Enhanced ER Ca2+ store filling by overexpression of SERCA2b promotes IP3-evoked puffs

Liberation of Ca²⁺ from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP₃R) is modulated by the ER Ca²⁺ content, and overexpression of SERCA2b to accelerate Ca²⁺ sequestration into the ER has been shown to potentiate the frequency and amplitude of IP₃-evoked Ca²⁺ waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP₃-evoked puffs to elucidate whether ER [Ca²⁺] may modulate IP₃R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca²⁺ release. SERCA2b and Ca²⁺ permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca²⁺ influx and thereby load ER stores. Puffs evoked by photoreleased IP₃ were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca²⁺ influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca²⁺ influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP₃R gating kinetics.     

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.     

Variable luminal sarcoplasmic reticulum Ca buffer capacity in smooth muscle cells

Sarcoplasmic reticulum contains the internal Ca²⁺ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca²⁺ level is the same all throughout the SR; however, whether the Ca²⁺ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca²⁺ buffer capacity of the SR by comparing the reduction in SR Ca²⁺ levels with the corresponding increase in [Ca²⁺]_i during activation of either IP₃Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca²⁺ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca²⁺ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca²⁺ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca²⁺ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca²⁺ influx, indicate the relevance of luminal SR Ca²⁺ buffer capacity in the [Ca²⁺]_i response. To further study the importance of luminal SR Ca²⁺ buffer capacity in the release process we used low levels of heparin to partially inhibit IP₃Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca²⁺ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca²⁺-binding proteins in the high-capacity, low-affinity conformation, particularly for IP₃R-mediated Ca²⁺ release.     

IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca²⁺ homeostasis. We have previously shown that the IP₃ receptor (IP₃R) plays a role in elevating basal cytoplasmic Ca²⁺ and that pharmacological blockade of IP₃R restores muscle function. Moreover, we have shown that the IP₃R pathway negatively regulates autophagy by controlling mitochondrial Ca²⁺ levels. Nevertheless, it remains unclear whether IP₃R is involved in abnormal mitochondrial Ca²⁺ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP₃R was downregulated in mdx fibers. Pharmacological blockade of IP₃R in mdx fibers restored both increased mitochondrial Ca²⁺ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP₃R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP₃R1 activity with changes in autophagy, mitochondrial Ca²⁺ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP₃R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP₃R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.     

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

In addition to its well established function in activating Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca²⁺ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca²⁺ signals via changes in ER Ca²⁺ store content, by imaging Ca²⁺ liberation through inositol trisphosphate receptors (IP₃R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca²⁺] was transiently elevated by applying voltage-clamp pulses to induce Ca²⁺ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca²⁺ signals evoked by strong photorelease of IP₃, and increased numbers of local Ca²⁺ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca²⁺ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca²⁺ on the IP₃R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca²⁺ may modulate IP₃R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.     

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.     

Analysis of IP3 receptors in and out of cells

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP₃R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca^2 + channels.

Scope of the review

In this brief review, we first consider a variety of complementary methods that allow the links between IP₃ binding and channel gating to be defined. How does IP₃ binding to the IP₃-binding core in each IP₃R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP₃, Ca^2 + signals and IP₃R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP₃R signalling.

Major conclusions

All IP₃R are regulated by both IP₃ and Ca^2 +. This allows them to initiate and regeneratively propagate intracellular Ca^2 + signals. The elementary Ca^2 + release events evoked by IP₃ in intact cells are mediated by very small numbers of active IP₃R and the Ca^2 +-mediated interactions between them. The spatial organization of these Ca^2 + signals and their stochastic dependence on so few IP₃Rs highlight the need for methods that allow the spatial organization of IP₃R signalling to be addressed with single-molecule resolution.

General significance

A variety of complementary methods provide insight into the structural basis of IP₃R activation and the contributions of IP₃-evoked Ca^2 + signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Probes for manipulating and monitoring IP3

Inositol 1,4,5-trisphosphate (IP₃) is an important second messenger produced via G-protein-coupled receptor- or receptor tyrosine kinase-mediated pathways. IP₃ levels induce Ca²⁺ release from the endoplasmic reticulum (ER) via IP₃ receptor (IP₃R) located in the ER membrane. The resultant spatiotemporal pattern of Ca²⁺ signals regulates diverse cellular functions, including fertilization, gene expression, synaptic plasticity, and cell death. Therefore, monitoring and manipulating IP₃ levels is important to elucidate not only the functions of IP₃-mediated pathways but also the encoding mechanism of IP₃R as a converter of intracellular signals from IP₃ to Ca²⁺.     

Fluvoxamine rescues mitochondrial Ca transport and ATP production through σ1-receptor in hypertrophic cardiomyocytes

Aims

We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ₁-receptor (σ₁R), ameliorates cardiac hypertrophy and dysfunction via σ₁R stimulation. Although σ₁R on non-cardiomyocytes interacts with the IP₃ receptor (IP₃R) to promote mitochondrial Ca^2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main methods

Here we performed Ca^2 + imaging and measured ATP production to define the role of σ₁Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca^2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key finding

These cardiomyocytes exhibited imbalances in expression levels of σ₁R and IP₃R and impairments in both phenylephrine-induced mitochondrial Ca^2 + mobilization from the SR and ATP production. Interestingly, σ₁R stimulation with fluvoxamine rescued impaired mitochondrial Ca^2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ₁R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ₁Rs suppressed intracellular Ca^2 + mobilization through IP₃Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance

These results suggest that σ₁R stimulation with fluvoxamine promotes SR-mitochondrial Ca^2 + transport and mitochondrial ATP production, whereas σ₁R stimulation suppresses intracellular Ca^2 + overload through IP₃Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ₁R agonists including fluvoxamine.     

Control of protein translation by IP3R-mediated Ca2+ release in Drosophila neuroendocrine cells

The inositol 1,4,5-trisphosphate receptor (IP₃R) is one of two Ca²⁺ channels that gates Ca²⁺ release from ER-stores. The ligand IP₃, generated upon specific G-protein coupled receptor activation, binds to IP₃R to release Ca²⁺ into the cytosol. IP₃R also mediates ER-store Ca²⁺ release into the mitochondria, under basal as well as stimulatory conditions; an activity that influences cellular bioenergetics and thus, cellular growth and proliferation. In Drosophila neuroendocrine cells expressing a hypomorphic mutant of IP₃R, we observed reduced protein translation levels. Here, we discuss the possible molecular mechanism for this observation. We hypothesize that the cellular energy sensor, AMPK connects IP₃R mediated Ca²⁺ release into the mitochondria, to protein translation, via the TOR pathway.     

Isoform- and Species-specific Control of Inositol 1,4,5-Trisphosphate (IP3) Receptors by Reactive Oxygen Species

Reactive oxygen species (ROS) stimulate cytoplasmic [Ca²⁺] ([Ca²⁺]_c) signaling, but the exact role of the IP₃ receptors (IP₃R) in this process remains unclear. IP₃Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP₃Rs at the ER-mitochondrial associations. Also, IP₃Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O₂^⨪) on IP₃R-mediated Ca²⁺ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed [Ca²⁺]_c spikes and frequency-modulated oscillations evoked by a O₂^⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The [Ca²⁺]_c signal was mediated by ER Ca²⁺ mobilization. X+XO added to permeabilized cells promoted the [Ca²⁺]_c rise evoked by submaximal doses of IP₃, indicating that O₂^⨪ directly sensitizes IP₃R-mediated Ca²⁺ release. In response to X+XO, DT40 cells lacking two of three IP₃R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP₃Rs (DKO2) showed a [Ca²⁺]_c signal, whereas DKO expressing type 3 IP₃R (DKO3) did not. By contrast, IgM that stimulates IP₃ formation, elicited a [Ca²⁺]_c signal in every DKO. X+XO also facilitated the Ca²⁺ release evoked by submaximal IP₃ in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP₃R (TKO). However, X+XO could also facilitate the effect of suboptimal IP₃ in TKO transfected with rat IP₃R3. Although in silico studies failed to identify a thiol missing in the chicken IP₃R3, an X+XO-induced redox change was documented only in the rat IP₃R3. Thus, ROS seem to specifically sensitize IP₃Rs through a thiol group(s) within the IP₃R, which is probably inaccessible in the chicken IP₃R3.     

Endoplasmic Reticulum Dysfunction and Ca<Superscript>2<Emphasis Type="Bold">+</Emphasis></Superscript> Deregulation in Isolated CA1 Neurons during Oxygen and Glucose Deprivation

Intracellular calcium ([Ca²⁺]_i) plays a pivotal role in neuronal ischemia. The aim of the present study was to investigate the routes of Ca²⁺ entry during non-excitotoxic oxygen and glucose deprivation (OGD) in acutely dissociated rat CA1 neurons. During OGD the fluo-3/fura red ratio reflecting [Ca²⁺]_i increased rapidly and irreversibly. [Ca²⁺]_i increased to the same degree in Ca²⁺ depleted medium, and also when both the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate (IP₃) receptors were blocked. When the endoplasmic reticulum (ER) Ca²⁺ stores were emptied with thapsigargin no increase in [Ca²⁺]_i was observed independent of extracellular Ca²⁺. The OGD induced Ca²⁺ deregulation in isolated CA1 neurons is not prevented by removing Ca²⁺, or by blocking the IP₃– or RyR receptors. However, when SERCA was blocked, no increase in [Ca²⁺]_i was observed suggesting that SERCA dysfunction represents an important mechanism for ischemic Ca²⁺ overload.     

Increased calcium release from sarcoplasmic reticulum stimulated by inositol trisphosphate in spontaneously hypertensive rat heart cells

It is known that inositol (1, 4, 5)-trisphosphate (IP₃) stimulates Ca²⁺ release from sarcoplasmic reticulum (SR) in several tissues, but in cardiac myocytes this phenomenon has not been confirmed. The purpose of the present study was to confirm the effect of (1, 4, 5)-IP₃ on Ca²⁺ release from SR in cardiac myocytes. The effect of IP₃ on Ca²⁺ release from SR in hypertrophic cardiac cells was also determined.We examined the effects of IP₃ on Ca²⁺ release from cardiac myocyte SR by the bigital-image method in a single cell. We also determined the effect of IP₃ on calcium release from isolated SR. SR was prepared from spontaneous hypertensive rat hearts and Wistar kyoto rat hearts. The SR was prelabeled with⁴⁵Ca²+, and then incubated with the indicated concentrations of IP³ for 1 min at 37°C. In cardiac myocytes treated with saponin, Ca²⁺ release stimulated by 10 M (1, 4, 5)-IP₃ was detected by fura-2. In⁴⁵Ca²⁺ prelabeled SR, the maximal Ca²⁺ release was achieved at 10 M IP₃ incubated for 1 min. The release of Ca²⁺ was higher in Sr of SHR than in the SR of WKY. IP₃ stimulates Ca²⁺ release from cardiac SR, and this release is greater in SHR than in WKY. However, it is uncertain whether this phenomenon plays a role in cardiac hypertrophy.     

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.     

Inhibition of mitochondrial calcium uptake rather than efflux impedes calcium release by inositol-1,4,5-trisphosphate-sensitive receptors

Mitochondria modulate cellular Ca²⁺ signals by accumulating the ion via a uniporter and releasing it via Na⁺- or H⁺-exchange. In smooth muscle, inhibition of mitochondrial Ca²⁺ uptake inhibits Ca²⁺ release from the sarcoplasmic reticulum (SR) via inositol-1,4,5-trisphosphate-sensitive receptors (IP₃R). At least two mechanisms may explain this effect. First, localised uptake of Ca²⁺ by mitochondria may prevent negative feedback by cytosolic Ca²⁺ on IP₃R activity, or secondly localised provision of Ca²⁺ by mitochondrial efflux may maintain IP₃R function or SR Ca²⁺ content. To distinguish between these possibilities the role of mitochondrial Ca²⁺ efflux on IP₃R function was examined. IP₃ was liberated in freshly isolated single colonic smooth muscle cells and mitochondrial Na⁺–Ca²⁺ exchanger inhibited with CGP-37157 (10 μM). Mitochondria accumulated Ca²⁺ during IP₃-evoked [Ca²⁺]_c rises and released the ion back to the cytosol (within 15 s) when mitochondrial Ca²⁺ efflux was active. When mitochondrial Ca²⁺ efflux was inhibited by CGP-37157, an extensive and sustained loading of mitochondria with Ca²⁺ occurred after IP₃-evoked Ca²⁺ release. IP₃-evoked [Ca²⁺]_c rises were initially unaffected, then only slowly inhibited by CGP-37157. IP₃R activity was required for inhibition to occur; incubation with CGP-37157 for the same duration without IP₃ release did not inhibit IP₃R. CGP-37157 directly inhibited voltage-gated Ca²⁺ channel activity, however SR Ca²⁺ content was unaltered by the drug. Thus, the gradual decline of IP₃R function that followed mitochondrial Na⁺–Ca²⁺ exchanger inhibition resulted from a gradual overload of mitochondria with Ca²⁺, leading to a reduced capacity for Ca²⁺ uptake. Localised uptake of Ca²⁺ by mitochondria, rather than mitochondrial Ca²⁺ efflux, appears critical for maintaining IP₃R activity.     

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.     

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.     

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.     

Pathophysiological consequences of isoform-specific IP3 receptor mutations

Ca²⁺ signaling governs a diverse range of cellular processes and, as such, is subject to tight regulation. A main component of the complex intracellular Ca²⁺-signaling network is the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R), a tetrameric channel that mediates Ca²⁺ release from the endoplasmic reticulum (ER) in response to IP₃. IP₃R function is controlled by a myriad of factors, such as Ca²⁺, ATP, kinases and phosphatases and a plethora of accessory and regulatory proteins. Further complexity in IP₃R-mediated Ca²⁺ signaling is the result of the existence of three main isoforms (IP₃R1, IP₃R2 and IP₃R3) that display distinct functional characteristics and properties. Despite their abundant and overlapping expression profiles, IP₃R1 is highly expressed in neurons, IP₃R2 in cardiomyocytes and hepatocytes and IP₃R3 in rapidly proliferating cells as e.g. epithelial cells. As a consequence, dysfunction and/or dysregulation of IP₃R isoforms will have distinct pathophysiological outcomes, ranging from neurological disorders for IP₃R1 to dysfunctional exocrine tissues and autoimmune diseases for IP₃R2 and -3. Over the past years, several IP₃R mutations have surfaced in the sequence analysis of patient-derived samples. Here, we aimed to provide an integrative overview of the clinically most relevant mutations for each IP₃R isoform and the subsequent molecular mechanisms underlying the etiology of the disease.     

A2A R mediated modulation in IP3 levels altering the [Ca2+]i through cAMP-dependent PKA signalling pathway

Stimulation of A_2A receptors (A_2A R) coupled to G_s/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A_2A R in the modulation of free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) involving IP₃, cAMP and PKA was investigated in HEK 293-A_2A R. The levels of IP₃ and cAMP were observed by enzyme immunoassay detection method and [Ca²⁺]_i using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A_2A R agonist NECA showed increased levels of cAMP, PKA, IP₃ and [Ca²⁺]_i levels. However, the reverse effect was observed with A_2A R antagonists (ZM241385 and caffeine). Blocking the G_αq/PLC/DAG/IP₃ pathway with neomycin, a PLC inhibitor did not affect the modulation of IP₃ and [Ca²⁺]_i levels in HEK 293-A_2A R cells. To investigate the G_αi/AC/cAMP/PKA, HEK 293-A_2A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A_2A R agonist (NECA) showed no effect on cAMP levels. Further, G_αs/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP₃ mediated [Ca²⁺]_i modulation. In the HEK 293-A_2A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP₃ and [Ca²⁺]_i levels. The study distinctly demonstrated that A_2A R modulates IP₃ levels to release the [Ca²⁺]_i via cAMP-dependent PKA. The role of A_2A R mediated G_αs pathway inducing IP₃ mediated [Ca²⁺]_i release may open new avenues in the therapy of neurodegenerative disorder.