Inositol 1,4,5-trisphosphate receptor 1, a widespread Ca2+ channel, is a novel substrate of polo-like kinase 1 in eggs

To initiate embryo development, the sperm induces in the egg release of intracellular calcium ([Ca²⁺]_i). During oocyte maturation, the inositol 1,4,5-trisphosphate receptor (IP₃R1), the channel implicated, undergoes modifications that enhance its function. We found that IP₃R1 becomes phosphorylated during maturation at an MPM-2 epitope and that this persists until the fertilization-associated [Ca²⁺]_i responses cease. We also reported that maturation without ERK activity diminishes IP₃R1 MPM-2 reactivity and [Ca²⁺]_i responses. Here, we show that IP₃R1 is a novel target for Polo-like kinase1 (Plk1), a conserved M-phase kinase, which phosphorylates it at an MPM-2 epitope. Plk1 and IP₃R1 interact in an M-phase preferential manner, and they exhibit close co-localization in the spindle/spindle poles area. This co-localization is reduced in the absence of ERK activity, as the ERK pathway regulates spindle organization and IP₃R1 cortical re-distribution. We propose that IP₃R1 phosphorylation by Plk1, and possibly by other M-phase kinases, underlies the delivery of spatially and temporally regulated [Ca²⁺]_i signals during meiosis/mitosis and cytokinesis.     

Redistribution and Increase in Cortical Inositol 1,4,5-Trisphosphate Receptors after Meiotic Maturation of the Mouse Oocyte

Mouse oocytes develop sensitivity to inositol 1,4,5-trisphosphate (IP₃) during oocyte maturation. We recently reported that a change in the organization of the endoplasmic reticulum (ER) during oocyte maturation may contribute to this enhanced sensitivity (Mehlmannet al.,1995,Dev. Biol.170, 607–615). Here, we investigated whether there is an increase in the number of available IP₃receptors after maturation and whether there is a redistribution of IP₃receptors similar to the redistribution of the ER that occurs during maturation. Western blot analysis of the IP₃receptor in oocytes and eggs demonstrated a 1.8-fold increase in immunoreactive mass of the IP₃receptor following oocyte maturation. Microinjection of the function-blocking monoclonal antibody 18A10 inhibited IP₃-induced Ca²⁺release in a concentration-dependent manner in both eggs and oocytes. More antibody was required to inhibit Ca²⁺release to the same extent in eggs compared to oocytes when both were injected with the same concentration of IP₃, suggesting that eggs contain a greater number of functional IP₃receptors. Immunolocalization of the IP₃receptor revealed that receptors were present in large clusters, 1–2 μm in diameter, in the cortex of the mature egg except in a ring-shaped band of cortex adjacent to the meiotic spindle. In contrast, receptor clusters were located around the entire cortex of the immature oocyte and were much smaller (<1 μm); larger patches were sometimes seen, but they did not display the same spherical organization as those in eggs. These results suggest that the number of cortical IP₃receptors increases during mouse oocyte maturation and that this increase may contribute to enhanced Ca²⁺release at fertilization.     

Modeling Ca2+ signaling differentiation during oocyte maturation

Ca²⁺ is a fundamental intracellular signal that mediates a variety of disparate physiological functions often in the same cell. Ca²⁺ signals span a wide range of spatial and temporal scales, which endow them with the specificity required to induce defined cellular functions. Furthermore, Ca²⁺ signaling is highly plastic as it is modulated dynamically during normal physiological development and under pathological conditions. However, the molecular mechanisms underlying Ca²⁺ signaling differentiation during cellular development remain poorly understood. Oocyte maturation in preparation for fertilization provides an exceptionally well-suited model to elucidate Ca²⁺ signaling regulation during cellular development. This is because a Ca²⁺ signal with specialized spatial and temporal dynamics is universally essential for egg activation at fertilization. Here we use mathematical modeling to define the critical determinants of Ca²⁺ signaling differentiation during oocyte maturation. We show that increasing IP₃ receptor (IP₃R) affinity replicates both elementary and global Ca²⁺ dynamics observed experimentally following oocyte maturation. Furthermore, our model reveals that because of the Ca²⁺ dependency of both SERCA and the IP₃R, increased IP₃R affinity shifts the system's equilibrium to a new steady state of high cytosolic Ca²⁺, which is essential for fertilization. Therefore our model provides unique insights into how relatively small alterations of the basic molecular mechanisms of Ca²⁺ signaling components can lead to dramatic alterations in the spatio-temporal properties of Ca²⁺ dynamics.     

Polycystin-1 Interacts with Inositol 1,4,5-Trisphosphate Receptor to Modulate Intracellular Ca2+ Signaling with Implications for Polycystic Kidney Disease

The PKD1 or PKD2 genes encode polycystins (PC) 1 and 2, which are associated with polycystic kidney disease. Previously we demonstrated that PC2 interacts with the inositol 1,4,5-trisphosphate receptor (IP₃R) to modulate Ca²⁺ signaling. Here, we investigate whether PC1 also regulates IP₃R. We generated a fragment encoding the last six transmembrane (TM) domains of PC1 and the C-terminal tail (QIF38), a section with the highest homology to PC2. Using a Xenopus oocyte Ca²⁺ imaging system, we observed that expression of QIF38 significantly reduced the initial amplitude of IP₃-induced Ca²⁺ transients, whereas a mutation lacking the C-terminal tail did not. Thus, the C terminus is essential to QIF38 function. Co-immunoprecipitation assays demonstrated that through its C terminus, QIF38 associates with the IP₃-binding domain of IP₃R. A shorter PC1 fragment spanning only the last TM and the C-terminal tail also reduced IP₃-induced Ca²⁺ release, whereas another C-terminal fragment lacking any TM domain did not. Thus, only endoplasmic reticulum-localized PC1 can modulate IP₃R. Finally, we show that in the polarized Madin-Darby canine kidney cells, heterologous expression of full-length PC1 resulted in a smaller IP₃-induced Ca²⁺ response. Overexpression of the IP₃-binding domain of IP₃R reversed the inhibitory effect of PC1, suggesting interaction of full-length PC1 (or its cleavage forms) with endogenous IP₃R in Madin-Darby canine kidney cells. These results indicate that the behavior of full-length PC1 in mammalian cells is congruent with that of PC1 C-terminal fragments in the oocyte system. These data demonstrate that PC1 inhibits Ca²⁺ release, perhaps opposing the effect of PC2, which facilitates Ca²⁺ release through the IP₃R.     

Kinase-dependent Regulation of Inositol 1,4,5-Trisphosphate-dependent Ca2+ Release during Oocyte Maturation

Fertilization induces a species-specific Ca²⁺ transient with specialized spatial and temporal dynamics, which are essential to temporally encode egg activation events such as the block to polyspermy and resumption of meiosis. Eggs acquire the competence to produce the fertilization-specific Ca²⁺ transient during oocyte maturation, which encompasses dramatic potentiation of inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release. Here we show that increased IP₃ receptor (IP₃R) sensitivity is initiated at the germinal vesicle breakdown stage of maturation, which correlates with maturation promoting factor (MPF) activation. Extensive phosphopeptide mapping of the IP₃R resulted in ∼70% coverage and identified three residues, Thr-931, Thr-1136, and Ser-114, which are specifically phos pho ryl a ted during maturation. Phospho-specific antibody analyses show that Thr-1136 phos pho ryl a tion requires MPF activation. Activation of either MPF or the mitogen-activated protein kinase cascade independently, functionally sensitizes IP₃-dependent Ca²⁺ release. Collectively, these data argue that the kinase cascades driving meiotic maturation potentiates IP₃-dependent Ca²⁺ release, possibly trough direct phos pho ryl a tion of the IP₃R.Egg activation refers to the cellular and molecular events that take place immediately following fertilization, transitioning the zygote into embryogenesis. In vertebrates, egg activation encompasses the block to polyspermy and the completion of oocyte meiosis, which is coupled to the extrusion of the second polar body. Interestingly, in all sexually reproducing organisms tested to date the cellular events associated with egg activation are Ca²⁺-dependent (1). Importantly the Ca²⁺ signal at fertilization encodes the progression of these cellular events in a defined temporal sequence that ensures a functional egg-to-embryo transition (2, 3). The first order of business for the fertilized egg is to block polyspermy, which could be lethal to the embryo. This presents a particularly difficult problem for the large Xenopus oocyte. Therefore, this species employs a fast and slow blocks to polyspermy, both of which are Ca²⁺-dependent (4). In addition, the Ca²⁺ release wave at fertilization releases the metaphase II cytostatic factor-dependent arrest in Xenopus oocytes. As is the case in other vertebrates, Xenopus eggs arrest at metaphase of meiosis II, an event that marks the completion of maturation.Therefore, Ca²⁺ dynamics at fertilization initiate and temporally encode critical cellular events for the egg-to-embryo transition. Specificity in Ca²⁺ signaling is encoded to a large extent in the spatial, temporal, and amplitude features of the Ca²⁺ signal. This endows Ca²⁺ signaling with its versatility and specificity, where in the same cell Ca²⁺ signals can mediate distinct cellular responses (5, 6).Ca²⁺ signaling pathways and intracellular organelles remodel during oocyte maturation, a complex cellular differentiation that prepares the egg for fertilization and egg activation (7, 8). In Xenopus the activity and distribution of multiple essential Ca²⁺-transporting proteins is modulated dramatically during oocyte maturation (8). Functional studies and mathematical modeling support the conclusion that the two critical determinants of Ca²⁺ signaling remodeling during Xenopus oocyte maturation are the internalization of the plasma-membrane Ca²⁺-ATPase, and the sensitization of inositol 1,4,5-trisphosphate (IP₃)²-dependent Ca²⁺ release (9–11). Indeed Ca²⁺ release from intracellular stores through the IP₃ receptor (IP₃R) represents the primary source for the initial Ca²⁺ rise at fertilization in vertebrates (12–14). The sensitivity of IP₃-dependent Ca²⁺ release is enhanced during maturation (10, 15). The IP₃R physically clusters during maturation (9, 16), and this is associated with functional clustering of elementary Ca²⁺ release events (10). IP₃R clustering is important for the slow and continuous nature of Ca²⁺ wave propagation in Xenopus eggs (10). In fact the potentiation of IP₃-dependent Ca²⁺ release is a hallmark of Ca²⁺ signaling differentiation during oocyte maturation in several vertebrate and invertebrate species (17–19). However, the mechanisms underlying enhanced IP₃-dependent Ca²⁺ release are not well understood.An attractive mechanism to explain increased IP₃R sensitivity during oocyte maturation is phosphorylation, given the critical role kinase cascades play in the initiation and progression of the meiotic cell cycle. Furthermore, the affinity of the IP₃R increases during mitosis apparently due to direct phosphorylation by maturation-promoting factor (MPF) (20, 21). In contrast, in starfish eggs, although the increase in Ca²⁺ release was dependent on MPF activation, MPF does not directly phosphorylate the IP₃R, but rather it appears to mediate its effect through the actin cytoskeleton (22, 23). More recently, the MAPK cascade has been shown to be important for shaping Ca²⁺ dynamics in mouse eggs (24). Together, these results argue that phosphorylation plays an important role in the sensitization of IP₃-dependent Ca²⁺ release during M-phase.Xenopus oocyte maturation is initiated by steroids that appear to act on a cell surface receptor (25). An important kinase cascade activated during maturation is the MAPK cascade that is initiated through the accumulation of Mos (Fig. 1A). This cascade culminates in the inhibition of Myt1, which phosphorylates and inhibits MPF. MPF is the key regulator of entry into M-phase and is composed of a Ser/Thr kinase subunit (cdk1) and cyclin B as a regulatory subunit. In addition, activation of Cdc25C is essential for oocyte maturation, because it represents the rate-limiting step in MPF activation (26). Cdc25C is phosphorylated by polo-like kinase through unknown upstream steps. In this work we analyze the functional regulation and phosphorylation pattern of the IP₃R during oocyte maturation to better understand the role of cell cycle kinases in modulating IP₃-dependent Ca²⁺ release.Open in a separate windowFIGURE 1.IP₃-dependent Ca²⁺ release dynamics during maturation. A, kinase cascades driving Xenopus oocyte maturation. B, oocytes were injected with caged-IP₃ and Oregan Green 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis 1 before imaging. Maturation was induced with progesterone, and cells were collected at different time points as indicated. Cells were imaged in line scan mode on a Zeiss LSM510 with the near UV 450 nm laser continuously on, at low intensity to produce a slow gradual IP₃ rise. After imaging each cell was lysed and analyzed individually for the activation state of MAPK and MPF. MPF was assayed using an anti-phospho-Tyr-15-cdk1 antibody (arrow). Dephosphorylation is indicative of MPF activation. MAPK activation was detected using a phospho-specific MAPK antibody (arrowhead). Tubulin was the loading control (dash). C, percent of cells at each time point that either exhibit no release for the duration of the line scan (No Rel., black), puffs only (puffs, green), puffs followed by a wave (Puff-Wave, blue), or only a Ca²⁺ wave (Wave, red). For each time point n = 11–23 cells. D, amplitude of the first peak during the line scan as compared with the maximal Ca²⁺ signal. Mean ± S.E. (n = 9–18). E, latency until the first Ca²⁺ signal (Time to first peak) as compared with the time required to reach maximal signal (Time to Max). Mean ± S.E. (n = 9–18). For C–E: oocytes (Ooc); cells treated with progesterone that have not undergone GVBD at 2 or more hours after progesterone (p > 2); cells at GVBD and up to 0.5 h after GVBD (GVBD 0–0.5); cells from 0.5 to 2.5 h after GVBD (GVBD 0.5–2.5); fully mature eggs at 3 or more hours after GVBD (>3 egg).     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

Expression of the zebrafish intermediate neurofilament Nestin in the developing nervous system and in neural proliferation zones at postembryonic stages

Background

At fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca²⁺ ([Ca²⁺]_i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca²⁺]_i oscillations through the generation of inositol 1,4,5-triphosphate (IP₃), which causes Ca²⁺ release by binding to IP₃ receptors located on the endoplasmic reticulum (ER) of the oocyte. Ability to respond to this activating stimulus develops during meiotic maturation of the oocyte. Here we examine how the development of this ability is perturbed when a single spermatozoon is introduced into the oocyte prematurely, i.e. during oocyte maturation.

Results

Mouse oocytes during maturation in vitro were fertilised by ICSI (intracytoplasmic sperm injection) 1 – 4 h after germinal vesicle break-down (GVBD) and were subsequently cultured until they reached metaphase II (MII) stage. At MII stage they were fertilised in vitro for the second time (refertilisation). We observed that refertilised oocytes underwent activation with similar frequency as control oocytes, which also went through maturation in vitro, but were fertilised only once at MII stage (87% and 93%, respectively). Refertilised MII oocytes were able to develop [Ca²⁺]_i oscillations in response to penetration by spermatozoa. We found however, that they generated a lower number of transients than control oocytes. We also showed that the oocytes, which were fertilised during maturation had a similar level of MPF activity as control oocytes, which were not subjected to ICSI during maturation, but had reduced level of IP₃ receptors.

Conclusion

Mouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca²⁺]_i transients, and to be activated after completion of maturation.     

A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation

Sustained elevation of intracellular calcium by Ca²⁺ release–activated Ca²⁺ channels is required for lymphocyte activation. Sustained Ca²⁺ entry requires endoplasmic reticulum (ER) Ca²⁺ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP₃R)/Ca²⁺ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca²⁺, and the mechanism that enables the prolonged activation of IP₃R1 required for lymphocyte activation is unclear. We show that IP₃R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP₃R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP₃ and renders the channel less sensitive to Ca²⁺-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca²⁺ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca²⁺ levels during lymphocyte activation.     

Pivotal role of type-1 inositol 1,4,5-trisphosphate receptor for glucagon-induced gluconeogenesis

We highlight a recent paper which documents the important role that Ca²⁺ release through type-1 Inositol 1,4,5-trisphosphate receptor (IP₃R1) plays in the acute regulation by glucagon of gluconeogenesis in hepatocytes. The specificity is likely the result of discrete localization close to mitochondria and PKA-dependent phosphorylation of IP₃R1 which enhances Ca²⁺ release.     

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release from the endoplasmic reticulum by AMP-activated kinase modulators

The 5' AMP-activated protein kinase (AMPK) is a nutrient-sensitive kinase that plays a key role in the control of cellular energy metabolism. We have explored here the relationship between AMPK and Ca²⁺ signaling by looking at the effect of an AMPK activator (A769662) and an AMPK inhibitor (dorsomorphin) on histamine-induced Ca²⁺-release from the endoplasmic reticulum (ER) in HeLa cells. Our data show that incubation with A769662 (EC₅₀ = 29 μM) inhibited histamine-induced Ca²⁺-release from the ER in intact cells, as well as inositol-1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release in permeabilized cells. On the contrary, dorsomorphin (EC₅₀ = 0.4 μM) activated both histamine and IP₃-induced Ca²⁺-release and reversed the effect of A769662. These results suggest a direct effect of AMPK regulation on IP₃ receptor (IP₃R) function. A phosphoproteomic study did not reveal changes in IP₃R phosphorylation, but showed significant changes in phosphorylation of proteins placed upstream in the IP₃R interactome and in several proteins related with Ca²⁺ metabolism, which could be candidates to mediate the effects observed. In conclusion, our data suggest that AMPK negatively regulates IP₃R. This effect constitutes a novel and very important link between Ca²⁺ signaling and the AMPK pathway.     

IP3-induced cytosolic and nuclear Ca2+ signals in HL-1 atrial myocytes: Possible role of IP3 receptor subtypes

HL-1 cells are the adult cardiac cell lines available that continuously divide while maintaining an atrial phenotype. Here we examined the expression and localization of inositol 1,4,5-trisphosphate receptor (IP₃R) subtypes, and investigated how pattern of IP₃-induced subcellular local Ca²⁺ signaling is encoded by multiple IP₃R subtypes in HL-1 cells. The type 1 IP₃R (IP₃R1) was expressed in the perinucleus with a diffuse pattern and the type 2 IP₃R (IP₃R2) was expressed in the cytosol with a punctate distribution. Extracellular ATP (1 mM) elicited transient intracellular Ca²⁺ releases accompanied by a Ca²⁺ oscillation, which was eliminated by the blocker of IP₃Rs, 2-APB, and attenuated by ryanodine. Direct introduction of IP₃ into the permeabilized cells induced Ca²⁺ transients with Ca²⁺ oscillations at ⩾ 20 μM of IP₃, which was removed by the inhibition of IP₃Rs using 2-APB and heparin. IP₃-induced local Ca²⁺ transients contained two distinct time courses: a rapid oscillation and a monophasic Ca²⁺ transient. The magnitude of Ca²⁺ oscillation was significantly larger in the cytosol than in the nucleus, while the monophasic Ca²⁺ transient was more pronounced in the nucleus. These results provide evidence for the molecular and functional expression of IP₃R1 and IP₃R2 in HL-1 cells, and suggest that such distinct local Ca²⁺ signaling may be correlated with the punctate distribution of IP₃R2s in the cytosol and the diffuse localization of IP₃R1 in the peri-nucleus.     

Mammalian target of rapamycin (mTOR) phosphorylates inositol 1,4,5-trisphosphate receptor type 2 and increases its Ca release activity

There is substantial evidence that crosstalk between the proliferation and Ca²⁺-signaling pathways plays a critical role in the regulation of normal physiological functions as well as in the pathogenesis of a variety of abnormal processes. In non-excitable cells, intracellular Ca²⁺ is mobilized through inositol 1,4,5-trisphosphate sensitive Ca²⁺ channels (IP₃R) expressed on the endoplasmic reticulum. Here we report that mTOR, a point of convergence for signals from mitogenic growth factors, nutrients and cellular energy levels, phosphorylates the IP₃R-2, the predominant isoform of IP₃R in AR4-2J cells. Pretreatment with the mTOR inhibitor rapamycin, decreased carbachol-induced Ca²⁺ release in AR4-2J cells. Rapamycin also decreased IP₃-induced Ca²⁺ release in permeabilized AR4-2J cells. We also showed that IGF-1 potentiates carbachol-induced Ca²⁺ release in AR4-2J cells, an effect that was prevented by rapamycin. Rapamycin also decreased carbachol-induced Ca²⁺ release in HEK 293A cells in which IP₃R-1 and IP₃R-3 had been knocked down. These results suggest that mTOR potentiates the activity of IP₃R-2 by a phosphorylation mechanism. This conclusion supports the concept of crosstalk between Ca²⁺ signaling and proliferation pathways and thus provides another way by which intracellular Ca²⁺ signals are finely encoded.     

Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-Trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs

The ability of inositol 1,4,5-trisphosphate receptors (IP₃R) to precisely initiate and generate a diverse variety of intracellular Ca²⁺ signals is in part mediated by the differential regulation of the three subtypes (R1, R2, and R3) by key functional modulators (IP₃, Ca²⁺, and ATP). However, the contribution of IP₃R heterotetramerization to Ca²⁺ signal diversity has largely been unexplored. In this report, we provide the first definitive biochemical evidence of endogenous heterotetramer formation. Additionally, we examine the contribution of individual subtypes within defined concatenated heterotetramers to the shaping of Ca²⁺ signals. Under conditions where key regulators of IP₃R function are optimal for Ca²⁺ release, we demonstrate that individual monomers within heteromeric IP₃Rs contributed equally toward generating a distinct ''blended'' sensitivity to IP₃ that is likely dictated by the unique IP₃ binding affinity of the heteromers. However, under suboptimal conditions where [ATP] were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. We show that R2 monomers within a heterotetramer were both necessary and sufficient to dictate the ATP regulatory properties. Finally, the ATP-binding site B in R2 critical for ATP regulation was mutated and rendered non-functional to address questions relating to the stoichiometry of IP₃R regulation. Two intact R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers. In summary, we demonstrate that heterotetrameric IP₃R do not necessarily behave as the sum of the constituent subunits, and these properties likely extend the versatility of IP₃-induced Ca²⁺ signaling in cells expressing multiple IP₃R isoforms.     

Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer’s Disease-Causing Mutants in Presenilins

Familial Alzheimer’s disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) Ca²⁺ release channels resulting in enhanced IP₃R channel gating in an amyloid beta (Aβ) production-independent manner. This gain-of-function enhancement of IP₃R activity is considered to be the main reason behind the upregulation of intracellular Ca²⁺ signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca²⁺ signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP₃R channel activity records obtained under optimal Ca²⁺ and multiple IP₃ concentrations to gain deeper insights into the enhancement of IP₃R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP₃R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP₃R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP₃ and Ca²⁺ concentrations and quantify the sensitivity of IP₃R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP₃ and Ca²⁺ and that very small amount of IP₃ is required to stimulate IP₃R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP₃ concentrations. We further demonstrate with simulations that the relatively longer time spent by IP₃R in the H mode leads to the observed higher frequency of local Ca²⁺ signals, which can account for the more frequent global Ca²⁺ signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca²⁺ signals in cells expressing FAD-causing mutant PS.     

Predominant role of type 1 IP3 receptor in aortic vascular muscle contraction

Inositol 1,4,5-trisphosphate receptor (IP₃R) plays a crucial role in generating Ca²⁺ signaling and three subtypes of IP₃R have been identified. In spite of a high degree of similarity among these subtypes, their effects on spatio-temporal Ca²⁺ patterns are specific and diverse; therefore the physiological significance of the differential expression levels of IP₃R subtypes in various tissues remains unknown. Here, we examined the relative contribution of the specific subtype of IP₃Rs to the agonist-induced Ca²⁺ signaling and contraction in IP₃R-deficient vascular smooth muscle cells and found that IP₃R1 deficient cells exclusively showed less sensitivity to the agonist, compared to those from the other genotypes. We also found that IP₃R1 dominantly expressed in vascular aortae on a consistent basis, and that phenylephrine (PE)-induced aortic muscle contraction was reduced specifically in IP₃R1-deficient aortae. Taken together, we concluded that IP₃R1 plays a predominant role in the function of the vascular smooth muscle in vivo.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling

Background information. The IP₃R (inositol 1,4,5‐trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca²⁺ release in virtually all cell types. We have previously demonstrated that caspase‐3‐mediated cleavage of IP₃R1 during cell death generates a C‐terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP₃R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase‐3‐cleaved C‐terminus of IP₃R1 increased the rate of thapsigargin‐mediated Ca²⁺ leak and decreased the rate of Ca²⁺ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca²⁺ stores. We detected the truncated IP₃R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP₃R1 blocked sperm factor‐induced Ca²⁺ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase‐3‐mediated truncation of IP₃R1 enhanced the Ca²⁺ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca²⁺ leak is largely compensated by enhanced Ca²⁺‐uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca²⁺ leak acts as a feed‐forward mechanism to divert the cell into apoptosis.     

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways

Regulation of bi-directional communication between intracellular Ca²⁺ pools and surface Ca²⁺ channels remains incompletely characterized. We report Ca²⁺ release mediated by inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca²⁺ release, Ca²⁺ influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP₃R induced Ca²⁺ release, whereas RyR-mediated Ca²⁺ release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP₃R and RyR linked Ca²⁺ influx even though Ca²⁺ pool sensitivities were different. These findings suggest discrete Ca²⁺ store/Ca²⁺ channel coupling mechanisms in the IP₃R and RyR pathways as revealed by the differential sensitivity to actin perturbation.     

Dynamic regulation of IP3 receptor clustering and activity by IP3

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels. Their regulation by both IP₃ and Ca²⁺ allows interactions between IP₃Rs to generate a hierarchy of intracellular Ca²⁺ release events. These can progress from openings of single IP₃R, through near-synchronous opening of a few IP₃Rs within a cluster to much larger signals that give rise to regenerative Ca²⁺ waves that can invade the entire cell. We have used patch-clamp recording from excised nuclear membranes of DT40 cells expressing only IP₃R3 and shown that low concentrations of IP₃ rapidly and reversibly cause IP₃Rs to assemble into small clusters. In addition to bringing IP₃Rs close enough to allow Ca²⁺ released by one IP₃R to regulate the activity of its neighbors, clustering also retunes the regulation of IP₃Rs by IP₃ and Ca²⁺. At resting cytosolic [Ca²⁺], lone IP₃R are more sensitive to IP₃ and the mean channel open time (~10ms) is twice as long as for clustered IP₃R. When the cytosolic free [Ca²⁺] is increased to 1µM, to mimic the conditions that might prevail when an IP₃R within a cluster opens, clustered IP₃R are no longer inhibited and their gating becomes coupled. IP₃, by dynamically regulating IP₃R clustering, both positions IP₃R for optimal interactions between them and it serves to exaggerate the effects of Ca²⁺ within a cluster. During the course of these studies, we have observed that nuclear IP₃R stably express one of two single channel K ⁺ conductances (γ_K ~120 or 200pS). Here we demonstrate that for both states of the IP₃R, the effects of IP₃ on clustering are indistinguishable. These observations reinforce our conclusion that IP₃ dynamically regulates assembly of IP₃Rs into clusters that underlie the hierarchical recruitment of elementary Ca²⁺ release events.     

Inositol 1,4,5-Trisphosphate Receptor Subtype-Specific Regulation of Calcium Oscillations

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca²⁺) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) are intracellular Ca²⁺ release channels that mediate Ca²⁺ release from endoplasmic reticulum (ER) Ca²⁺ stores. The three IP₃R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP₃R by the endogenous modulators IP₃, Ca²⁺, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP₃R subtype in shaping cytosolic Ca²⁺ oscillations.     

Atrial local Ca signaling and inositol 1,4,5-trisphosphate receptors

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca²⁺ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca²⁺ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in atrial myocytes, and show our new findings on the role of IP₃R subtype in the regulation of spontaneous focal Ca²⁺ releases in the compartmentalized areas of atrial myocytes. The Ca²⁺ sparks, representing focal Ca²⁺ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca²⁺ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP₃Rs compared with ventricular cells and possess significant levels of type 1 IP₃R (IP₃R1) and type 2 IP₃R (IP₃R2). Over the last decade the roles of atrial IP₃R on the enhancement of Ca²⁺-induced Ca²⁺ release and arrhythmic Ca²⁺ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca²⁺ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP₃R1 in the maintenance of peri-nuclear and non-junctional Ca²⁺ sparks via stimulating a posttranslational organization of RyR clusters.