Protein kinase A increases the binding affinity and the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

Background information. In endocrine cells, IP₃R (inositol 1,4,5‐trisphosphate receptor), a ligand‐gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺ concentration. There are three subtypes of IP₃R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP₃R‐3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP₃R‐3 in RINm5F cells. Results. We show that immunoprecipitated IP₃R‐3 is a good substrate for PKA. Using a back‐phosphorylation approach, we show that endogenous PKA phosphorylates IP₃R‐3 in intact RINm5F cells. [³H]IP₃ (inositol 1,4,5‐trisphosphate) binding affinity and IP₃‐induced Ca²⁺ release activity were enhanced in permeabilized cells that were pre‐treated with forskolin or PKA. The PKA‐induced enhancement of IP₃R‐3 activity was also observed in intact RINm5F cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. Conclusion. The results of the present study reveal a converging step where the cAMP and the Ca²⁺ signalling systems act co‐operatively in endocrine cell responses to external stimuli.     

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.     

KRAS-induced actin-interacting protein regulates inositol 1,4,5-trisphosphate-receptor-mediated calcium release

KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous- actin-interacting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) and is responsible for the proper subcellular localization of IP₃R. Since it remains unknown whether KRAP regulates the IP₃R-mediated Ca²⁺ signaling, we herein examined the effects of KRAP on the IP₃R-mediated Ca²⁺ release by Ca²⁺ imagings in the cultured HEK293 or MCF7 cells. Reduction of KRAP protein by KRAP-specific siRNA diminishes ATP-induced Ca²⁺ release and the ATP-induced Ca²⁺ release is completely quenched by the pretreatment with the IP₃R inhibitor but not with the ryanodine receptor inhibitor, indicating that KRAP regulates IP₃R-mediated Ca²⁺ release. To further reveal mechanistic insights into the regulation of IP₃R-mediated Ca²⁺ release by KRAP, we examined the effects of the KRAP-knockdown on the releasable Ca²⁺ content of intracellular Ca²⁺ stores. Consequently, reduction of KRAP does not affect the amount of ionophore- or Ca²⁺-ATPase inhibitor-induced Ca²⁺ release in the HEK293 cells, indicating that releasable Ca²⁺ content of intracellular Ca²⁺ stores is not altered by KRAP. Thus, KRAP is involved in the proper regulation of IP₃R-mediated Ca²⁺ release.     

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.     

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.     

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.     

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.     

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.     

Agonist‐evoked Ca2+ wave progression requires Ca2+ and IP3

Smooth muscle responds to IP₃‐generating agonists by producing Ca²⁺ waves. Here, the mechanism of wave progression has been investigated in voltage‐clamped single smooth muscle cells using localized photolysis of caged IP₃ and the caged Ca²⁺ buffer diazo‐2. Waves, evoked by the IP₃‐generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) over a single though substantial length (～30 µm) of the cell. During regenerative propagation, the wave‐front was about 1/3 the length (～9 µm) of the initiation site. The wave‐front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP₃ may explain the amplitude changes. Ca²⁺ was required for IP₃‐mediated wave progression to occur. Increasing the Ca²⁺ buffer capacity in a small (2 µm) region immediately in front of a CCh‐evoked Ca²⁺ wave halted progression at the site. However, the wave front does not progress by Ca²⁺‐dependent positive feedback alone. In support, colliding [Ca²⁺]_c increases from locally released IP₃ did not annihilate but approximately doubled in amplitude. This result suggests that local IP₃‐evoked [Ca²⁺]_c increases diffused passively. Failure of local increases in IP₃ to evoke waves appears to arise from the restricted nature of the IP₃ increase. When IP₃ was elevated throughout the cell, a localized increase in Ca²⁺ now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP₃ and Ca²⁺ are required for active propagation of the wave front to occur. J. Cell. Physiol. 224: 334–344, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

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.     

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.     

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.     

Termination of calcium puffs and coupled closings of inositol trisphosphate receptor channels

Calcium puffs are localized Ca²⁺ signals mediated by Ca²⁺ release from the endoplasmic reticulum (ER) through clusters of inositol trisphosphate receptor (IP₃R) channels. The recruitment of IP₃R channels during puffs depends on Ca²⁺-induced Ca²⁺ release, a regenerative process that must be terminated to maintain control of cell signaling and prevent Ca²⁺ cytotoxicity. Here, we studied puff termination using total internal reflection microscopy to resolve the gating of individual IP₃R channels during puffs in intact SH-SY5Y neuroblastoma cells. We find that the kinetics of IP₃R channel closing differ from that expected for independent, stochastic gating, in that multiple channels tend to remain open together longer than predicted from their individual open lifetimes and then close in near-synchrony. This behavior cannot readily be explained by previously proposed termination mechanisms, including Ca²⁺-inhibition of IP₃Rs and local depletion of Ca²⁺ in the ER lumen. Instead, we postulate that the gating of closely adjacent IP₃Rs is coupled, possibly via allosteric interactions, suggesting an important mechanism to ensure robust puff termination in addition to Ca²⁺-inactivation.     

Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function

We recently reported key physiologic roles for Ca²⁺-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca²⁺-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca²⁺, inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated Ca²⁺ release from the sarcoplasmic reticulum represents a potential Ca²⁺ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP₃Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP₃Rs and are activated by IP₃R-mediated Ca²⁺ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP₃Rs in human DSM cells. As the TRPM4 channels and IP₃Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca²⁺-mediated TRPM4-IP₃R regulatory mechanism. To investigate IP₃R regulation of TRPM4 channel activity, we sought to determine the consequences of IP₃R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP₃Rs with the selective IP₃R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP₃Rs have a key role in mediating the Ca²⁺-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP₃Rs are spatially and functionally coupled in human DSM.     

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.     

Role of Inositol 1,4,5-Trishosphate Receptors in Pathogenesis of Huntington’s Disease and Spinocerebellar Ataxias

Huntington’s disease (HD) and spinocerebellar ataxias (SCAs) are autosomal-dominant neurodegenerative disorders. HD is caused by polyglutamine (polyQ) expansion in the amino-terminal region of a protein huntingtin (Htt) and primarily affects medium spiny striatal neurons (MSN). Many SCAs are caused by polyQ-expansion in ataxin proteins and primarily affect cerebellar Purkinje cells. The reasons for neuronal dysfunction and death in HD and SCAs remain poorly understood and no cure is available for the patients. Our laboratory discovered that mutant huntingtin, ataxin-2 and ataxin-3 proteins specifically bind to the carboxy-terminal region of the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1), an intracellular Ca²⁺ release channel. Moreover, we found that association of mutant huntingtin or ataxins with IP₃R1 causes sensitization of IP₃R1 to activation by IP₃ in planar lipid bilayers and in neuronal cells. These results suggested that deranged neuronal Ca²⁺ signaling might play an important role in pathogenesis of HD, SCA2 and SCA3. In support of this idea, we demonstrated a connection between abnormal Ca²⁺ signaling and neuronal cell death in experiments with HD, SCA2 and SCA3 transgenic mouse models. Additional data in the literature indicate that abnormal neuronal Ca²⁺ signaling may also play an important role in pathogenesis of SCAl, SCA5, SCA6, SCA14 and SCA15/16. Based on these results I propose that IP₃R and other Ca²⁺ signaling proteins should be considered as potential therapeutic targets for treatment of HD and SCAs.     

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca²⁺ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP₃R), an intracellular Ca²⁺ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP₃R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2^−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca²⁺ release in intact cells and IP₃-induced Ca²⁺ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca²⁺ signal by a local Ca²⁺-induced Ca²⁺-release mechanism, which only occurred in the presence of the TRPP2-IP₃R interaction, and not via altered IP₃R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca²⁺ microdomains necessary for initiating Ca²⁺-induced Ca²⁺ release. The data strongly suggest that defects in this mechanism may account for the altered Ca²⁺ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.     

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.