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1.
《Cell calcium》2017
The Ca2+-selective tetrameric Transient Receptor Potential Vanilloid 6 (TRPV6) channel is an inwardly rectifying ion channel. The constitutive current endures Ca2+-induced inactivation as a result of the activation of phospholipase C followed depletion of phosphatidylinositol 4,5-bisphosphate, and calmodulin binding. Replacing a glycine residue within the cytosolic S4-S5 linker of the human TRPV6 protein, glycine 516, which is conserved in all TRP channel proteins, by a serine residue forces the channels into an open conformation thereby enhancing constitutive Ca2+ entry and preventing inactivation. Introduction of a second mutation (T621A) into TRPV6G516S reduces constitutive activity and partially rescues the TRPV6 function. According to the recently revealed crystal structure of the rat TRPV6 the T621 is adjacent to the distal end of the transmembrane segment 6 (S6) within a short linker between S6 and the helix formed by the TRP domain. These results indicate that the S4-S5 linker and the S6-TRP-domain linker are critical constituents of TRPV6 channel gating and that disturbance of their sequences foster constitutive Ca2+ entry. 相似文献
2.
Theun de Groot Sjoerd Verkaart Qi Xi René J. M. Bindels Joost G. J. Hoenderop 《The Journal of biological chemistry》2010,285(37):28481-28487
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. 相似文献
3.
Maiwase Tembo Rachel E. Bainbridge Crystal Lara-Santos Kayla M. Komondor Grant J. Daskivich Jacob D. Durrant Joel C. Rosenbaum Anne E. Carlson 《The Journal of biological chemistry》2022,298(8)
TransMEMbrane 16A (TMEM16A) is a Ca2+-activated Cl− channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca2+, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P2 head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca2+ to excised inside–out patches, we found that TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P2 analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P2 reduces TMEM16A-conducted currents. Application of PIP2 analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 4′ position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P2 binds to and potentiates TMEM16A channels. 相似文献
4.
Kuai Yu Jinqiu Zhu Zhiqiang Qu Yuan-Yuan Cui H. Criss Hartzell 《The Journal of general physiology》2014,143(2):253-267
The Ca2+-activated Cl channel anoctamin-1 (Ano1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. Ano1 is activated by increases in intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to Ano1 or whether phosphorylation or additional Ca2+-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of Ano1 by Ca2+ for the following reasons. (a) Exogenous CaM has no effect on Ano1 currents in inside-out excised patches. (b) Overexpression of Ca2+-insensitive mutants of CaM have no effect on Ano1 currents, whereas they eliminate the current mediated by the small-conductance Ca2+-activated K+ (SK2) channel. (c) Ano1 does not coimmunoprecipitate with CaM, whereas SK2 does. Furthermore, Ano1 binds very weakly to CaM in pull-down assays. (d) Ano1 is activated in excised patches by low concentrations of Ba2+, which does not activate CaM. In addition, we conclude that reversible phosphorylation/dephosphorylation is not required for current activation by Ca2+ because the current can be repeatedly activated in excised patches in the absence of ATP or other high-energy compounds. Although Ano1 is blocked by the CaM inhibitor trifluoperazine (TFP), we propose that TFP inhibits the channel in a CaM-independent manner because TFP does not inhibit Ano1 when applied to the cytoplasmic side of excised patches. These experiments lead us to conclude that CaM is not required for activation of Ano1 by Ca2+. Although CaM is not required for channel opening by Ca2+, work of other investigators suggests that CaM may have effects in modulating the biophysical properties of the channel. 相似文献
5.
Hydrolysis of phosphatidylinositol 4,5-bisphosphate mediates calcium-induced inactivation of TRPV6 channels 总被引:2,自引:0,他引:2
TRPV6 is a member of the transient receptor potential superfamily of ion channels that facilitates Ca(2+) absorption in the intestines. These channels display high selectivity for Ca(2+), but in the absence of divalent cations they also conduct monovalent ions. TRPV6 channels have been shown to be inactivated by increased cytoplasmic Ca(2+) concentrations. Here we studied the mechanism of this Ca(2+)-induced inactivation. Monovalent currents through TRPV6 substantially decreased after a 40-s application of Ca(2+), but not Ba(2+). We also show that Ca(2+), but not Ba(2+), influx via TRPV6 induces depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2) or PIP(2)) and the formation of inositol 1,4,5-trisphosphate. Dialysis of DiC(8) PI(4,5)P(2) through the patch pipette inhibited Ca(2+)-dependent inactivation of TRPV6 currents in whole-cell patch clamp experiments. PI(4,5)P(2) also activated TRPV6 currents in excised patches. PI(4)P, the precursor of PI(4,5)P(2), neither activated TRPV6 in excised patches nor had any effect on Ca(2+)-induced inactivation in whole-cell experiments. Conversion of PI(4,5)P(2) to PI(4)P by a rapamycin-inducible PI(4,5)P(2) 5-phosphatase inhibited TRPV6 currents in whole-cell experiments. Inhibiting phosphatidylinositol 4 kinases with wortmannin decreased TRPV6 currents and Ca(2+) entry into TRPV6-expressing cells. We propose that Ca(2+) influx through TRPV6 activates phospholipase C and the resulting depletion of PI(4,5)P(2) contributes to the inactivation of TRPV6. 相似文献
6.
Drosophila TRPML Forms PI(3,5)P2-activated Cation Channels in Both Endolysosomes and Plasma Membrane
Xinghua Feng Yu Huang Yungang Lu Jian Xiong Ching-On Wong Pu Yang Jintang Xia De Chen Guangwei Du Kartik Venkatachalam Xuefeng Xia Michael X. Zhu 《The Journal of biological chemistry》2014,289(7):4262-4272
Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca2+ and Fe2+ release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1–3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P2-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca2+, Mn2+, and Fe2+, but not Fe3+. The TRPML currents were inhibited by trivalent cations Fe3+, La3+, and Gd3+. These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology. 相似文献
7.
Rainer Schindl Reinhard Fritsch Isaac Jardin Irene Frischauf Heike Kahr Martin Muik Maria Christine Riedl Klaus Groschner Christoph Romanin 《The Journal of biological chemistry》2012,287(42):35612-35620
TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca2+ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca2+ and Na+ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca2+ influx in HEK293 cells. 相似文献
8.
Horacio Poblete Ingrid Oyarzún Pablo Olivero Jeffrey Comer Matías Zu?iga Romina V. Sepulveda David Báez-Nieto Carlos González Leon Fernando González-Nilo Ramón Latorre 《The Journal of biological chemistry》2015,290(4):2086-2098
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P2 is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P2 binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P2 interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P2 behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P2 binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P2 in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P2 binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate. 相似文献
9.
Sarabeth Graham Min Ding Yanfeng Ding Sherry Sours-Brothers Rafal Luchowski Zygmunt Gryczynski Thomas Yorio Haiying Ma Rong Ma 《The Journal of biological chemistry》2010,285(30):23466-23476
This study examined the effect of H2O2 on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H2O2 significantly stimulated Ca2+ entry in a dose-dependent manner. Electrophysiological experiments showed that H2O2 significantly increased TRPC6 channel open probability and whole-cell currents. H2O2 also evoked a robust inward current in A7r5 vascular smooth muscle cells, which was nearly abolished by knockdown of TRPC6 using a small interfering RNA. Catalase substantially attenuated arginine vasopressin (AVP)-induced Ca2+ entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H2O2 response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H2O2-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-sn-glycerol effect on TRPC6 was significantly greater in the presence of H2O2. Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H2O2. Similarly, in cells transfected with TRPC6-EGFP, confocal microscopy showed a significant increase in fluorescence intensity in the region of the cell membrane and adjacent to the membrane. AVP also increased the fluorescence intensity on the surface of the cells co-transfected with TRPC6-EGFP and V1 receptor, and this response was inhibited by catalase. These data indicate that H2O2 activates TRPC6 channels via modification of thiol groups of intracellular proteins. This cysteine oxidation-dependent pathway not only stimulates the TRPC6 channel by itself but also sensitizes the channels to diacylglycerol and promotes TRPC6 trafficking to the cell surface. 相似文献
10.
Ca2+ (calcium) homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum) and Ca2+ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER–PM contacts via CaM binding. 相似文献
11.
Takaaki Sokabe Tomoko Fukumi-Tominaga Shigenobu Yonemura Atsuko Mizuno Makoto Tominaga 《The Journal of biological chemistry》2010,285(24):18749-18758
Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca2+ dynamics. We found that TRPV4 interacts with β-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca2+-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca2+ increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin. 相似文献
12.
《Cell calcium》2019
Recently there has been a flurry of interest in the regulation of the homo-dimeric calcium-activated chloride channel ANO1 (also known as TMEM16A) by phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). These recent studies show that upon Ca2+ binding, PI(4,5)P2 cooperates to maintain the conductive state of ANO1. PI(4,5)P2 does so by binding to sites or modules on the protein’s cytosolic side. These findings add a new function to the PI(4,5)P2 repertoire and a new dimension to ANO1 gating. 相似文献
13.
Haruka Yamazaki Jenny Chan Mitsuhiko Ikura Takayuki Michikawa Katsuhiko Mikoshiba 《The Journal of biological chemistry》2010,285(46):36081-36091
The N-terminal ∼220-amino acid region of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/Ca2+ release channel has been referred to as the suppressor/coupling domain because it is required for both IP3 binding suppression and IP3-induced channel gating. Measurements of IP3-induced Ca2+ fluxes of mutagenized mouse type 1 IP3R (IP3R1) showed that the residues responsible for IP3 binding suppression in this domain were not essential for channel opening. On the other hand, a single amino acid substitution of Tyr-167 to alanine completely impaired IP3-induced Ca2+ release without reducing the IP3 binding activity. The corresponding residue in type 3 IP3R (IP3R3), Trp-168, was also critical for channel opening. Limited trypsin digestion experiments showed that the trypsin sensitivities of the C-terminal gatekeeper domain differed markedly between the wild-type channel and the Tyr-167 mutant under the optimal conditions for channel opening. These results strongly suggest that the Tyr/Trp residue (Tyr-167 in IP3R1 and Trp-168 in IP3R3) is critical for the functional coupling between IP3 binding and channel gating by maintaining the structural integrity of the C-terminal gatekeeper domain at least under activation gating. 相似文献
14.
Venkat G. Magupalli Sumiko Mochida Jin Yan Xin Jiang Ruth E. Westenbroek Angus C. Nairn Todd Scheuer William A. Catterall 《The Journal of biological chemistry》2013,288(7):4637-4648
Ca2+/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of CaV2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca2+/CaM stimulus. Autophosphorylated CaMKII can bind the CaV2.1 channel and synapsin-1 simultaneously. CaMKII binding to CaV2.1 channels induces Ca2+-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of CaV2.1 channels by binding of Ca2+/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to CaV2.1 channels. These results define the functional properties of a signaling complex of CaMKII and CaV2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity. 相似文献
15.
Maria J. Sarmento Ana Coutinho Aleksander Fedorov Manuel Prieto Fabio Fernandes 《生物化学与生物物理学报:生物膜》2014
Calcium has been shown to induce clustering of PI(4,5)P2 at high and non-physiological concentrations of both the divalent ion and the phosphatidylinositol, or on supported lipid monolayers. In lipid bilayers at physiological conditions, clusters are not detected through microscopic techniques. Here, we aimed to determine through spectroscopic methodologies if calcium plays a role in PI(4,5)P2 lateral distribution on lipid bilayers under physiological conditions. Using several different approaches which included information on fluorescence quantum yield, polarization, spectra and diffusion properties of a fluorescent derivative of PI(4,5)P2 (TopFluor(TF)-PI(4,5)P2), we show that Ca2 + promotes PI(4,5)P2 clustering in lipid bilayers at physiological concentrations of both Ca2 + and PI(4,5)P2. Fluorescence depolarization data of TF-PI(4,5)P2 in the presence of calcium suggests that under physiological concentrations of PI(4,5)P2 and calcium, the average cluster size comprises ~ 15 PI(4,5)P2 molecules. The presence of Ca2 +-induced PI(4,5)P2 clusters is supported by FCS data. Additionally, calcium mediated PI(4,5)P2 clustering was more pronounced in liquid ordered (lo) membranes, and the PI(4,5)P2-Ca2 + clusters presented an increased affinity for lo domains. In this way, PI(4,5)P2 could function as a lipid calcium sensor and the increased efficiency of calcium-mediated PI(4,5)P2 clustering on lo domains might provide targeted nucleation sites for PI(4,5)P2 clusters upon calcium stimulus. 相似文献
16.
Elizabeth H. P. Leunissen Anil V. Nair Christian Büll Dirk J. Lefeber Floris L. van Delft René J. M. Bindels Joost G. J. Hoenderop 《The Journal of biological chemistry》2013,288(41):29238-29246
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 (TRPV5N358Q) 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. 相似文献
17.
Daniel P. Poole Silvia Amadesi Nicholas A. Veldhuis Fe C. Abogadie TinaMarie Lieu William Darby Wolfgang Liedtke Michael J. Lew Peter McIntyre Nigel W. Bunnett 《The Journal of biological chemistry》2013,288(8):5790-5802
G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR2), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR2 regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR2 agonist stimulated a transient increase in [Ca2+]i. TRPV4 expression led to a markedly sustained increase in [Ca2+]i. Removal of extracellular Ca2+ and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A2 and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR2 generates arachidonic acid-derived lipid mediators, such as 5′,6′-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR2-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR2 was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR2 signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR2-stimulated neurogenic inflammation. Thus, PAR2 activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR2. 相似文献
18.
Vidisha Kini Alejandra Chavez Dolly Mehta 《The Journal of biological chemistry》2010,285(43):33082-33091
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. 相似文献
19.
Claudia Fecher-Trost Ulrich Wissenbach Andreas Beck Pascal Schalkowsky Christof Stoerger Janka Doerr Anna Dembek Martin Simon-Thomas Armin Weber Peter Wollenberg Thomas Ruppert Ralf Middendorff Hans H. Maurer Veit Flockerzi 《The Journal of biological chemistry》2013,288(23):16629-16644
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. 相似文献
20.
Sonia Gallego-Sand��n Arancha Rodr��guez-Garc��a Mar��a Teresa Alonso Javier Garc��a-Sancho 《The Journal of biological chemistry》2009,284(47):32591-32601
Transient receptor potential vanilloid type 1 (TRPV1) is a plasma membrane Ca2+ channel involved in transduction of painful stimuli. Dorsal root ganglion (DRG) neurons express ectopic but functional TRPV1 channels in the endoplasmic reticulum (ER) (TRPV1ER). We have studied the properties of TRPV1ER in DRG neurons and HEK293T cells expressing TRPV1. Activation of TRPV1ER with capsaicin or other vanilloids produced an increase of cytosolic Ca2+ due to Ca2+ release from the ER. The decrease of [Ca2+]ER was directly revealed by an ER-targeted aequorin Ca2+ probe, expressed in DRG neurons using a herpes amplicon virus. The sensitivity of TRPV1ER to capsaicin was smaller than the sensitivity of the plasma membrane TRPV1 channels. The low affinity of TRPV1ER was not related to protein kinase A- or C-mediated phosphorylations, but it was due to inactivation by cytosolic Ca2+ because the sensitivity to capsaicin was increased by loading the cells with the Ca2+ chelator BAPTA. Decreasing [Ca2+]ER did not affect the sensitivity of TRPV1ER to capsaicin. Disruption of the TRPV1 calmodulin-binding domains at either the C terminus (Δ35AA) or the N terminus (K155A) increased 10-fold the affinity of TRPV1ER for capsaicin, suggesting that calmodulin is involved in the inactivation. The lack of TRPV1 sensitizers, such as phosphatylinositol 4,5-bisphosphate, in the ER could contribute to decrease the affinity for capsaicin. The low sensitivity of TRPV1ER to agonists may be critical for neuron health, because otherwise Ca2+ depletion of ER could lead to ER stress, unfolding protein response, and cell death. 相似文献