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1.
Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and (45)Ca(2+) flux from permeabilised COS cells, we demonstrated that CaBPs decrease the sensitivity of InsP(3)-induced Ca(2+) release (IICR). In addition, we found a Ca(2+)-independent interaction between CaBP1 and the NH(2)-terminal 159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased InsP(3) binding to the receptor reminiscent of that observed for CaM. Unlike CaM, however, CaBPs do not inhibit ryanodine receptors, have a higher affinity for InsP(3)Rs and more potently inhibited IICR. We also show that phosphorylation of CaBP1 at a casein kinase 2 consensus site regulates its inhibition of IICR. Our data suggest that CaBPs are endogenous regulators of InsP(3)Rs tuning the sensitivity of cells to InsP(3).  相似文献   

2.
The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca2+ channel, in store-operated Ca2+ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3' and 5' rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-l polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca2+ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP3F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC50 = 0.5 microM), indicating that InsP3F and LPA principally activate store-operated Ca2+ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP3F-, LPA- or thapsigargin-stimulated Ca2+ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP3F-stimulated Ca2+ inflow and only a very small enhancement of LPA-stimulated Ca2+ in-flow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca2+ inflow through the previously-characterised Ca2+ -specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.  相似文献   

3.
An unusual inward current which is slowly elicited in the Xenopus oocyte membrane during sustained depolarization is reportedly carried by Na+. It is thought that Na+ selective channels are in some way induced to become voltage-sensitive by the depolarization. Earlier studies report that the induction process involves a phospholipase C and a protein kinase C as well as calcium ions. The present work investigated the origins of this calcium in the oocyte. We show that injection of the powerful Ca2+ chelator (BAPTA) in the oocyte, before induction of the Na+ channels, prevented the appearance of the Na+ current, confirming an important role for [Ca2+]i. However, in oocytes perfused with Ca2+ -free medium, induction of the channels could still be obtained, indicating that induction did not depend upon the entry of external Ca2+. Downmodulation of Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive stores with caffeine and with a low molecular weight heparin resulted in decreased or no Na+ currents. The results are discussed in terms of the contributions from other endogenous calcium-dependent conductances which can influence the Na+ current amplitudes and time courses. The results presented support the idea that intracellular Ca2+ increase principally due to Ca2+ released from InsP3-sensitive stores is needed by the enzyme systems to produce the depolarization-induced activation of the Na+ conductance in the Xenopus oocyte.  相似文献   

4.
The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of [Ca(2+)](i) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.  相似文献   

5.
Rapid and precise control of Na(+)/Ca(2+) exchanger (NCX1) activity is essential in the maintenance of beat-to-beat Ca(2+) homeostasis in cardiac myocytes. Here, we show that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, is a novel endogenous protein inhibitor of cardiac NCX1. Using a heterologous expression system that is devoid of both endogenous PLM and NCX1, we first demonstrated by confocal immunofluorescence studies that both exogenous PLM and NCX1 co-localized at the plasma membrane. Reciprocal co-immunoprecipitation studies revealed specific protein-protein interaction between PLM and NCX1. The functional consequences of direct association of PLM with NCX1 was the inhibition of NCX1 activity, as demonstrated by whole-cell patch clamp studies to measure NCX1 current density and radiotracer flux assays to assess Na(+)-dependent (45)Ca(2+) uptake. Inhibition of NCX1 by PLM was specific, because a single mutation of serine 68 to alanine in PLM resulted in a complete loss of inhibition of NCX1 current, although association of the PLM mutant with NCX1 was unaltered. In native adult cardiac myocytes, PLM co-immunoprecipitated with NCX1. We conclude that PLM, a member of the FXYD family of small ion transport regulators known to modulate Na(+)-K(+)-ATPase, also regulates Na(+)/Ca(2+) exchange in the heart.  相似文献   

6.
The effect of Ca2+ on inositol (1,4,5)-trisphosphate 3-kinase (3-kinase) activity was measured on Xenopus oocyte cytosolic extracts. The Ca2+-evoked elevation in 3-kinase activity appeared to be mediated by calmodulin (CaM) and the calmodulin-dependent protein kinase II (CaMKII). The results observed in vitro were totally retrieved in intact oocytes and tend to demonstrate the involvement of a CaMKII-mediated phosphorylation in the regulation of 3-kinase activity. Finally, electrophysiological recordings of InsP3-elicited chloride current transients in the presence of CaM/CaMKII inhibitors allowed to postulate an involvement of 3-kinase activity in the regulation of InsP3-mediated Ca2+ release.  相似文献   

7.
Mouse embryonic stem (mES) cells have the potential to differentiate into all types of cells, but the physiological properties of undifferentiated mES cells, including Ca2+ signaling systems, are not fully understood. In this study, we investigated Ca2+ signaling pathways in mES cells by using confocal Ca2+ imaging systems, patch clamp techniques and RT-PCR. The stimulations with ATP and histamine (His) induced a transient increase of intracellular Ca2+ concentration ([Ca2+]i), which were prevented by the pretreatment of 2-amino-ethoxydiphenyl borate (2-APB), a blocker for inositol-1,4,5-triphosphate receptors (InsP3Rs). The application of caffeine (Caff) or ryanodine (Ry) did not change [Ca2+]i. When stores were depleted with Ca2+ -ATPase blocker, thapsigargin (TG), or histamine, the capacitative Ca2+ entry (CCE) was observed. In whole cell patch clamp mode, store-operated Ca2+ currents could be recorded in cells treated with histamine and thapsigargin. On the other hand, voltage-operated Ca2+ channels (VOCCs) could not be elicited. The application of blockers for plasma membrane Ca2+ pump (PMCAs) (carboxeosin or caloxin2A1) induced a large increase of [Ca2+]i. When the Na+/Ca2+ exchangers (NCXs) were blocked by Na+ free solution or KBR7943, [Ca2+]i was also elevated. Using RT-PCR, mRNAs for InsP3Rs type-1, -2, and -3, PMCA-1 and -4, NCX-1, -2, and -3 could be detected. From these results, we conclude that Ca2+ release from ER is mediated by InsP3Rs in mES cells before differentiation and Ca2+ entry through plasma membrane is mainly mediated by the store-operated Ca2+ channels (SOCs). For the Ca2+ extrusion systems, both NCXs and PMCAs play important roles for maintaining the low level of [Ca2+]i.  相似文献   

8.
The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3) to InsP3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca2+ signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca2+ response to InsP3. We have found that the development of increased sensitivity of the Ca2+ stores to InsP3 receptors together with the Ca2+ signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP3 receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca2+ increase induced by InsP3 under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF.  相似文献   

9.
Inositol 1,4,5-trisphosphate receptors in the heart   总被引:2,自引:0,他引:2  
Inositol 1,4,5-trisphosphate (InsP3) is an established calcium-mobilizing messenger, which is well-known to activate Ca2+ signaling in many cell types. Contractile cardiomyocytes express hormone receptors that are coupled to the production of InsP3. Such cardioactive hormones, including endothelin, may have profound inotropic and arrhythmogenic actions, but it is unclear whether InsP3 underlies any of these effects. We have examined the expression and localization of InsP3 receptors (InsP3Rs), and the potential role of InsP3 in modulating cardiac excitation-contraction coupling (EC coupling). Stimulation of electrically-paced atrial and ventricular myocytes with a membrane-permeant InsP3 ester was found to evoke an increase in the amplitudes of action potential-evoked Ca2+ transients and to cause pro-arrhythmic diastolic Ca2+ transients. All the effects of the InsP3 ester could be blocked using a membrane-permeant antagonist of InsP3Rs (2-aminoethoxydiphenyl borate; 2-APB). Furthermore, 2-APB blocked arrhythmias evoked by endothelin and delayed the onset of positive inotropic responses. Our data indicate that atrial and ventricular cardiomyocytes express functional InsP3Rs, and these channels have the potential to influence EC coupling.  相似文献   

10.
The cardiac Na+-Ca2+ exchanger (NCX) is an important regulator of intracellular ion homeostasis and cardiac function. Gaining insight into modulation of the NCX is therefore important in order to understand ion handling in the heart under physiological and pathological conditions. Typically, the functional contribution of the NCX is often regarded as "secondary" to the changes in luminal Na+ and Ca2+. Whilst it is well accepted that the NCX can be regulated by various factors, including the concentrations of transported ions, direct receptor-mediated modulation of the cardiac NCX is more controversial. Evidence from several different laboratories supports the notion that the cardiac NCX is a direct target of neurotransmitters and hormones and their downstream signalling pathways; however, the issue remains unresolved due to conflicting data showing a lack of direct modulation. The present review summarizes overall findings regarding the modulation of the cardiac NCX, in particular on molecular mechanisms of direct phosphorylation of NCX by beta-adrenergic/adenylate cyclase/protein kinase A and (for comparative purposes) on endothelin-1/protein kinase C signalling pathways. It also aims to consider whether it is currently possible to reconcile discrepancies between studies in the interpretation of the regulation of the cardiac NCX by agents stimulating the beta-adrenoceptor/PKA pathway.  相似文献   

11.
The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.  相似文献   

12.
Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.  相似文献   

13.
Elementary Ca2+ release signals in nerve growth factor- (NGF-) differentiated PC12 cells and hippocampal neurons, functionally analogous to the "Ca2+ sparks" and "Ca2+ puffs" identified in other cell types, were characterized by confocal microscopy. They either occurred spontaneously or could be activated by caffeine and metabotropic agonists. The release events were dissimilar to the sparks and puffs described so far, as many arose from clusters of both ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Increasing either the stimulus strength or loading of the intracellular stores enhanced the frequency of and coupling between elementary release sites and evoked global Ca2+ signals. In the PC12 cells, the elementary Ca2+ release preferentially occurred around the branch points. Spatio-temporal recruitment of such elementary release events may regulate neuronal activities.  相似文献   

14.
Calreticulin (CRT) is a highly conserved Ca(2+)-binding protein that resides in the lumen of the endoplasmic reticulum (ER). We overexpressed CRT in Xenopus oocytes to determine how it could modulate inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) influx. Under conditions where it did not affect the spatially complex elevations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) due to InsP(3)-induced Ca(2+) release, overexpressed CRT decreased by 46% the Ca(2+)-gated Cl(-) current due to Ca(2+) influx. Deletion mutants revealed that CRT requires its high capacity Ca(2+)-binding domain to reduce the elevations of [Ca(2+)](i) due to Ca(2+) influx. This functional domain was also required for CRT to attenuate the InsP(3)-induced decline in the free Ca(2+) concentration within the ER lumen ([Ca(2+)](ER)), as monitored with a "chameleon" indicator. Our data suggest that by buffering [Ca(2+)](ER) near resting levels, CRT may prevent InsP(3) from depleting the intracellular stores sufficiently to activate Ca(2+) influx.  相似文献   

15.
We studied the role of the Na+/Ca2+ exchanger (NCX) in modulating oocyte postovulatory aging by observing changes in NCX contents and activities in aging mouse and rat oocytes. Whereas the NCX activity was measured by observing oocyte activation following culture with NCX inhibitor or activator, the NCX contents were determined by immunohistochemical quantification. Although NCX was active in freshly-ovulated rat oocytes recovered 13 h post hCG injection and in aged oocytes recovered 19 h post hCG in both species, it was not active in freshly-ovulated mouse oocytes. However, NCX became active when the freshly-ovulated mouse oocytes were activated with ethanol before culture. Measurement of cytoplasmic Ca2+ revealed Ca2+ increases always before NCX activation. Whereas levels of the reactive oxygen species (ROS) and the activation susceptibility increased, the density of NCX member 1 (NCX1) decreased significantly with oocyte aging in both species. While culture with H2O2 decreased the density of NCX1 significantly, culture with NaCl supplementation sustained the NCX1 density in mouse oocytes. It was concluded that (a) the NCX activity was involved in the modulation of oocyte aging and spontaneous activation; (b) ROS and Na+ regulated the NCX activity in aging oocytes by altering its density as well as functioning; and (c) cytoplasmic Ca2+ elevation was essential for NCX activation in the oocyte.  相似文献   

16.
The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.  相似文献   

17.
The inositol 1,4,5-trisphosphate receptor (InsP3R) is an integral membrane protein in the endoplasmic reticulum (ER) which functions as a ligand-gated Ca2+ release channel. InsP3-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+]i), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP3R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP3R (InsP3R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP3R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was approximately 4 x 10(-10) cm2/s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (approximately 75%) of channels mobile. D was modestly increased at 37 degrees C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP3R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP3R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATP- and microtubule-facilitated motility of the channel.  相似文献   

18.
Fertilization in mammals stimulates a series of Ca(2+) oscillations that continue for 3-4 h. Cell-cycle-dependent changes in the ability to release Ca(2+) are one mechanism that leads to the inhibition of Ca(2+) transients after fertilization. The downregulation of InsP(3)Rs at fertilization may be an additional mechanism for inhibiting Ca(2+) transients. In the present study we examine the mechanism of this InsP(3)R downregulation. We find that neither egg activation nor Ca(2+) transients are necessary or sufficient for the stimulation of InsP(3)R downregulation. First, parthenogenetic activation fails to stimulate downregulation. Second, downregulation persists when fertilization-induced Ca(2+) transients and egg activation are inhibited using BAPTA. Third, downregulation can be induced in immature oocytes that do not undergo egg activation. Other than fertilization, the only stimulus that downregulated InsP(3)Rs was microinjection of the potent InsP(3)R agonist adenophostin A. InsP(3)R downregulation was inhibited by the cysteine protease inhibitor ALLN but MG132 and lactacystin were not effective. Finally, we have injected maturing oocytes with adenophostin A and produced MII eggs depleted of InsP(3)Rs. We show that sperm-induced Ca(2+) signaling is inhibited in such InsP(3)R-depleted eggs. These data show that InsP(3)R binding is sufficient for downregulation and that Ca(2+) signaling at fertilization is mediated via the InsP(3)R.  相似文献   

19.
The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the epsilon and zeta isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca2+ was investigated by co-expressing NCX2 and 14-3-3epsilon in HeLa cells together with the recombinant Ca2+ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3epsilon to dispose of a Ca2+ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3epsilon protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity.  相似文献   

20.
We previously demonstrated in the mouse oocyte that in vivo postovulatory aging significantly suppresses activity of the endoplasmic reticulum (ER) Ca(2+)-ATPase (Igarashi et al. 1997. Mol Reprod Dev 48:383-390). We undertook the present study to further examine the effects of oocyte aging on Ca(2+) release from the inositol 1,4,5-triphosphate (InsP(3))-sensitive Ca(2+) channels of the ER membrane, because not only Ca(2+) reuptake, but also Ca(2+) release from the ER, substantially affect Ca(2+) oscillations in fertilized oocytes. A transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was induced by photolysis of caged InsP(3) microinjected into the cytoplasm in both fresh (14 hr post hCG) and aged (20 hr or 24 hr post hCG) oocytes, where the maximum rate of increase in [Ca(2+)](i) significantly decreased in the aged oocytes. Reduced ER Ca(2+) release in the aged oocyte may not be attributable to aging-related desensitization of the InsP(3)-sensitive Ca(2+) channels in the ER because concentrations of caged InsP(3) for half maximal [Ca(2+)](i) increase were identical for fresh and aged oocytes. The peak [Ca(2+)](i) response following administration of 5 microM thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, was significantly reduced in the aged oocyte, suggesting reduction of the ER Ca(2+) stores. We conclude from these results that reduction of Ca(2+) release from the InsP(3)-sensitive Ca(2+) stores in the aged oocyte arises from depletion of the ER Ca(2+) stores with aging. These aging-related changes in Ca(2+) release and reuptake may account for alterations in Ca(2+) oscillations in aged fertilized oocytes.  相似文献   

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