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

Abstract

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.

Introduction

Intracellular calcium (Ca²⁺) dynamics play pivotal roles in numerous physiological processes, including fertilization, cell proliferation and differentiation, apoptosis, embryonic development, secretion, muscle contraction, immunity, brain function, chemical senses, and light transduction [1, 2]. Two main Ca²⁺ mobilizing systems co-exist in the cell: Ca²⁺ influx from the extracellular medium and Ca²⁺ release from internal stores. The inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) is a tetrameric intracellular IP₃-gated Ca²⁺ release channel that is predominantly located on the membrane of the endoplasmic reticulum (ER). It is present in almost all cell types and plays a crucial role in converting extracellular stimuli into intracellular signals [1, 3]. Upon extracellular stimulation by various agonists, such as hormones, growth factors, neurotransmitters, neurotrophins, odorants, and light, Phospholipase-C (PLC) is activated and phosphatidylinositol 4,5-bisphosphate is hydrolysated, generating IP₃. IP₃ binds to the IP₃R, leading to the IP₃-induced Ca²⁺ release (IICR) from the ER. Thirty years ago, Mikoshiba et al. found a mutant mouse with deficient Purkinje cells that had very low expression of P₄₀₀, a glycoprotein that was later uncovered as one of the IP₃R subtypes (IP₃R1) [4, 5]. In 1989, Mikoshiba and co-workers were the first group to reveal that IP₃R is a trans-membrane protein and determine the primary sequence of IP₃R1, at the time the second largest molecule successfully cloned [6]. So far, three IP₃R subtypes (IP₃R1, IP₃R2, IP₃R3) as well as alternative splicing variants of IP₃R1 and IP₃R2 have been identified and cloned in mammals [1]. The expression patterns of the three subtypes are distinct but overlapping, and most cells express more than one subtype [7–9]. The three IP₃R subtypes share 65–85% homology and can be separated into five functional domains [1, 10–12]. The NH₂-terminal region contains a ligand coupling/suppressor domain, which suppresses IP₃-binding activity and determines different IP₃-binding affinity for each subtype [13], and an IP₃-binding core domain that is the minimum region required for specific IP₃ binding [14]. The ligand coupling/suppressor domain and the IP₃-binding core are often referred to as the IP₃-binding domain. Besides the IP₃-binding domain is the internal coupling domain, which confers regulation by various intracellular modulators (Ca²⁺, calmodulin (CaM), ATP) and phosphorylation by several protein kinases (cAMP-dependent protein kinase (PKA), protein kinase C (PKC), cGMP-dependent protein kinase, Ca²⁺/CaM-dependent protein kinase II (CamKII), and tyrosine kinase) [3]. The COOH-terminal region has a six membrane-spanning channel domain and a short cytoplasmic COOH-terminal tail (CTT), called the “gatekeeper domain”, which is critical for IP₃R channel opening [10]. The Ca²⁺ release activity of the IP₃R channel is therefore regulated by many intracellular modulators (IP₃, Ca²⁺, ATP, CaM), protein kinases, and IP₃R-binding proteins [1, 3], leading to various spatiotemporal cytosolic Ca²⁺ patterns and diverse cellular responses [1, 2]. The relatively low homology in the three IP₃R subtypes may underlie subtype-specific properties, that will affect Ca²⁺ signaling and in particular the spatiotemporal features of Ca²⁺ responses.

A prolonged elevation in the cytosolic Ca²⁺ concentration is considered toxic to the cell and in some cases may result in cell death. However, the cell can protect itself by temporally limiting the cytosolic Ca²⁺ elevation, often resulting in one of the most delicate patterns of Ca²⁺ signals, that being the oscillatory change in the cytosolic Ca²⁺ concentration, or Ca²⁺ oscillations [15–17]. Extensive studies over the past 30 years have revealed that cytosolic Ca²⁺ oscillations are ubiquitous and diverse cellular signals that control multiple processes in the cell. With cytosolic Ca²⁺ oscillations, cells not only avoid deleterious effects of sustained cytosolic Ca²⁺ concentrations, but also send out information encoded in the frequency and/or the amplitude of the oscillations to modulate cellular activity [15]. This review focuses on the separate role of the IP₃R subtypes in generating Ca²⁺ oscillations and on the molecular mechanisms responsible for the specific role of each subtype in regulating this ubiquitous signal.

A General Mechanism Generating Ca²⁺ Oscillations Based on Regulation of IP₃R

Many studies have indicated that IP₃R is involved in generating cytosolic Ca²⁺ oscillations [15–17]. For instance, the FGF-induced Ca²⁺ oscillations in mice fibroblasts are inhibited by an IP₃R antagonist [18]. Ca²⁺ oscillations are thought to arise due to periodic release of Ca²⁺ from intracellular Ca²⁺ stores via IP₃R [19]. Early studies using reconstituted IP₃R in lipid bilayers have indicated that Ca²⁺ can both activate and inhibit IP₃R [20, 21]. The IP₃R is activated at low cytosolic Ca²⁺ concentrations, elevating the cytosolic Ca²⁺ concentration through a process often referred to as Ca²⁺-induced Ca²⁺ release (CICR). High cytosolic Ca²⁺ concentration can instead inhibit IP₃R, leading to a decrease in intracellular Ca²⁺ release. In vivo, the binding of IP₃ together with fluctuating cytosolic Ca²⁺ concentrations can trigger successive cycles of IP₃R activation and inhibition, which result in cytosolic Ca²⁺ oscillations. Accordingly, Ca²⁺ oscillations can be produced by application of IP₃ to permeabilized hepatocytes [22] and blowfly salivary gland cells [23] and by injecting IP₃ analogs into fertilized ascidians eggs [24]. Moreover, DT40 cells expressing a mutant IP₃R with reduced sensitivity to Ca²⁺ do not exhibit Ca²⁺ oscillations upon application of cross-linked B-cell receptors [25]. Finally, thimerosal, which sensitizes IP₃R for lower IP₃ levels, potentiates IP₃-induced Ca²⁺ oscillations in sea urchin eggs [26]. These data, together with mathematical models [27, 28], have confirmed that the cross-talk between Ca²⁺ and IP₃ in regulating the IP₃R is critical for generating Ca²⁺ oscillations. However, in Madin-Darby canine kidney epithelial cells [29] and Chinese hamster ovary cells [30–32], each peak of the oscillatory Ca²⁺ signal is preceded by elevated IP₃, as measured by means of a pleckstrin homology domain of PLC-δ₁ tagged with a fluorescent protein indicator. Therefore, it has been proposed that dynamic IP₃ production may produce cytosolic Ca²⁺ oscillations. Nevertheless, other studies using different cells and methods reported opposite conclusions [33–35]. For example, expression of an IP₃ binding domain of IP₃R1 together with two different fluorescent proteins in HeLa cells does not reveal fluctuations in the intracellular IP₃ concentration during Ca²⁺ oscillations [33].

Subtype Specificity of Ca²⁺ Oscillations

Numerous studies using cells endogenously or exogenously expressing single or combined IP₃R subtypes indicate that the subtle distinctions in the properties of each subtype contribute differently to the regulation of cytosolic Ca²⁺ oscillations [3].

Miyakawa et al. [36] first described IP₃R subtype-specific Ca²⁺ oscillations using genetically engineered B cells that express either single or combined IP₃R subtypes. They found that Ca²⁺-signaling patterns depend on the expression levels of IP₃R subtypes, probably because of their specific response to endogenous modulators, such as IP₃, Ca²⁺ and ATP. IP₃R2 is the most sensitive to IP₃ and is required for robust, long lasting, and regular Ca²⁺ oscillations that occur upon activation of B-cell receptors. IP₃R1 mediates less regular Ca²⁺ oscillations. IP₃R3 is the least sensitive to IP₃ as well as Ca²⁺ and generates only monophasic Ca²⁺ transients. Morel et al. [37] examined the roles of IP₃R1 and IP₃R2 in Ca²⁺ oscillations using vascular myocytes and found that acetylcholine induces Ca²⁺ oscillations in cells expressing both subtypes, and fails to do so in cells expressing only IP₃R1. The oscillations are inhibited by intracellular infusion of heparin, anti-IP₃R2 antibody or antisense oligonucleotides targeting IP₃R2, suggesting that the IP₃R2 subtype is required for acetylcholine-induced Ca²⁺ oscillations in vascular myocytes. Using HeLa cells, which express comparable amounts of IP₃R1 and IP₃R3, Mikoshiba and co-workers showed that knockdown of IP₃R1 terminates Ca²⁺ oscillations, whereas knockdown of IP₃R3 leads to more robust and long lasting Ca²⁺ oscillations [38]. These IP₃R3 knockdown effects were similar in COS-7 cells that predominantly express IP₃R3, suggesting that IP₃R3 functions as an anti Ca²⁺-oscillatory unit. Almirza et al. reported similar results using normal kidney fibroblasts, which expresses IP₃R1 and IP₃R3 [39]. When IP₃R1 or IP₃R3 are knocked-down, the frequency of prostaglandin F_2α-induced Ca²⁺ oscillations is significantly decreased or increased, respectively. In NIH-3T3 cells, which predominantly express IP₃R2 and IP₃R3, ATP activates Ca²⁺ oscillations [40]. Ca²⁺ oscillations were induced by application of carbachol in AR4-2J cells, which predominantly expresses IP₃R2, and in HEK293A cells in which both IP₃R1 and IP₃R3 were knocked-down [41]. The contribution of IP₃R2 to Ca²⁺ oscillations is further confirmed by the fact that IP₃-dependent Ca²⁺ oscillations were abolished in osteoclasts of IP₃R2 knockout mice [42]. In rat insulinoma RINm5F cells, which almost exclusively express IP₃R3, application of carbachol or EGF, two agonists that activate PLC through different receptors, or application of IP₃ to permeabilized cells, elicit transient Ca²⁺ release and does not induce Ca²⁺ oscillations [43]. Several reports, including mathematical modeling studies, have indicated that the specific intracellular localization of the IP₃R is crucial for the generation of Ca²⁺ oscillations [44–46]. For instance, Kim et al. [45] found that HL-1 cells derived from mouse cardiac myocytes express both IP₃R1 and IP₃R2. IP₃R1 is expressed diffusely in the perinucleus and IP₃R2 is expressed in the cytosol with a punctuated distribution. Both application of ATP to intact cells and direct introduction of IP₃ into permeabilized cells evoke IP₃-dependent transient intracellular Ca²⁺ release accompanied by Ca²⁺ oscillations. The magnitude of Ca²⁺ oscillations is significantly larger in the cytosol than in the nucleus, while the monophasic Ca²⁺ transient is more pronounced in the nucleus. These results suggest that subtype specificity as well as specific localization of the IP₃R contribute to distinct local Ca²⁺ signaling. Altogether, these data suggest that IP₃R1 and IP₃R2, in particular IP₃R2, crucially contribute in generating Ca²⁺ oscillations, whereas IP₃R3 is an anti-oscillatory unit. Nevertheless, in A7r5 cells derived from rat embryonic thoracic aorta muscle cells, which express IP₃R1 and IP₃R3, knockdown of IP₃R1 only reduces the frequency of arginine vasopressin-induced Ca²⁺ oscillations without affecting the number of cells exhibiting Ca²⁺ oscillations [47]. Moreover, both acetylcholine and cholecystokinin octapeptide activate IP₃R2- and IP₃R3-dependent Ca²⁺ oscillations in pancreatic acinar cells. However, unlike IP₃R2-dependent oscillations, the amplitude of IP₃R3-dependent oscillations decreases throughout the stimulation [48]. The IP₃R subtype-specific Ca²⁺ oscillations are summarized in Table 1.

Table 1 The occurrence of Ca²⁺ oscillations and the expression of the different IP₃R subtypes

The IP₃R exists as a homo- or hetero-tetrameric complex to form a functional Ca²⁺ release channel [49–51]. The influence of homo- or hetero-tetrameric channels on intracellular Ca²⁺ oscillations has been investigated. Studies on genetically engineered DT40 cells that express a single IP₃R subtype and therefore a homo-tetrameric receptor demonstrate Ca²⁺ oscillations [36]. Cells with all subtypes, which should at least partially express hetero-tetrameric IP₃Rs, also exhibit Ca²⁺ oscillations [37–43, 45, 47]. Taken together these data suggest that both homo- and hetero-tetrameric IP₃Rs can generate intracellular Ca²⁺ oscillations.

In conclusion, it appears that IP₃R subtype-specific expression crucially shapes cytosolic Ca²⁺ signaling patterns. IP₃R2 is the main pro-oscillatory subtype, whereas IP₃R1 can induce a transient Ca²⁺ signal or an oscillatory Ca²⁺ signal. IP₃R3 mainly shows an anti-oscillatory behavior, but could underlie short-term oscillations depending on the cell type and stimulus. Further characterization of homo- and hetero-tetrameric IP₃R-dependent Ca²⁺ oscillations are needed for fully understanding the intricacies of each IP₃R subunit in shaping Ca²⁺ oscillations.

Subtype Specificity of IP₃-Binding Affinity to IP₃R

As summarized earlier, cytosolic Ca²⁺ oscillations are IP₃R subtype-dependent. IP₃ and Ca²⁺ are the two key modulators of IP₃R and the distinct subtype properties determine the diverse regulatory effects. Each subtype has different IP₃ binding affinity. Sudhof et al. were first to report, using an equilibrium IP₃ binding assay, that the order of IP₃-binding affinity was IP₃R2 > IP₃R1 > IP₃R3 [9, 52]. Applying the same method, Wojcikiewicz et al. [53] and Nerou et al. [54] later claimed a different order, IP₃R1 > IP₃R2 > IP₃R3. Mikoshiba and co-workers performed a detailed molecular analysis of the IP₃ binding affinity of all three subtypes [11, 13]. They found that the IP₃-binding affinities of purified IP₃-binding domains are close to the intrinsic IP₃-binding affinity of all three IP₃R subtypes, and describe the following order IP₃R2 > IP₃R1 > IP₃R3. They also showed that IP₃-binding core fragments, which do not contain the ligand coupling/suppressor domain, display an almost identical IP₃-binding affinity for all three subtypes. By a serious and compelling molecular analysis, they concluded that the functional diversity in ligand sensitivity among IP₃R subtypes arises from structural differences in the ligand coupling/suppressor domain, which attenuate the IP₃-binding affinity of the IP₃-binding core domain through an intramolecular mechanism. Tu et al. recorded single-channel activities of the recombinant IP₃R1, IP₃R2, and IP₃R3 reconstituted into planar lipid bilayers [55]. This report had a similar conclusion with IP₃R2 showing the highest apparent IP₃-affinity, followed by IP₃R1, and then by IP₃R3.

Differences amongst IP₃R subtypes in terms of IP₃-binding affinities do not reflect intrinsic differences in the properties of the channels to regulate Ca²⁺ oscillations. Instead differences in the state of phosphorylation and/or association with interacting proteins exist. Nevertheless, IP₃R1 and IP₃R2 are most sensitive to IP₃, a property that could contribute in their function as Ca²⁺ oscillatory unit. The exact contribution of subtype specific IP₃-binding affinities on Ca²⁺ oscillations remains to be further investigated.

Subtype Specificity of Ca²⁺ Inhibition and Induction

As mentioned earlier, repeated activation and inhibition of IICR by fluctuating cytosolic Ca²⁺ levels have been proposed as central molecular mechanisms for IP₃R-dependent Ca²⁺ oscillations [56]. Several stimulatory and inhibitory Ca²⁺ binding sites on the IP₃R have been identified and characterized. For instance, two sites are localized in the IP₃ binding core and another site is located close to the transmembrane domain [57], exemplifying the complex synergy between IP₃ and Ca²⁺ in the regulation of the IICR [12]. Ca²⁺ regulation of IP₃R activity may result in changed IP₃ binding affinity, alteration of channel open probability, or indirect influence on IP₃R associated proteins, such as the CaM. Interestingly, this can occur specifically on one IP₃R subtype, making Ca²⁺ regulation of IICR one of the major mechanisms to produce versatile signals, as confirmed by mathematical modeling studies [58].

The complex regulation of the IP₃R subtypes′ activity by Ca²⁺ has been recently reviewed in detail [3] and we will therefore mainly focus on how Ca²⁺ itself modulates Ca²⁺ oscillations. Everyone in the field agrees that all three subtypes are activated by Ca²⁺. Inhibition of the IP₃R by Ca²⁺, however, is more controversial. In single channel studies, each subtype is inhibited by high Ca²⁺ concentrations, even though the threshold and speed of inhibition differs [3]. Moreover, Ca²⁺ inhibition of IICR sometimes depends on the addition of an extra factor, for example ATP for IP₃R3 [55]. Therefore, all three subtypes can potentially support Ca²⁺ oscillations based on the model described previously, where concerted actions of IP₃ and Ca²⁺ stimulates IP₃R. Accordingly, IP₃R1-, IP₃R2-, and IP₃R3-dependent Ca²⁺ oscillations have been observed, although IP₃R3-dependent Ca²⁺ oscillations are less likely to occur and are also less frequently observed (see previous sections).

In most cases, cells express more than one IP₃R subtype. Interestingly, when several IP₃R subtypes are expressed, one of them becomes dominant regarding Ca²⁺ regulation of IICR [36]. This result also calls for caution when drawing conclusions on the subtype specificity of Ca²⁺ signaling, since expression of even a small amount of one subtype could critically affect the Ca²⁺ signaling pattern [59].

Taken together, Ca²⁺ activation and inhibition properties of IP₃R1 and IP₃R2 make them likely to support Ca²⁺ oscillations in physiological conditions [60], whereas specific cellular circumstances are required for activation of IP₃R3-dependent Ca²⁺ oscillations.

Subtype Specificity of Phosphorylation of IP₃R

Phosphorylation of the IP₃R is involved in many Ca²⁺ signaling pathways [61] and the different subtypes are interacting with protein kinases and phosphatases differently [62]. Many of the phosphorylation sites are subtype-specific, increasing the diversity in regulatory fine tuning of Ca²⁺ oscillations. The functional consequences of these regulatory modifications are only partially understood, and in some cases remain controversial. Therefore we will here focus on those protein kinases known to modulate Ca²⁺ oscillations through phosphorylation of IP₃R.

PKA-dependent phosphorylation of IP₃R has been demonstrated extensively. Phosphomimetic mutations of IP₃R1 expressed in DT40 cells showed that PKA-mediated phosphorylation decreases the threshold for Ca²⁺ oscillations, without affecting the amplitude or frequency [63]. PKA phosphorylates two distinct sites in IP₃R1 internal coupling domain (S1588 and S1755) [64]. Although these sites are not conserved in IP₃R2 and IP₃R3, PKA-dependent phosphorylation of these subtypes has been demonstrated [65]. In parotid acinar cells [66] and the pancreatic AR4-2J cell line [67], PKA directly phosphorylates IP₃R2, dramatically potentiating Ca²⁺ release. Interestingly, raising cAMP during sub-threshold agonist stimulation resulted in an oscillatory Ca²⁺ signal, while raising cAMP during an Ca²⁺ oscillation converted the response into a peak and plateau-like signal [66], probably because of a shift in the concentration dependency in IICR. CaMKII has been proposed to be involved in the control of the Ca²⁺-dependent regulation of IICR and in the occurrence of Ca²⁺ oscillations [68]. The most extensive information regarding CaMKII regulation of IP₃R is derived from studies performed on IP₃R2 [69, 70], which is the predominant subtype in cardiac ventricular myocytes. CaMKII-dependent phosphorylation significantly decreased the open probability of IP₃R2 in lipid bilayers, which suggests a Ca²⁺-dependent negative feedback mechanism on IP₃R2 activity in the cardiomyocyte nuclear envelope [71]. This may also result in a Ca²⁺-dependent inhibitory loop of Ca²⁺ oscillations [72]. Functional effects of PKC-mediated phosphorylation of the IP₃R were first studied in isolated rat liver nuclei [73]. PKC-mediated phosphorylation of IP₃R1 in vitro is in addition regulated by Ca²⁺ and CaM [74]. As both Ca²⁺ and CaM inhibit the PKC-mediated phosphorylation of IP₃R1, it is possible that this process may contribute to the negative slope of the Ca²⁺-dependent bell-shaped regulation of IP₃R by Ca²⁺, consequently affecting Ca²⁺ oscillations. Recent demonstrations suggest a role for PKC-mediated phosphorylation of IP₃R2 [75] and IP₃R3 [43]. These reports show that when IP₃R2 or IP₃R3 are phosphorylated by PKC, IP₃-dependent Ca²⁺ oscillations are decreased in cells expressing only those subtypes. Thus, PKC may act as a subtype specific regulator of IP₃R-mediated cytosolic Ca²⁺ oscillations. These differences are not unexpected since IP₃R subtypes possess different potential phosphorylation sites [43, 76]. How phosphorylation of IP₃R subtypes by distinct protein kinases affect Ca²⁺ oscillations are summarized in Table 2.

Table 2 The IP₃R subtype specificity of protein kinases and IP₃R-associated proteins and their modulating effects on Ca²⁺ oscillations

The subtype specific regulation of IP₃R by phosphorylation and its relation to Ca²⁺ oscillations are not fully understood. These processes are likely to be dependent on specific IP₃R subtypes expression levels and protein kinases activation, and need to be further investigated.

Regulation of IP₃R Activity by Accessory Proteins

About forty proteins have been reported to interact with IP₃R, most of which modulate IP₃R channel activity [1, 3, 77–81]. There is a lack of data regarding IP₃R2 and IP₃R3 specific binding proteins since most of these proteins are identified by co-immunoprecipitation studies with one or two IP₃R subtypes or using IP₃R1 probes. Few reports show that some of these associated proteins modulate Ca²⁺ oscillations differently. Therefore, we summarize here the proteins that bind to IP₃R and modulate Ca²⁺ oscillations, whether they bind to a specific IP₃R subtype or not (Table 2; Fig. 1).

Fig. 1

Cartoon illustrating the three IP₃R subtypes (IP₃R1, IP₃R2, and IP₃R3) and related protein kinases and interacting proteins involved in the regulation of cytosolic Ca²⁺ oscillations

CaBP1, one of the neuronal Ca²⁺ binding proteins, was co-immunoprecipitated with IP₃R1 and IP₃R3 [82]. The CaBP1-binding site was mapped in the ligand coupling/suppressor domain of IP₃R1. This interaction functionally inhibits IP₃-dependent Ca²⁺ oscillations in COS-7 cells expressing CaBP1, in permeabilized COS-7 cells exposed to recombinant CaBP1, and in Xenopus oocytes injected with recombinant CaBP1.

Na,K-ATPase, a plasma membrane ion pump, directly binds to the IP₃ binding-domain of all three IP₃R subtypes through its NH₂-terminal tail [83, 84]. In the presence of ouabain, Na,K-ATPase triggers IP₃-dependent Ca²⁺ oscillations in COS-7 cells and in primary culture of rat renal proximal tubule cells. Overexpression of a peptide corresponding to the wild type NH₂-terminal tail of Na,K-ATPase decreased the number of cells exhibiting Ca²⁺ oscillations, an effect not observed when a mutant type that does not bind to IP₃R was used.

IRBIT was identified to bind to the IP₃ binding core of IP₃R1 [85]. This interaction suppresses the activation of IP₃R by regulating the IP₃ sensitivity of IP₃R1. Knockdown of IRBIT in HeLa cells increases ATP-induced cytosolic Ca²⁺ oscillations.

AKAP9, one of the neuronal PKA-anchoring adaptor proteins, binds to the leucine/isoleucine zipper (LIZ) motif in the internal coupling domain of IP₃R1 [86]. Expression of a 36-residues LIZ fragment, which can disrupt the IP₃R1-AKAP9 association, reduces the frequency of Ca²⁺ oscillations induced by application of dopamine in primary culture of medium spiny neuron [87].

Presenilins (PS), including PS1 and PS2, are proteins bound to the gamma-secretase protease complex. Mutations in the genes encoding PS1 and PS2 are the major cause of familial Alzheimer’s disease (FAD). Wildtype and FAD-mutants of PS1 and PS2 have been co-immunoprecipitated with IP₃R1 and IP₃R3 [88, 89]. These interactions exert profound stimulatory effects on the IP₃R gating activity. Mutated PSs were demonstrated to increase frequency of both spontaneous Ca²⁺ oscillations and Ca²⁺ oscillations triggered by cross-linking the B cell receptor with IgM antibody in both DT40 cells and FAD patient B cells.

ERp44 is an ER lumenal protein of the thioredoxin family. Depending on the oxidative status in the ER lumen, it can interact directly with the third IP₃R1 lumenal loop and inhibit its activity [90]. Knockdown of ERp44 in HeLa cells increases ATP-triggered cytosolic Ca²⁺ oscillations.

GRP78, another ER lumenal protein, also interacts with the third lumenal loop of the IP₃R1 [91]. In contrast to ERp44, GRP78 enhances IP₃R1 channel activity. Knockdown of GRP78 in HeLa cells decreases ATP-triggered Ca²⁺ oscillations, which is restored by re-expression of the protein.

Bcl-2, Bcl-X_L, and Mcl-1, three anti-apoptotic proteins that belong to Bcl-2 family, have been reported to bind to the CTT and/or the internal coupling domain of all three IP₃R subtypes [78, 92–95]. Bcl-2 enhances IP₃-mediated Ca²⁺ oscillations induced by T cell receptor activation in WEHI7.2 cells, Jurkat cells, and wild type DT40 cells [78, 92, 94, 96], whereas Ca²⁺ oscillations induced by serum withdrawal in NIH-3T3 murine fibroblasts are dampened [97]. Expression of Bcl-X_L in wild type DT40 cells or in DT40 cells engineered to express each IP₃R subtype increases the number of the cells exhibiting Ca²⁺ oscillations as well as the oscillatory frequency [93, 95]. Interaction of Mcl-1 with IP₃R increases the number of DT40 cells exhibiting anti-B cell receptor antibody induced Ca²⁺ oscillations [78]. Bcl-2 and Mcl-1 also increase the number of cells exhibiting Ca²⁺ oscillations and the amplitude and/or the frequency of spontaneous Ca²⁺ oscillations in DT 40 cells [78].

Cytochrome C, one of the key components of the apoptotic cascade, was found to selectively and directly bind to IP₃R1 CTT during early apoptosis via a cluster of glutamic acid residues (binding to IP₃R2 and IP₃R3 were not confirmed), resulting in staurosporine-induced sustained Ca²⁺ oscillations [98, 99].

G-protein-coupled receptor kinase-interacting proteins (GIT), including GIT1 and GIT2, bind to the CTT of all three IP₃R subtypes, but have stronger binding affinity to IP₃R2 (more than 10- and 20-fold as compared to IP₃R1 and IP₃R3, respectively), and inhibit IICR [81]. Knockdown of GIT proteins in HeLa or COS-7 cells increases the number of cells exhibiting Ca²⁺ oscillations.

Neuronal Ca²⁺ sensor 1 (NCS-1), a Ca²⁺ binding protein whose expression could be enhanced by application of Taxol, a natural product for the treatment of solid tumors, was co-immunoprecipitated with all three subtypes of IP₃R [80, 100]. The NCS-1-IP₃R interaction increases the number of cells exhibiting IP₃R-dependent Ca²⁺ oscillations in SH-SY5Y human neuroblastoma cells [100] and the frequency of spontaneous Ca²⁺ oscillations in rat ventricular cardiomyocytes [80].

The diversity in distribution of associated proteins and/or IP₃R subtypes is essential for the versatility of IP₃R subtype-dependent Ca²⁺ oscillations in different cell types. More information, however, is required for determining the individual role of each separate subtype in modulating cytosolic Ca²⁺ oscillations.

Conclusion and Future Directions

It is evident that the different IP₃R subtypes are regulated by a large number of cellular mechanisms that varies in a cell type-specific manner. In this review we have focused on IP₃R subtype-specific modulation of Ca²⁺ oscillations. Ca²⁺ oscillations are repetitive increases in the cytosolic Ca²⁺ concentration that are used by the cell to convey information within or between cells. The oscillatory Ca²⁺ signal is known to be initiated at the onset of fertilization [101–103] and to continue throughout life to control a vast array of cellular processes as diverse as proliferation, differentiation, development, learning and memory, contraction, secretion, and cell death [1, 15]. Altered intracellular Ca²⁺ signaling has been linked to many diseases, such as Hungtington’s, Alzheimer’s and Parkinson’s diseases, amyotrophic lateral sclerosis, schizophrenia, spinocerebellar ataxias, heart failure, polycystic kidney disease, and human immunodeficiency virus infection [104–107]. It is therefore essential to determine the molecular mechanisms involved in the generation of intracellular Ca²⁺ oscillations. Additionally, Ca²⁺ oscillations are known to encode information in their frequency and amplitude to activate various specific downstream targets [15–17]. Efforts to understand the nature of these “cellular radio signals” started at the same time as Ca²⁺ oscillations were discovered and have resulted in a large number of publications [16–18, 22, 28, 30, 32, 35, 37, 39, 42, 47, 56, 60, 83, 100–102], most of which is cell type- and agonist-specific. To determine the associations between (1) stimulus, (2) Ca²⁺ oscillation, and (3) activation of a specific downstream cellular process, future studies will have to consider the molecular partners involved in each step. The recent rapid development of sophisticated molecular and genetic tools, such as small interfering RNA [108] and optogenetics [109], will surely advance our future knowledge about IP₃R subtype-specific regulation of Ca²⁺ oscillations.