Novel identification and characterisation of Transient receptor potential melastatin 3 ion channels on Natural Killer cells and B lymphocytes: effects on cell signalling in Chronic fatigue syndrome/Myalgic encephalomyelitis patients.

BACKGROUND: Transient receptor potential melastatin 3 (TRPM3) cation channels are ubiquitously expressed by multiple cells and have an important regulatory role in calcium-dependent cell signalling to help maintain cellular homeostasis. TRPM3 protein expression has yet to be determined on Natural Killer (NK) cells and B lymphocytes. Multiple single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) patients and have been proposed to correlate with illness presentation. The object of the study was to assess TRPM3 surface expression on NK and B lymphocytes from healthy controls, followed by a comparative investigation examining TRPM3 surface expression, and cytoplasmic and mitochondrial calcium influx in CD19(+) B cells, CD56(bright) and CD56(dim) cell populations from CFS/ME patients. RESULTS: TRPM3 cell surface expression was identified for NK and B lymphocytes in healthy controls (CD56(bright) TRPM3 35.72 % ± 7.37; CD56(dim) 5.74 % ± 2.00; B lymphocytes 2.05 % ± 0.19, respectively). There was a significant reduction of TRPM3 surface expression on CD19(+) B cells (1.56 ± 0.191) and CD56(bright) NK cells (17.37 % ± 5.34) in CFS/ME compared with healthy controls. Anti-CD21 and anti-IgM conjugated biotin was cross-linked with streptavidin,and subsequently treatment with thapsigargin. This showed a significant reduction in cytoplasmic calcium ion concentration in CD19(+) B lymphocytes. CD56(bright) NK cells also had a significant decrease in cytoplasmic calcium in the presence of 2-APB and thapsigargin in CFS/ME patients. CONCLUSIONS: The results from this preliminary investigation identify, for the first time, TRPM3 surface expression on both NK and B lymphocytes in healthy controls. We also report for the first time, significant reduction in TRPM3 cell surface expression in NK and B lymphocytes, as well as decreased intracellular calcium within specific conditions in CFS/ME patients. This warrants further examination of these pathways to elucidate whether TRPM3 and impaired calcium mobilisation has a role in CFS/ME.

Novel identification and characterisation of Transient receptor potential melastatin 3 ion channels on Natural Killer cells and B lymphocytes: effects on cell signalling in Chronic fatigue syndrome/Myalgic encephalomyelitis patients

BACKGROUND: Transient receptor potential melastatin 3 (TRPM3) cation channels are ubiquitously expressed by multiple cells and have an important regulatory role in calcium-dependent cell signalling to help maintain cellular homeostasis. TRPM3 protein expression has yet to be determined on Natural Killer (NK) cells and B lymphocytes. Multiple single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) patients and have been proposed to correlate with illness presentation. The object of the study was to assess TRPM3 surface expression on NK and B lymphocytes from healthy controls, followed by a comparative investigation examining TRPM3 surface expression, and cytoplasmic and mitochondrial calcium influx in CD19+ B cells, CD56bnght and CD56dim cell populations from CFS/ME patients. RESULTS: TRPM3 cell surface expression was identified for NK and B lymphocytes in healthy controls (CD56bright TRPM3 35.72 % ± 7.37; CD56dim 5.74 % ± 2.00; B lymphocytes 2.05 % ± 0.19, respectively). There was a significant reduction of TRPM3 surface expression on CD19+ B cells (1.56 ± 0.191) and CD56bright NK cells (17.37 % ± 5.34) in CFS/ME compared with healthy controls. Anti-CD21 and anti-IgM conjugated biotin was cross-linked with streptavidin,and subsequently treatment with thapsigargin. This showed a significant reduction in cytoplasmic calcium ion concentration in CD19+ B lymphocytes. CD56bright NK cells also had a significant decrease in cytoplasmic calcium in the presence of 2-APB and thapsigargin in CFS/ME patients. CONCLUSIONS: The results from this preliminary investigation identify, for the first time, TRPM3 surface expression on both NK and B lymphocytes in healthy controls. We also report for the first time, significant reduction in TRPM3 cell surface expression in NK and B lymphocytes, as well as decreased intracellular calcium within specific conditions in CFS/ME patients. This warrants further examination of these pathways to elucidate whether TRPM3 and impaired calcium mobilisation has a role in CFS/ME.

The identification of a volume-regulated anion channel: an amazing Odyssey.

The volume-regulated anion channel (VRAC) plays a pivotal role in cell volume regulation in essentially all cell types studied. Additionally, VRAC appears to contribute importantly to a wide range of other cellular functions and pathological events, including cell motility, cell proliferation, apoptosis and excitotoxic glutamate release in stroke. Although biophysically, pharmacologically and functionally thoroughly described, VRAC has until very recently remained a genetic orphan. The search for the molecular identity of VRAC has been long and has yielded multiple potential candidates, all of which eventually turned out to have properties not fully compatible with those of VRAC. Recently, two groups have independently identified the protein leucine-rich repeats containing 8A (LRRC8A), belonging to family of proteins (LRRC8A-E) distantly related to pannexins, as the likely pore-forming subunit of VRAC. In this brief review, we summarize the history of the discovery of VRAC, outline its basic biophysical and pharmacological properties, link these to several cellular functions in which VRAC appears to play important roles, and sketch the amazing search for the molecular identity of this channel. Finally, we describe properties of the LRRC8 proteins, highlight some features of the LRRC8A knockout mouse and discuss the impact of the discovery of LRRC8 as VRAC on future research.

On the putative role of transient receptor potential cation channels in asthma.

The mammalian transient receptor potential (TRP) superfamily consists of 28 mammalian TRP cation channels, which can be subdivided into six main subfamilies: the TRPC ('Canonical'), TRPV ('Vanilloid'), TRPM ('Melastatin'), TRPP ('Polycystin'), TRPML ('Mucolipin') and the TRPA ('Ankyrin') groups. Increasing evidence has accumulated during the previous few years that links TRP channels to the cause of several diseases or to critically influence and/or determine their progress. This review focuses on the possible role of TRP channels in the aetiology of asthmatic lung disease.

The calcium-conducting ion channel transient receptor potential canonical 6 is involved in macrophage inflammatory protein-2-induced migration of mouse neutrophils.

AIM: The role of the calcium-conducting ion channel transient receptor potential canonical 6 (TRPC6) in macrophage inflammatory protein-2 (MIP-2) induced migration of mouse neutrophils was investigated. METHODS: Neutrophil granulocytes isolated from murine bone marrow of wild-type (TRPC6+/+) and TRPC6 knockout (TRPC6)/)) mice were tested for the presence of TRPC6 channel expression using quantitative real-time polymerase chain reactions and immunocytochemistry. The effect of different stimuli (e.g. MIP-2, 1-oleoyl-2-acetyl-sn-glycerol, formyl-methionyl-leucyl-phenylalanin) on migration of isolated neutrophils was tested by two-dimensional (2D) migration assays, phalloidin staining and intracellular calcium imaging. RESULTS: We found that neutrophil granulocytes express TRPC6 channels. MIP-2 induced fast cell migration of isolated neutrophils in a 2D celltracking system. Strikingly, MIP-2 was less potent in neutrophils derived from TRPC6)/) mice. These cells showed less phalloidin-coupled fluorescence and the pattern of cytosolic calcium transients was altered. CONCLUSIONS: We describe in this paper for the first time a role for transient receptor potential (TRP) channels in migration of native lymphocytes as a new paradigm for the universal functional role of TRPs. Our data give strong evidence that TRPC6 operates downstream to CXC-type Gq-protein-coupled chemokine receptors upon stimulation with MIP-2 and is crucial for the arrangement of filamentous actin in migrating neutrophils. This is a novel cell function of TRP channel beyond their well-recognized role as universal cell sensors.

TRPC1 channels regulate directionality of migrating cells.

Cell migration depends on the generation of structural asymmetry and on different steps: protrusion and adhesion at the front and traction and detachment at the rear part of the cell. The activity of Ca(2+) channels coordinate these steps by arranging intracellular Ca(2+) signals along the axis of movement. Here, we investigated the role of the putative mechanosensitive canonical transient receptor potential channel 1 (TRPC1) in cell migration. We analyzed its function in transformed renal epithelial (Madin-Darby canine kidney-focus) cells with variation of TRPC1 expression. As shown by time lapse video microscopy, TRPC1 knockdown cells have partially lost their polarity and the ability to persistently migrate into a given direction. This failure is linked to the suppression of a local Ca(2+) gradient at the front of migrating TRPC1 knockdown cells, whereas TRPC1 overexpression leads to steeper Ca(2+) gradients. We propose that the Ca(2+) signaling events regulated by TRPC1 within the lamellipodium determine polarity and directed cell migration.

Role of caveolar compartmentation in endothelium-derived hyperpolarizing factor-mediated relaxation: Ca2+ signals and gap junction function are regulated by caveolin in endothelial cells.

BACKGROUND: In endothelial cells, caveolin-1, the structural protein of caveolae, acts as a scaffolding protein to cluster lipids and signaling molecules within caveolae and, in some instances, regulates the activity of proteins targeted to caveolae. Specifically, different putative mediators of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation are located in caveolae and/or regulated by the structural protein caveolin-1, such as potassium channels, calcium regulatory proteins, and connexin 43, a molecular component of gap junctions. METHODS AND RESULTS: Comparing relaxation in vessels from caveolin-1 knockout mice and their wild-type littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from caveolin-1 knockout mice. The absence of caveolin-1 is associated with an impairment of calcium homeostasis in endothelial cells, notably, a decreased activity of Ca2+-permeable TRPV4 cation channels that participate in nitric oxide- and EDHF-mediated relaxation. Moreover, morphological characterization of caveolin-1 knockout and wild-type arteries showed fewer gap junctions in vessels from knockout animals associated with a lower expression of connexins 37, 40, and 43 and altered myoendothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolar compartment of the plasma membrane. CONCLUSIONS: We demonstrated that expression of caveolin-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway.

Modulation of synaptic plasticity and Tau phosphorylation by wild-type and mutant presenilin1.

The function of presenilin1 (PS1) in intra-membrane proteolysis is undisputed, as is its role in neurodegeneration in FAD, in contrast to its exact function in normal conditions. In this study, we analyzed synaptic plasticity and its underlying mechanisms biochemically in brain of mice with a neuron-specific deficiency in PS1 (PS1(n-/-)) and compared them to mice that expressed human mutant PS1[A246E] or wild-type PS1. PS1(n-/-) mice displayed a subtle impairment in Schaffer collateral hippocampal long-term potentiation (LTP) as opposed to normal LTP in wild-type PS1 mice, and a facilitated LTP in mutant PS1[A246E] mice. This finding correlated with, respectively, increased and reduced NMDA receptor responses in PS1[A246E] mice and PS1(n-/-) mice in hippocampal slices. Postsynaptically, levels of NR1/NR2B NMDA-receptor subunits and activated alpha-CaMKII were reduced in PS1(n-/-) mice, while increased in PS1[A246E] mice. In addition, PS1(n-/-) mice, displayed reduced paired pulse facilitation, increased synaptic fatigue and lower number of total and docked synaptic vesicles, implying a presynaptic function for wild-type presenilin1, unaffected by the mutation in PS1[A246E] mice. In contrast to the deficiency in PS1, mutant PS1 activated GSK-3beta by decreasing phosphorylation on Ser-9, which correlated with increased phosphorylation of protein tau at Ser-396-Ser-404 (PHF1/AD2 epitope). The synaptic functions of PS1, exerted on presynaptic vesicles and on postsynaptic NMDA-receptor activity, were concluded to be independent of alterations in GSK-3beta activity and phosphorylation of protein tau.

Effect of prolonged c-kit receptor inhibition by imatinib mesylate on the uterine contractility of pregnant rabbits.

BACKGROUND: The c-kit receptor expressed by interstitial cells in the gastrointestinal tract is crucial to their pacemaking function. The function of similar c-kit-expressing myometrial cells is unknown. METHODS: Imatinib mesylate, a specific c-kit receptor antagonist, was administered to pregnant New Zealand white rabbits (term = 31 days, n = 35) from day 27 gestation by intramuscular injection twice daily at high (50 microg/kg) or medium (10 microg/kg) dose and compared with a control group injected with vehicle only. In a second phase, two further groups received imatinib at medium or low (1 mug/kg) dose for a longer duration starting from day 18 until delivery. Three does from the latter groups as well as controls underwent myometrial biopsy under general anesthesia after spontaneous vaginal birth. Contractility was recorded by isometric tensiometry. The outcome measures were delay of parturition and in vitro contractility characteristics. RESULTS: High-dose imatinib induced early delivery when compared with the control group (28.6 vs. 30.7 days, p < 0.001). The other groups delivered at term. No effect on in vitro contractility was apparent in any of the groups. CONCLUSIONS: c-kit receptor inhibition in pregnant rabbits does not delay significantly the length of gestation or change myometrial contractility in vitro.

Insights into TRPM4 function, regulation and physiological role.

In the current review we will summarise data from the recent literature describing molecular and functional properties of TRPM4. Together with TRPM5, these channels are up till now the only molecular candidates for a class of non-selective, Ca(2+)-impermeable cation channels which are activated by elevated Ca2+ levels in the cytosol. Apart from intracellular Ca2+, TRPM4 activation is also dependent on membrane potential. Additionally, channel activity is modulated by ATP, phosphatidylinositol bisphosphate (PiP2), protein kinase C (PKC) phosphorylation and heat. The molecular determinants for channel activation, permeation and modulation are increasingly being clarified, and will be discussed here in detail. The physiological role of Ca(2+)-activated non-selective cation channels is unclear, especially in the absence of gene-specific knock-out mice, but evidence indicates a role as a regulator of membrane potential, and thus the driving force for Ca2+ entry from the extracellular medium.

TRPM8.

Originally cloned as a prostate-specific protein, TRPM8 is now best known as a cold- and menthol-activated channel implicated in thermosensation. In this chapter we provide a brief review of current knowledge concerning the biophysical properties, gating mechanisms, pharmacology and (patho)physiology of this TRP channel.

Regulation of TRP channels: a voltage-lipid connection.

TRP (transient receptor potential) channels respond to a plethora of stimuli in a fine-tuned manner. We show here that both membrane potential and the level of PI (phosphatidylinositol) phosphates are efficient regulators of TRP channel gating. Recent work has shown that this regulation applies to several members of the TRPV (TRP vanilloid) subfamily (TRPV1 and TRPV5) and the TRPM (TRP melastatin) subfamily (TRPM4/TRPM5/TRPM7/TRPM8), whereas regulation of members of the TRPC subfamily is still disputed. The mechanism whereby PIP(2) (PI 4,5-bisphosphate) acts on TRPM4, a Ca(2+)- and voltage-activated channel, is shown in detail in this paper: (i) PIP(2) may bind directly to the channel, (ii) PIP(2) induces sensitization to activation by Ca(2+), and (iii) PIP(2) shifts the voltage dependence towards negative and physiologically more meaningful potentials. A PIP(2)-binding pocket seems to comprise a part of the TRP domain and especially pleckstrin homology domains in the C-terminus.

Influence of temperature on taste perception.

Daily experience tells us that temperature has a strong influence on how we taste. Despite the longstanding interest of many specialists in this aspect of taste, we are only starting to understand the molecular mechanisms underlying the temperature dependence of different taste modalities. Recent research has led to the identification of some strong thermosensitive molecules in the taste transduction pathway. The cold activation of the epithelial Na(+) channel and the heat activation of the taste variant of the vanilloid receptor (TRPV1t) may underlie the temperature dependence of salt responses. Heat activation of the transient receptor potential channel TRPM5 explains the enhancement of sweet taste perception by warm temperatures. Current development of methods to study taste cell physiology will help to determine the contribution of other temperature-sensitive events in the taste transduction pathways. Vice versa, the analysis of the thermodynamic properties of these events may assist to unveil the nature of several taste processes.

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration.

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.

The effect of imatinib mesylate on the contractility of isolated rabbit myometrial strips.

BACKGROUND: C-kit receptor expressing interstitial cells generate and coordinate the electrical signals that control peristalsis in the gut. However, the function of interstitial cells in the myometrium is not known. METHODS: (1) Sections of rabbit myometrium were subjected to immunohistochemical staining for the c-kit receptor. (2) Spontaneously contracting myometrial strips from New Zealand White rabbits near term were mounted in an organ bath and attached to a tension-recording device. The effect of increased concentrations of the c-kit receptor antagonist imatinib mesylate on these contractions was observed. The main outcome measures were the change in frequency, amplitude and duration of contraction. RESULTS: (1) Multipolar cells expressing c-kit were identified in the fibromuscular septum confirming the presence of interstitial cells in rabbit myometrium. (2) Imatinib decreased the amplitude of contractions by approximately 20% at 100 microM. No effect was seen at lower concentrations. No effect of imatinib on frequency or duration of contractions was observed at any of the concentrations studied. CONCLUSIONS: In isolated rabbit myometrium, acute inhibition of the c-kit receptor by imatinib mesylate affects only the amplitude of spontaneous contractions at concentrations, the equivalent of x10-100 the normal therapeutic concentration.

Structure-function relationship of the TRP channel superfamily.

Transient receptor potential (TRP) channels are involved in the perception of a wide range of physical and chemical stimuli, including temperature and osmolarity changes, light, pain, touch, taste and pheromones, and in the initiation of cellular responses thereupon. Since the last decade, rapid progress has been made in the identification and characterization of new members of the TRP superfamily. They constitute a large superfamily of cation channels that are expressed in almost all cell types in both invertebrates and vertebrates. This review summarizes and discusses the current knowledge on the TRP protein structure and its impact on the regulation of the channel function.

Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium.

TRPV4 is a broadly expressed Ca2+-permeable cation channel in the vanilloid subfamily of transient receptor potential channels. TRPV4 gates in response to a large variety of stimuli, including cell swelling, warm temperatures, the synthetic phorbol ester 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), and the endogenous lipid arachidonic acid (AA). Activation by cell swelling and AA requires cytochrome P450 (CYP) epoxygenase activity to convert AA to epoxyeicosatrienoic acids (EETs) such as 5,6-EET, 8,9-EET, which both act as direct TRPV4 agonists. To evaluate the role of TRPV4 and its modulation by the CYP pathway in vascular endothelial cells, we performed Ca2+ imaging and patch-clamp measurements on mouse aortic endothelial cells (MAECs) isolated from wild-type and TRPV4(-/-) mice. All TRPV4-activating stimuli induced robust Ca2+ responses in wild-type MAECs but not in MAECs isolated from TRPV4(-/-) mice. Upregulation of CYP2C expression by preincubation with nifedipine enhanced the responses to AA and cell swelling in wild-type MAECs, whereas responses to other stimuli remained unaffected. Conversely, inhibition of CYP2C9 activity with sulfaphenazole abolished the responses to AA and hypotonic solution (HTS). Moreover, suppression of EET hydrolysis using 1-adamantyl-3-cyclo-hexylurea or indomethacin, inhibitors of soluble epoxide hydrolases (sEHs), and cyclooxygenases, respectively, enhanced the TRPV4-dependent responses to AA, HTS, and EETs but not those to 4alpha-PDD or heat. Together, our data establish that CYP-derived EETs modulate the activity of TRPV4 channels in endothelial cells and shows the unraveling of novel modulatory pathways via CYP2C modulation and sEH inhibition.

Cell volume response to hyposmotic shock and elevated cAMP in bovine trabecular meshwork cells.

PURPOSE: Hyposmolar perfusion of intact trabecular meshwork (TM) induces a decrease in its hydraulic conductivity (Lp). However, exposure to agents that elevate intracellular cAMP in TM cells increases Lp. Since volume of TM cells could directly influence porosity of the TM and hence Lp, this study has investigated changes in volume in response to acute hyposmotic shock (i.e. regulatory volume decrease or RVD) and elevated cAMP in cultured TM cells. METHODS: Bovine trabecular meshwork cells (BTMC), grown on glass coverslips and loaded with the fluorescent dye MQAE, were used to measure rapid changes in cell volume using the principle of dynamic fluorescence quenching. Activation of volume-regulated anion channels (VRAC) was assessed by measuring volume-sensitive Cl(-) currents (I(Cl,swell)) in the whole cell configuration of the patch clamp technique and by determining the swelling-induced enhancement in I(-) permeability using the halide-sensitivity of MQAE. Expressions of ClC (chloride channels of the ClC gene family), P-glycoprotein (Pgp), and cystic fibrosis transmembrane regulator (CFTR) Cl(-) channels were examined by RT-PCR. Elevation of cAMP in response to forskolin was confirmed by determining the phosphorylation of cAMP response element-binding protein and activating transcription factor-1 (CREB, ATF-1), which form the downstream targets of protein kinase A. RESULTS: As a response to hyposmotic shock, there was an acute increase in cell volume but there was no robust RVD. Patch clamp experiments showed activation of a characteristic Cl(-) current in response to cell swelling. This Cl(-) current was inhibited by NPPB (100microM) and fluoxetine (50microM), both of which are known blockers of VRAC. Experiments, which used the halide-sensitivity of MQAE, also indicated a 9-fold increase in I(-) influx upon cell swelling (8.9+/-4.6; n=9), consistent with activation of a VRAC-like Cl(-) current. To examine whether RVD is limited by K(+) conductance, the swollen cells were exposed to gramicidin, which is known to induce cation channel activity. Such a maneuver led to secondary swelling with [Na(+)](o)=140mM but a rapid shrinkage [Na(+)](o)=8mM indicating that the RVD is limited by cationic conductance necessary for K(+) efflux. Exposure to forskolin, which resulted in CREB and ATF-1 phosphorylation, caused a reversible decrease in cell volume (14.5+/-5%; n=20) under isosmotic and hyposmotic conditions. RT-PCR analysis confirmed expression of ClC-2, ClC-5, and Pgp Cl(-) channels in bovine TM cells. However, ClC-3 and CFTR were not expressed. CONCLUSIONS: TM cells respond to acute hyposmotic shock in an osmometric manner, but their RVD is limited by K(+) conductance. The lack of CFTR expression and decrease in cell volume in response to forskolin concomitant with hyposmolarity suggest that elevated cAMP activates a K(+) conductance. Thus, the altered resistance to aqueous outflow in response to hyposmotic perfusion of the TM and elevated cAMP may be attributed to persistent cell swelling and cell shrinkage, respectively.

Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4.

TRPV4 is a Ca(2+)- and Mg(2+)-permeable cation channel within the vanilloid receptor subgroup of the transient receptor potential (TRP) family, and it has been implicated in Ca(2+)-dependent signal transduction in several tissues, including brain and vascular endothelium. TRPV4-activating stimuli include osmotic cell swelling, heat, phorbol ester compounds, and 5',6'-epoxyeicosatrienoic acid, a cytochrome p450 epoxygenase metabolite of arachidonic acid (AA). It is presently unknown how these distinct activators converge on opening of the channel. Here, we demonstrate that blockers of phospholipase A(2) (PLA(2)) and cytochrome p450 epoxygenase inhibit activation of TRPV4 by osmotic cell swelling but not by heat and 4alpha-phorbol 12,13-didecanoate. Mutating a tyrosine residue (Tyr-555) in the N-terminal part of the third transmembrane domain to an alanine strongly impairs activation of TRPV4 by 4alpha-phorbol 12,13-didecanoate and heat but has no effect on activation by cell swelling or AA. We conclude that TRPV4-activating stimuli promote channel opening by means of distinct pathways. Cell swelling activates TRPV4 by means of the PLA(2)-dependent formation of AA, and its subsequent metabolization to 5',6'-epoxyeicosatrienoic acid by means of a cytochrome p450 epoxygenase-dependent pathway. Phorbol esters and heat operate by means of a distinct, PLA(2)- and cytochrome p450 epoxygenase-independent pathway, which critically depends on an aromatic residue at the N terminus of the third transmembrane domain.

TRPs make sense.

Drosophila flies with the trp mutation exhibit impaired vision due to the lack of a specific Ca2+ influx pathway in the photoreceptors. The identification of the trp gene product as a Ca2+-permeable ion channel and the search for TRP homologues in flies, worms and mammals has opened the way to the discovery of a whole superfamily of cation channels, baptized TRP channels. In contrast to voltage-gated K+, Na+, or Ca2+ channels, with whom they share their transmembrane architecture, TRP channels are not activated by voltage but by a variety of signals including intra- and extracellular ligands, Ca2+-store depletion and mechanical or thermal stress. Due to the promiscuity of these gating mechanisms, TRP channels are privileged candidates as primary sensing molecules for the recognition and integration of physical and chemical signals from the environment. In this review we discuss recent evidence that implicates members of the TRP superfamily in sensory signal transduction.