InsP3R Isoforms Research Articles

Post-translational Regulation of the Type III Inositol 1,4,5-Trisphosphate Receptor by miRNA-506

The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP3R3) is apically localized and triggers Ca(2+) waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP3R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP3R3 in cholangiocyte Ca(2+) signaling and secretion is well established and because loss of InsP3R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3'-UTR of human InsP3R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP3R3-3'UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP3R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP3R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP3-mediated Ca(2+) signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP3R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP3R3 expression and InsP3R3-mediated Ca(2+) signaling and secretion.

Generation and Application of a Novel InsP3R1 Mono-Antibody from Mouse

Inositol 1, 4, 5-Trisphosphate Receptor (InsP3R) is an intracellular Ca2+ release channel, which widely participates in cellular processes. Three isoforms of InsP3R were identified as InsP3R1, InsP3R2, and InsP3R3. They share 60–0% protein sequence homology and form a channel in a manner of homotetramer or heterotetramer. Several InsP3R isoform-specific rabbit antibodies have been generated to distinguish their localization and functions. However, there is no report of such a valid antibody raised from other species. In his article, we prepare a mouse monoclonal antibody against a synthetic peptide with rat InsP3R1-specific carboxyl terminus sequence. This monoclonal antibody of InsP3R1 (R1-mAb) was purified and characterized as IgG2b, which can recognize InsP3R1 by Western-blot (WB) analysis and immunoprecipitate (IP) InsP3R1 from moue brain lysate tested. Applied in immunofluorescent (IF) and immunohistochemical (IHC) assays, this antibody and rabbit polyclonal antibody could give the consistent results in SH-SY5Y cells, human sperm, and mouse brain paraffin section. In summary, we generate a mouse InsP3R1-specific IgG 2b antibody identifying InsP3R1 in WB, IF, IHC, and IP analysis, which provides a possible choice for detection of InsP3R1, especially in application of co-localization analysis with other InsP3R isoforms or other proteins.

Apical localization of inositol 1,4,5-trisphosphate receptors is independent of extended synaptotagmins in hepatocytes.

Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.

Preparation of Microsomes to Study Ca2+ Channels

Native cerebellar microsomes are used to study the properties of native cerebellar inositol(1,4,5)-trisphosphate receptor 1 (InsP3R1) and ryanodine receptor 1 (RyanR1) Ca(2+) release channels. Additionally, microsomes prepared from Sf9 cells infected with InsP3R- or RyanR-expressing recombinant baculoviruses can be used to compare properties of different InsP3R and RyanR isoforms and to perform structure-function studies of both types of receptors. This protocol describes how to prepare ER microsomes from native cerebellar tissue or Sf9 cells infected with InsP3R- or RyanR-expressing recombinant baculoviruses for planar lipid bilayer (also called black lipid membranes or BLM) experiments. Prepared material is aliquoted and can be stored at -80°C freezer for many months before BLM experiments.

The inositol 1,4,5‐trisphosphate receptor in C. elegans

AbstractThe soil nematode Caenorhabditis elegans is a genetic model organism whose cellular physiology is closely related to that of mammals, with many signaling cascades and second messengers mirroring those found in higher organisms. Due to the genetic, anatomical, and behavioral simplicity of worms, integrative physiological techniques are relatively straightforward and represent a powerful approach to understand the molecular mechanisms underlying more complex system functions. Studies of the nematode inositol 1,4,5‐trisphosphate receptor (InsP3R) have led to advances in our understanding of its role in development and behavior. Unlike mammals, which express three InsP3R isoforms, nematodes express only a single InsP3R coded for by the itr‐1 gene. Forward genetic screens have resulted in the generation of conditional loss‐ and gain‐of‐function alleles of itr‐1, and genetic epistasis analyses have provided insight into how the InsP3R integrates various signals to regulate morphogenesis, sensation, and rhythmic behaviors. Novel techniques in the worm model have helped to define cell‐specific regulatory pathways that control InsP3 signaling and have shed light on new roles for the InsP3R itself in systems physiology. WIREs Membr Transp Signal 2012,1:321–328. doi: 10.1002/wmts.25For further resources related to this article, please visit the WIREs website.

Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes

In hepatocytes, as in other cell types, Ca 2+ signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP 3R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca 2+ signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP 3 receptors (InsP 3R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP 3R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP 3-induced Ca 2+ release in hepatocytes. This can be explained by the rather low expression level expression of InsP 3R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca 2+ signaling via an inhibitory effect on InsP 3 synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP 3 synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.

Isoform-specific Regulation of the Inositol 1,4,5-Trisphosphate Receptor by O-Linked Glycosylation

The inositol 1,4,5-trisphosphate receptor (InsP(3)R), an intracellular calcium channel, has three isoforms with >65% sequence homology, yet the isoforms differ in their function and regulation by post-translational modifications. We showed previously that InsP(3)R-1 is functionally modified by O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) (Rengifo, J., Gibson, C. J., Winkler, E., Collin, T., and Ehrlich, B. E. (2007) J. Neurosci. 27, 13813-13821). We now report the effect of O-GlcNAcylation on InsP(3)R-2 and InsP(3)R-3. Analysis of AR4-2J cells, a rat pancreatoma cell line expressing predominantly InsP(3)R-2, showed no detectable O-GlcNAcylation of InsP(3)R-2 and no significant functional changes despite the presence of the enzymes for addition (O-β-N-acetylglucosaminyltransferase) and removal (O-β-N-acetylglucosaminidase) of the monosaccharide. In contrast, InsP(3)R-3 in Mz-ChA-1 cells, a human cholangiocarcinoma cell line expressing predominantly InsP(3)R-3, was functionally modified by O-GlcNAcylation. Interestingly, the functional impact of O-GlcNAcylation on the InsP(3)R-3 channel was opposite the effect measured with InsP(3)R-1. Addition of O-GlcNAc by O-β-N-acetylglucosaminyltransferase increased InsP(3)R-3 single channel open probability. Incubation of Mz-ChA-1 cells in hyperglycemic medium caused an increase in the InsP(3)-dependent calcium release from the endoplasmic reticulum. The dynamic and inducible nature of O-GlcNAcylation and the InsP(3)R isoform specificity suggest that this form of modification of InsP(3)R and subsequent changes in intracellular calcium transients are important in physiological and pathophysiological processes.

The type III inositol 1,4,5-trisphosphate receptor is associated with aggressiveness of colorectal carcinoma

The inositol 1,4,5-trisphosphate receptor (InsP3R) mediates Ca 2+ signaling in epithelia and regulates cellular functions such as secretion, apoptosis and cell proliferation. Loss of one or more InsP3R isoform has been implicated in disease processes such as cholestasis. Here we examined whether gain of expression of InsP3R isoforms also may be associated with development of disease. Expression of all three InsP3R isoforms was evaluated in tissue from colorectal carcinomas surgically resected from 116 patients. Type I and II InsP3Rs were seen in both normal colorectal mucosa and colorectal cancer, while type III InsP3R was observed only in colorectal cancer. Type III InsP3R expression in the advancing margins of tumors correlated with depth of invasion, lymph node metastasis, liver metastasis, and TNM stage. Heavier expression of type III InsP3R also was associated with decreased 5-year survival. shRNA knockdown of type III InsP3R in CACO-2 colon cancer cells enhanced apoptosis, while over-expression of the receptor decreased apoptosis. Thus, type III InsP3R becomes expressed in colon cancer, and its expression level is directly related to aggressiveness of the tumor, which may reflect inhibition of apoptosis by the receptor. These findings suggest a previously unrecognized role for Ca 2+ signaling via this InsP3R isoform in colon cancer.

Redox-Regulated Heterogeneous Thresholds for Ligand Recruitment among InsP3R Ca2+-Release Channels

To clarify the molecular mechanisms behind quantal Ca2+ release, the graded Ca2+ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP3R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP3R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP3R gene or from cells solely expressing one recombinant InsP3R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels. In contrast, sulfhydryl reduction had limited effects on channel open probability. Thus, sulfhydryl redox modification can regulate various aspects of intracellular Ca2+ signaling, including quantal Ca2+ release, by tuning ligand sensitivities of InsP3R channels. No intrinsic termination of channel activity with a timescale comparable to that for quantal Ca2+ release was observed under any steady ligand conditions, indicating that this process is unlikely to contribute.

ATP Regulation of Type-1 Inositol 1,4,5-Trisphosphate Receptor Activity Does Not Require Walker A-type ATP-binding Motifs

ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.

Differential Modulation of Type-1, Type-2 and Type-3 Inositol (1,4,5)-Trisphosphate Receptors by ATP

Inositol (1,4,5)-trisphosphate receptors (InsP3R) are the predominant route of Ca2+ release in non-excitable cells and they play a vital role in regulating intracellular Ca2+ signals. There are three isoforms (InsP3R-1, InsP3R-2 and InsP3R-3) of InsP3R expressed in mammalian cells. This sequence diversity along with varied tissue distributions suggests that there are isoform-specific regulatory mechanisms. One such regulatory mechanism is the modulation of Ca2+ release from InsP3R by cytosolic ATP. ATP positively regulates all three InsP3R isoforms, but with distinct functional characteristics. We found that ATP was required for maximal InsP3-induced Ca2+ release from InsP3R-1 and InsP3R-3 while InsP3R-2 attained maximal activity in the absence of ATP. Furthermore, InsP3R-2 was more sensitive to ATP modulation than either InsP3R-1 or InsP3R-3. All three isoforms contain putative ATP binding domains, but the contributions of these sites to ATP modulation of InsP3R are poorly understood. InsP3R-1 contains two predicted ATP binding domains (ATPA, and ATPB) while InsP3R-2 and InsP3R-3 each express a single ATPB site. We examined the contributions of these ATP binding sites to the subtype-specific effects of ATP on InsP3R isoforms. ER Ca2+ measurements from permeabilized DT40 cells and single channel recordings of InsP3R were used to measure the effects of ATP on wild-type and mutated InsP3R. We found that ablation of the ATPB site in InsP3R-2 eliminated the enhancing effects of ATP on this isoform. Surprisingly, the positive effects of ATP were retained in InsP3R-1 and InsP3R-3 devoid of their respective ATP binding sites. ATP, therefore, differentially regulates the three InsP3R isoforms and likely regulates InsP3R-1 and InsP3R-3 via novel ATP binding sites. The implications of this differential regulation on Ca2+ signals would likely be determined by the relative ratios of the three isoforms expressed in a given cell.

Regulation Of Inositol 1,4,5-Trisphosphate Receptor Isoforms By O-Linked Glycosylation

The inositiol 1,4,5 trisphospate receptor (InsP3R), an intracellular calcium channel, is a family of three isoforms. All three isoforms display a significant level of sequence identity yet they differ in expression level, localization and many functional aspects. We previously showed that InsP3R type 1 is modified by O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylated). Through this dynamic and inducible modification a single monosaccharide is covalently attached to serine and threonine residues of the protein backbone, providing protein regulation similar to O-phosphorylation. We also reported that increased O-GlcNAcylation of the InsP3R type 1 reduced the percent of cells that responded to addition of extracellular agonists and those that did respond had a decreased InsP3 dependent calcium release from the endoplasmic reticulum (ER). We now report that the InsP3R type 3 is also O-GlcNAcylated. Interestingly, the functional impact of O-GlcNAcylation on InsP3R type 3 channel is opposite to the effect measured with the InsP3R type 1. Human cholangiocytoma cells (MzChA-1) contain >90% InsP3R type 3. When these cells were incubated in hyperglycemic media there was an increase in the percent cells responding to InsP3 generating stimuli and there was an increase in the InsP3 dependent calcium release from the ER. A difference in functional response between InsP3R isoforms was reported previously for phosphorylation by cyclic AMP dependent protein kinase (PKA). In contrast, the InsP3R type 2 showed no detectable O-GlcNAc glycosylation and no significant functional changes even though the enzymes necessary for both the addition and removal of the monosccharide are present in all cell types tested. The dynamic and inducible nature of O-GlcNAcylation and the isoform specificity suggests that this form of modification of the InsP3R and subsequent changes in intracellular calcium transients will be important in physiological and pathophysiological processes.

An antidepressant behaviour in mice carrying a gene-specific InsP3R1, InsP3R2 and InsP3R3 protein knockdown

Evidence has accumulated for the involvement of Ca 2+ in the pathophysiology of mood disorders. Elevations in both resting and stimulated intracellular Ca 2+ levels in patients with affective disorders have been reported. The role of inositol-1,4,5-trisphosphate receptors (InsP3Rs), which allow mobilization of intracellular Ca 2+ stores, was, then, investigated in the mouse forced swimming test. InsP3R antagonists (heparin, xestospongin C) as well as an inositol monophosphatase inhibitor (LiCl) showed an antidepressant activity of intensity comparable to clinically used antidepressants. InsP3Rl, InsP3R2 and InsP3R3 knockdown mice were obtained to investigate the role of InsP3R isoforms. We generated mice carrying a cerebral knockdown of InsP3Rl, InsP3R2 and InsP3R3 proteins by administering antisense oligonucleotides complementary to the sequence of InsP3Rl, InsP3R2 and InsP3R3. These antisense-treated mice showed a specific InsP3R protein level reduction in the mouse cerebral cortex and hippocampus, demonstrated by immunoblotting, immunoprecipitation and immunocytochemistry experiments. Knockdown mice for each InsP3R isoforms showed an antidepressant behaviour and the induced phenotype was reversible disappearing 7 days after the end of the treatment. The absence of impairment of locomotor activity and spontaneous mobility in InsP3R knockdown mice was revealed. These results indicate the involvement of the InsP3R-mediated pathway in the modulation of depressive conditions and may be useful for the development of new therapeutical strategies for the treatment of mood disorders.

Cyclic AMP Regulates Bicarbonate Secretion in Cholangiocytes Through Release of ATP Into Bile

Bicarbonate secretion is a primary function of cholangiocytes. Either adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca(2+) can mediate bicarbonate secretion, but these are thought to act through separate pathways. We examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R) in mediating bicarbonate secretion because this is the only intracellular Ca(2+) release channel in cholangiocytes. Intrahepatic bile duct units (IBDUs) were microdissected from rat liver then luminal pH was examined by confocal microscopy during IBDU microperfusion. Cyclic AMP was increased using forskolin or secretin, and Ca(2+) was increased using acetylcholine (ACh) or adenosine triphosphate (ATP). Apyrase was used to hydrolyze extracellular ATP, and suramin was used to block apical P2Y ATP receptors. In selected experiments, IBDUs were pretreated with short interfering RNA (siRNA) to silence expression of specific InsP3R isoforms. Both cAMP and Ca(2+) agonists increased luminal pH. The effect of ACh on luminal pH was reduced by siRNA for basolateral (types I and II) but not apical (type III) InsP3R isoforms. The effect of forskolin on luminal pH was reduced by a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and by siRNA for the type III InsP3R. Luminal apyrase or suramin blocked the effects of forskolin but not ACh on luminal pH. Cyclic AMP-induced ductular bicarbonate secretion depends on an autocrine signaling pathway that involves CFTR, apical release of ATP, stimulation of apical nucleotide receptors, and then activation of apical, type III InsP3Rs. The primary role of CFTR in bile duct secretion may be to regulate secretion of ATP rather than to secrete chloride and/or bicarbonate.

Apoptosis regulation by Bcl-x L modulation of mammalian inositol 1,4,5-trisphosphate receptor channel isoform gating

Members of the Bcl-2 family of proteins regulate apoptosis, with some of their physiological effects mediated by their modulation of endoplasmic reticulum (ER) Ca(2+) homeostasis. Antiapoptotic Bcl-x(L) binds to the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel to enhance Ca(2+)- and InsP(3)-dependent regulation of channel gating, resulting in reduced ER [Ca(2+)], increased oscillations of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), and apoptosis resistance. However, it is controversial which InsP(3)R isoforms mediate these effects and whether reduced ER [Ca(2+)] or enhanced [Ca(2+)](i) signaling is most relevant for apoptosis protection. DT40 cell lines engineered to express each of the three mammalian InsP(3)R isoforms individually displayed enhanced apoptosis sensitivity compared with cells lacking InsP(3)R. In contrast, coexpression of each isoform with Bcl-x(L) conferred enhanced apoptosis resistance. In single-channel recordings of channel gating in native ER membranes, Bcl-x(L) increased the apparent sensitivity of all three InsP(3)R isoforms to subsaturating levels of InsP(3). Expression of Bcl-x(L) reduced ER [Ca(2+)] in type 3 but not type 1 or 2 InsP(3)R-expressing cells. In contrast, Bcl-x(L) enhanced spontaneous [Ca(2+)](i) signaling in all three InsP(3)R isoform-expressing cell lines. These results demonstrate a redundancy among InsP(3)R isoforms in their ability to sensitize cells to apoptotic insults and to interact with Bcl-x(L) to modulate their activities that result in enhanced apoptosis resistance. Furthermore, these data suggest that modulation of ER [Ca(2+)] is not a specific requirement for ER-dependent antiapoptotic effects of Bcl-x(L). Rather, apoptosis protection is conferred by enhanced spontaneous [Ca(2+)](i) signaling by Bcl-x(L) interaction with all isoforms of the InsP(3)R.

Lipid Rafts Establish Calcium Waves in Hepatocytes

Polarity is critical for hepatocyte function. Ca(2+) waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca(2+) waves depends on lipid rafts. Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mbetaCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca(2+) waves were examined by confocal microscopy. Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mbetaCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mbetaCD treatment. Vasopressin-induced Ca(2+) signals began as apical-to-basal Ca(2+) waves, and mbetaCD slowed the wave speed and prolonged the rise time. MbetaCD had a similar effect on Ca(2+) waves in acinar cells but did not affect Ca(2+) signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mbetaCD. Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca(2+) waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity.

The Spatial Distribution of Inositol 1,4,5-Trisphosphate Receptor Isoforms Shapes Ca2+ Waves

Cytosolic Ca(2+) is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca(2+) waves and other spatial patterns of Ca(2+) signals. To investigate the mechanism responsible for the formation of Ca(2+) waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca(2+) wave formation. Ca(2+) signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca(2+) signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca(2+) waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca(2+) waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca(2+) waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca(2+) signals.

Modelling the effect of specific inositol 1,4,5‐trisphosphate receptor isoforms on cellular Ca2+ signals

Oscillations of cytosolic Ca2+ are well-known to rely on the regulatory properties of the InsP3R (inositol 1,4,5-trisphosphate receptor). Three isoforms of this channel have been identified. They differ in their regulatory properties by Ca2+ and InsP3. Experiments in different cell types clearly indicate that the relative amounts of each isoform affect the time course of Ca2+ changes after agonist stimulation. In the present study, we investigate whether different steady-state curves for the open probability of the InsP3Rs as a function of Ca2+ imply different dynamical behaviours when these receptors are present in a cellular environment. We therefore describe by a specific phenomenological model the three main types of curves that have been reported: (i) the classical bell-shaped curve, (ii) the bell-shaped curve that is shifted towards higher Ca2+ concentrations when InsP3 is increased, and (iii) a monotonous increasing function of cytosolic Ca2+. We show that, although these types of curves can be ascribed to slight differences in the channel regulation by Ca2+ and InsP3, they can indicate important variations as to the receptor role in cellular Ca2+ control. Thus the receptor associated with the classical bell-shaped curve appears to be the most robust Ca2+ oscillator. If the steady-state curve is supposed to be a monotonous increasing function of cytosolic Ca2+, the modelled receptor cannot sustain Ca2+ oscillations in the absence of Ca2+ exchanges with the extracellular medium. When the bell-shaped curve is shifted towards higher Ca2+ concentrations with increasing InsP3 levels, the model predicts that the receptor is less robust to changes in density; this receptor, however, provides a finer control of the steady-state level of Ca2+ when varying the InsP3 concentration. Our model allows us to propose an explanation for the experimental observations about the effect of selectively expressing or down-regulating InsP3R isoforms, as well as to make theoretical predictions.

The Type III Inositol 1,4,5-Trisphosphate Receptor Preferentially Transmits Apoptotic Ca2+ Signals into Mitochondria

There are three isoforms of the inositol 1,4,5- trisphosphate receptor (InsP(3)R), each of which has a distinct effect on Ca(2+) signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca(2+)-mediated events. To investigate this question, we examined the effects of each InsP(3)R isoform on transmission of Ca(2+) signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP(3)R isoforms. ATP-induced cytosolic Ca(2+) signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca(2+) signals in mitochondria, which were inhibited more effectively by silencing the type III InsP(3)R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP(3)R. These findings provide evidence that the type III isoform of the InsP(3)R plays a special role in induction of apoptosis by preferentially transmitting Ca(2+) signals into mitochondria.

The inositol 1,4,5-trisphosphate receptors

The inositol (1,4,5)-trisphosphate receptors (InsP 3R) are the intracellular calcium (Ca 2+) release channels that play a key role in Ca 2+ signaling in cells. Three InsP 3R isoforms—InsP 3R type 1 (InsP 3R1), InsP 3R type 2 (InsP 3R2), and InsP 3R type 3 (InsP 3R3) are expressed in mammals. A single InsP 3R isoform is expressed in Drosophila melanogaster (DmInsP 3R) and Caenorhabditis elegans (CeInsP 3R). The progress made during last decade towards understanding the function and the properties of the InsP 3R is briefly reviewed in this chapter. The main emphasis is on studies that revealed structural determinants responsible for the ligand recognition by the InsP 3R, ion permeability of the InsP 3R, modulation of the InsP 3R by cytosolic Ca 2+, ATP and PKA phosphorylation and on the recently identified InsP 3R-binding partners. The main focus is on the InsP 3R1, but the recent information about properties of other InsP 3R isoforms is also discussed.