Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+ -induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.     

Cell signaling microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations

Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic alpha1-subunit abolished Ca2+ oscillations and downstream activation of NF-kappaB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.     

Functional and biochemical analysis of the type 1 inositol (1,4,5)-trisphosphate receptor calcium sensor

Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP(3)R1) by cytosolic calcium (Ca(2+)) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP(3)R1 regulation by Ca(2+) are poorly understood. Using DT40 cell expression system and Ca(2+) imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP(3)R1 modulation by Ca(2+). By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP(3)R1 Ca(2+)-sensor region (E1932-R2270) binds Ca(2+) with 0.16 micro M affinity. We further established that E2100D and E2100Q mutations decrease Ca(2+)-binding affinity of the putative InsP(3)R1 Ca(2+)-sensor region to 1 micro M. In planar lipid bilayer experiments with recombinant InsP(3)R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP(3)R1 bell-shaped Ca(2+) dependence from 0.2 micro M to 1.5 micro M Ca(2+). In agreement with the biochemical data, we found that the apparent affinities of Ca(2+) activating and inhibitory sites of the InsP(3)R1 were 0.2 micro M for the wild-type channels and 1-2 micro M Ca(2+) for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP(3)R1 Ca(2+) sensor.     

Dual regulation of calcium mobilization by inositol 1,4, 5-trisphosphate in a living cell

Changes in cytosolic free calcium ([Ca(2+)](i)) often take the form of a sustained response or repetitive oscillations. The frequency and amplitude of [Ca(2+)](i) oscillations are essential for the selective stimulation of gene expression and for enzyme activation. However, the mechanism that determines whether [Ca(2+)](i) oscillates at a particular frequency or becomes a sustained response is poorly understood. We find that [Ca(2+)](i) oscillations in rat megakaryocytes, as in other cells, results from a Ca(2+)-dependent inhibition of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. Moreover, we find that this inhibition becomes progressively less effective with higher IP(3) concentrations. We suggest that disinhibition, by increasing IP(3) concentration, of Ca(2+)-dependent inhibition is a common mechanism for the regulation of [Ca(2+)](i) oscillations in cells containing IP(3)-sensitive Ca(2+) stores.     

Effects of inositol 1,4,5-trisphosphate receptor-mediated intracellular stochastic calcium oscillations on activation of glycogen phosphorylase

In various cell types cytosolic calcium (Ca(2+)) is an important regulator. The possible role of Ca(2+) release from the inositol 1,4,5-trisphosphate (IP(3)) receptor channel in the regulation of the phosphorylation-dephosphorylation cycle process involved in glycogen degradation by glycogen phosphorylase have theoretically investigated by using the Li-Rinzel model for cytosolic Ca(2+) oscillations. For the case of deterministic cytosolic Ca(2+) oscillations, there exists an optimal frequency of cytosolic Ca(2+) oscillations at which the average fraction of active glycogen phosphorylase reaches a maximum value, and a mutation for the average fraction of active glycogen phosphorylase occurs at the higher bifurcation point of Ca(2+) oscillations. For the case of stochastic cytosolic Ca(2+) oscillations, the fraction of active phosphorylase is strongly affected by the number of IP(3) receptor channels and the level of IP(3) concentration. Small number of IP(3) receptor channels can potentiate the sensitivity of the activity of glycogen phosphorylase. The average frequency and amplitude of active phosphorylase stochastic oscillations are increased with the level of increasing IP(3) stimuli. The various distributions for the amplitude of active glycogen phosphorylase oscillations in parameters plane are discussed.     

Immunogold localization of inositol 1, 4, 5-trisphosphate (InsP3) receptor in mouse cerebellar Purkinje cells using three monoclonal antibodies

Ultrastructural localization of InsP3 receptor in mouse cerebellar Purkinje cells was investigated by immunogold technique using three monoclonal antibodies (mab 10A6, 4C11 and 18A10). The epitopes of the three antibodies were numerously detected on the smooth endoplasmic reticulum (ER) (especially, on the stacks of flattened smooth ER, subsurface cisterns and spine apparatus), scantily on the rough ER and on the outer nuclear membrane, but were not detectable on either the plasmalemma, synaptic densities, mitochondria or Golgi apparatus. Not only mab 4C11 and 10A6 which bind to the N-terminal region of the receptor but also 18A10 which binds to the C-terminal region were localized on the cytoplasmic surface of the ER membranes. This indicates that the C terminus of InsP3 receptor is localized on the cytoplasmic surface of the ER. We noticed that gold particles are usually localized on the fuzzy structure of the cytoplasmic surface of smooth ER, which is suggested to correspond to the feet structure of the ryanodine receptor. In the Nissl body, gold particles were found not only on the ER membranes but also in the cytoplasmic matrix between the rough ER cisterns. We suggest that the peculiar structure of Nissl body, which is composed of parallel cisterns of rough ER, sandwiching a number of free polyribosomes between the cisternal elements, is due to the fact that the major proteins like InsP3 receptor are synthesized mostly on the free polyribosomes and become membrane bound only at the later stage of the biosynthesis.     

Microinjection of inositol 1,2-(cyclic)-4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate into intact Xenopus oocytes can induce membrane currents independent of extracellular calcium

Inositol phosphate action in an intact cell has been investigated by intracellular microinjection of eight inositol phosphate derivatives into Xenopus laevis oocytes. These cells have calcium-regulated chloride channels but do not have a calcium-induced calcium release system. Microinjection of inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,2-(cyclic)-4,5-trisphosphate (cIP3), inositol 1,4,5-trisphosphate (IP3), or inositol 4,5-bisphosphate [(4,5)IP2], open chloride channels to induce a membrane depolarization. However, inositol 1-phosphate (IP1), inositol 1,3,4,5,6-pentakisphosphate (IP5), inositol 1,4-bisphosphate, or inositol 3,4-bisphosphate are unable to induce this depolarization. The depolarization is mimicked by calcium microinjection, inhibited by EGTA coinjection, and is insensitive to removal of extracellular calcium. By means of the depolarization response, the efficacy of various inositol phosphate derivatives are compared. IP3 and cIP3 induce similar half-maximal, biphasic depolarization responses at an intracellular concentration of approximately 90 nM, whereas IP4 induces a mono- or biphasic depolarization at approximately 3400 nM. At concentrations similar to that required for IP3 and cIP3, (4,5)IP2 induces a long-term (greater than 40 min) depolarization. The efficacy (cIP3 = IP3 = (4,5)IP2 much greater than IP4) and action of the various inositol phosphates in an intact cell and their inability to induce meiotic cell division are discussed.     

A mathematical model of spontaneous calcium(II) oscillations in astrocytes

Astrocytes exhibit oscillations and waves of Ca²⁺ ions within their cytosol and it appears that this behavior helps facilitate the astrocyte's interaction with its environment, including its neighboring neurons. Often changes in the oscillatory behavior are initiated by an external stimulus such as glutamate, recently however, it has been observed that oscillations are also initiated spontaneously. We propose here a mathematical model of how spontaneous Ca²⁺ oscillations arise in astrocytes. This model uses the calcium-induced calcium release and inositol cross-coupling mechanisms coupled with a receptor-independent method for producing inositol (1,4,5)-trisphosphate as the heart of the model. By computationally mimicking experimental constraints we have found that this model provides results that are qualitatively similar to experiment.     

内质网蛋白44(ERp44)通过1, 4, 5-三磷酸肌醇受体介导基因转录

细胞核内钙离子浓度的增加可以引起包括钙离子激活的基因转录在内的很多生理功能.运用Western blot、免疫荧光、实时定量聚合酶链反应、钙成像以及外源三磷酸腺苷刺激细胞释放钙离子等试验方法,发现1,4,5-三磷酸肌醇受体和内质网蛋白44(ERp44)在内质网和核膜上都有很好的共定位.外源三磷酸腺苷可以通过1,4,5-三磷酸肌醇受体刺激核内钙瞬变并磷酸化环磷酸腺苷反应原件结合蛋白(CREB)、刺激原癌基因c-Myc的表达.但是,这些功能都能被1,4,5-三磷酸肌醇受体抑制剂2-氨乙氧基二苯酯硼酸(2-APB)和过表达内质网蛋白44(ERp44)所抑制.这些结果均提示在子宫颈癌HeLa细胞中内质网蛋白44(ERp44)通过1,4,5-三磷酸肌醇受体而介导基因转录.     

The correlation between Ca2+ influx and inositol 1, 4, 5-trisphosphate (IP3) formation in platelets stimulated by various agonists

The relationship between Ca2+ influx (delta [Ca2+]i) and the formation of inositol 1,4,5-trisphosphate (IP3) was investigated in human platelets stimulated by various agonists. Both delta [Ca2+]i and IP3 were increased in proportion to the amount of the agonists (thrombin, ADP, PAF, STA2), the receptors of which were demonstrated in platelets, and were correlated with each other. However, the ratio of delta [Ca2+]i to IP3 was significantly varied among agonists. Furthermore, in thrombin stimulated platelets, IP3 was small at low temperature (20 degrees C) compared with that at high temperature (37 degrees C) in spite of the similar delta [Ca2+]i. Thus, Ca2+ influx in human platelets seems to be regulated directly through the receptor operated mechanism and IP3 may not be involved in it.     

Cell cycle-coupled [Ca(2+)](i) oscillations in mouse zygotes and function of the inositol 1,4,5-trisphosphate receptor-1

Sperm entry in mammalian eggs initiates oscillations in the concentration of free calcium ([Ca(2+)](i)). In mouse eggs, oscillations start at metaphase II (MII) and conclude as the zygotes progress into interphase and commence pronuclear (PN) formation. The inositol 1,4,5-trisphosphate receptor (IP(3)R-1), which underlies the oscillations, undergoes degradation during this transition, suggesting that one or more of the eggs' Ca(2+)-releasing machinery components may be regulated in a cell cycle-dependent manner, thereby coordinating [Ca(2+)](i) responses with the cell cycle. To ascertain the site(s) of interaction, we initiated oscillations at different stages of the cell cycle in zygotes with different IP(3)R-1 mass. In addition to sperm, we used two other agonists: porcine sperm factor (pSF), which stimulates production of IP(3), and adenophostin A, a non-hydrolyzable analogue of IP(3). None of the agonists tested induced oscillations at interphase, suggesting that neither decreased IP(3)R-1 mass nor lack of production or excessive IP(3) degradation can account for the insensitivity to IP(3) at this stage. Moreover, the releasable Ca(2+) content of the stores did not change by interphase, but it did decrease by first mitosis. More importantly, experiments revealed that IP(3)R-1 sensitivity and possibly IP(3) binding were altered at interphase, and our data demonstrate stage-specific IP(3)R-1 phosphorylation by M-phase kinases. Accordingly, increasing the activity of M-phase kinases restored the oscillatory-permissive state in zygotes. We therefore propose that the restriction of oscillations in mouse zygotes to the metaphase stage may be coordinated at the level of IP(3)R-1 and that this involves cell cycle stage-specific receptor phosphorylation.     

Interaction of luminal calcium and cytosolic ATP in the control of type 1 inositol (1,4,5)-trisphosphate receptor channels

Ca(2+) within intracellular stores (luminal Ca(2+)) is believed to play a role in regulating Ca(2+) release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P(3))-gated Ca(2+) channel (or Ins(1,4,5)P(3) receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P(3) receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca(2+)] using K(+) as the current carrier. At a high luminal [Ca(2+)] and in the presence of optimal [Ins(1,4,5)P(3)] and cytosolic [Ca(2+)], a short burst of Ins(1,4,5)P(3) receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca(2+)] caused the channel to reactivate indefinitely. At luminal [Ca(2+)], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca(2+)] caused reactivation. We provide evidence that luminal Ca(2+) is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca(2+) overload is prevented when the energy state of the cell is compromised.     

Kinetics of inositol 1,4,5-trisphosphate and inositol cyclic 1:2,4,5-trisphosphate metabolism in intact rat parotid acinar cells. Relationship to calcium signalling

Stimulation of rat parotid acinar cells by the muscarinic cholinergic receptor agonist methacholine results in the formation of inositol 1,4,5-trisphosphate [1,4,5)IP3) and inositol cyclic 1:2,4,5-trisphosphate [c1:2,4,5)IP3) which, after 40 min, accumulate to a ratio of 1:0.57. The turnover rates of these inositol trisphosphates have been determined in cholinergically stimulated rat parotid cells by measuring the degradation of the 3H-labeled compounds following receptor blockade. (1,4,5)IP3 is rapidly metabolized, with a half-time of 7.6 s; (c1:2,4,5)IP3 declines much more slowly with a half-time of almost 10 min. Because the formation and metabolism of (c1:2,4,5)IP3 are so slow, (c1:2,4,5)IP3 gradually accumulates upon prolonged receptor activation. Inositol trisphosphate turnover was compared to the receptor-mediated changes in cytoplasmic Ca2+ concentration, as measured by the fluorescent Ca2+ indicator, fura-2. The Ca2+ signal decays upon termination of inositol phosphate formation and returns to base line within 30 s. Thus, while (c1:2,4,5)IP3 may have some yet unknown biological effects on Ca2+ homeostasis, its metabolism seems far too slow to be the primary regulator of cytosolic Ca2+ levels under long term stimulatory conditions. The rate at which the Ca2+ signal decays is, however, somewhat slowed after prolonged agonist stimulation. Furthermore, the capacity of the cells to mobilize intracellular Ca2+ in response to a second agonist stimulation is slightly delayed when the duration of the first stimulus is prolonged. The results suggest that the regulation of cytoplasmic Ca2+ levels may be more complicated than initially realized and could depend on the combined actions of more than one inositol polyphosphate.     

The inositol 1,4,5-trisphosphate (InsP3) receptor

Conclusion In this review, we have described the functional properties and regulation of the InsP₃R. Not all aspects of InsP₃R function and regulation were covered, the main focus was on the most recent and physiologically important data. Information about the structure, heterogeneity, functional properties, and regulation of the InsP₃R is useful for understanding the spatiotemporal aspects of Ca signaling. The combination of biochemical, biophysical and molecular biological techniques has revealed the intricacies of the InsP₃R over the past decade. However, questions about the functional differences between various isoforms and splice variants of the InsP₃R, the structural determinants responsible for regulation of InsP₃R by Ca and ATP, the functional effects of InsP₃R phosphorylation and many others remain to be elucidated. Future investigations can be expected to provide answers to these important questions.We thank S. Bezprozvannaya for expert technical assistance. This work was supported by National Institutes of Health grants HL 33026 and GM 39029, and a Grant-in-Aid from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.     

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.     

Two-state conformational changes in inositol 1,4,5-trisphosphate receptor regulated by calcium

Inositol 1,4,5-trisphosphate receptor (IP3R) is a highly controlled calcium (Ca2+) channel gated by inositol 1,4,5-trisphosphate (IP3). Multiple regulators modulate IP3-triggered pore opening by binding to discrete allosteric sites within IP3R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca2+, the most pivotal regulator, induced marked structural changes in the tetrameric IP3R purified from mouse cerebella. Electron microscopy of the IP3R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca2+ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP3-binding domains, assigned by binding to heparin-gold. Ca2+-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca2+ appeared to regulate IP3 gating activity through the rearrangement of functional domains.     

Ligation of low-density lipoprotein receptor-related protein with antibodies elevates intracellular calcium and inositol 1,4, 5-trisphosphate in macrophages

We have probed the signaling characteristics of the macrophage low-density lipoprotein receptor-related protein (LRP) with monoclonal antibody 8G1, its Fab and F(ab')(2) fragments directed against the ligand binding heavy chain, and monoclonal antibody 5A6 directed against the membrane-spanning light chain of LRP. Ligation of LRP with 8G1, its Fab and F(ab')(2) fragments, or 5A6 increased intracellular Ca(2+) levels two- to threefold. Prior ligation of LRP with 8G1 did not affect the increase in [Ca(2+)](i) observed on subsequent ligation of LRP with lactoferrin, P. exotoxin A, or lipoprotein lipase. Binding to LRP by 8G1, its Fab and F(ab')(2) fragments, or 5A6 increased inositol 1,4,5-trisphosphate (IP(3)) levels by 50 to 100%. Incubation of macrophages with guanosine 5', 3'-O(thio)-triphosphate (GTP-gamma-S) before treatment with antibody potentiated and sustained the 8G1-induced increase in IP(3) levels. Treatment of macrophages with guanyl-5'-yl thiophosphate prior to GTP-gamma-S treatment abolished the GTP-gamma-S-potentiated increase in IP(3) levels in 8G1-treated macrophages. Antibody-induced increases in IP(3) and [Ca(2+)](i) in macrophages on ligation of LRP were pertussis toxin sensitive. Binding of 8G1 or its Fab or F(ab')(2) fragments to LRP stimulated macrophage protein kinase C (PKC) activity as evaluated by histone IIIs phosphorylation by about two- to sevenfold. Staurosporin inhibited the anti-LRP antibody-induced increase in PKC activity. Ligation of LRP with 8G1 increased cellular cAMP levels about twofold. Preincubation of macrophage with the LRP-binding protein receptor-associated protein suppressed the 8G1-induced increase in cAMP levels. Thus, binding of antibodies directed against either chain of LRP triggers complex signaling cascades.     

Expression of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) and its role in neural tube defects

ITPK1 is the rate-limiting enzyme in the pathway leading to formation of the highly phosphorylated inositol phosphates including IP₆ and the inositol pyrophosphates. One or more of these metabolites are essential for life as deletion of either of the kinases that form IP₅ or IP₆ in mice results in embryonic lethality. We have produced mice harboring a hypomorphic allele for Itpk1, and mice homozygous for this gene trap allele produce low but detectable levels of active enzyme. We have studied the expression of Itpk1 in various tissues and found that the enzyme is highly expressed in smooth muscle of vessels and other tissues. In addition, these mice have neural tube defects in 12% of homozygous embryos. Since the levels of enzyme expression vary greatly in homozygous animals, we speculate that relative deficiency of one or more inositol phosphates accounts for these defects. We plan to feed an inositol deficient diet or one with supplemental inositol to animals to demonstrate altered prevalence of neural tube defects.     

Heterooligomer of type 1 and type 2 inositol 1, 4, 5-trisphosphate receptor expressed in rat liver membrane fraction exists as tetrameric complex

Functional IP(3)-sensitive intracellular Ca(2+) release channel is considered to be a tetramer of IP(3)R. Heterooligomeric complexes composed of distinct types of IP(3)R have been reported, however, crucial evidences for them being tetramer have not appeared. Here we report that the heterooligomer composed of IP(3)R1 and IP(3)R2 also exists as tetramer. Cross-linked heterooligomer was immunoprecipitated with IP(3)R1-specific antibody and detected by agarose-PAGE/Western blot analysis with IP(3)R2-specific antibody. Tetramer, trimer, dimer, and possibly monomer were detected. The trimer, dimer, and monomer were likely to be originated from the tetramer, since: (1) the immunoprecipitating antibody (IP(3)R1-specific) does not recognize IP(3)R2, therefore IP(3)R2 monomer itself could not have been immunoprecipitated; and (2) tetramer was the major native product of IP(3)R complex containing type 2 isoform in liver membrane fraction. Thus we conclude tetramer is the native form of heterooligomer composed of IP(3)R1 and IP(3)R2.     

Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium release through the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1.

Our previous studies have demonstrated that calmodulin binds to IP3R type I (IP3R1) in a Ca2+ dependent manner, which suggests that calmodulin regulates the IP3R1 channel. In the present study, we investigated real-time kinetics of interactions between calmodulin and IP3R1 as well as effects of calmodulin on IP3-induced Ca2+ release by purified and reconstituted IP3R1. Kinetic analysis revealed that calmodulin binds to IP3R1 in a Ca2+ dependent manner and that both association and dissociation phase consist of two components with time constants of k(a) = 4.46 x 10(2) and > 10(4) M(-1) s(-1) k(d) = 1.44 x 10(-2) and 1.17 x 10(-1) s(-1). The apparent dissociation constant was calculated to be 27.3 microM. The IP3-induced Ca2+ release through the purified and reconstituted IP3R1 was inhibited by Ca2+/calmodulin, in a dose dependent manner. We interpret our findings to mean that calmodulin binds to IP3R1 in a Ca2+ dependent manner to exert inhibitory effect on IP3R channel activity. This event may be one of the mechanisms governing the negative feedback regulation of IP3-induced Ca2+ release by Ca2+.