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.     

Alpha-Helical Destabilization of the Bcl-2-BH4-Domain Peptide Abolishes Its Ability to Inhibit the IP3 Receptor

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP₃R), the primary Ca²⁺-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP₃R-mediated Ca²⁺ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP₃R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP ₃R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine “hinges” replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP ₃R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP₃R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP₃R with significant pharmacological implications.     

Tyr-167/Trp-168 in Type 1/3 Inositol 1,4,5-Trisphosphate Receptor Mediates Functional Coupling between Ligand Binding and Channel Opening

The N-terminal ∼220-amino acid region of the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R)/Ca²⁺ release channel has been referred to as the suppressor/coupling domain because it is required for both IP₃ binding suppression and IP₃-induced channel gating. Measurements of IP₃-induced Ca²⁺ fluxes of mutagenized mouse type 1 IP₃R (IP₃R1) showed that the residues responsible for IP₃ binding suppression in this domain were not essential for channel opening. On the other hand, a single amino acid substitution of Tyr-167 to alanine completely impaired IP₃-induced Ca²⁺ release without reducing the IP₃ binding activity. The corresponding residue in type 3 IP₃R (IP₃R3), Trp-168, was also critical for channel opening. Limited trypsin digestion experiments showed that the trypsin sensitivities of the C-terminal gatekeeper domain differed markedly between the wild-type channel and the Tyr-167 mutant under the optimal conditions for channel opening. These results strongly suggest that the Tyr/Trp residue (Tyr-167 in IP₃R1 and Trp-168 in IP₃R3) is critical for the functional coupling between IP₃ binding and channel gating by maintaining the structural integrity of the C-terminal gatekeeper domain at least under activation gating.     

The Inositol Trisphosphate Receptor in the Control of Autophagy

The second messenger myo-inositol-1,4,5-trisphosphate (IP₃) acts on the IP₃ receptor (IP₃R), an IP3-activated Ca²⁺ channel of the endoplasmic reticulum (ER). The IP₃R agonist IP3 inhibits starvation-induced autophagy. The IP₃R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-X_L, two proteins that physically interact with IP₃R. Autophagy can also be induced by depletion of the IP₃R by small interfering RNAs. Autophagy induction by IP3R blockade cannot be explained by changes in steady state levels of Ca²⁺ in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP₃R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

Addendum to:

Regulation of Autophagy by the Inositol Trisphosphate Receptor

A. Criollo, M.C. Maiuri, E. Tasdemir, I. Vitale, A.A. Fiebig, D. Andrews, J. Molgo, J. Diaz, S. Lavandero, F. Harper, G. Pierron, D. di Stefano, R. Rizzuto, G. Szabadkai and G. Kroemer

Cell Death Differ 2007; In press     

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

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

Structural Studies of Inositol 1,4,5-Trisphosphate Receptor: COUPLING LIGAND BINDING TO CHANNEL GATING*

The three isoforms of the inositol 1,4,5-trisphosphate receptor (IP₃R) exhibit distinct IP₃ sensitivities and cooperativities in calcium (Ca²⁺) channel function. The determinants underlying this isoform-specific channel gating mechanism have been localized to the N-terminal suppressor region of IP₃R. We determined the 1.9 Å crystal structure of the suppressor domain from type 3 IP₃R (IP₃R3_SUP, amino acids 1–224) and revealed structural features contributing to isoform-specific functionality of IP₃R by comparing it with our previously determined structure of the type 1 suppressor domain (IP₃R1_SUP). The molecular surface known to associate with the ligand binding domain (amino acids 224–604) showed marked differences between IP₃R3_SUP and IP₃R1_SUP. Our NMR and biochemical studies showed that three spatially clustered residues (Glu-20, Tyr-167, and Ser-217 in IP₃R1 and Glu-19, Trp-168, and Ser-218 in IP₃R3) within the N-terminal suppressor domains of IP₃R1_SUP and IP₃R3_SUP interact directly with their respective C-terminal fragments. Together with the accompanying paper (Yamazaki, H., Chan, J., Ikura, M., Michikawa, T., and Mikoshiba, K. (2010) J. Biol. Chem. 285, 36081–36091), we demonstrate that the single aromatic residue in this region (Tyr-167 in IP₃R1 and Trp-168 in IP₃R3) plays a critical role in the coupling between ligand binding and channel gating.     

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.     

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.     

G-protein-coupled Receptor Kinase-interacting Proteins Inhibit Apoptosis by Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Signal Regulation

The inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) is an intracellular IP₃-gated calcium (Ca²⁺) release channel and plays important roles in regulation of numerous Ca²⁺-dependent cellular responses. Many intracellular modulators and IP₃R-binding proteins regulate the IP₃R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP₃R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP₃R. The interaction was favored by the cytosolic Ca²⁺ concentration and it functionally inhibited IP₃R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP₃R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP₃R-mediated Ca²⁺ signal through a direct interaction with IP₃R in a cytosolic Ca²⁺-dependent manner.The inositol 1,4,5-trisphosphate (IP₃)³ receptor (IP₃R) consisting of three subtypes, IP₃R1, IP₃R2, and IP₃R3, is a tetrameric intracellular IP₃-gated calcium (Ca²⁺) release channel localized at the endoplasmic reticulum (ER) with its NH₂ terminus and COOH-terminal tail (CTT) exposed to the cytoplasm (1, 2; see Fig. 1A). IP₃Rs are composed of five functional domains. The long NH₂-terminal cytoplasmic region contains three domains, a coupling/suppressor domain, an IP₃-binding core domain, and an internal coupling domain. The COOH-terminal region has a six-membrane spanning channel domain and a short cytoplasmic CTT “gatekeeper domain” that is critical for IP₃R channel opening (2, 3). Ca²⁺ release activity of the IP₃R channel is regulated by many intracellular modulators (ATP, calmodulin, and Ca²⁺), protein kinases, and IP₃R-binding proteins (2, 4), and the tight regulation of IP₃R channel activity by these factors generates various spatial and temporal intracellular Ca²⁺ patterns such as Ca²⁺ spikes and Ca²⁺ oscillations, leading to numerous cellular responses (1, 2, 5, 6).Open in a separate windowFIGURE 1.GIT1 and GIT2 bind to all three subtypes of IP₃R. A, schematic of ER residential IP₃R. The CTT of IP₃R1 is used as bait in a yeast two-hybrid screen. B, schematic representation of GIT1, GIT2, and two GIT1 fragments identified from the yeast two-hybrid screen. Functional domains are indicated. ARF-GAP, ARF-specific GTPase-activating protein domain; ANK-REP, ankyrin repeats; CC, coiled-coil domains; SHD, the Spa2-homology domain; EF, EF-hand; IQ, IQ-like motifs; aa, amino acid. C, GIT1 binds to IP₃R1 in vitro. GST and GST-IP₃R1/CTT were incubated with mouse brain lysate for a pull-down assay. The input and pulled-down samples were probed with α-GIT1. D and E, GIT1 binds to IP₃R1 in vivo. Mouse brain lysates were processed to control IgG and α-IP₃R1 (D) or α-GIT1 (E) for IP. The input and IP samples were probed with α-GIT1 and α-IP₃R1. F and G, both GIT1 and GIT2 bind to all three IP₃R subtypes. HeLa cells coexpressing GFP-fused IP₃R1, IP₃R2, or IP₃R3 and mRFP-fused GIT1 (F) or GIT2 (G) were processed for IP using α-RFP. The input and IP samples were blotted with α-GFP (top) and α-RFP (bottom).One of the physiological roles of IP₃R-mediated Ca²⁺ signaling is a pro-apoptotic regulator during apoptosis. Ca²⁺ released from ER can stimulate several key enzymes activated during apoptosis such as endonucleases (7) and calpain (8). In addition, the close proximity of ER to mitochondria may facilitate the mitochondrial overload of Ca²⁺ released from the IP₃Rs with certain apoptotic stimuli, triggering the opening of the mitochondrial permeability transition pore and the release of apoptotic signaling molecules, such as cytochrome c and apoptosis-inducing factor, which leads to the activation of caspases (5, 6). Moreover, several key components of apoptotic cascades, such as cytochrome c (9) and anti-apoptosis proteins Bcl-2 (10, 11) and Bcl-X_L (12), have been reported to interact with the internal coupling domain and/or the CTT of IP₃R and enhance the Ca²⁺-release activity of IP₃Rs during apoptosis. In this study, we identified the ubiquitously expressed G-protein-coupled receptor kinase-interacting proteins (GIT) (13), GIT1 and GIT2, as novel IP₃R-binding proteins that bind to the CTT of IP₃R and inhibit apoptosis by regulation of IP₃R-mediated Ca²⁺ signal.     

Distinct subcellular localisations of the putative inositol 1,3,4,5-tetrakisphosphate receptors GAP1 and GAP1 result from the GAP1 PH domain directing plasma membrane targeting

Inositol 1,3,4,5-tetrakisphosphate (IP₄), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP₄ receptor, termed GAP1^IP4BP[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1^IP4BP, called GAP1^m, has been identified [2] and here we describe the cloning of a GAP1^m cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1^m, in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP₄ with a similar affinity and specificity to that of the corresponding GAP1^IP4BP mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1^IP4BP is located solely at the plasma membrane, GAP1^m seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP₄.     

Inositol-1,4,5-trisphosphate mass content in isolated perfused rat heart during alpha-1-adrenoceptor stimulation

Inositol-1,4,5-trisphosphate (IP₃) has been proposed to be a second messenger in response to alpha-1-adrenoceptor stimulation also in myocardial cells. We studied the effect of alpha-1-adrenoceptor stimulation (5 × 10^–5 mol/l phenylephrine or 5 × 10^–5 mol/l noradrenaline both in the presence of 10^–6 mol/l timolol) on IP₃ mass content in isolated perfused rat hearts. IP₃ content was determined by a specific receptor-binding assay-kit (TRK 1000, Amersham) after validating the method. For comparison also the effect of muscarinic stimulation (10^–4 mol/l carbachol in the presence of 10^–6 mol/l timolol) on IP₃ content was measured in corresponding preparations. A basal IP₃ level of about 75 pmol/mg protein was found. There were no prominent effects of alpha-1-adrenoceptor stimulation on total IP₃ content in isolated perfused rat hearts. Phenylephrine gave a statistically significant increase of about 40% at 1/4 min and a statistically significant decrease of about 25% at 4 min after start of exposure. Noradrenaline, however, gave no statistically significant change of IP₃ at the time-points studied. Muscarinic stimulation caused a slight, statistically insignificant, increase of IP₃ at 1/4 min. The results are compatible with an assumption that agonist stimulation evokes a localized increase of IP3 which may be masked by a relatively high total IP₃ mass content. The IP₃ peak after phenylephrine coincided with the early positive inotropic phase of the response reported earlier in perfused rat hearts for alpha-1-adrenoceptor stimulation by phenylephrine. Although this might be compatible with a role for IP₃ in this early and transient phase, a mediator function of IP₃ in the inotropic response is not established.     

Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP₃) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP₃ levels by rapid photolysis of caged IP₃ has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP₃ uncaging restricted to the nucleus elicites ‘mini’-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.     

Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP₃) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP₃ levels by rapid photolysis of caged IP₃ has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP₃ uncaging restricted to the nucleus elicites ‘mini’-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.     

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.     

Genomic organization of mouse orexin receptors: characterization of two novel tissue-specific splice variants

In humans and rat, orexins orchestrate divergent actions through their G protein-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R). Orexins also play an important physiological role in mouse, but the receptors through which they function are not characterized. To characterize the physiological role(s) of orexins in the mouse, we cloned and characterized the mouse orexin receptor(s), mOX1R and mOX2R, using rapid amplification of cDNA (mouse brain) ends, RT-PCR, and gene structure analysis. The mOX1R cDNA encodes a 416-amino acid (aa) receptor. We have identified two alternative C terminus splice variants of the mOX2R; mOX2 alpha R (443 aa) and mOX2 beta R (460 aa). Binding studies in human embryonic kidney 293 cells transfected with mOX1R, mOX2 alpha R, and the mOX2 beta R revealed specific, saturable sites for both orexin-A and -B. Activation of these receptors by orexins induced inositol triphosphate (IP(3)) turnover. However, human embryonic kidney 293 cells transfected with mOXRs demonstrated no cAMP response to either orexin-A or orexin-B challenge, although forskolin and GTP gamma S revealed a dose-dependent increase in cAMP. Although, orexin-A and -B showed no difference in binding characteristics between the splice variants; interestingly, orexin-B led to an increase in IP(3) production at all concentrations in the mOX2 beta R variant. Orexin-A, however, showed no difference in IP(3) production between the two variants. Additionally, in the mouse, we demonstrate that these splice variants are distributed in a tissue-specific manner, where OX2 alpha R mRNA was undetectable in skeletal muscle and kidney. Moreover, food deprivation led to a greater increase in hypothalamic mOX2 beta R gene expression, compared with both mOX1R and mOX2 alpha R. This potentially implicates a fundamental physiological role for these splice variants.     

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.     

Non-inositol 1,4,5-trisphosphate (IP3) receptor IP3-binding proteins

Conventionally, myo-D-inositol 1, 4,5-trisphosphate (IP₃) is thought to exert its second messenger effects through the gating of IP₃R Ca²⁺ release channels, located in Ca²⁺-storage organelles like the endoplasmic reticulum. However, there is considerable indirect evidence to support the concept that IP₃ might interact with other, non-IP₃R proteins within cells. To explore this possibility further, the Protein Data Bank was searched using the term “IP3”. This resulted in the retrieval of 203 protein structures, the majority of which were members of the IP₃R/ryanodine receptor superfamily of channels. Only 49 of these structures were complexed with IP₃. These were inspected for their ability to interact with the carbon-1 phosphate of IP₃, since this is the least accessible phosphate group of its precursor, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). This reduced the number of structures retrieved to 35, of which 9 were IP₃Rs. The remaining 26 structures represent a diverse range of proteins, including inositol-lipid metabolizing enzymes, signal transducers, PH domain containing proteins, cytoskeletal anchor proteins, the TRPV4 ion channel, a retroviral Gag protein and fibroblast growth factor 2. Such proteins may impact on IP₃ signalling and its effects on cell-biology. This represents an area open for exploration in the field of IP₃ signalling.     

Granule neurons in cerebellum express distinct splice variants of the inositol trisphosphate receptor that are modulated by calcium

Primary cultures of granule cells (GC) from rat cerebellar cortex were used to determine whether bioelectric activity, via a Ca²⁺/calmodulin-dependent kinase (CaMK) signaling cascade, modulates expression and exon selection in the inositol trisphosphate receptor type 1 (IP₃R1). IP₃R1 contains or lacks three exons (S1, S2, and S3) that are regulated in a regionally and temporally specific manner. The neuronal, or long, form of IP₃R1 is distinguished from peripheral tissues by inclusion of the S2 exon. Although previous studies indicated that IP₃R1 are undetectable in the cerebellar granular layer in vivo, receptor protein and mRNA are induced in cultured GC grown in medium supplemented with 25 mM KCl or NMDA, two trophic agents that promote long-term survival, compared with GC grown in 5 mM KCl. IP₃R1 induction in response to 25 mM KCl or NMDA is attenuated by coaddition of voltage-sensitive calcium channel or NMDA receptor antagonists, respectively. Actinomycin D, CaMK, and calcineurin antagonists likewise suppress induction. Unlike the major variants of IP₃R1 in Purkinje neurons, which lack S1 and S3, GC grown with trophic agents express mRNA containing these exons. Both neuronal types contain S2. Evidence obtained using mutant mice with Purkinje cell lesions, laser-microdissected GC neurons from slices, and explant cultures indicates that GC predominantly express the S1-containing variant of IP₃R1 in vivo. calmodulin; exon selection     

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.