Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.     

Receptor-operated Ca2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line

Prostate smooth muscle cells predominantly express alpha1-adrenoceptors (alpha1-AR). alpha1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca(2+) entry pathways associated with the activation of alpha1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous alpha1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca(2+) influx which depended on the extracellular Ca(2+) and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca(2+) influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca(2+)-permeable channels mediated both alpha-agonist- and OAG-evoked Ca(2+) influx. Following the sarcoplasmic reticulum (SR) Ca(2+) store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca(2+) influx. However, OAG failed to enhance Ca(2+) influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-induced Ca(2+) influx required TRPC3 and TRPC6, whereas the Tg-activated ("capacitative") Ca(2+) entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor- and store-operated Ca(2+) entry pathways.     

Mast-cell degranulation induced by physical stimuli involves the activation of transient-receptor-potential channel TRPV2

A characteristic of mast cells is the degranulation in response to various stimuli. Here we have investigated the effects of various physical stimuli in the human mast-cell line HMC-1. We have shown that HMC-1 express the transient receptor potential channels TRPV1, TRPV2 and TRPV4. In the whole-cell patch-clamp configuration, increasing mechanical stress applied to the mast cell by hydrostatic pressure (-30 to -90 cm H(2)O applied via the patch pipette) induced a current that could be inhibited by 10 microM of ruthenium red. This current was also inhibited by 20 microM SKF96365, an inhibitor that is among TRPV channels specific for the TRPV2. A characteristic of TRPV2 is its activation by high noxious temperature; temperatures exceeding 50 °C induced a similar ruthenium-red-sensitive current. As another physical stimulus, we applied laser light of 640 nm. Here we have shown for the first time that the application of light (at 48 mW for 20 min) induced an SKF96365-sensitive current. All three physical stimuli that led to activation of SKF96365-sensitive current also induced pronounced degranulation in the mast cells, which could be blocked by ruthenium red or SKF96365. The results suggest that TRPV2 is activated by the three different types of physical stimuli. Activation of TRPV2 allows Ca(2+) ions to enter the cell, which in turn will induce degranulation. We, therefore, suggest that TRPV2 plays a key role in mast-cell degranulation in response to mechanical, heat and red laser-light stimulation.     

TRPC通道对PC12细胞缺氧缺糖再灌注后氧化损伤的保护作用

瞬时受体电位C通道(transient receptor potential canonical,TRPC)属于瞬时受体电位（TRP）离子通道家族成员之一,与Ca2+ 调控及氧化应激关系密切．然而,TRPC通道在缺血缺氧性脑损伤中的作用仍有争议．本实验采用MTT法、台盼蓝排斥实验、LDH漏出实验联合Annexin/PI染色流式分析等显示,当采用通道阻断剂-SKF96365特异阻断TRPC通道时,PC12细胞对缺氧缺糖再灌注（oxygen-glucose deprivation／reperfusion,OGD-R）引起的损伤变得更敏感,细胞凋亡增加．尽管同时采用SKF96365、NMDA受体、AMPA受体和L-型钙通道阻断剂可引起细胞损伤和死亡减弱,但仍呈现明显的SKF96365浓度依懒性,提示TRPC通道参与细胞对缺氧缺糖再灌注耐受的调节,具有保护作用．钙指示剂Fluo-3AM荧光标记结合激光共聚焦显微镜分析显示,SKF96365可明显降低缺氧缺糖再灌注后细胞内的Ca2+浓度．总之,实验结果提示,TRPC通道对缺氧缺糖再灌注引起的细胞损伤和凋亡具有保护作用,其机制可能涉及TRPC通道对细胞内钙浓度的调节,详尽机制有待进一步研究．     

Nitric oxide activates TRP channels by cysteine S-nitrosylation

Transient receptor potential (TRP) proteins form plasma-membrane cation channels that act as sensors for diverse cellular stimuli. Here, we report a novel activation mechanism mediated by cysteine S-nitrosylation in TRP channels. Recombinant TRPC1, TRPC4, TRPC5, TRPV1, TRPV3 and TRPV4 of the TRPC and TRPV families, which are commonly classified as receptor-activated channels and thermosensor channels, induce entry of Ca(2+) into cells in response to nitric oxide (NO). Labeling and functional assays using cysteine mutants, together with membrane sidedness in activating reactive disulfides, show that cytoplasmically accessible Cys553 and nearby Cys558 are nitrosylation sites mediating NO sensitivity in TRPC5. The responsive TRP proteins have conserved cysteines on the same N-terminal side of the pore region. Notably, nitrosylation of native TRPC5 upon G protein-coupled ATP receptor stimulation elicits entry of Ca(2+) into endothelial cells. These findings reveal the structural motif for the NO-sensitive activation gate in TRP channels and indicate that NO sensors are a new functional category of cellular receptors extending over different TRP families.     

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca²⁺ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca²⁺ imaging, we found that the depletion of ER/SR Ca²⁺ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca²⁺ ([Ca²⁺]_i), followed by sustained increase depending on extracellular Ca²⁺. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na⁺/Ca²⁺ exchanger inhibitors, inhibited [Ca²⁺]_i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl₃) or by an increased extracellular Ca²⁺([Ca²⁺]_o) increased the concentration of intracelluar Ca²⁺, whereas, the sustained elevation of [Ca²⁺]_i was reduced in the presence of SKF96365. Similarly, the duration of [Ca²⁺]_i increase was also shortened in the absence of extracellular Ca²⁺. Western blot analysis showed that GdCl₃ increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl₃. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca²⁺-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.     

TRPC4 can be activated by G-protein-coupled receptors and provides sufficient Ca(2+) to trigger exocytosis in neuroendocrine cells

Transient receptor potential (TRP) channels form a large family of plasma membrane cation channels. Mammalian members of the "short" TRP family (TRP channel (TRPC) 1-7 are Ca(2+)-permeant, non-selective cation channels that are widely expressed in various cell types, including neurons. TRPC activity is linked through unknown mechanisms to G-protein-coupled receptors or receptor tyrosine kinases that activate phospholipase C. To investigate the properties and function of TRPC4 in neuronally derived cells, we transiently expressed mouse TRPC4 and histamine H(1) receptor in mouse adrenal chromaffin cells and PC12 cells. Histamine, but not thapsigargin, stimulated Mn(2+) influx in transfected cells. In the whole-cell patch clamp mode, histamine triggered a transient current in TRPC4-expressing cells. No current was evoked by perfusion with inositol 1,4,5-trisphosphate. When exocytosis was monitored with the capacitance detection technique, the magnitude of the membrane capacitance increase (Delta C(m)) on application of histamine in H(1) receptor/TRPC4-expressing chromaffin cells was comparable with that triggered by a train of depolarizing pulses. Our results indicate that TRPC4 channels behave as receptor, but not store-operated, channels in neuronally derived cells. TRPC4 channels can provide sufficient Ca(2+) influx to trigger a robust secretory response in voltage-clamped neurosecretory cells. Similar mechanisms may modulate exocytosis in other neuronal systems.     

A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2

The coupling between receptor-mediated Ca2+ store release and the activation of "store-operated" Ca2+ entry channels is an important but so far poorly understood mechanism. The transient receptor potential (TRP) superfamily of channels contains several members that may serve the function of store-operated channels (SOCs). The 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2, is a recently described inhibitor of SOC activity in T-lymphocytes. We compared its action on SOC activation in a number of cell types and evaluated its modification of three specific TRP channels, canonical transient receptor potential 3 (TRPC3), TRPC5, and TRPV6, to throw light on any link between SOC and TRP channel function. Using HEK293 cells, DT40 B cells, and A7r5 smooth muscle cells, BTP2 blocked store-operated Ca2+ entry within 10 min with an IC50 of 0.1-0.3 microM. Store-operated Ca2+ entry induced by Ca2+ pump blockade or in response to muscarinic or B cell receptor activation was similarly sensitive to BTP2. Using the T3-65 clonal HEK293 cell line stably expressing TRPC3 channels, TRPC3-mediated Sr2+ entry activated by muscarinic receptors was also blocked by BTP2 with an IC50 of <0.3 microM. Importantly, direct activation of TRPC3 channels by diacylglycerol was also blocked by BTP2 (IC50 approximately 0.3 microM). BTP2 still blocked TRPC3 in medium with N-methyl-D-glucamine-chloride replacing Na+, indicating BTP2 did not block divalent cation entry by depolarization induced by activating monovalent cation entry channels. Whereas whole-cell carbachol-induced TRPC3 current was blocked by 3 microM BTP2, single TRPC3 channel recordings revealed persistent short openings suggesting BTP2 reduces the open probability of the channel rather than its pore properties. TRPC5 channels transiently expressed in HEK293 cells were blocked by BTP2 in the same range as TRPC3. However, function of the highly Ca(2+)-selective TRPV6 channel, with many channel properties akin to SOCs, was entirely unaffected by BTP2. The results indicate a strong functional link between the operation of expressed TRPC channels and endogenous SOC activity.     

Rapid vesicular translocation and insertion of TRP channels

The broadly expressed transient receptor potential (TRP) family of ion channels are permeant to cations, most resulting in increased intracellular calcium. However, their regulation and gating is not well understood. Here, we report that growth factor stimulation initiates the rapid translocation of the transient receptor potential ion channel, TRPC5, from vesicles held in reserve just under the plasma membrane. This process, which we term 'rapid vesicular insertion of TRP' (RiVIT), dramatically increases membrane-associated TRPC5 channels and functional TRPC5 current, resulting in tight spatial-temporal control of these Ca(2+)-permeant nonselective channels. Epidermal growth factor (EGF)-induced incorporation of functional TRP channels requires phosphatidylinositide 3-kinase (PI(3)K), the Rho GTPase Rac1 and phosphatidylinositol 4-phosphate 5-kinase (PIP(5)K alpha). The increase in TRPC5 availability affects neurite extension rates in cultured hippocampal neurons, and may be a general mechanism for initiating Ca(2+) influx and cell morphological changes in response to stimuli.     

Insulin increases surface expression of TRPC6 channels in podocytes: role of NADPH oxidases and reactive oxygen species

Insulin receptors in podocytes are essential for normal kidney function. Here, we show that insulin evokes a rapid increase in the surface expression of canonical transient receptor potential-6 channel (TRPC6) channels in cultured podocytes, but caused a decrease in surface expression of TRPC5. These effects are accompanied by a marked increase in outwardly rectifying cationic currents that can be blocked by 10 μM SKF96365 or 100 μM La(3+). Application of oleoyl-2-acetyl-sn-glycerol (OAG) also increased SKF96365- and La(3+)-sensitive cationic currents in podocytes. Importantly, current responses to a combination of OAG and insulin were the same amplitude as those evoked by either agent applied alone. This occlusion effect suggests that OAG and insulin are targeting the same population of channels. In addition, shRNA knockdown of TRPC6 markedly reduced cationic currents stimulated by insulin. The effects of insulin on TRPC6 were mimicked by treating podocytes with H(2)O(2). Insulin treatment rapidly increased the generation of H(2)O(2) in podocytes, and it increased the surface expression of the NADPH oxidase NOX4 in cultured podocytes. Basal and insulin-stimulated surface expression of TRPC6 were reduced by pretreatment with diphenylene iodonium, an inhibitor of NADPH oxidases and other flavin-dependent enzymes, by siRNA knockdown of NOX4, and by manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, a membrane-permeable mimetic of superoxide dismutase and catalase. These observations suggest that insulin increases generation of ROS in part through activation of NADPH oxidases, and that this step contributes to modulation of podocyte TRPC6 channels.     

Endogenous TRPC1, TRPC3, and TRPC7 proteins combine to form native store-operated channels in HEK-293 cells

Endogenously expressed canonical transient receptor potential (TRPC) homologs were investigated for their role in forming store-operated, 1-oleoyl-2-acetyl-sn-glycerol-stimulated, or carbachol (CCh)-stimulated calcium entry pathways in HEK-293 cells. Measurement of thapsigargin-stimulated Ba(2+) entry indicated that the individual suppression of TRPC1, TRPC3, or TRPC7 protein levels, by small interfering RNA (siRNA) techniques, dramatically inhibited (52-68%) store-operated calcium entry (SOCE), whereas suppression of TRPC4 or TRPC6 had no effect. Combined suppression of TRPC1-TRPC3, TRPC1-TRPC7, TRPC3-TRPC7, or TRPC1-TRPC3-TRPC7 gave only slightly more inhibition of SOCE (74-78%) than seen with suppression of TRPC1 alone (68%), suggesting that these three TRPC homologs work in tandem to mediate a large component of SOCE. Evidence from co-immunoprecipitation experiments indicates that a TRPC1-TRPC3-TRPC7 complex, predicted from siRNA results, does exist. The suppression of either TRPC3 or TRPC7, but not TRPC1, induced a high Ba(2+) leak flux that was inhibited by 2-APB and SKF96365, suggesting that the influx is via leaky store-operated channels. The high Ba(2+) leak flux is eliminated by co-suppression of TRPC1-TRPC3 or TRPC1-TRPC7. For 1-oleoyl-2-acetyl-sn-glycerol-stimulated cells, siRNA data indicate that TRPC1 plays no role in mediating Ba(2+) entry, which appears to be mediated by the participation of TRPC3, TRPC4, TRPC6, and TRPC7. CCh-stimulated Ba(2+) entry, on the other hand, could be inhibited by suppression of any of the five endogenously expressed TRPC homologs, with the degree of inhibition being consistent with CCh stimulation of both store-operated and receptor-operated channels. In summary, endogenous TRPC1, TRPC3, and TRPC7 participate in forming heteromeric store-operated channels, whereas TRPC3 and TRPC7 can also participate in forming heteromeric receptor-operated channels.     

Anandamide acts as an intracellular messenger amplifying Ca2+ influx via TRPV1 channels

The endocannabinoid anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca2+ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca(2+)-ATPase inhibitor thapsigargin leads to formation of anandamide, and subsequent TRPV1-dependent Ca2+ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1-mediated Ca2+ entry. In DRG neurons, this mechanism was found to lead to TRPV1-mediated currents that were enhanced by selective blockade of anandamide cellular efflux. Thus, endogenous anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5-triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release.     

The HECT ubiquitin ligase AIP4 regulates the cell surface expression of select TRP channels

TRPV4 is a widely expressed member of the transient receptor potential (TRP) family that facilitates Ca(2+) entry into nonexcitable cells. TRPV4 is activated by several stimuli, but it is largely unknown how the activity of this channel is terminated. Here, we show that ubiquitination represents an important mechanism to control the presence of TRPV4 at the plasma membrane. Ubiquitination of TRPV4 is dramatically increased by the HECT (homologous to E6-AP carboxyl terminus)-family ubiquitin ligase AIP4 without inducing degradation of this channel. Instead, AIP4 promotes the endocytosis of TRPV4 and decreases its amount at the plasma membrane. Consequently, the basal activity of TRPV4 is reduced despite an overall increase in TRPV4 levels. This mode of regulation is not limited to TRPV4. TRPC4, another member of the TRP channel family, is also strongly ubiquitinated in the presence of AIP4, leading to the increased intracellular localization of TRPC4 and the reduction of its basal activity. However, ubiquitination of several other TRP channels is not affected by AIP4, demonstrating that AIP4-mediated regulation is a unique property of select TRP channels.     

Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons

The lipid diacylglycerol (DAG) analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) was used to verify the existence of DAG‐sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca²⁺ ([Ca²⁺]i) in nearly 35% of the KCl‐responsive cells. These Ca²⁺ responses disappeared in a Ca²⁺‐free medium supplemented with EGTA. Mn²⁺ quench experiments showed that OAG activated Ca²⁺‐conducting channels that were also permeant to Ba²⁺. The OAG‐induced Ca²⁺ responses were unaffected by nifedipine or omega‐conotoxin GVIA (Sigma‐Aldrich, Saint‐Quentin Fallavier, France) but blocked by 1‐[β‐(3‐(4‐Methoxyphenyl)propoxy)‐4‐methoxyphenethyl]‐1H‐imidazole hydrochloride (SKF)‐96365 and Gd³⁺. Replacing Na⁺ ions with N‐methyl‐d ‐glucamine diminished the amplitude of the OAG‐induced Ca²⁺ responses showing that the Ca²⁺ entry was mediated via Na⁺‐dependent and Na⁺‐independent mechanisms. Experiments carried out with the fluorescent Na⁺ indicator CoroNa Green showed that OAG elevated [Na⁺]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca²⁺]i but not the protein kinase C activator phorbol 12‐myristate 13‐acetate. Moreover, the OAG‐induced Ca²⁺ responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C‐type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca²⁺]i. Whole‐cell patch‐clamp recordings showed that hyperforin activated non‐selective cation channels. They were blocked by SKF‐96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin‐ and OAG‐sensitive Ca²⁺‐permeable channels displaying TRPC6‐like properties. This is the first report revealing the existence of second messenger‐operated channels in cortical neurons.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.     

Role of Oxidative Stress and Ca2+ Signaling on Molecular Pathways of Neuropathic Pain in Diabetes: Focus on TRP Channels

Diabetes mellitus, a debilitating chronic disease, affects ~100?million people. Peripheral neuropathy is one of the most common early complications of diabetes in ~66?% of these patients. Altered Ca(2+) handling and Ca(2+) signaling were detected in a huge variety of preparations isolated from animals with experimentally induced type 1 and 2 diabetes as well as patients suffering from the disease. We reviewed the role of Ca(2+) signaling through cation channels and oxidative stress on diabetic neuropathic pain in sensory neurons. The pathogenesis of diabetic neuropathy involves the polyol pathway, advanced glycation end products, oxidative stress, protein kinase C activation, neurotrophism, and hypoxia. Experimental studies with respect to oxidative stress and Ca(2+) signaling, inhibitor roles of antioxidants in diabetic neuropathic pain are also summarized in the review. We hypothesize that deficits in insulin, triggers alterations of sensory neurone phenotype that are critical for the development of abnormal Ca(2+) homeostasis and oxidative stress and associated mitochondrial dysfunction. The transient receptor potential channels are a large family of proteins with six main subfamilies. The sheer number of different TRPs with distinct functions supports the statement that these channels are involved in a wide range of processes ranging in diabetic neuropathic pain and it seems that the TRPC, TRPM and TRPV groups are mostly responsible from diabetic neuropathic pain. In conclusion, the accumulating evidence implicating Ca(2+) dysregulation and over production of oxidative stress products in diabetic neuropathic pains, along with recent advances in understanding of genetic variations in cation channels such as TRP channels, makes modulation of neuronal Ca(2+) handling an increasingly viable approach for therapeutic interventions against the painful and degenerative aspects of many diabetic neuropathies.     

Angiotensin-2-Mediated Ca2+ Signaling in the Retinal Pigment Epithelium: Role of Angiotensin-Receptor- Associated-Protein and TRPV2 Channel

Angiotensin II (AngII) receptor (ATR) is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE) expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap), and transient-receptor-potential channel-V2 (TRPV2). AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE) cells by AngII results in biphasic increases in intracellular free Ca²⁺inhibited by losartan. Xestospongin C (xest C) and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca²⁺response. RPE cells from Atrap^−/− mice showed smaller AngII-evoked Ca²⁺peak (by 22%) and loss of sustained Ca²⁺elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD) at 15 µM stimulates intracellular Ca²⁺-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (a TRPV channel inhibitor) reduced the cannabidiol-induced Ca²⁺-rise. Application of {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca²⁺transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca²⁺transients in the RPE by releasing Ca²⁺from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca²⁺elevation.