highly variable and carry possible deleterious mutations in human population. These unexpected findings may explain the individual differences in terms of complex sensory functions.

Sensory Nerve Terminal Mitochondrial Dysfunction Induces Hyperexcitability in Airway Nociceptors via Protein Kinase C

Airway sensory nerve excitability is a key determinant of respiratory disease-associated reflexes and sensations such as cough and dyspnea. Inflammatory signaling modulates mitochondrial function and produces reactive oxygen species (ROS). Peripheral terminals of sensory nerves are densely packed with mitochondria; thus, we hypothesized that mitochondrial modulation would alter neuronal excitability. We recorded action potential firing from the terminals of individual bronchopulmonary C-fibers using a mouse ex vivo lung-vagal ganglia preparation. C-fibers were characterized as nociceptors or non-nociceptors based upon conduction velocity and response to transient receptor potential (TRP) channel agonists. Antimycin A (mitochondrial complex III Qi site inhibitor) had no effect on the excitability of non-nociceptors. However, antimycin A increased excitability in nociceptive C-fibers, decreasing the mechanical threshold by 50% and increasing the action potential firing elicited by a P2X2/3 agonist to 270% of control. Antimycin A-induced nociceptor hyperexcitability was independent of TRP ankyrin 1 or TRP vanilloid 1 channels. Blocking mitochondrial ATP production with oligomycin or myxothiazol had no effect on excitability. Antimycin A-induced hyperexcitability was dependent on mitochondrial ROS and was blocked by intracellular antioxidants. ROS are known to activate protein kinase C (PKC). Antimycin A-induced hyperexcitability was inhibited by the PKC inhibitor bisindolylmaleimide (BIM) I, but not by its inactive analog BIM V. In dissociated vagal neurons, antimycin A caused ROS-dependent PKC translocation to the membrane. Finally, H2O2 also induced PKC-dependent nociceptive C-fiber hyperexcitability and PKC translocation. In conclusion, ROS evoked by mitochondrial dysfunction caused nociceptor hyperexcitability via the translocation and activation of PKC.

Considering channel activation and inhibition

Considering channel activation and inhibition

5,6-EET Is Released upon Neuronal Activity and Induces Mechanical Pain Hypersensitivity via TRPA1 on Central Afferent Terminals

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidonic acid that act as endogenous signaling molecules in multiple biological systems. Here we have investigated the specific contribution of 5,6-EET to transient receptor potential (TRP) channel activation in nociceptor neurons and its consequence for nociceptive processing. We found that, during capsaicin-induced nociception, 5,6-EET levels increased in dorsal root ganglia (DRGs) and the dorsal spinal cord, and 5,6-EET is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux (100 nm) in cultured DRG neurons that was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices, 5,6-EET dose dependently enhanced the frequency, but not the amplitude, of spontaneous EPSCs (sEPSCs) in lamina II neurons that also responded to mustard oil (allyl isothiocyanate), indicating a presynaptic action. Furthermore, 5,6-EET-induced enhancement of sEPSC frequency was abolished in TRPA1-null mice, suggesting that 5,6-EET presynaptically facilitated spinal cord synaptic transmission by TRPA1. Finally, in vivo intrathecal injection of 5,6-EET caused mechanical allodynia in wild-type but not TRPA1-null mice. We conclude that 5,6-EET is synthesized on the acute activation of nociceptors and can produce mechanical hypersensitivity via TRPA1 at central afferent terminals in the spinal cord.

Metabotropic Glutamate Receptor 1 Mediates the Electrophysiological and Toxic Actions of the Cycad Derivative β-N-Methylamino-l-Alanine on Substantia Nigra Pars Compacta DAergic Neurons

Amyotrophic lateral sclerosis-Parkinson dementia complex (ALS-PDC) is a neurodegenerative disease with ALS, parkinsonism, and Alzheimer's symptoms that is prevalent in the Guam population. beta-N-Methylamino alanine (BMAA) has been proposed as the toxic agent damaging several neuronal types in ALS-PDC, including substantia nigra pars compacta dopaminergic (SNpc DAergic) neurons. BMAA is a mixed glutamate receptor agonist, but the specific pathways activated in DAergic neurons are not yet known. We combined electrophysiology, microfluorometry, and confocal microscopy analysis to monitor membrane potential/current, cytosolic calcium concentration ([Ca(2+)](i)) changes, cytochrome-c (cyt-c) immunoreactivity, and reactive oxygen species (ROS) production induced by BMAA. Rapid toxin applications caused reversible membrane depolarization/inward current and increase of firing rate and [Ca(2+)](i) in DAergic neurons. The inward current (I(BMAA)) was mainly mediated by activation of metabotropic glutamate receptor 1 (mGluR1), coupled to transient receptor potential (TRP) channels, and to a lesser extent, AMPA receptors. Indeed, mGluR1 (CPCCOEt) and TRP channels (SKF 96365; Ruthenium Red) antagonists reduced I(BMAA), and a small component of I(BMAA) was reduced by the AMPA receptor antagonist CNQX. Calcium accumulation was mediated by mGluR1 but not by AMPA receptors. Application of a low concentration of NMDA potentiated the BMAA-mediated calcium increase. Prolonged exposure to BMAA caused significant modifications of membrane properties, calcium overload, cell shrinkage, massive cyt-c release into the cytosol and ROS production. In SNpc GABAergic neurons, BMAA activated only AMPA receptors. Our study identifies the mGluR1-activated mechanism induced by BMAA that may cause the neuronal degeneration and parkinsonian symptoms seen in ALS-PDC. Moreover, environmental exposure to BMAA might possibly also contribute to idiopathic PD.

Overexpression of Trpp5 contributes to cell proliferation and apoptosis probably through involving calcium homeostasis

Trpp5 is one member of the polycystic kidney disease (PKD) family, which belongs to transient receptor potential (TRP) superfamily. Our previous study has shown that Trpp5 is developmentally expressed in mouse testis and overexpression of Trpp5 increases intracellular free calcium concentration in MDCK cells. However, the roles of this protein in cellular processes are largely unknown. Here, we demonstrated that Trpp5 resided in both cytoplasm and cell membrane of HEK293 cells. We found that overexpression of Trpp5 slightly increased the calcium current amplitude of HEK293 cells and shifted the reversal potential to a more negative value. Meanwhile, overexpression of Trpp5 suppressed proliferation of Hela cells via inhibiting DNA replication and induced apoptosis of Hela cells with morphological changes and accumulation of fragmented DNA. Collectively, these findings suggest that Trpp5 might involve calcium homeostasis contributing to cell proliferation and apoptosis.

TRP channels and ASICs mediate mechanical hyperalgesia in models of inflammatory muscle pain and delayed onset muscle soreness

The roles of ion channels in sensory neurons were examined in experimental models of muscle pain in the rat. Rats were injected with 50 μl of 4% carrageenan or subjected to an eccentric exercise (ECC) of the gastrocnemius muscle (GM). The Randall–Selitto and von Frey tests were performed on the calves to evaluate mechanical hyperalgesia of the muscle. The changes in expression of four genes and proteins of ion channels in dorsal root ganglia were examined using quantitative PCR and immunohistochemistry, respectively. Effects of antagonists to transient receptor potential (TRP) channels and acid sensing ion channels (ASICs) on the mechanical hyperalgesia induced by carrageenan injection or ECC were evaluated. The mechanical hyperalgesia was observed 6–24 h after carrageenan injection and 1–3 days after ECC in the Randall–Selitto test. Infiltrations of the inflammatory cells in the GM were seen in carrageenan-injected animals but not in those subjected to ECC. Expressions of genes and proteins in sensory neurons showed no changes. Intramuscular injection of antagonists to TRPV1 showed an almost complete suppressive effect on ECC-induced muscle hyperalgesia but not a carrageenan-induced one. Antagonists to TRP channels and ASICs showed suppressive effects for both carrageenan- and ECC-induced muscle hyperalgesia. The carrageenan injection and ECC models are useful models of acute inflammatory pain and delayed onset muscle soreness (DOMS), respectively, and the time course and underlying etiology might be different. TRP channels and ASICs are closely related to the development of muscle mechanical hyperalgesia, and TRPV1 is involved in ECC-induced DOMS.

Overexpression of TRPC3 increases apoptosis in response to ischemia/reperfusion but not TNF‐α in adult mouse cardiomyocytes

Capacitative calcium entry (CCE), attributed to transient receptor potential (TRP) proteins, has been shown to be important in regulating cardiomyocyte hypertrophy. Increased cytosolic Ca2+ also plays a critical role in mediating cell death in response to ischemia/reperfusion (I/R) injury. Therefore we tested the hypothesis that overexpression of TRPC3 in cardiomyocytes will increase sensitivity to I/R injury. Adult cardiomyocytes isolated from wild-type (WT) mice and from mice over expressing TRPC3 in the heart (TG) were subject to 90 min ischemia and 3hr reperfusion. After I/R viability was 51±1% in WT and 42±5% in TG (p<0.05). There was no significant difference in necrosis between WT and TG groups; however, apoptosis assessed by annexin-V was markedly increased in TG (32±1%) compared to WT (21±3%; p<0.05). Treatment with SKF96365 (0.5μM), an inhibitor of CCE, significantly improved cellular viability (54±4%) and decreased apoptosis (15±4%) in TG group, whereas the L-type Ca2+ channel inhibitor verapamil (10μM) had no effect. Calpain-mediated cleavage of α-fodrin was increased ~3-fold in the TG group following I/R compared to WT (p<0.05). In contrast to I/R, there was no difference between WT and TG groups in apoptosis induced by TNF-α (10ng/ml for 2 and 18 hrs). These results suggest that Ca2+ entry via TRP may play a role in cardiomyocyte apoptosis following I/R due at least in part by increased calpain activation. (Supported by NIH grants HL076165, HL079364, HL-077100).

Calcineurin‐dependent cardiomyopathy is activated by TRPC in the adult mouse heart

The manner in which Ca2+-sensitive signaling proteins are activated in contracting cardiomyocytes is an intriguing theoretical problem given that the cytoplasm is continually bathed with systolic Ca2+ concentrations that should maximally activate most Ca2+-sensitive signaling kinases and phosphatases. Store-operated Ca2+ entry, partially attributed to transient receptor potential (TRP) proteins, can mediate activation of the Ca2+-sensitive phosphatase calcineurin in nonexcitable cells. Here we investigated the gain-of-function phenotype associated with TRPC3 expression in the mouse heart using transgenesis to examine the potential role of store-operated Ca2+ entry in regulating cardiac calcineurin activation and ensuing hypertrophy/myopathy. Adult myocytes isolated from TRPC3 transgenic mice showed abundant store-operated Ca2+ entry that was inhibited with SKF96365 but not verapamil or KB-R7943. Associated with this induction in store-operated Ca2+ entry, TRPC3 transgenic mice showed increased calcineurin-nuclear factor of activated T cells (NFAT) activation in vivo, cardiomyopathy, and increased hypertrophy after neuroendocrine agonist or pressure overload stimulation. The cardiomyopathic phenotype and increased hypertrophy after pressure overload stimulation were blocked by targeted disruption of the calcineurin Abeta gene. Thus, enhanced store-operated Ca2+ entry in the heart can regulate calcineurin-NFAT signaling in vivo, which could secondarily impact the hypertrophic response and cardiomyopathy.

Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices

During a period of acute ischemia in vivo or oxygen–glucose deprivation (OGD) in vitro, CA1 neurons depolarize, swell and become overloaded with calcium. Our aim was to test the hypothesis that the initial responses to OGD are at least partly due to transient receptor potential (TRP) channel activation. As some TRP channels are temperature-sensitive, we also compared the effects of pharmacological blockade of the channels with the effects of reducing temperature. Acute hippocampal slices (350 μm) obtained from Wistar rats were submerged in ACSF at 36 °C. CA1 neurons were monitored electrophysiologically using extracellular, intracellular or whole-cell patch-clamp recordings. Cell swelling was assessed by recording changes in relative tissue resistance, and changes in intracellular calcium were measured after loading neurons with fura-2 dextran. Blockers of TRP channels (ruthenium red, La 3+, Gd 3+, 2-APB) or lowering temperature by 3 °C reduced responses to OGD. This included: (a) an increased delay to negative shifts of extracellular DC potential; (b) reduction in rate of the initial slow membrane depolarization, slower development of OGD-induced increase in cell input resistance and slower development of whole-cell inward current; (c) reduced tissue swelling; and (d) a smaller rise in intracellular calcium. Mild hypothermia (33 °C) and La 3+ or Gd 3+ (100 μM) showed an occlusion effect when delay to extracellular DC shifts was measured. Expression of TRPM2/TRPM7 (oxidative stress-sensitive) and TRPV3/TRPV4 (temperature-sensitive) channels was demonstrated in the CA1 subfield with RT-PCR. These results indicate that TRP or TRP-like channels are activated by cellular stress and contribute to ischemia-induced membrane depolarization, intracellular calcium accumulation and cell swelling. We also hypothesize that closing of some TRP channels (TRPV3 and/or TRPV4) by lowering temperature may be partly responsible for the neuroprotective effect of hypothermia.

Alpha1-adrenergic receptors activate Ca(2+)-permeable cationic channels in prostate cancer epithelial cells.

The prostate gland is a rich source of alpha1-adrenergic receptors (alpha1-ARs). alpha1-AR antagonists are commonly used in the treatment of benign prostatic hyperplasia symptoms, due to their action on smooth muscle cells. However, virtually nothing is known about the role of alpha1-ARs in epithelial cells. Here, by using two human prostate cancer epithelial (hPCE) cell models - primary cells from resection specimens (primary hPCE cells) and an LNCaP (lymph node carcinoma of the prostate) cell line - we identify an alpha1A subtype of adrenergic receptor (alpha1A-AR) and show its functional coupling to plasmalemmal cationic channels via direct diacylglycerol (DAG) gating. In both cell types, agonist-mediated stimulation of alpha1A-ARs and DAG analogues activated similar cationic membrane currents and Ca(2+) influx. These currents were sensitive to the alpha1A-AR antagonists, prazosin and WB4101, and to transient receptor potential (TRP) channel blockers, 2-aminophenyl borate and SK&F 96365. Chronic activation of alpha1A-ARs enhanced LNCaP cell proliferation, which could be antagonized by alpha1A-AR and TRP inhibitors. Collectively, our results suggest that alpha1-ARs play a role in promoting hPCE cell proliferation via TRP channels.

Internal Ca(2+) release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue.

Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. In mammalian cells, the release of Ca2+ from intracellular compartments is required for signaling and involves the regulated opening of ryanodine and inositol-1,4,5-trisphosphate (IP3) receptors. However, in budding yeast, no signaling pathway has been shown to involve Ca2+ release from internal stores, and no homologues of ryanodine or IP3 receptors exist in the genome. Here we show that hyperosmotic shock provokes a transient increase in cytosolic Ca2+ in vivo. Vacuolar Ca2+, which is the major intracellular Ca2+ store in yeast, is required for this response, whereas extracellular Ca2+ is not. We aimed to identify the channel responsible for this regulated vacuolar Ca2+ release. Here we report that Yvc1p, a vacuolar membrane protein with homology to transient receptor potential (TRP) channels, mediates the hyperosmolarity induced Ca2+ release. After this release, low cytosolic Ca2+ is restored and vacuolar Ca2+ is replenished through the activity of Vcx1p, a Ca2+/H+ exchanger. These studies reveal a novel mechanism of internal Ca2+ release and establish a new function for TRP channels.