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1.
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
2.
Seung-Jae Noh Myung-Jun Kim Sangwoo Shim Jin-Kwan Han 《Journal of cellular physiology》1998,176(2):412-423
In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca2+-dependent oscillatory Cl− currents by acting through membrane-bound receptors. External application of 50 μM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-μM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca2+-dependent Cl− currents were observed in the absence of extracellular Ca2+, were blocked by intracellular injection of the Ca2+ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca2+ ATPase. Intracellular Ca2+ release appeared to be from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, because Cl− currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xβ), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xβ, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein α subunits that were identified in Xenopus laevis; Gqα, G11α, G0α, and Gi1α. Among AS-ODNs against the Gαs tested, AS-Gqα and AS-Gi1α to S1P and AS-Gqα and AS-G11α to LPA specifically reduced current responses, respectively, to about 20–30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca2+ from the IP3-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes. J. Cell. Physiol. 176:412–423, 1998. © 1998 Wiley-Liss, Inc. 相似文献
3.
Elena Bossi Elena Centinaio Andrea Moriondo Antonio Peres 《Journal of cellular physiology》1998,174(2):154-159
The role of Ca2+ on the depolarization-induced appearance of a Na+ current in Xenopus oocytes was studied. Oocytes were voltage-clamped and the induction of the Na+ current was tested under various conditions. In oocytes pre-injected with 400 pmol EGTA to increase the intracellular Ca2+ buffering power, the current was significantly reduced. Conversely, when intracellular Ca2+ was made to increase by injecting an analogue of inositol 1,4,5-trisphosphate (3-F InsP3), to cause Ca2+ release from internal stores, the induction of the Na+ current was potentiated. The depolarization-inducible Na+ channels of the Xenopus oocyte membrane appear, therefore, to be Ca2+ sensitive, as well as depolarization-activated. J. Cell. Physiol. 174:154–159, 1998. © 1998 Wiley-Liss, Inc. 相似文献
4.
We studied the blocking action of neuroleptic drugs, haloperidol, pimozide, and fluspirilene, on three types of cloned low voltage-activated (T-type) Ca2+ channels, 1G, 1H, and 1I, functionally expressed in Xenopus oocytes. Fluspirilene and pimozide (members of the diphenylbutylpiperidine group) and haloperidol (belonging to butyrophenones) inhibited Ca2+ currents with different values of the K
d constant and maximum intensity of blocking. Effects of the neuroleptics were voltage-dependent and were accompanied by slowing-down of the kinetics of the currents. The mechanism of blocking is probably based on interaction between the neuroleptics and the channels in the activated and inactivated states. The difference in efficiency and specificity of blockade of various T-channel subtypes by neuroleptics should be considered when estimating the therapeutic significance of the tested pharmacological agents. 相似文献
5.
Over twenty years ago it was shown that depletion of the intracellular Ca2+ store in smooth muscle triggered a Ca2+ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca2+ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca2+ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca2+ ions. Two activation mechanisms have been identified which involve Ca2+ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of α-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes. 相似文献
6.
Background
Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP3/Ca2+ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca2+ signaling and InsP3-induced Ca2+ release from the endoplasmic reticulum (ER).Results
Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca2+]i changes into a sustained increase. Intraluminal Ca2+ measurements in the ER of β-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP3-induced Ca2+ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP3 receptor Ca2+ channel for InsP3. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP3-induced Ca2+ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP3/Ca2+ signaling pathway for 5-HT, because IBMX potentiated Ca2+-dependent Cl- transport activated by a threshold 5-HT concentration.Conclusion
This report shows, for the first time for an insect system, that cAMP can potentiate InsP3-induced Ca2+ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP3/Ca2+ signaling pathways enhances transepithelial electrolyte transport. 相似文献7.
This review will focus on the recent advance in the study of effect of transmembrane Ca2+ gradient on the function of membrane proteins. It consits of two parts: 1. Transmembrane Ca2+ gradient and sarcoplasmic reticulum Ca2+-ATPase; 2. Effect of transmembrane Ca2+ gradient on the components and coupling of cAMP signal transduction pathway. The results obtained indicate that a proper transmembrane Ca2+ gradient may play an important role in modulating the conformation and activity of SR Ca2+-ATPase and the function of membrane proteins involved in the cAMP signal transduction by mediating the physical state change of the membrane phospholipids.Abbreviations Cai
Ca2+ inside vesicles
- Ca0
Ca2+ outside vesicles
- SR
sarcoplasmic reticulum
- PC
phosphatidylcholine
- PS
phosphatidylserine
- PG
phosphatidylglycerol
- PE
phosphatidylethanolamine
- DPH
1,6-diphenyl-1,3,5-hexatriene
- n-AS
n-(9-anthroyloxy) fatty acids
- TMA-DPH
1-(4-trimethylammoniumphenyl)-6)-phenyl-1,3,5-hexatriene
- FCCP
carbonylcyanide-p-trifluoromethoxyphenylhydrazone
- -AR
-adrenergic receptors
- DHA
dihydroalprenolol
- AC
adenylate cyclase
- AC·Lca+–
higher Ca2+ inside vesicles
- AC·Lca– –
lower Ca2+ on both side of vesicles
- AC·Lca++
higher Ca2+ on both side of vesicles
- AC·Lca– +
higher Ca2+ outside vesicles
- cAMP
cyclic adenosine monophosphate
- Gs
stimulatory GTP-binding protein
- GTP
guanosine triposphate
- GTPS
guanosine 50-(3-thiotriphosphate) 相似文献
8.
Congmin Li Jenny Chan Franciose Haeseleer Katsuhiko Mikoshiba Krzysztof Palczewski Mitsuhiko Ikura James B. Ames 《The Journal of biological chemistry》2009,284(4):2472-2481
Calcium-binding protein 1 (CaBP1), a neuron-specific member of the
calmodulin (CaM) superfamily, modulates Ca2+-dependent activity of
inositol 1,4,5-trisphosphate receptors (InsP3Rs). Here we present
NMR structures of CaBP1 in both Mg2+-bound and
Ca2+-bound states and their structural interaction with
InsP3Rs. CaBP1 contains four EF-hands in two separate domains. The
N-domain consists of EF1 and EF2 in a closed conformation with Mg2+
bound at EF1. The C-domain binds Ca2+ at EF3 and EF4, and exhibits
a Ca2+-induced closed to open transition like that of CaM. The
Ca2+-bound C-domain contains exposed hydrophobic residues
(Leu132, His134, Ile141, Ile144,
and Val148) that may account for selective binding to
InsP3Rs. Isothermal titration calorimetry analysis reveals a
Ca2+-induced binding of the CaBP1 C-domain to the N-terminal region
of InsP3R (residues 1-587), whereas CaM and the CaBP1 N-domain did
not show appreciable binding. CaBP1 binding to InsP3Rs requires
both the suppressor and ligand-binding core domains, but has no effect on
InsP3 binding to the receptor. We propose that CaBP1 may regulate
Ca2+-dependent activity of InsP3Rs by promoting
structural contacts between the suppressor and core domains.Calcium ion (Ca2+) in the cell functions as an important
messenger that controls neurotransmitter release, gene expression, muscle
contraction, apoptosis, and disease processes
(1). Receptor stimulation in
neurons promotes large increases in intracellular Ca2+ levels
controlled by Ca2+ release from intracellular stores through
InsP3Rs (2). The
neuronal type-1 receptor
(InsP3R1)2
is positively and negatively regulated by cytosolic Ca2+
(3-6),
important for the generation of repetitive Ca2+ transients known as
Ca2+ spikes and waves
(1). Ca2+-dependent
activation of InsP3R1 contributes to the fast rising phase of
Ca2+ signaling known as Ca2+-induced Ca2+
release (7).
Ca2+-induced inhibition of InsP3R1, triggered at higher
cytosolic Ca2+ levels, coordinates the temporal decay of
Ca2+ transients (6).
The mechanism of Ca2+-dependent regulation of InsP3Rs is
complex (8,
9), and involves direct
Ca2+ binding sites
(5,
10) as well as remote sensing
by extrinsic Ca2+-binding proteins such as CaM
(11,
12), CaBP1
(13,
14), CIB1
(15), and NCS-1
(16).Neuronal Ca2+-binding proteins (CaBP1-5
(17)) represent a new
sub-branch of the CaM superfamily
(18) that regulate various
Ca2+ channel targets. Multiple splice variants and isoforms of
CaBPs are localized in different neuronal cell types
(19-21)
and perform specialized roles in signal transduction. CaBP1, also termed
caldendrin (22), has been
shown to modulate the Ca2+-sensitive activity of InsP3Rs
(13,
14). CaBP1 also regulates
P/Q-type voltage-gated Ca2+ channels
(23), L-type channels
(24), and the transient
receptor potential channel, TRPC5
(25). CaBP4 regulates
Ca2+-dependent inhibition of L-type channels in the retina and may
be genetically linked to retinal degeneration
(26). Thus, the CaBP proteins
are receiving increased attention as a family of Ca2+ sensors that
control a variety of Ca2+ channel targets implicated in neuronal
degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in
CaM and troponin C (18)
(Fig. 1). By analogy to CaM
(27), the four EF-hands are
grouped into two domains connected by a central linker that is four residues
longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain
non-conserved amino acids within the N-terminal region that may confer target
specificity. Another distinguishing property of CaBPs is that the second
EF-hand lacks critical residues required for high affinity Ca2+
binding (17). CaBP1 binds
Ca2+ only at EF3 and EF4, whereas it binds Mg2+ at EF1
that may serve a functional role
(28). Indeed, changes in
cytosolic Mg2+ levels have been detected in cortical neurons after
treatment with neurotransmitter
(29). Other neuronal
Ca2+-binding proteins such as DREAM
(30), CIB1
(31), and NCS-1
(32) also bind Mg2+
and exhibit Mg2+-induced physiological effects. Mg2+
binding in each of these proteins helps stabilize their Ca2+-free
state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary
structural elements (α-helices and β-strands) were derived from NMR
analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted
green, red, cyan, and yellow. Residues in the 12-residue
Ca2+-binding loops are underlined and chelating residues
are highlighted bold. Non-conserved residues in the hydrophobic patch
are colored red.Despite extensive studies on CaBP1, little is known about its structure and
target binding properties, and regulation of InsP3Rs by CaBP1 is
somewhat controversial and not well understood. Here, we present the NMR
solution structures of both Mg2+-bound and Ca2+-bound
conformational states of CaBP1 and their structural interactions with
InsP3R1. These CaBP1 structures reveal important
Ca2+-induced structural changes that control its binding to
InsP3R1. Our target binding analysis demonstrates that the C-domain
of CaBP1 exhibits Ca2+-induced binding to the N-terminal cytosolic
region of InsP3R1. We propose that CaBP1 may regulate
Ca2+-dependent channel activity in InsP3Rs by promoting
a structural interaction between the N-terminal suppressor and ligand-binding
core domains that modulates Ca2+-dependent channel gating
(8,
33,
34). 相似文献
9.
Matthew J. Betzenhauser Jenna L. Fike Larry E. Wagner II David I. Yule 《The Journal of biological chemistry》2009,284(37):25116-25125
Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP3Rs) represents a mechanism for shaping intracellular Ca2+ signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca2+ release in cells that express predominantly InsP3R2. PKA is known to phosphorylate InsP3R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP3R2 in DT40-3KO cells that are devoid of endogenous InsP3R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca2+ signals and augmented the single channel open probability of InsP3R2. A PKA phosphorylation site unique to the InsP3R2 was identified at Ser937. The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser937, since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca2+ signaling following PKA activation in cells that express predominantly InsP3R2.Hormones, neurotransmitters, and growth factors stimulate the production of InsP33 and Ca2+ signals in virtually all cell types (1). The ubiquitous nature of this mode of signaling dictates that this pathway does not exist in isolation; indeed, a multitude of additional signaling pathways can be activated simultaneously. A prime example of this type of “cross-talk” between independently activated signaling systems results from the parallel activation of cAMP and Ca2+ signaling pathways (2, 3). Interactions between these two systems occur in numerous distinct cell types with various physiological consequences (3–6). Given the central role of InsP3R in Ca2+ signaling, a major route of modulating the spatial and temporal features of Ca2+ signals following cAMP production is potentially through PKA phosphorylation of the InsP3R isoform(s) expressed in a particular cell type.There are three InsP3R isoforms (InsP3R1, InsP3R2, and InsP3R3) expressed to varying degrees in mammalian cells (7, 8). InsP3R1 is the major isoform expressed in the nervous system, but it is less abundant compared with other subtypes in non-neuronal tissues (8). Ca2+ release via InsP3R2 and InsP3R3 predominate in these tissues. InsP3R2 is the major InsP3R isoform in many cell types, including hepatocytes (7, 8), astrocytes (9, 10), cardiac myocytes (11), and exocrine acinar cells (8, 12). Activation of PKA has been demonstrated to enhance InsP3-induced Ca2+ signaling in hepatocytes (13) and parotid acinar cells (4, 14). Although PKA phosphorylation of InsP3R2 is a likely causal mechanism underlying these effects, the functional effects of phosphorylation have not been determined in cells unambiguously expressing InsP3R2 in isolation. Furthermore, the molecular determinants of PKA phosphorylation of this isoform are not known.PKA-mediated phosphorylation is an efficient means of transiently and reversibly regulating the activity of the InsP3R. InsP3R1 was identified as a major substrate of PKA in the brain prior to its identification as the InsP3R (15, 16). However, until recently, the functional consequences of phosphorylation were unresolved. Initial conflicting results were reported indicating that phosphoregulation of InsP3R1 could result in either inhibition or stimulation of receptor activity (16, 17). Mutagenic strategies were employed by our laboratory to clarify this discrepancy. These studies unequivocally assigned phosphorylation-dependent enhanced Ca2+ release and InsP3R1 activity at the single channel level, through phosphorylation at canonical PKA consensus motifs at Ser1589 and Ser1755. The sites responsible were also shown to be specific to the particular InsP3R1 splice variant (18). These data were also corroborated by replacing the relevant serines with glutamates in a strategy designed to construct “phosphomimetic” InsP3R1 by mimicking the negative charge added by phosphorylation (19, 20). Of particular note, however, although all three isoforms are substrates for PKA, neither of the sites phosphorylated by PKA in InsP3R1 are conserved in the other two isoforms (21). Recently, three distinct PKA phosphorylation sites were identified in InsP3R3 that were in different regions of the protein when compared with InsP3R1 (22). To date, no PKA phosphorylation sites have been identified in InsP3R2.Interactions between Ca2+ and cAMP signaling pathways are evident in exocrine acinar cells of the parotid salivary gland. In these cells, both signals are important mediators of fluid and protein secretion (23). Multiple components of the [Ca2+]i signaling pathway in these cells are potential substrates for modulation by PKA. Previous work from this laboratory established that activation of PKA potentiates muscarinic acetylcholine receptor-induced [Ca2+]i signaling in mouse and human parotid acinar cells (4, 24, 25). A likely mechanism to explain this effect is that PKA phosphorylation increases the activity of InsP3R expressed in these cells. Consistent with this idea, activation of PKA enhanced InsP3-induced Ca2+ release in permeabilized mouse parotid acinar cells and also resulted in the phosphorylation of InsP3R2 (4).Invariably, prior work examining the functional effects of PKA phosphorylation on InsP3R2 has been performed using cell types expressing multiple InsP3R isoforms. For example, AR4-2J cells are the preferred cell type for examining InsP3R2 in relative isolation, because this isoform constitutes more than 85% of the total InsP3R population (8). InsP3R1, however, contributes up to ∼12% of the total InsP3R in AR4-2J cells. An initial report using InsP3-mediated 45Ca2+ flux suggested that PKA activation increased InsP3R activity in AR4-2J cells (21). A similar conclusion was made in a later study, which documented the effects of PKA activation on agonist stimulated Ca2+ signals in AR4-2J cells (26). Any effects of phosphorylation observed in these experiments could plausibly have resulted from phosphorylation of the residual InsP3R1.Although PKA enhances InsP3-induced calcium release in cells expressing predominantly InsP3R2, including hepatocytes, parotid acinar cells, and AR4-2J cells (4, 13, 21, 26, 27), InsP3R2 is not phosphorylated at stoichiometric levels by PKA (21). This observation has called into question the physiological significance of PKA phosphorylation of InsP3R2 (28). The apparent low levels of InsP3R2 phosphorylation are clearly at odds with the augmented Ca2+ release observed in cells expressing predominantly this isoform. The equivocal nature of these findings probably stems from the fact that, to date, all of the studies demonstrating positive effects of PKA activation on Ca2+ release were conducted in cells that also express InsP3R1. The purpose of the current experiments was to analyze the functional effects of phosphorylation on InsP3R2 expressed in isolation on a null background. We report that InsP3R2 activity is increased by PKA phosphorylation under these conditions, and furthermore, we have identified a unique phosphorylation site in InsP3R2 at Ser937. In total, these results provide a direct mechanism for the cAMP-induced activation of InsP3R2 via PKA phosphorylation of InsP3R2. 相似文献
10.
Staurosporine (Stp) is an inhibitor of protein kinase C (PKC) that has been used to address the role of this enzyme in a variety of cells. However, Stp can also inhibit protein tyrosine kinases (PTK). We have investigated the effects of Stp on the InsP3- (using mAb C305 directed against the β chain of the T cell receptor (TcR)/CD3 complex) and the thapsigargin (Tg)-dependent release and influx of Ca2+ in human (Jurkat) T cells. The addition of Stp (200 nM) during the sustained phase of the TcR-dependent Ca2+ response resulted in a rapid inhibition of the influx of Ca2+ that was not seen when Ca2+ mobilization was triggered by Tg (1 μM). When the cells were preincubated with Stp (200 nM), there was an inhibition of the mAb C305- but not the Tg-dependent Ca2+ response. The effect of Stp was not the result of the inhibition of PKC as shown by down-regulation of PKC and with the use of the specific PKC inhibitor bis-indolyl maleimide GF 109203X. The effect of Stp on the entry of Ca2+ in activated (mAb C305) Jurkat lymphocytes was dose-related and was not the result of a direct inhibition of plasma membrane Ca2+ channels based on an absence of effect on the Tg-dependent entry of Ca2+ and the use of Ca2+ channel blockers (econazole and Ni2+). These blockers terminated the influx of Ca2+ but the Tg-sensitive Ca2+ reserves were not refilled in marked contrast to the effect of Stp. Quantification of InsP3 revealed that the addition of Stp resulted in an approximate 40% reduction in mAb C305-activated Jurkat cells. The effects of Stp can be explained as follows. Stp decreases the mAb C305-induced production of InsP3 by inhibiting the TcR/CD3-dependent activation of PTK associated with the stimulation of phospholipase C-γ1. A decrease in [InsP3] without a return to baseline is sufficient to close the InsP3 Ca2+ channel, endoplasmic Ca2+ ATPases use the incoming Ca2+ to refill the Ca2+ pools and that terminates the capacitative entry of Ca2+. A simple kinetic model reproduced the experimental data. 相似文献
11.
P. Damiani R.A. Fissore J.B. Cibelli C.R. Long J.J. Balise J.M. Robl R.T. Duby 《Molecular reproduction and development》1996,45(4):521-534
In this study we evaluated nuclear and ooplasmic maturation of prepuberal calf oocytes to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the MII stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following IVF, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. These anomalies were characterized by delayed formation of sperm aster and asynchronous pronuclear formation. Microfluorometry was used to characterize the Ca2+ responses of calf oocytes to the addition of agonists or after IVF. The addition of thimerosal demonstrated the presence of Ca2+ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trisphosphate (InsP3), used to test the sensitivity of the InsP3R, released significantly less Ca2+ in calf than in cow oocytes, whereas higher concentrations of InsP3 (500 μM) released maximal [Ca2+]i in both oocytes. These results suggested that the Ca2+ content of intracellular stores was similar, but the sensitivity of the InsP3R may be different. Following insemination, calf oocytes exhibiting [Ca2+]i oscillations displayed comparable amplitude and intervals to cow oocytes; however, a significantly higher number of fertilized calf oocytes failed to show oscillations. Our findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation. © 1996 Wiley-Liss, Inc. 相似文献
12.
The ubiquitous InsP3/Ca2+ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca2+ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca2+] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP3-induced Ca2+ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP3/Ca2+ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca2+ signals; (2) cyclosporin A and FK506, inhibitors of Ca2+/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca2+ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca2+ responses does not involve Ca2+ entry into the cells; (4) cyclosporin A increases InsP3-dependent Ca2+ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca2+ responses, indicating that PKA and calcineurin act antagonistically on the InsP3/Ca2+ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP3/Ca2+ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations. 相似文献
13.
Matthew J. Betzenhauser Larry E. Wagner II Hyung Seo Park David I. Yule 《The Journal of biological chemistry》2009,284(24):16156-16163
ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.Inositol 1,4,5-trisphosphate receptors (InsP3R)3 are a family of large, tetrameric, InsP3-gated cation channels. The three members of this family (InsP3R1, InsP3R2, and InsP3R3) are nearly ubiquitously expressed and are localized primarily to the endoplasmic reticulum (ER) membrane (1–3). Numerous hormones, neurotransmitters, and growth factors bind to receptors that stimulate phospholipase C-induced InsP3 production (4). InsP3 subsequently binds to the InsP3R and induces channel opening. This pathway represents a major mechanism for Ca2+ liberation from ER stores (5). All three InsP3R isoforms are dynamically regulated by cytosolic factors in addition to InsP3 (1). Ca2+ is perhaps the most important determinant of InsP3R activity besides InsP3 itself and is known to regulate InsP3R both positively and negatively (6). ATP, in concert with InsP3 and Ca2+, also regulates InsP3R as do numerous kinases, phosphatases, and protein-binding partners (7–10). This intricate network of regulation allows InsP3R activity to be finely tuned by the local cytosolic environment (9). As a result, InsP3-induced Ca2+ signals can exhibit a wide variety of spatial and temporal patterns, which likely allows Ca2+ to control many diverse cellular processes.Modulation of InsP3-induced Ca2+ release (IICR) by ATP and other nucleotides provides a direct link between intracellular Ca2+ signaling and the metabolic state of the cell. Metabolic fluctuations could, therefore, impact Ca2+ signaling in many cell types given that InsP3R are expressed in all cells (11, 12). Consistent with this, ATP has been shown to augment IICR in many diverse cell types including primary neurons (13), smooth muscle cells (14), and exocrine acinar cells (15) as well as in immortalized cell lines (16–18). The effects of ATP on InsP3R function do not require hydrolysis because non-hydrolyzable ATP analogues are as effective as ATP (7, 14). ATP is thought to bind to distinct regions in the central, coupling domain of the receptors and to facilitate channel opening (2, 19). ATP is not required for channel gating, but instead, increases InsP3R activity in an allosteric fashion by increasing the open probability of the channel in the presence of activating concentrations of InsP3 and Ca2+ (7, 8, 20).Despite a wealth of knowledge regarding the functional effects of ATP on InsP3R function, there is relatively little known about the molecular determinants of these actions. ATP is thought to exert effects on channel function by direct binding to glycine-rich regions containing the consensus sequence GXGXXG that are present in the receptors (2). These sequences were first proposed to be ATP-binding domains due to their similarity with Walker A motifs (21). The neuronal S2+ splice variant of InsP3R1 contains two such domains termed ATPA and ATPB. A third site, ATPC, is formed upon removal of the S2 splice site (2, 22). The ATPB site is conserved in InsP3R2 and InsP3R3, while the ATPA and ATPC sites are unique to InsP3R1. Our prior work examining the functional consequences of mutating these ATP-binding sites has yielded unexpected results. For example, mutating the ATPB site in InsP3R2 completely eliminated the enhancing effects of ATP on this isoform while mutating the analogous site in InsP3R3 failed to alter the effects of ATP (23). This indicated the presence of an additional locus for ATP modulation of InsP3R3. In addition, mutation of the ATPC in the S2− splice variant of InsP3R1 did not alter the ability of ATP to modulate Ca2+ release, but instead impaired the ability of protein kinase A to phosphorylate Ser-1755 of this isoform (22).The ATPA and ATPB sites in InsP3R1 were first identified as putative nucleotide-binding domains after the cloning of the full-length receptor (24). Early binding experiments with 8-azido-[α-32P]ATP established that ATP cross-linked with receptor purified from rat cerebellum at one site per receptor monomer (19). Later, more detailed, binding experiments on trypsinized recombinant rat InsP3R1 showed cross-linking of ATP to two distinct regions of the receptor that corresponded with the ATPA and ATPB sites (17). We and others (16, 22, 23) have also reported the binding of ATP analogues to purified GST fusions of small regions of InsP3R1 surrounding the ATPA and ATPB sites. It is widely accepted, in the context of the sequence similarity to Walker A motifs and biochemical data, that the ATPA and ATPB sites are the loci where ATP exerts its positive functional effects on InsP3R1 function (1–3, 16). Furthermore, the higher affinity of the ATPA site to ATP is thought to confer the higher sensitivity of InsP3R1 to ATP versus InsP3R3, which contains the ATPB site exclusively (25, 26). The purpose of this study, therefore, was to examine the contributions of the ATPA and ATPB sites to ATP modulation of the S2+ splice variant of InsP3R1. We compared the effects of ATP on InsP3R1 and on ATP-binding site mutated InsP3R1 using detailed functional analyses in permeabilized cells and in single channel recordings. Here we report that InsP3R1 is similar to InsP3R3 in that ATP modulates IICR even at maximal InsP3 concentrations and that neither the ATPA nor the ATPB site is required for this effect. 相似文献
14.
The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca2+ release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca2+ following Ca2+ transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP3), and by Ca2+ influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca2+ transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH2-cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca2+ sequestration by enhancing SERCA pump activity, in addition to its well-established action on RyRs to liberate Ca2+. 相似文献
15.
Tyson S. Ikeda Eun-Sil Hwang Michael J. Coady Bruce A. Hirayama Matthias A. Hediger Ernest M. Wright 《The Journal of membrane biology》1989,110(1):87-95
Summary The Na+/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed inXenopus oocytes. Injection of cloned RNA into oocytes increased Na+/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed inXenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of14C--methyl-d-glucopyranoside uptake into brush border membrane vesicles andXenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na+-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (K
i
6 and 9 m), and saturable (-methyl-d-glucopyranosideK
m
110 and 570 m). The sugar specificity was examined by competition experiments, and in both cases the selectivity wasd-glucose>-methyl-d-glucopyranoside>d-galactose>3-O-methyl-d-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na+/glucose cotransporter. 相似文献
16.
Shcheglovitov A. K. Zhelay T. I. Kondratskii A. P. Naidenov V. G. Shuba Ya. M. 《Neurophysiology》2004,36(2):93-101
We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca2+ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC50 = 98 and 243 M, maximum blocking intensity Amax = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC50 = 5 M and Amax = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca2+ channels in native cells. 相似文献
17.
R. C. Hardie 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1995,177(6):707-721
In order to test the hypothesis that excitation in Drosophila photoreceptors is mediated by Ca2+ released from internal stores, the Ca2+ buffers EGTA, BAPTA and di-bromo-BAPTA (DBB) were introduced into dissociated photoreceptors via whole-cell recording pipettes. All buffers were preloaded with Ca2+ to provide the same free Ca2+ concentration (250 nM). EGTA (up to 18 mM free buffer) had only weak effects upon voltage-clamped flash responses in normal Ringer's solution (1.5 mM Ca
0
2+
), and no effect in Ca2+-free solution. The maximum BAPTA concentration tested (14.4 mM free BAPTA) reduced the initial rate of rise by ca. 5000-fold in normal Ringer's solution; by ca. 500-fold in Ca2+free solution; and only ca. 60-fold in the absence of Mg2+, which preferentially blocks one component of the light-sensitive current. Although BAPTA delayed the time-to-peak in normal Ringer's solution, responses in Ca2+ free Ringer's solution were accelerated. These results support the role of Ca2+ influx in regulating sensitivity and response kinetics; however, in view of the high concentrations required to attenuate responses in Ca2+ free Ringer's solution, the role of Ca2+ release in excitation remains unclear. DBB was ca. 2–3 fold more potent than BAPTA, and at concentrations > 5 mM had a qualitatively different action, greatly delaying the time-to-peak. This suggests DBB may have distinct pharmacological actions or access to compartments inaccessible to BAPTA.The only current activated by introducing 5–500 M Ca2+ (buffered with nitrilo-triacetic acid) was electrogenic Na+/Ca2+ exchange. When this was blocked by removing Nao
0
+
, a novel cationic conductance was activated. However, its properties did not resemble those the light-activated conductance, and thus do not support the hypothesis that Ca2+ is sufficient for excitation.Abbreviations
BAPTA
bis-(o-aminophenoxy)-ethane-N,N,N-tetracetic acid
-
DBB
Di-bromo-bapta
-
NTA
nitrilo-triacetic acid
-
InsP
3
inositol 1,4,5-trisphosphate 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.
Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes 总被引:2,自引:0,他引:2
Kyozuka K Chun JT Puppo A Gragnaniello G Garante E Santella L 《Developmental biology》2008,320(2):426-435
Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP3-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP3, despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis. 相似文献
20.
M. Tshipamba H. De Smedt L. Missiaen B. Himpens L. Van Den Bosch R. Borghgraef 《Journal of cellular physiology》1993,155(1):96-103
We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release in renal epithelial LLC-PK1 cells. AVP-induced changes in the intracellular free calcium concentration ([Ca2+]i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca2+ mobilization was also observed in the absence of extracellular Ca2+, but was completely abolished after depletion of the intracellular Ca2+ stores by 2 μM thapsigargin. Using 45Ca2+ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP3 affected the Ca2+ content of nonmitochondrial Ca2+ pools in different loading and release conditions. Less than 10% of the Ca2+ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca2+. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca2+, but Ca2+ sequestered in this pool could not be released by InsP3. The thapsigargin-sensitive Ca2+ pool, in contrast, was nearly completely InsP3 sensitive. A submaximal [InsP3], however, released only a fraction of the sequestered Ca2+. This fraction was dependent on the cytosolic as well as on the luminal [Ca2+]. The cytosolic free [Ca2+] affected the InsP3-induced Ca2+ release in a biphasic way. Maximal sensitivity toward InsP3 was found at a free cytosolic [Ca2+] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca2+] decreased the InsP3 sensitivity. Other divalent cations or La3+ did not provoke similar inhibitory effects on InsP3-induced Ca2+ release. The luminal free [Ca2+] was manipulated by varying the time of incubation of Ca2+ -loaded cells in an EGTA-containing medium. Reduction of the Ca2+ content to one-third of its initial value resulted in a fivefold decrease in the InsP3 sensitivity of the Ca2+ release. © 1993 Wiley-Liss, Inc. 相似文献