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1.
Human immunodeficiency virus type 1 (HIV-1) release efficiency is directed by late (L) domain motifs in the viral structural precursor polyprotein Gag, which serve as links to the ESCRT (endosomal sorting complex required for transport) machinery. Linkage is normally through binding of Tsg101, an ESCRT-1 component, to the P7TAP motif in the p6 region of Gag. In its absence, budding is directed by binding of Alix, an ESCRT adaptor protein, to the LY36PXnL motif in Gag. We recently showed that budding requires activation of the inositol 1,4,5-triphosphate receptor (IP3R), a protein that “gates” Ca2+ release from intracellular stores, triggers Ca2+ cell influx and thereby functions as a major regulator of Ca2+ signaling. In the present study, we determined whether the L domain links Gag to Ca2+ signaling machinery. Depletion of IP3R and inactivation of phospholipase C (PLC) inhibited budding whether or not Tsg101 was bound to Gag. PLC hydrolysis of phosphatidylinositol-(4,5)-bisphosphate generates inositol (1,4,5)-triphosphate, the ligand that activates IP3R. However, with Tsg101 bound, Gag release was independent of Gq-mediated activation of PLC, and budding was readily enhanced by pharmacological stimulation of PLC. Moreover, IP3R was redistributed to the cell periphery and cytosolic Ca2+ was elevated, events indicative of induction of Ca2+ signaling. The results suggest that L domain function, ESCRT machinery and Ca2+ signaling are linked events in Gag release. 相似文献
2.
Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition. 相似文献
3.
Ma YH Wei HW Su KH Ives HE Morris RC 《American journal of physiology. Cell physiology》2004,286(1):C112-C118
Cl is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca2+ concentration ([Ca2+]i) is Cl dependent. After incubating the cells at different extracellular Cl concentration ([Cl]e) for 40 min, the ANG II-induced Ca2+ transients at 120 meq/l Cl were more than twice those at either 80 or 20 meq/l Cl. Replacing Cl with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca2+, ANG II-induced as well as platelet-derived growth factor-induced Ca2+ release exhibited Cl dependency. The difference of Ca2+ release with high vs. low [Cl]e was not affected by acutely altering [Cl]e 1 min before administration of ANG II when [Cl]i was yet to be equilibrated with [Cl]e. Pretreatment of a Cl channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca2+ release and entry at 20 meq/l Cl but did not alter those at 120 meq/l Cl. However, after equilibration, a reduced [Cl]e did not affect thapsigargin-induced Ca2+ release, suggesting that Cl may not affect the size of intracellular Ca2+ stores. Nevertheless, at high [Cl], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] induced by ANG II was approximately sixfold that at low [Cl]. Thus the Cl-dependent effects of ANG II on Ca2+ transients may be mediated, at least in part, by a Cl-dependent Ins(1,4,5)P3 accumulation in VSMC. anion; inositol 1,4,5-trisphosphate; Ca2+ release 相似文献
4.
Abstract: Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3–4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2+-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+], changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information. 相似文献
5.
The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated intracellular Ca2+ channels. We previously identified an IP3R binding protein, IRBIT, which binds to the IP3 binding domain of IP3R and is dissociated from IP3R in the presence of IP3. In the present study, we showed that IRBIT suppresses the activation of IP3R by competing with IP3 by [3H]IP3 binding assays, in vitro Ca2+ release assays, and Ca2+ imaging of intact cells. Multiserine phosphorylation of IRBIT was essential for the binding, and 10 of the 12 key amino acids in IP3R for IP3 recognition participated in binding to IRBIT. We propose a unique mode of IP3R regulation in which IP3 sensitivity is regulated by IRBIT acting as an endogenous "pseudoligand" whose inhibitory activity can be modulated by its phosphorylation status. 相似文献
6.
Kanae Sasaki-Osugi Chiaki Imoto Terunao Takahara Hideki Shibata Masatoshi Maki 《The Journal of biological chemistry》2013,288(46):33361-33375
7.
Joshua T. Maxwell Sankar Natesan Gregory A. Mignery 《The Journal of biological chemistry》2012,287(47):39419-39428
InsP3-mediated calcium release through the type 2 inositol 1,4,5-trisphosphate receptor (InsP3R2) in cardiac myocytes results in the activation of associated CaMKII, thus enabling the kinase to act on downstream targets, such as histone deacetylases 4 and 5 (HDAC4 and HDAC5). The CaMKII activity also feedback modulates InsP3R2 function by direct phosphorylation and results in a dramatic decrease in the receptor-channel open probability (Po). We have identified S150 in the InsP3R2 core suppressor domain (amino acids 1–225) as the specific residue that is phosphorylated by CaMKII. Site-directed mutagenesis reveals that S150 is the CaMKII phosphorylation site responsible for modulation of channel activity. Nonphosphorylatable (S150A) and phosphomimetic (S150E) mutations were studied in planar lipid bilayers. The InsP3R2 S150A channel showed no decrease in activity when treated with CaMKII. Conversely, the phosphomimetic (S150E) channel displayed a very low Po under normal recording conditions in the absence of CaMKII (2 μm InsP3 and 250 nm [Ca2+]FREE) and mimicked a WT channel that has been phosphorylated by CaMKII. Phopho-specific antibodies demonstrate that InsP3R2 Ser-150 is phosphorylated in vivo by CaMKIIδ. The results of this study show that serine 150 of the InsP3R2 is phosphorylated by CaMKII and results in a decrease in the channel open probability. 相似文献
8.
We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca2+]i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca2+]i. In cells clamped at −60 mV, ANG II (10 μM) or ATP (100 μM) induced an oscillatory inward current. Caffeine (5 μM) induced only one transient inward current. In control conditions, the reversal potential (Erev) of these currents was close to the equilibrium potential for Cl− ions (ECl = −2.1 mV) and was shifted towards more positive values in low-Cl− solutions. Niflumic acid (10–50 μM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca2+]i by Indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca2+]i, whereas the caffeine-induced current was accompanied by only one transient increase in [Ca2+]i Niflumic acid (25 μM) had no effect on agonist-induced [Ca2+]i responses, whereas thapsigargin (1 μM) abolished both membrane current and the [Ca2+]i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca2+]i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 μM) or nifedipine (1 μM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca2+]i and that both oscillatory phenomena are primarily IP3 mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca2+ channels. The resulting Ca2+ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade. 相似文献
9.
Hideaki Ando Katsuhiro KawaaiKatsuhiko Mikoshiba 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2014
IRBIT (also called AHCYL1) was originally identified as a binding protein of the intracellular Ca2 + channel inositol 1,4,5-trisphosphate (IP3) receptor and functions as an inhibitory regulator of this receptor. Unexpectedly, many functions have subsequently been identified for IRBIT including the activation of multiple ion channels and ion transporters, such as the Na+/HCO3− co-transporter NBCe1-B, the Na+/H+ exchanger NHE3, the Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), and the Cl−/HCO3− exchanger Slc26a6. The characteristic serine-rich region in IRBIT plays a critical role in the functions of this protein. In this review, we describe the evolution, domain structure, expression pattern, and physiological roles of IRBIT and discuss the potential molecular mechanisms underlying the coordinated regulation of these diverse ion channels/transporters through IRBIT. 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
10.
Protein kinase C contributes to desensitization of ANG II signaling in adult rat cardiac fibroblasts 总被引:1,自引:0,他引:1
Meszaros JG Raphael R Lio FM Brunton LL 《American journal of physiology. Cell physiology》2000,279(6):C1978-C1985
We have studiedGq-linked ANG II signaling [inositol phosphate (IP)accumulation, Ca2+ mobilization] in primary cultures ofrat cardiac fibroblasts (CFs) and have found that ANG II initiates aprotein kinase C (PKC)-mediated negative feedback loop that rapidlyterminates the ANG II response. Pharmacological inhibition of PKC bystaurosporine and GF-109203X doubled IP production over that achievedin response to ANG II alone. Inhibition of PKC also led to largerCa2+ transients in response to ANG II, suggesting thatCa2+ mobilization was proportional toGq-phospholipase C-IP3 activity underthe conditions studied. Depletion of cellular PKC by overnight treatment with phorbol 12-myristate 13-acetate (PMA) similarly augmented ANG II-induced IP production. Acute activation of PKC by PMAhalved IP formation, with an EC501 nM; 4-PMA wasinactive. Time course data demonstrated that ANG II-mediated IPproduction fully desensitized within 30 s; PKC inhibition reducedthe rate and extent of this desensitization. In cells desensitized toANG II, a purinergic agonist still mobilized intracellularCa2+, indicating that desensitization was homologous. TheANG II-induced Ca2+ signal was fully resensitized within 30 min. The data demonstrate that a large portion of theIP-Ca2+ responses of rat CFs to ANG II are short-livedbecause of rapid, PKC-mediated desensitization. 相似文献
11.
12.
《Journal of molecular biology》2019,431(7):1440-1459
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93–based mechanism-of-action studies and drug discovery efforts. 相似文献
13.
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
14.
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
15.
Michiko Yamasaki-Mann Angelo Demuro Ian Parker 《The Journal of biological chemistry》2010,285(32):25053-25061
In addition to its well established function in activating Ca2+ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca2+ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca2+ signals via changes in ER Ca2+ store content, by imaging Ca2+ liberation through inositol trisphosphate receptors (IP3R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca2+] was transiently elevated by applying voltage-clamp pulses to induce Ca2+ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca2+ signals evoked by strong photorelease of IP3, and increased numbers of local Ca2+ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca2+ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca2+ on the IP3R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca2+ may modulate IP3R function, in addition to simply determining the size of the available store and the electrochemical driving force for release. 相似文献
16.
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
17.
《Cell calcium》2020
Type 3 Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R3s) have been identified as anti-oncogenic channels by propelling pro-apoptotic Ca2+ signals to mitochondria. Yet, recent studies (Rezuchova et al, Cell Death Dis, 2019; Ueasilamongkol et al, Hepathology, 2019; Guerra et al, Gut, 2019) revealed that IP3R3 upregulation drives oncogenesis via ER-mitochondrial Ca2+ crosstalk, adding complexity to IP3R3’s role in cancer. 相似文献
18.
Wataru Masuda Matthew J. Betzenhauser David I. Yule 《The Journal of biological chemistry》2010,285(48):37927-37938
Ca2+ release through inositol 1,4,5-trisphosphate receptors (InsP3R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca2+ signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca2+ release via InsP3R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP3R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP3R-1 subtype resulted in enhanced Ca2+ release in the absence of IRAG expression. In contrast, IRAG bound to each InsP3R subtype, and phosphorylation of IRAG by PKG attenuated Ca2+ release through all InsP3R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca2+ release through InsP3R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP3R-2. Phosphorylation of IRAG resulted in reduced Ca2+ release through all InsP3R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways. 相似文献
19.
Yu-Mi Yang Jiae Lee Hae Jo Soonhong Park Inik Chang Shmuel Muallem Dong Min Shin 《The Journal of biological chemistry》2014,289(36):24971-24979
Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca2+-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca2+ signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca2+ signaling in parotid gland acinar cells using Homer2-deficient (Homer2−/−) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca2+-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca2+-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca2+ extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca2+ clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca2+ signaling in parotid acinar cells. 相似文献
20.
《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