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1.
Transient receptor potential protein 1 (Trp1) has been proposed as a component of the store-operated Ca(2+) entry (SOCE) channel. However, the exact mechanism by which Trp1 is regulated by store depletion is not known. Here, we examined the role of the Trp1 C-terminal domain in SOCE by expressing hTrp1alpha lacking amino acids 664-793 (DeltaTrp1alpha) or full-length hTrp1alpha in the HSG (human submandibular gland) cell line. Both carbachol (CCh) and thapsigargin (Tg) activated sustained Ca(2+) influx in control (nontransfected), DeltaTrp1alpha-, and Trp1alpha-expressing cells. Sustained [Ca(2+)](i), following stimulation with either Tg or CCh in DeltaTrp1alpha-expressing cells, was about 1.5-2-fold higher than in Trp1alpha-expressing cells and 4-fold higher than in control cells. Importantly, (i) basal Ca(2+) influx and (ii) Tg- or CCh-stimulated internal Ca(2+) release were similar in all the cells. A similar increase in Tg-stimulated Ca(2+) influx was seen in cells expressing Delta2Trp1alpha, lacking the C-terminal domain amino acid 649-793, which includes the EWKFAR sequence. Further, both inositol 1,4,5-trisphosphate receptor-3 and caveolin-1 were immunoprecipitated with DeltaTrp1alpha and Trp1alpha. In aggregate, these data suggest that (i) the EWKFAR sequence does not contribute significantly to the Trp1-associated increase in SOCE, and (ii) the Trp1 C-terminal region, amino acids 664-793, is involved in the modulation of SOCE. 相似文献
2.
Coassembly of Trp1 and Trp3 proteins generates diacylglycerol- and Ca2+-sensitive cation channels 总被引:4,自引:0,他引:4
Lintschinger B Balzer-Geldsetzer M Baskaran T Graier WF Romanin C Zhu MX Groschner K 《The Journal of biological chemistry》2000,275(36):27799-27805
To analyze the functional consequences of coassembly of transient receptor potential 1 (Trp1) and Trp3 channel proteins, we characterized membrane conductances and divalent cation entry derived by separate overexpression and by coexpression of both Trp isoforms. Trp1 expression generated a 1-oleoyl-2-acetyl-sn-glycerol (OAG)-activated conductance that was detectable only in Ca(2+)-free extracellular solution. Trp3 expression gave rise to an OAG-activated conductance that was suppressed but clearly detectable at physiological Ca(2+) concentrations. Coexpression of both species resulted in a constitutively active, OAG-sensitive conductance, which exhibited distinctive cation selectivity and high sensitivity to inhibition by intracellular Ca(2+). Trp1-expressing cells displayed only modest carbachol-induced Ca(2+) entry and lacked OAG-induced Sr(2+) entry, whereas Trp3-expressing cells responded to both agents with a substantial divalent cation entry. Coexpression of Trp1 plus Trp3 suppressed carbachol-induced Ca(2+) entry compared with Trp3 expression and abolished OAG-induced Sr(2+) entry signals. We concluded that coassembly of Trp1 and Trp3 resulted in the formation of oligomeric Trp channels that are subject to regulation by phospholipase C and Ca(2+). The distinguished Ca(2+) sensitivity of these Trp1/Trp3 hetero-oligomers appeared to limit Trp-mediated Ca(2+) signals and may be of importance for negative feedback control of Trp function in mammalian cells. 相似文献
3.
Physical interaction between transient receptor potential (Trp) channels and inositol 1,4,5-trisphosphate receptors (IP(3)Rs) has been presented as a candidate mechanism for the activation of store-mediated Ca(2+) entry. The role of a human homologue of Drosophila transient receptor potential channel, hTrp1, in the conduction of store-mediated Ca(2+) entry was examined in human platelets. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 557-571 of hTrp1, inhibited both store depletion-induced Ca(2+) and Mn(2+) entry in a concentration-dependent manner. Stimulation of platelets with the physiological agonist thrombin activated coupling between the IP(3) receptor type II and endogenously expressed hTrp1. This event was reversed by refilling of the internal Ca(2+) stores but maintained after removal of the agonist if the stores were not allowed to refill. Inhibition of IP(3) recycling using Li(+) or inhibition of IP(3)Rs with xestospongin C or treatment with jasplakinolide, to stabilize the cortical actin filament network, abolished thrombin-induced coupling between hTrp1 and IP(3)R type II. Incubation with the anti-hTrp1 antibody inhibited thrombin-evoked Ca(2+) entry without affecting Ca(2+) release from intracellular stores. These results provide evidence for the involvement of hTrp1 in the activation of store-mediated Ca(2+) entry by coupling to IP(3)R type II in normal human cells. 相似文献
4.
Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, I(SOC), in A549 and endothelial cells. Twenty different 'chimeric' 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a I(SOC), which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 microM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca(2+) response to thapsigargin by 25% and similarly reduced I(SOC) by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion. 相似文献
5.
Thyagarajan B Poteser M Romanin C Kahr H Zhu MX Groschner K 《The Journal of biological chemistry》2001,276(51):48149-48158
The role of Trp3 in cellular regulation of Ca(2+) entry by NO was studied in human embryonic kidney (HEK) 293 cells. In vector-transfected HEK293 cells (controls), thapsigargin (TG)-induced (capacitative Ca(2+) entry (CCE)-mediated) intracellular Ca(2+) signals and Mn(2+) entry were markedly suppressed by the NO donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (3 microm) or by authentic NO (100 microm). In cells overexpressing Trp3 (T3-9), TG-induced intracellular Ca(2+) signals exhibited an amplitude similar to that of controls but lacked sensitivity to inhibition by NO. Consistently, NO inhibited TG-induced Mn(2+) entry in controls but not in T3-9 cells. Moreover, CCE-mediated Mn(2+) entry into T3-9 cells exhibited a striking sensitivity to inhibition by extracellular Ca(2+), which was not detectable in controls. Suppression of mitochondrial Ca(2+) handling with the uncouplers carbonyl cyanide m-chlorophenyl hydrazone (300 nm) or antimycin A(1) (-AA(1)) mimicked the inhibitory effect of NO on CCE in controls but barely affected CCE in T3-9 cells. T3-9 cells exhibited enhanced carbachol-stimulated Ca(2+) entry and clearly detectable cation currents through Trp3 cation channels. NO as well as carbonyl cyanide m-chlorophenyl hydrazone slightly promoted carbachol-induced Ca(2+) entry into T3-9 cells. Simultaneous measurement of cytoplasmic Ca(2+) and membrane currents revealed that Trp3 cation currents are inhibited during Ca(2+) entry-induced elevation of cytoplasmic Ca(2+), and that this negative feedback regulation is blunted by NO. Our results demonstrate that overexpression of Trp3 generates phospholipase C-regulated cation channels, which exhibit regulatory properties different from those of endogenous CCE channels. Moreover, we show for the first time that Trp3 expression determines biophysical properties as well as regulation of CCE channels by NO and mitochondrial Ca(2+) handling. Thus, we propose Trp3 as a subunit of CCE channels. 相似文献
6.
Ca(2+) influx via plasma membrane Trp3 channels is proposed to be regulated by a reversible interaction with inositol trisphosphate receptor (IP(3)R) in the endoplasmic reticulum. Condensation of the cortical actin layer has been suggested to physically disrupt this interaction and inhibit Trp3-mediated Ca(2+) influx. This study examines the effect of cytoskeletal reorganization on the localization and function of Trp3 and key Ca(2+) signaling proteins. Calyculin-A treatment resulted in formation of condensed actin layer at the plasma membrane; internalization of Trp3, Galpha(q/11), phospholipase Cbeta, and caveolin-1; and attenuation of 1-oleoyl-2-acetyl-sn-glycerol- and ATP-stimulated Sr(2+) influx. Importantly, Trp3 and IP(3)R-3 remained co-localized inside the cell and were co-immunoprecipitated. Jasplakinolide also induced internalization of Trp3 and caveolin-1. Pretreatment of cells with cytochalasin D or staurosporine did not affect Trp3 but prevented calyculin-A-induced effects. Based on these data, we suggest that Trp3 is assembled in a caveolar Ca(2+) signaling complex with IP(3)R, SERCA, Galpha(q/11), phospholipase Cbeta, caveolin-1, and ezrin. Furthermore, our data demonstrate that conditions which stabilize cortical actin induce loss of Trp3 activity due to internalization of the Trp3-signaling complex, not disruption of IP(3)R-Trp3 interaction. This suggests that localization of the Trp3-associated signaling complex, rather than Trp3-IP(3)R coupling, depends on the status of the actin cytoskeleton. 相似文献
7.
Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells 总被引:4,自引:0,他引:4
Liu X Wang W Singh BB Lockwich T Jadlowiec J O'Connell B Wellner R Zhu MX Ambudkar IS 《The Journal of biological chemistry》2000,275(5):3403-3411
The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells. 相似文献
8.
Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains 总被引:12,自引:0,他引:12
Lockwich TP Liu X Singh BB Jadlowiec J Weiland S Ambudkar IS 《The Journal of biological chemistry》2000,275(16):11934-11942
Trp1 has been proposed as a component of the store-operated Ca(2+) entry (SOC) channel. However, neither the molecular mechanism of SOC nor the role of Trp in this process is yet understood. We have examined possible molecular interactions involved in the regulation of SOC and Trp1 and report here for the first time that Trp1 is assembled in signaling complex associated with caveolin-scaffolding lipid raft domains. Endogenous hTrp1 and caveolin-1 were present in low density fractions of Triton X-100-extracted human submandibular gland cell membranes. Depletion of plasma membrane cholesterol increased Triton X-100 solubility of Trp1 and inhibited carbachol-stimulated Ca(2+) signaling. Importantly, thapsigargin stimulated Ca(2+) influx, but not internal Ca(2+) release, and inositol 1,4,5-triphosphate (IP(3))-stimulated I(soc) were also attenuated. Furthermore, both anti-Trp1 and anti-caveolin-1 antibodies co-immunoprecipitated hTrp1, caveolin-1, Galpha(q/11), and IP(3) receptor-type 3 (IP(3)R3). These results demonstrate that caveolar microdomains provide a scaffold for (i) assembly of key Ca(2+) signaling proteins into a complex and (ii) coordination of the molecular interactions leading to the activation of SOC. Importantly, we have shown that Trp1 is also localized in this microdomain where it interacts with one or more components of this complex, including IP(3)R3. This finding is potentially important in elucidating the physiological function of Trp. 相似文献
9.
The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.
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The transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5-trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, we find that Trp is associated into a multiprotein complex with the norpA-encoded phospholipase C, an eye-specific protein kinase C (InaC) and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein-protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog analyzed here is isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1-S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel. 相似文献
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12.
Conigrave AD Mun HC Delbridge L Quinn SJ Wilkinson M Brown EM 《The Journal of biological chemistry》2004,279(37):38151-38159
Parathyroid hormone (PTH) secretion is acutely regulated by the extracellular Ca(2+)-sensing receptor (CaR). Thus, Ca(2+) ions, and to a lesser extent Mg(2+) ions, have been viewed as the principal physiological regulators of PTH secretion. Herein we show that in physiological concentrations, l-amino acids acutely and reversibly activated the extracellular Ca(2+)-sensing receptor in normal human parathyroid cells and inhibited parathyroid hormone secretion. Individual l-amino acids, especially of the aromatic and aliphatic classes, as well as plasma-like amino acid mixtures, stereoselectively mobilized Ca(2+) ions in normal human parathyroid cells in the presence but not the absence of the CaR agonists, extracellular Ca(2+) (Ca(2+)(o)), or spermine. The order of potency was l-Trp = l-Phe > l-His > l-Ala > l-Glu > l-Arg = l-Leu. CaR-active amino acids also acutely and reversibly suppressed PTH secretion at physiological ionized Ca(2+) concentrations. At a Ca(2+)(o) of 1.1 mm and an amino acid concentration of 1 mm, CaR-active amino acids (l-Phe = l-Trp > l-His = l-Ala), but not CaR-inactive amino acids (l-Leu and l-Arg), stereoselectively suppressed PTH secretion by up to 40%, similar to the effect of raising Ca(2+)(o) to 1.2 mm. A physiologically relevant increase in the -fold concentration of the plasma-like amino acid mixture (from 1x to 2x) also reversibly suppressed PTH secretion in the Ca(2+)(o) concentration range 1.05-1.25 mm. In conclusion, l-amino acids acutely and reversibly activate endogenous CaRs and suppress PTH secretion at physiological concentrations. The results indicate that l-amino acids are physiological regulators of PTH secretion and thus whole body calcium metabolism. 相似文献
13.
L-Arginine uptake and Ca(2+) changes in unstirred platelets activated by thrombin, collagen and Ca(2+) ionophore A23187 were evaluated. Thrombin did not affect L-arginine uptake at short incubation times (2-15 min), but at prolonged times slowed down the amino acid transport. Collagen was ineffective. A23187 decreased the L-arginine uptake in a dose-dependent manner, producing the maximal inhibition at 5 microM. In FURA 2-loaded platelets collagen did not modify Ca(2+) basal level, thrombin induced a late Ca(2+) rise and A23187 dose-dependently increased cytosolic Ca(2+), eliciting the highest increase at 5 microM. It is likely that L-arginine uptake is inversely modulated by Ca(2+) concentrations and is inhibited during platelet stimulation with agonists which induce cytosolic Ca(2+) elevation. 相似文献
14.
Yang M Gupta A Shlykov SG Corrigan R Tsujimoto S Sanborn BM 《Biology of reproduction》2002,67(3):988-994
Capacitative Ca2+ entry plays a role in thapsigargin- and oxytocin-mediated increases in intracellular free Ca2+ in human myometrium. Members of the Trp protein family have been implicated in capacitative Ca2+ entry in a number of tissues. Pregnant human myometrium and the human myometrial cell line PHM1-41 expressed mRNA for hTrp1, hTrp3, hTrp4, hTrp6, and hTrp7. A number of known splice variants of hTrp1 and hTrp4 were expressed in these cells. In addition, novel splice variants for hTrp1 and hTrp3 were discovered. hTrp1gamma1 and hTrp1gamma2 contain insertions between previously described exons 9 and 10 that would alter reading frame and produce Trp proteins truncated in the membrane spanning region if expressed. The hTrp3 variant introduces sequence between exons 8 and 9 that would insert 16 amino acids in the C-terminal region of the protein upstream of the calmodulin and inositol 1,4,5-triphosphate receptor interaction domain. hTrp1, hTrp3, and hTrp4 proteins were detected in both pregnant human myometrial and PHM1-41 membranes; a weak band consistent with hTrp6 expression was detected in pregnant human myometrium. These data are consistent with the presence of proteins that could form putative capacitative Ca2+ channels in human myometrium. Control of the activity of these channels may be important for the control of uterine contractile activity. 相似文献
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Silve C Petrel C Leroy C Bruel H Mallet E Rognan D Ruat M 《The Journal of biological chemistry》2005,280(45):37917-37923
The Ca(2+)-sensing receptor (CaSR) belongs to the class III G-protein-coupled receptors (GPCRs), which include receptors for pheromones, amino acids, sweeteners, and the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). These receptors are characterized by a long extracellular amino-terminal domain called a Venus flytrap module (VFTM) containing the ligand binding pocket. To elucidate the molecular determinants implicated in Ca(2+) recognition by the CaSR VFTM, we developed a homology model of the human CaSR VFTM from the x-ray structure of the metabotropic glutamate receptor type 1 (mGluR1), and a phylogenetic analysis of 14 class III GPCR VFTMs. We identified critical amino acids delineating a Ca(2+) binding pocket predicted to be adjacent to, but distinct from, a cavity reminiscent of the binding site described for amino acids in mGluRs, GABA-B receptor, and GPRC6a. Most interestingly, these Ca(2+)-contacting residues are well conserved within class III GPCR VFTMs. Our model was validated by mutational and functional analysis, including the characterization of activating and inactivating mutations affecting a single amino acid, Glu-297, located within the proposed Ca(2+) binding pocket of the CaSR and associated with autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia, respectively, genetic diseases characterized by perturbations in Ca(2+) homeostasis. Altogether, these data define a Ca(2+) binding pocket within the CaSR VFTM that may be conserved in several other class III GPCRs, thereby providing a molecular basis for extracellular Ca(2+) sensing by these receptors. 相似文献
17.
A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP 总被引:65,自引:0,他引:65
J D Clark L L Lin R W Kriz C S Ramesha L A Sultzman A Y Lin N Milona J L Knopf 《Cell》1991,65(6):1043-1051
We report the cloning and expression of a cDNA encoding a high molecular weight (85.2 kd) cytosolic phospholipase A2 (cPLA2) that has no detectable sequence homology with the secreted forms of PLA2. We show that cPLA2 selectively cleaves arachidonic acid from natural membrane vesicles and demonstrate that cPLA2 translocates to membrane vesicles in response to physiologically relevant changes in free calcium. Moreover, we demonstrate that an amino-terminal 140 amino acid fragment of cPLA2 translocates to natural membrane vesicles in a Ca(2+)-dependent fashion. Interestingly, we note that this 140 amino acid domain of cPLA2 contains a 45 amino acid region with homology to PKC, p65, GAP, and PLC. We suggest that this homology delineates a Ca(2+)-dependent phospholipid-binding motif, providing a mechanism for the second messenger Ca2+ to translocate and activate cytosolic proteins. 相似文献
18.
Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid. 总被引:23,自引:0,他引:23
K Bandoh J Aoki H Hosono S Kobayashi T Kobayashi K Murakami-Murofushi M Tsujimoto H Arai K Inoue 《The Journal of biological chemistry》1999,274(39):27776-27785
Lysophosphatidic acid (LPA), together with sphingosine 1-phosphate, is a bioactive lipid mediator that acts on G-protein-coupled receptors to evoke multiple cellular responses, including Ca(2+) mobilization, modulation of adenylyl cyclase, and mitogen-activated protein (MAP) kinase activation. In this study, we isolated a human cDNA encoding a novel G-protein-coupled receptor, designated EDG7, and characterized it as a cellular receptor for LPA. The amino acid sequence of the EDG7 protein is 53.7 and 48.8% identical to those of the human functional LPA receptors EDG2 and EDG4, respectively, previously identified. LPA (oleoyl) but not other lysophospholipids induced an increase in the [Ca(2+)](i) of EDG7-overexpressing Sf9 cells. Other LPA receptors, EDG4 but not EDG2, transduced the Ca(2+) response by LPA when expressed in Sf9 cells. LPAs with an unsaturated fatty acid but not with a saturated fatty acid induced an increase in the [Ca(2+)](i) of EDG7-expressing Sf9 cells, whereas LPAs with both saturated and unsaturated fatty acids elicited a Ca(2+) response in Sf9 cells expressing EDG4. In EDG7- or EDG4-expressing Sf9 cells, LPA stimulated forskolin-induced increase in intracellular cAMP levels, which was not observed in EDG2-expressing cells. In PC12 cells, EDG4 but not EDG2 or EDG7 mediated the activation of MAP kinase by LPA. Neither the EDG7- nor EDG4-transduced Ca(2+) response or cAMP accumulation was inhibited by pertussis toxin. In conclusion, the present study demonstrates that EDG7, a new member of the EDG family of G-protein-coupled receptors, is a specific LPA receptor that shows distinct properties from known cloned LPA receptors in ligand specificities, Ca(2+) response, modulation of adenylyl cyclase, and MAP kinase activation. 相似文献
19.
Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K(+)), calcium (Ca(2+)) and transient receptor potential (Trp) channels, but not sodium (Na(+)) channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K(+) and Trp channel subunits, and genes encoding novel homologues of voltage-gated K(v) channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca(2+) channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca(2+) channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca(2+) uniporter (MCU). In contrast to humans, which express many K(+), Ca(2+) and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K(+), Ca(2+) and Trp channel homologues. Furthermore, the sequences of fungal K(+), Ca(2+), Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs. 相似文献
20.
Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells 总被引:7,自引:0,他引:7
Chu ST Cheng HH Huang CJ Chang HC Chi CC Su HH Hsu SS Wang JL Chen IS Liu SI Lu YC Huang JK Ho CM Jan CR 《Life sciences》2007,80(4):364-369
Melittin, a peptide from bee venom, is thought to be a phospholipase A(2) activator and Ca(2+) influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca(2+)](i) and killed cells in MG63 human osteosarcoma cells. [Ca(2+)](i) changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was abolished by removing extracellular Ca(2+). Melittin-induced Ca(2+) entry was confirmed by Mn(2+) quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca(2+)-insensitive. The melittin-induced Ca(2+) influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A(2) with AACOCF(3) and aristolochic acid; but was substantially inhibited by blocking L-type Ca(2+) channels. At concentrations of 0.5 microM and 1 microM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 microM melittin was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca(2+)](i) increase by causing Ca(2+) entry through L-type Ca(2+) channels in a manner independent of protein kinase-C and phospholipase A(2) activity; and this [Ca(2+)](i) increase subsequently caused apoptosis. 相似文献