共查询到20条相似文献,搜索用时 15 毫秒
1.
Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. 相似文献
2.
Bernard T. Drumm Gerard P. Sergeant Mark A. Hollywood Keith D. Thornbury Noel G. McHale Brian J. Harvey 《Cell calcium》2014
Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca2+ waves that rely on Ca2+ release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca2+ waves in ICC. Intracellular [Ca2+] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10 μM) significantly inhibited the generation of spontaneous Ca2+ waves in ICC and this was associated with a significant decrease in the ER Ca2+ load, measured with 10 mM caffeine responses. Ca2+ waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1 mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP3Rs) with 10 μM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca2+ wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca2+ load sufficient to inactivate IP3Rs but not RyRs. 相似文献
3.
Aims
We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ1-receptor (σ1R), ameliorates cardiac hypertrophy and dysfunction via σ1R stimulation. Although σ1R on non-cardiomyocytes interacts with the IP3 receptor (IP3R) to promote mitochondrial Ca2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.Main methods
Here we performed Ca2 + imaging and measured ATP production to define the role of σ1Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.Key finding
These cardiomyocytes exhibited imbalances in expression levels of σ1R and IP3R and impairments in both phenylephrine-induced mitochondrial Ca2 + mobilization from the SR and ATP production. Interestingly, σ1R stimulation with fluvoxamine rescued impaired mitochondrial Ca2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ1R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ1Rs suppressed intracellular Ca2 + mobilization through IP3Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.Significance
These results suggest that σ1R stimulation with fluvoxamine promotes SR-mitochondrial Ca2 + transport and mitochondrial ATP production, whereas σ1R stimulation suppresses intracellular Ca2 + overload through IP3Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ1R agonists including fluvoxamine. 相似文献4.
Joseph SK Hajnóczky G 《Apoptosis : an international journal on programmed cell death》2007,12(5):951-968
Inositol 1,4,5-trisphosphate receptors (IP3Rs) serve to discharge Ca2+ from ER stores in response to agonist stimulation. The present review summarizes the role of these receptors in models of
Ca2+-dependent apoptosis. In particular we focus on the regulation of IP3Rs by caspase-3 cleavage, cytochrome c, anti-apoptotic proteins and Akt kinase. We also address the evidence that some of the effects of IP3Rs in apoptosis may be independent of their ion-channel function. The role of IP3Rs in delivering Ca2+ to the mitochondria is discussed from the perspective of the factors determining inter-organellar dynamics and the spatial
proximity of mitochondria and ER membranes. 相似文献
5.
Yaping Lin-Moshier Peter J. Sebastian LeeAnn Higgins Natalie D. Sampson Jane E. Hewitt Jonathan S. Marchant 《The Journal of biological chemistry》2013,288(1):355-367
Changes in cytoplasmic Ca2+ concentration, resulting from activation of intracellular Ca2+ channels within the endoplasmic reticulum, regulate several aspects of cellular growth and differentiation. Ca2+ homeostasis endoplasmic reticulum protein (CHERP) is a ubiquitously expressed protein that has been proposed as a regulator of both major families of endoplasmic reticulum Ca2+ channels, inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), with resulting effects on mitotic cycling. However, the manner by which CHERP regulates intracellular Ca2+ channels to impact cellular growth is unknown. Here, we challenge previous findings that CHERP acts as a direct cytoplasmic regulator of IP3Rs and RyRs and propose that CHERP acts in the nucleus to impact cellular proliferation by regulating the function of the U2 snRNA spliceosomal complex. The previously reported effects of CHERP on cellular growth therefore are likely indirect effects of altered spliceosomal function, consistent with prior data showing that loss of function of U2 snRNP components can interfere with cell growth and induce cell cycle arrest. 相似文献
6.
A rise in the intracellular concentration of ionized calcium ([Ca2+]i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca2+ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca2+ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca2+]i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP3Rs) is an important determinant of the [Ca2+]i dynamics and recruits neighboring Ca2+-release sites to generate [Ca2+]i waves; (v) [Ca2+]i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca2+-dependent K+ and Cl- channels sense the local changes in [Ca2+]i during a Ca2+ spark and thereby may couple changes in [Ca2+]i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI
cat) does not mirror changes in [Ca2+]i, thus reflecting the complexity of [Ca2+]i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca2+ release, either spontaneous or caffeine-induced, does not augment mI
cat; (ix) intracellular flash release of Ca2+ is less effective in augmentation of mI
cat than flash release of IP3, suggesting that IP3 may sensitize muscarinic cationic channels to Ca2+; (x) intracellular flash release of IP3 fails to augment mI
cat in SMCs, in which [Ca2+]i was strongly buffered, suggesting that IP3 exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca2+]i rather than depletion of the IP3-dependent Ca2+ store; and (xi) predominant expression of IP3R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP3R-mediated Ca2+ release and muscarinic cationic channel opening.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 455–465, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year. 相似文献
7.
Joon-Chul Kim Krishna P. Subedi Joung Real Ahn 《Progress in biophysics and molecular biology》2010,103(1):59-70
In atrial myocytes lacking t-tubules, action potential triggers junctional Ca2+ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca2+ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP3Rs) in atrial myocytes, and show our new findings on the role of IP3R subtype in the regulation of spontaneous focal Ca2+ releases in the compartmentalized areas of atrial myocytes. The Ca2+ sparks, representing focal Ca2+ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca2+ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP3Rs compared with ventricular cells and possess significant levels of type 1 IP3R (IP3R1) and type 2 IP3R (IP3R2). Over the last decade the roles of atrial IP3R on the enhancement of Ca2+-induced Ca2+ release and arrhythmic Ca2+ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca2+ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP3R1 in the maintenance of peri-nuclear and non-junctional Ca2+ sparks via stimulating a posttranslational organization of RyR clusters. 相似文献
8.
György Hajnóczky György Csordás Rajeshwari Krishnamurthy Gábor Szalai 《Journal of bioenergetics and biomembranes》2000,32(1):15-25
Many agonists bring about their effects on cellular functions through a rise incytosolic [Ca2+]([Ca2+]c) mediated by the second messenger inositol 1,4,5-trisphosphate (IP3). Imaging studiesof single cells have demonstrated that [Ca2+]c signals display cell specific spatiotemporalorganization that is established by coordinated activation of IP3 receptor Ca2+ channels.Evidence emerges that cytosolic calcium signals elicited by activation of the IP3 receptors areefficiently transmitted to the mitochondria. An important function of mitochondrial calciumsignals is to activate the Ca2+-sensitive mitochondrial dehydrogenases, and thereby to meetdemands for increased energy in stimulated cells. Activation of the permeability transitionpore (PTP) by mitochondrial calcium signals may also be involved in the control of cell death.Furthermore, mitochondrial Ca2+ transport appears to modulate the spatiotemporal organizationof [Ca2+]c responses evoked by IP3 and so mitochondria may be important in cytosolic calciumsignaling as well. This paper summarizes recent research to elucidate the mechanisms andsignificance of IP3-dependent mitochondrial calcium signaling. 相似文献
9.
《Cell calcium》2017
The inositol trisphosphate (IP3) signaling pathway evokes local Ca2+ signals (Ca2+ puffs) that arise from the concerted openings of clustered IP3 receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP3 into the cytosol. Photorelease of IP3 from a caged precursor provides a convenient and widely employed means to study the final stage of IP3-mediated Ca2+ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP3. Here, we address whether Ca2+ puffs evoked by photoreleased IP3 fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP3 analog, i-IP3. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1 μm), suggesting that IP3 from both sources accesses the same, or closely co-localized clusters of IP3Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP3 uniformly throughout the cytoplasm, our results imply that IP3 generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP3Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain. 相似文献
10.
Hristina Ivanova Tim Vervliet Ludwig Missiaen Jan B. ParysHumbert De Smedt Geert Bultynck 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2014
Cell-death and -survival decisions are critically controlled by intracellular Ca2 + homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a pivotal role in these processes by mediating Ca2 + flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca2 + signaling by directly targeting IP3R channels, which can happen in an IP3R-isoform-dependent manner. In this review, we will focus on how the different IP3R isoforms (IP3R1, IP3R2 and IP3R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP3Rs by cellular factors like IP3, Ca2 +, Ca2 +-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP3R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca2 + store in cell death and survival and how IP3Rs and pro-survival/pro-death proteins can modulate the basal ER Ca2 + leak. Third, we will review the regulation of the Ca2 +-flux properties of the IP3R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP3R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau. 相似文献
11.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2019,1866(7):1092-1100
Inositol 1,4,5-trisphosphate receptors (IP3R) are the most widely expressed intracellular Ca2+ release channels. Their activation by IP3 and Ca2+ allows Ca2+ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca2+] may directly regulate cytosolic effectors or fuel Ca2+ uptake by other organelles, while the decrease in ER luminal [Ca2+] stimulates store-operated Ca2+ entry (SOCE). We are close to understanding the structural basis of both IP3R activation, and the interactions between the ER Ca2+-sensor, STIM, and the plasma membrane Ca2+ channel, Orai, that lead to SOCE. IP3Rs are the usual means through which extracellular stimuli, through ER Ca2+ release, stimulate SOCE. Here, we review evidence that the IP3Rs most likely to respond to IP3 are optimally placed to allow regulation of SOCE. We also consider evidence that IP3Rs may regulate SOCE downstream of their ability to deplete ER Ca2+ stores. Finally, we review evidence that IP3Rs in the plasma membrane can also directly mediate Ca2+ entry in some cells. 相似文献
12.
Sarcoplasmic reticulum contains the internal Ca2+ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca2+ level is the same all throughout the SR; however, whether the Ca2+ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca2+ buffer capacity of the SR by comparing the reduction in SR Ca2+ levels with the corresponding increase in [Ca2+]i during activation of either IP3Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca2+ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca2+ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca2+ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca2+ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca2+ influx, indicate the relevance of luminal SR Ca2+ buffer capacity in the [Ca2+]i response. To further study the importance of luminal SR Ca2+ buffer capacity in the release process we used low levels of heparin to partially inhibit IP3Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca2+ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca2+-binding proteins in the high-capacity, low-affinity conformation, particularly for IP3R-mediated Ca2+ release. 相似文献
13.
D. R. Laver L. D. Roden G. P. Ahern K. R. Eager P. R. Junankar A. F. Dulhunty 《The Journal of membrane biology》1995,147(1):7-22
Ca2+-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca2+ inhibited cardiac RyRs with an average K m = 15 mm, skeletal RyRs with K m = 0.7 mm and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca2+ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [3H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca2+-inhibition in cardiac RyRs (Chu, Fill, Stefani &; Entman (1993) J. Membrane Biol. 135, 49–59). Ryanodine prevented Ca2+ from inhibiting either cardiac or skeletal RyRs. Ca2+-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mm Cs+, but not by 500 mm K+, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca2+-activation. Ca2+-inhibition in skeletal RyRs was retained in the presence of 500 mm Cs+. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mm Cs+ in the solutions fail to demonstrate Ca2+-inhibition, while Ca2+-inhibition of Ca2+ release is observed in vesicle studies where K+ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca2+-inhibition in skeletal RyRs and cardiac RyRs, with different Ca2+ affinities for inhibition. We conclude that differences in the Ca2+-inhibition in cardiac and skeletal channels depends on their Ca2+ binding properties. 相似文献
14.
Jean-Paul DecuypereGiovanni Monaco Geert BultynckLudwig Missiaen Humbert De SmedtJan B. Parys 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2011,1813(5):1003-1013
The amount of Ca2+ taken up in the mitochondrial matrix is a crucial determinant of cell fate; it plays a decisive role in the choice of the cell between life and death. The Ca2+ ions mainly originate from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores of the endoplasmic reticulum (ER). The uptake of these Ca2+ ions in the mitochondria depends on the functional properties and the subcellular localization of the IP3 receptor (IP3R) in discrete domains near the mitochondria. To allow for an efficient transfer of the Ca2+ ions from the ER to the mitochondria, structural interactions between IP3Rs and mitochondria are needed. This review will focus on the key proteins involved in these interactions, how they are regulated, and what are their physiological roles in apoptosis, necrosis and autophagy. This article is part of a Special Issue entitled: 11th European Symposium on Calcium. 相似文献
15.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2021,1868(12):119121
Recently, a functional IP3R ortholog (CO.IP3R-A) capable of IP3-induced Ca2+ release has been discovered in Capsaspora owczarzaki, a close unicellular relative to Metazoa. In contrast to mammalian IP3Rs, CO.IP3R-A is not modulated by Ca2+, ATP or PKA. Protein-sequence analysis revealed that CO.IP3R-A contained a putative binding site for anti-apoptotic Bcl-2, although Bcl-2 was not detected in Capsaspora owczarzaki and only appeared in Metazoa. Here, we examined whether human Bcl-2 could form a complex with CO.IP3R-A channels and modulate their Ca2+-flux properties using ectopic expression approaches in a HEK293 cell model in which all three IP3R isoforms were knocked out. We demonstrate that human Bcl-2 via its BH4 domain could functionally interact with CO.IP3R-A, thereby suppressing Ca2+ flux through CO.IP3R-A channels. The BH4 domain of Bcl-2 was sufficient for interaction with CO.IP3R-A channels. Moreover, mutating the Lys17 of Bcl-2's BH4 domain, the residue critical for Bcl-2-dependent modulation of mammalian IP3Rs, abrogated Bcl-2's ability to bind and inhibit CO.IP3R-A channels. Hence, this raises the possibility that a unicellular ancestor of animals already had an IP3R that harbored a Bcl-2-binding site. Bcl-2 proteins may have evolved as controllers of IP3R function by exploiting this pre-existing site, thereby counteracting Ca2+-dependent apoptosis. 相似文献
16.
Diptiman D. Bose 《Biochemical and biophysical research communications》2009,379(2):594-133
Regulation of bi-directional communication between intracellular Ca2+ pools and surface Ca2+ channels remains incompletely characterized. We report Ca2+ release mediated by inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca2+ release, Ca2+ influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP3R induced Ca2+ release, whereas RyR-mediated Ca2+ release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP3R and RyR linked Ca2+ influx even though Ca2+ pool sensitivities were different. These findings suggest discrete Ca2+ store/Ca2+ channel coupling mechanisms in the IP3R and RyR pathways as revealed by the differential sensitivity to actin perturbation. 相似文献
17.
We recently reported key physiologic roles for Ca2+-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca2+-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca2+, inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the sarcoplasmic reticulum represents a potential Ca2+ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP3Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca2+ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca2+-mediated TRPM4-IP3R regulatory mechanism. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP3Rs with the selective IP3R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP3Rs have a key role in mediating the Ca2+-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM. 相似文献
18.
Using the voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea
pigtaenia coli. In our study, we identified and studied a charibdotoxin-sensitive component of Ca2+-dependent K+ current carried through the channels of high conductance (in most publications called “big conductance,”I
BK(Ca)). This component was completely blocked by 100 nM charibdotoxin and by tetraethylammonium in concentrations as low as 1 mM.I
BK(Ca) demonstrated fast kinetics of inactivation, which nearly coincided with that of Ca2+ current. In addition to the dependence on Ca2+ concentration, this current also showed voltage-dependent properties: with a rise in the level of depolarization its amplitude
increased. In many cells, depolarizing shifts in the membrane potential evoke spontaneous outward currents. Such currents
probably represent the secondary effect of cyclic Ca2+ release from the caffeine-sensitive intracellular stores that result in short-term activation of charibdotoxin-sensitive
Ca2+-dependent K+ channels. 相似文献
19.
Puffs are local Ca2+ signals that arise by Ca2+ liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP3Rs). The locations of puff sites observed by Ca2+ imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP3Rs have shown that the majority of IP3Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP3Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ∼70% of the IP3R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ∼0.095 μm s−1, whereas the remaining molecules were essentially immotile. A fraction of the immotile IP3Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP3R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP3Rs were apparent following activation of IP3/Ca2+ signaling. We conclude that stable clusters of small numbers of immotile IP3Rs may underlie local Ca2+ release sites, whereas the more numerous motile IP3Rs appear to be functionally silent. 相似文献
20.
Dionisio N Albarran L Berna-Erro A Hernandez-Cruz JM Salido GM Rosado JA 《Cellular signalling》2011,23(11):1850-1856