Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knock-out (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch clamp recordings in primary urothelial cells, we observed that Mg²⁺-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in cells from control mice. Assessment of voiding behavior indicated a significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice and 0.36 ± 0.04 g in control mice, p < 0.05, n = 6–8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1β was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knock-out mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo.     

Furanocoumarins Are a Novel Class of Modulators for the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Channel

Furanocoumarin imperatorin is the major active component of Angelica dahurica root extracts, widely used in traditional medicine to treat headache, toothache, and orbital eye pain. In this study, we investigated the mechanisms that may underlie the pain-relieving effects of the compound. We found that imperatorin significantly inhibited formalin- and capsaicin-induced nocifensive responses but did not alter baseline thermal withdrawal thresholds in the rat. We established that imperatorin is a weak agonist of TRPV1, a channel implicated in detecting several noxious stimuli, exhibiting a 50% effective concentration (EC₅₀) of 12.6 ± 3.2 μm. A specific TRPV1 antagonist, JNJ-17203212 (0.5 μm), potently inhibited imperatorin-induced TRPV1 activation. Site-directed mutagenesis studies revealed that imperatorin most likely acted via a site adjacent to or overlapping with the TRPV1 capsaicin-binding site. TRPV1 recovery from desensitization was delayed in the presence of imperatorin. Conversely, imperatorin sensitized TRPV1 to acid activation but did not affect the current amplitude and/or the activation-inactivation properties of Na_v1.7, a channel important for transmission of nociceptive information. Thus, our data indicate that furanocoumarins represent a novel group of TRPV1 modulators that may become important lead compounds in the drug discovery process aimed at developing new treatments for pain management.     

A New Role for PTEN in Regulating Transient Receptor Potential Canonical Channel 6-mediated Ca2+ Entry,Endothelial Permeability,and Angiogenesis

Phosphatase and tensin homologue (PTEN) is a dual lipid-protein phosphatase that catalyzes the conversion of phosphoinositol 3,4,5-triphosphate to phosphoinositol 4,5-bisphosphate and thereby inhibits PI3K-Akt-dependent cell proliferation, migration, and tumor vascularization. We have uncovered a previously unrecognized role for PTEN in regulating Ca²⁺ entry through transient receptor potential canonical channel 6 (TRPC6) that does not require PTEN phosphatase activity. We show that PTEN tail-domain residues 394–403 permit PTEN to associate with TRPC6. The inflammatory mediator thrombin promotes this association. Deletion of PTEN residues 394–403 prevents TRPC6 cell surface expression and Ca²⁺ entry. However, PTEN mutant, C124S, which lacks phosphatase activity, did not alter TRPC6 activity. Thrombin failed to increase endothelial monolayer permeability in the endothelial cells, transducing the Δ394–403 PTEN mutant. Paradoxically, we also show that thrombin failed to induce endothelial cell migration and tube formation in cells transducing the Δ394–403 PTEN mutant. Our results demonstrate that PTEN, through residues 394–403, serves as a scaffold for TRPC6, enabling cell surface expression of the channel. Ca²⁺ entry through TRPC6 induces an increase in endothelial permeability and directly promotes angiogenesis. Thus, PTEN is indicated to play a role beyond suppressing PI3K signaling.     

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca²⁺ channel TRPV5 constitutes the apical entry gate for Ca²⁺ transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca²⁺ reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2 analysis. Removal of amino acid His⁷¹² elevated the [Ca²⁺]_i, indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His⁷¹² for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His⁷¹² was confirmed by patch clamp analysis, which demonstrates increased Na⁺ and Ca²⁺ currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca²⁺-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca²⁺]_i. Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His⁷¹² plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.     

Wild-type and Brachyolmia-causing Mutant TRPV4 Channels Respond Directly to Stretch Force

Whether animal ion channels functioning as mechanosensors are directly activated by stretch force or indirectly by ligands produced by the stretch is a crucial question. TRPV4, a key molecular model, can be activated by hypotonicity, but the mechanism of activation is unclear. One model has this channel being activated by a downstream product of phospholipase A₂, relegating mechanosensitivity to the enzymes or their regulators. We expressed rat TRPV4 in Xenopus oocytes and repeatedly examined >200 excised patches bathed in a simple buffer. We found that TRPV4 can be activated by tens of mm Hg pipette suctions with open probability rising with suction even in the presence of relevant enzyme inhibitors. Mechanosensitivity of TRPV4 provides the simplest explanation of its various force-related physiological roles, one of which is in the sensing of weight load during bone development. Gain-of-function mutants cause heritable skeletal dysplasias in human. We therefore examined the brachyolmia-causing R616Q gain-of-function channel and found increased whole-cell current densities compared with wild-type channels. Single-channel analysis revealed that R616Q channels maintain mechanosensitivity but have greater constitutive activity and no change in unitary conductance or rectification.     

Forward Genetic Analysis Reveals Multiple Gating Mechanisms of TRPV4

TRPV4 is a polymodal cation channel gain-of-function (GOF) allele which causes skeletal dysplasia in humans. To better understand its gating, we screened for additional GOF alleles based on their ability to block yeast proliferation. Repeatedly, only a limited number of such growth-blocking mutations were isolated. Expressed in oocytes, wild-type channels can be strongly activated by either hypotonicity or exposure to the potent agonist 4αPDD, although the GOF channels behaved as if they were fully prestimulated as well as lacking a previously uncharacterized voltage-dependent inactivation. Five of six mutations occurred at or near the inner ends of the predicted core helices, giving further direct evidence that this region indeed forms the main intracellular gate in TRP channels. Surprisingly, both wild-type channels as well as these GOF channels maintain strong steady-state outward rectification that is not due to a Ca²⁺ block, as has been proposed elsewhere. We conclude that TRPV4 contains an additional voltage-dependent gating mechanism in series with the main intracellular gate.     

Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.     

WNK4 Kinase Stimulates Caveola-mediated Endocytosis of TRPV5 Amplifying the Dynamic Range of Regulation of the Channel by Protein Kinase C

WNK4 (with-no-lysine (K) kinase-4) is present in the distal nephron of the kidney and plays an important role in the regulation of renal ion transport. The epithelial Ca²⁺ channel TRPV5 (transient receptor potential vanilloid 5) is the gatekeeper of transcellular Ca²⁺ reabsorption in the distal nephron. Previously, we reported that activation of protein kinase C (PKC) increases cell-surface abundance of TRPV5 by inhibiting caveola-mediated endocytosis of the channel. Here, we report that WNK4 decreases cell-surface abundance of TRPV5 by enhancing its endocytosis. Deletion analysis revealed that stimulation of endocytosis of TRPV5 involves amino acids outside the kinase domain of WNK4. We also investigated interplay between WNK4 and PKC regulation of TRPV5. The maximal level of TRPV5 current density stimulated by the PKC activator 1-oleoyl-acetyl-sn-glycerol (OAG) is the same with or without WNK4. The relative increase of TRPV5 current stimulated by OAG, however, is greater in the presence of WNK4 compared with that without WNK4 (∼215% increase versus 60% increase above the level without OAG). Moreover, the rate of increase of TRPV5 by OAG is faster with WNK4 than without WNK4. The enhanced increase of TRPV5 in the presence of WNK4 is also observed when PKC is activated by parathyroid hormones. Thus, WNK4 exerts tonic inhibition of TRPV5 by stimulating caveola-mediated endocytosis. The lower basal TRPV5 level in the presence of WNK4 allows amplification of the stimulation of channel by PKC. This interaction between WNK4 and PKC regulation of TRPV5 may be important for physiological regulation of renal Ca²⁺ reabsorption by parathyroid hormones or the tissue kallikrein in vivo.     

The chimeric approach reveals that differences in the TRPV1 pore domain determine species-specific sensitivity to block of heat activation

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.     

Function of transient receptor potential cation channel subfamily V member 4 (TRPV4) as a mechanical transducer in flow-sensitive segments of renal collecting duct system

The TRPV4 Ca(2+)-permeable channel is sensitive to mechanical stimuli. In the current study we have employed immunocytochemical staining in kidney slices and functional assessments (Ca(2+) imaging) in isolated, split-opened, tubule segments to define TRPV4 sites of expression and flow-dependent function in the collecting duct system. Staining patterns revealed strong expression of TRPV4 along the entire collecting duct system with highest levels at the apical (luminal)/subapical region of the principal cells (PCs), the dominant cell type, with more diffuse staining in intercalated cells (ICs). Using fluorescence Ca(2+) imaging and the selective TRPV4 agonist, GSK1016790A, we demonstrated functional TRPV4 channels in PCs and ICs of split-opened cortical collecting ducts and connecting tubules. The agonist was ineffective in inducing a rise in [Ca(2+)](i) in the absence of extracellular Ca(2+) or in tubules from TRPV4-deficient animals. Most importantly, a 10-fold elevation in luminal (apical) fluid flow induced a rapid and sustained influx of Ca(2+) that was abolished by the TRPV channel inhibitor, ruthenium red, or in tubules isolated from TRPV4 deficient animals. We concluded that TRPV4 is highly expressed along the entire collecting duct system where it appears to function as a sensor/transducer of flow-induce mechanical stresses.     

The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase

The transient receptor potential vanilloid type 5 (TRPV5) Ca²⁺ channel facilitates transcellular Ca²⁺ transport in the distal convoluted tubule (DCT) of the kidney. The channel is glycosylated with a complex type N-glycan and it has been postulated that hydrolysis of the terminal sialic acid(s) stimulate TRPV5 activity. The present study delineates the role of the N-glycan in TRPV5 activity using biochemical assays in Human Embryonic Kidney 293 cells expressing TRPV5, isoelectric focusing and total internal reflection fluorescent microscopy. The anti-aging hormone klotho and other glycosidases stimulate TRPV5-dependent Ca²⁺ uptake. Klotho was found to increase the plasma membrane stability of TRPV5, via the TRPV5 N-glycan. Sialidase mimicked this stimulatory action. However, this effect was independent of the N-glycosylation state of TRPV5, since the N-glycosylation mutant (TRPV5^N358Q) was activated to the same extent. We showed that the increased TRPV5 activity after sialidase treatment is caused by inhibition of lipid raft-mediated internalization. In addition, sialidase modified the N-glycan of transferrin, a model glycoprotein, differently from klotho. Previous studies showed that after klotho treatment, galectin-1 binds the TRPV5 N-glycan and thereby increases TRPV5 activity. However, galectin-3, but not galectin-1, was expressed in the DCT. Furthermore, an increase in TRPV5-mediated Ca²⁺ uptake was detected after galectin-3 treatment. In conclusion, two distinct TRPV5 stimulatory mechanisms were demonstrated; a klotho-mediated effect that is dependent on the N-glycan of TRPV5 and a sialidase-mediated stimulation that is lipid raft-dependent and independent of the N-glycan of TRPV5.     

Activation of Polycystic Kidney Disease-2-like 1 (PKD2L1)-PKD1L3 Complex by Acid in Mouse Taste Cells

Five basic tastes (bitter, sweet, umami, salty, and sour) are detected in the four taste areas where taste buds reside. Although molecular mechanisms for detecting bitter, sweet, and umami have been well clarified, those for sour and salty remain poorly understood. Several channels including acid-sensing ion channels have been proposed as candidate sour receptors, but they do not encompass all sour-sensing abilities in vivo. We recently reported a novel candidate for sour sensing, the polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 channel complex. This channel is not a traditional ligand-gated channel and is gated open only after removal of an acid stimulus, called an off response. Here we show that off responses upon acid stimulus are clearly observed in native taste cells from circumvallate, but not fungiform papillae, of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice, from which Type III taste cells can be visualized, using Ca²⁺ imaging and patch clamp methods. Off responses were detected in most cells where PKD2L1 immunoreactivity was observed. Interestingly, the pH threshold for acid-evoked intracellular Ca²⁺ increase was around 5.0, a value much higher than that observed in HEK293 cells expressing the PKD2L1-PKD1L3 complex. Thus, PKD2L1-PKD1L3-mediated acid-evoked off responses occurred both in HEK293 cells and in native taste cells, suggesting the involvement of the PKD2L1-PKD1L3 complex in acid sensing in vivo.     

Blood Vessel Epicardial Substance (Bves) Regulates Epidermal Tight Junction Integrity through Atypical Protein Kinase C

Bves is widely observed in the cell junction of the skin, epicardium, intestine, and cornea of both developmental embryos and mature adults. However, it is not clear how Bves confers its role in intercellular adhesion. Here, we identified the zebrafish bves (zBves) and found that the epidermal barrier function could be disrupted after knockdown of Bves, and these zBves morphants were sensitive to osmotic stress. A loss of zBves would affect the partitioning defective protein (PAR) junctional complex identified by the rescue experiment with tjp-2/ZO-2 or the PAR complex (par-3, par-6, and prkci/atypical (a)PKC) mRNAs, in which the survival rate of embryos increased 11, 24, 25, and 28%, respectively, after injection with junctional components; the tjp-2 and aPKC mRNA-rescued embryos also had 24 and 45% decreases in the defective rate. Immunofluorescent studies demonstrated that the aggregation of aPKC around the cell junctions had disintegrated in zBves morphants. However, the expression and assembly of zBves were not influenced by aPKC-MO. These results indicate that a loss of zBves affects the proteins involved in the pathway of the PAR junctional complex, especially aPKC, and both aPKC and Bves are indispensable to claudin expression.     

The in Vivo TRPV6 Protein Starts at a Non-AUG Triplet,Decoded as Methionine,Upstream of Canonical Initiation at AUG

TRPV6 channels function as epithelial Ca²⁺ entry pathways in the epididymis, prostate, and placenta. However, the identity of the endogenous TRPV6 protein relies on predicted gene coding regions and is only known to a certain level of approximation. We show that in vivo the TRPV6 protein has an extended N terminus. Translation initiates at a non-AUG codon, at ACG, which is decoded by methionine and which is upstream of the annotated AUG, which is not used for initiation. The in vitro properties of channels formed by the extended full-length TRPV6 proteins and the so-far annotated and smaller TRPV6 are similar, but the extended N terminus increases trafficking to the plasma membrane and represents an additional scaffold for channel assembly. The increased translation of the smaller TRPV6 cDNA version may overestimate the in vivo situation where translation efficiency may represent an additional mechanism to tightly control the TRPV6-mediated Ca²⁺ entry to prevent deleterious Ca²⁺ overload.     

Conserved Gating Elements in TRPC4 and TRPC5 Channels

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca²⁺-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4–S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4_G503S and TRPC5_G504S mutants causes cell death, which could be prevented by buffering the Ca²⁺ of the culture medium. Current-voltage relationships of the TRPC4_G503S and TRPC5_G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4_G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.     

Potentiation of the Transient Receptor Potential Vanilloid 1 Channel Contributes to Pruritogenesis in a Rat Model of Liver Disease

Persistent pruritus is a common disabling dermatologic symptom associated with different etiologic factors. These include primary skin conditions, as well as neuropathic, psychogenic, or systemic disorders like chronic liver disease. Defective clearance of potential pruritogenic substances that activate itch-specific neurons innervating the skin is thought to contribute to cholestatic pruritus. However, because the underlying disease-specific pruritogens and itch-specific neuronal pathways and mechanism(s) are unknown, symptomatic therapeutic intervention often leads to no or only limited success. In the current study, we aimed to first validate rats with bile duct ligation (BDL) as a model for hepatic pruritus and then to evaluate the contribution of inflammation, peripheral neuronal sensitization, and specific signaling pathways and subpopulations of itch-responsive neurons to scratching behavior and thermal hypersensitivity. Chronic BDL rats displayed enhanced scratching behavior and thermal hyperalgesia indicative of peripheral neuroinflammation. BDL-induced itch and hypersensitivity involved a minor contribution of histaminergic/serotonergic receptors, but significant activation of protein-activated receptor 2 (PAR₂) receptors, prostaglandin PGE₂ formation, and potentiation of transient receptor potential vanilloid 1 (TRPV1) channel activity. The sensitization of dorsal root ganglion nociceptors in BDL rats was associated with increased surface expression of PAR₂ and TRPV1 proteins and an increase in the number of PAR₂- and TRPV1-expressing peptidergic neurons together with a shift of TRPV1 receptor expression to medium sized dorsal root ganglion neurons. These results suggest that pruritus and hyperalgesia in chronic cholestatic BDL rats are associated with neuroinflammation and involve PAR₂-induced TRPV1 sensitization. Thus, pharmacological modulation of PAR₂ and/or TRPV1 may be a valuable therapeutic approach for patients with chronic liver pruritus refractory to conventional treatments.     

Identification of a Membrane-targeting Domain of the Transient Receptor Potential Canonical (TRPC)4 Channel Unrelated to Its Formation of a Tetrameric Structure

Canonical transient receptor potential (TRPC) channels are Ca²⁺-permeable nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). The TRPC4 channel is expressed in a punctate distribution in the membrane. To identify the regulating region of the channel trafficking to the membrane, we generated deletion mutants of the TRPC4 channel. We determined that when either region that was downstream of the 20 amino acids of the N terminus or the 700–730 amino acids was deleted, the mutants were retained in the endoplasmic reticulum. By coexpression of the wild-type TRPC4 with deletion mutants, we found that the 23–29 amino acids of the N terminus regulate a membrane trafficking. Additionally, by the fluorescence resonance energy transfer (FRET) method, we found that the regions downstream of the 99 amino acid region of the N terminus and upstream of the 730 amino acid region in the C terminus produce assembly of the TRPC4 tetramers. We inferred the candidate proteins that regulate or interact with the 23–29 domain of TRPC4.     

Identification of a binding motif in the S5 helix that confers cholesterol sensitivity to the TRPV1 ion channel

The TRPV1 ion channel serves as an integrator of noxious stimuli with its activation linked to pain and neurogenic inflammation. Cholesterol, a major component of cell membranes, modifies the function of several types of ion channels. Here, using measurements of capsaicin-activated currents in excised patches from TRPV1-expressing HEK cells, we show that enrichment with cholesterol, but not its diastereoisomer epicholesterol, markedly decreased wild-type rat TRPV1 currents. Substitutions in the S5 helix, rTRPV1-R579D, and rTRPV1-F582Q, decreased this cholesterol response and rTRPV1-L585I was insensitive to cholesterol addition. Two human TRPV1 variants, with different amino acids at position 585, had different responses to cholesterol with hTRPV1-Ile(585) being insensitive to this molecule. However, hTRPV1-I585L was inhibited by cholesterol addition similar to rTRPV1 with the same S5 sequence. In the absence of capsaicin, cholesterol enrichment also inhibited TRPV1 currents induced by elevated temperature and voltage. These data suggest that there is a cholesterol-binding site in TRPV1 and that the functions of TRPV1 depend on the genetic variant and membrane cholesterol content.     

The Contribution of TRPV4-Mediated Calcium Signaling to Calcium Homeostasis in Endothelial Cells

A large variety of cation transport systems are involved in the regulation of calcium homeostasis in endothelial cells. The focus of the present study is to determine the contribution of nonselective cation channels from the TRP (transient receptor potential) family to cellular calcium homeostasis of porcine aortic endothelial cells (PAEC). One member of the TRPV (vanniloid) subfamily, TRPV4, has previously been shown to be involved in cation transport induced by a large variety of stimulations including osmolarity, temperature, mechanical stress, and phosphorylation. Here, we demonstrate the existence of several TRP proteins, including TRPV4, in PAEC using RT-PCR. To test whether this channel is functional, we performed FURA-2 calcium measurements and whole-cell patch-clamp experiments. We observed the induction of large calcium signals following mechanical stress, altered extracellular temperature, and the selective TRPV4 activator 4-α -PDD. These effects were diminished in the presence of the TRPV4 inhibitor miconazole, suggesting the involvement of this channel in mediating endothelial calcium signals. The large amounts of transported calcium and the short signaling ways suggest a potentially important role of this channel in many physiological processes.