InsP3R Isoforms Research Articles

Modulation of Mammalian Inositol 1,4,5-Trisphosphate Receptor Isoforms by Calcium: A Role of Calcium Sensor Region

In the accompanying article, we compared main functional properties of the three mammalian inositol 1,4,5-trisphosphate receptors (InsP3R) isoforms. In this article we focused on modulation of mammalian InsP3R isoforms by cytosolic Ca2+. We found that: 1), when recorded in the presence of 2μM InsP3 and 0.5mM ATP all three mammalian InsP3R isoforms display bell-shaped Ca2+ dependence in physiological range of Ca2+ concentrations (pCa 8–5); 2), in the same experimental conditions InsP3R3 is most sensitive to modulation by Ca2+ (peak at 107nM Ca2+), followed by InsP3R2 (peak at 154nM Ca2+), and then by InsP3R1 (peak at 257nM Ca2+); 3), increase in ATP concentration to 5mM had no significant effect of Ca2+ dependence of InsP3R1 and InsP3R2; 4), increase in ATP concentration to 5mM converted Ca2+ dependence of InsP3R3 from “narrow” shape to “square” shape; 5), ATP-induced change in the shape of InsP3R3 Ca2+ dependence was mainly due to an >200-fold reduction in the apparent affinity of the Ca2+-inhibitory site; 6), the apparent Ca2+ affinity of the Ca2+ sensor region (Cas) determined in biochemical experiments is equal to 0.23μM Ca2+ for RT1-Cas, 0.16μM Ca2+ for RT2-Cas, and 0.10μM Ca2+ for RT3-Cas; and 7), Ca2+ sensitivity of InsP3R1 and InsP3R3 isoforms recorded in the presence of 2μM InsP3 and 0.5mM ATP or 2μM InsP3 and 5mM ATP can be exchanged by swapping their Cas regions. Obtained results provide novel information about functional properties of mammalian InsP3R isoforms and support the importance of the Ca2+ sensor region (Cas) in determining the sensitivity of InsP3R isoforms to modulation by Ca2+.

Functional Characterization of Mammalian Inositol 1,4,5-Trisphosphate Receptor Isoforms

Inositol 1,4,5-trisphosphate receptors (InsP3R) play a key role in intracellular calcium (Ca2+) signaling. Three mammalian InsP3R isoforms—InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) are expressed in mammals, but the functional differences between the three mammalian InsP3R isoforms are poorly understood. Here we compared single-channel behavior of the recombinant rat InsP3R1, InsP3R2, and InsP3R3 expressed in Sf9 cells, reconstituted into planar lipid bilayers and recorded with 50mM Ba2+ as a current carrier. We found that: 1), for all three mammalian InsP3R isoforms the size of the unitary current is 1.9 pA and single-channel conductance is 74–80 pS; 2), in optimal recording conditions the maximal single-channel open probability for all three mammalian InsP3R isoforms is in the range 30–40%; 3), in optimal recording conditions the mean open dwell time for all three mammalian InsP3R isoforms is 7–8ms, the mean closed dwell time is ∼10ms; 4), InsP3R2 has the highest apparent affinity for InsP3 (0.10μM), followed by InsP3R1 (0.27μM), and then by InsP3R3 (0.40μM); 5), InsP3R1 has a high-affinity (0.13mM) ATP modulatory site, InsP3R2 gating is ATP independent, and InsP3R3 has a low-affinity (2mM) ATP modulatory site; 6), ATP modulates InsP3R1 gating in a noncooperative manner (nHill=1.3); 7), ATP modulates InsP3R3 gating in a highly cooperative manner (nHill=4.1). Obtained results provide novel information about functional properties of mammalian InsP3R isoforms.

The role of inositol 1,4,5-trisphosphate receptors in the regulation of bile secretion in health and disease

Ca 2+ signaling via the inositol 1,4,5-trisphosphate receptor (InsP3R) is a ubiquitous mechanism for regulation of cell function, yet very little is known about the role of the InsP3R in specific disease states. Converging lines of evidence suggest that the liver may provide a model for the role of the InsP3R in health and disease. Ca 2+ signaling is mediated entirely by the InsP3R in hepatocytes and cholangiocytes, the two types of epithelia in the liver. Here we review the role of specific InsP3R isoforms and the physiological effects of InsP3R-mediated Ca 2+ signals in both of these types of epithelia. In addition, we review evidence that the InsP3R is lost from cholangiocytes in cholestatic forms of liver disease, and discuss this as a possible final common pathway for cholestasis.

Modulation of Ca2+ oscillations by phosphorylation of Ins(1,4,5)P3 receptors.

Activation of InsP(3)Rs (InsP(3) receptors) represents the major mechanism underlying intracellular calcium release in non-excitable cells such as hepatocytes and exocrine cells from the pancreas and salivary glands. Modulation of calcium release through InsP(3)Rs is therefore a major route whereby the temporal and spatial characteristics of calcium waves and oscillations can potentially be 'shaped'. In this study, the functional consequences of phosphoregulation of InsP(3)Rs were investigated. Pancreatic and parotid acinar cells express all three types of InsP(3)R in differing abundance, and all are potential substrates for phosphoregulation. PKA (protein kinase A)-mediated phosphorylation of InsP(3)Rs in pancreatic acinar cells resulted in slowed kinetics of calcium release following photo-release of InsP(3). In contrast, activation of PKA in parotid cells resulted in a marked potentiation of calcium release. In pancreatic acinar cells the predominant InsP(3)R isoform phosphorylated was the type 3 receptor, while the type 2 receptor was markedly phosphorylated in parotid acinar cells. In order to further decipher the effects of phosphorylation on individual InsP(3)R subtypes, DT-40 cell lines expressing homotetramers of a single isoform of InsP(3)R were utilized. These data demonstrate that phosphoregulation of InsP(3)Rs results in subtype-specific effects and may play a role in the specificity of calcium signals by 'shaping' the spatio-temporal profile of the response.

Regulation of Ca 2+ signaling in rat bile duct epithelia by inositol 1,4,5-trisphosphate receptor isoforms

Cytosolic Ca 2+ (Ca i 2+) regulates secretion of bicarbonate and other ions in the cholangiocyte. In other cell types, this second messenger acts through Ca 2+ waves, Ca 2+ oscillations, and other subcellular Ca 2+ signaling patterns, but little is known about the subcellular organization of Ca 2+ signaling in cholangiocytes. Therefore, we examined Ca 2+ signaling and the subcellular distribution of Ca 2+ release channels in cholangiocytes and in a model cholangiocyte cell line. The expression and subcellular distribution of inositol 1,4,5-trisphosphate (InsP 3) receptor (InsP 3R) isoforms and the ryanodine receptor (RyR) were determined in cholangiocytes from normal rat liver and in the normal rat cholangiocyte (NRC) polarized bile duct cell line. Subcellular Ca 2+ signaling in cholangiocytes was examined by confocal microscopy. All 3 InsP 3R isoforms were expressed in cholangiocytes, whereas RyR was not expressed. The type III InsP 3R was the most heavily expressed isoform at the protein level and was concentrated apically, whereas the type I and type II isoforms were expressed more uniformly. The type III InsP 3R was expressed even more heavily in NRC cells but was concentrated apically in these cells as well. Adenosine triphosphate (ATP), which increases Ca 2+ via InsP 3 in cholangiocytes, induced Ca 2+ oscillations in both cholangiocytes and NRC cells. Acetylcholine (ACh) induced apical-to-basal Ca 2+ waves. In conclusion, Ca 2+ signaling in cholangiocytes occurs as polarized Ca 2+ waves that begin in the region of the type III InsP 3R. Differential subcellular localization of InsP 3R isoforms may be an important molecular mechanism for the formation of Ca 2+ waves and oscillations in cholangiocytes. Because Ca i 2+ is in part responsible for regulating ductular secretion, these findings also may have implications for the molecular basis of cholestatic disorders. (H EPATOLOGY 2002;36:284-296.)

Inositol 1,4,5-trisphosphate receptor isoforms show similar Ca2+release kinetics

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+release channel which upon activation initiates many cellular functions. Multiple InsP3R subtypes are expressed in most cell types but the physiological significance of this heterogeneity is poorly understood. This study has directly compared the functional properties of the three different InsP3R isoforms by analyzing their InsP3-induced Ca2+release (IICR) properties in cell lines which predominantly express each isoform subtype. The InsP3-dependence of the amount or extent of IICR was InsP3R isoform-specific, with the type III isoform having the lowest affinity with respect to Ca2+release. The transient kinetics of IICR, measured using stopped-flow spectrofluorimetry, however, were similar for all three InsP3R isoforms. At maximal InsP3concentrations (20μM) the rate constants where between 0.8 and 1.0s−1for the fast phase and 0.25–0.45s−1for the slow phase. The concentration of InsP3required to induce half-maximal rates of Ca2+release (EC50) were also similar for the three isoforms (0.2–0.4μM for the fast phase and 0.75–0.95μM for the slow phase). These results indicate the InsP3R channel does not significantly differ functionally in terms of Ca2+release rates between isoforms. The temporal and spatial features of intracellular Ca2+signals are thus probably achieved through InsP3R isoform-specific regulation or localization rather than their intrinsic Ca2+efflux properties.

Calcium channels in lymphocytes.

Calcium channels in lymphocytes.

Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart.

The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.

Single-channel properties in endoplasmic reticulum membrane of recombinant type 3 inositol trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of [Ca(2+)](i) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.

Type III InsP3 receptor channel stays open in the presence of increased calcium.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is the main calcium(Ca2+) release channel in most tissues. Three isoforms have been identified, but only types I and II InsP3R have been characterized. Here we examine the functional properties of the type III InsP3R because this receptor is restricted to the trigger zone from which Ca2+ waves originate and it has distinctive InsP3-binding properties. We find that type III InsP3R forms Ca2+ channels with single-channel currents that are similar to those of type I InsP3R; however, the open probability of type III InsP3R isoform increases monotonically with increased cytoplasmic Ca2+ concentration, whereas the type I isoform has a bell-shaped dependence on cytoplasmic Ca2+. The properties of type III InsP3R provide positive feedback as Ca2+ is released; the lack of negative feedback allows complete Ca2+ release from intracellular stores. Thus, activation of type III InsP3R in cells that express only this isoform results in a single transient, but global, increase in the concentration of cytosolic Ca2+. The bell-shaped Ca2+-dependence curve of type I InsP3R is ideal for supporting Ca2+ oscillations, whereas the properties of type III InsP3R are better suited to signal initiation.

Characterization of inositol 1,4,5-trisphosphate receptor isoform mRNA expression and regulation in rat pancreatic islets, RINm5F cells and betaHC9 cells.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ channel that plays a role in the regulation of insulin secretion. In rat isolated pancreatic islets the expression of types I, II and III InsP3R mRNA was identified by reverse transcriptase-polymerase chain reaction and confirmed by cDNA cloning and sequencing. The islet ratios of types I, II and III InsP3R mRNA to beta-actin mRNA were 0.08 +/- 0.02, 0.08 +/- 0.03 and 0.25 +/- 0.04 respectively. Types I, II and III InsP3R mRNA were also expressed in rat (RINm5F) and mouse (betaHC9) pancreatic beta-cell lines, and rat cerebellum. Type III InsP3R mRNA was quantitatively the most abundant form in rat islets and RINm5F cells. In betaHC9 cells, types II and III InsP3R mRNA were expressed at similar levels, and in much greater abundance than type I mRNA. Type III was the least abundant InsP3R mRNA in cerebellum. Culture of betaHC9 cells for 5 days at 2.8 and 25 mM glucose, or RINm5F cells for 7 days at 5.5 and 20 mM glucose, resulted in significantly enhanced expression of type III, but not types I and II, InsP3R mRNA in the cells at the higher glucose concentrations. During short-term (0.5-2 h) incubations, betaHC9 cell type III InsP3R mRNA levels increased in response to glucose in a time- and concentration-dependent manner. Actinomycin D inhibited the glucose response. Alpha-ketoisocaproic acid also stimulated betaHC9 cell type III InsP3R mRNA expression in a concentration-dependent manner, whereas 2-deoxyglucose and 3-O-methylglucose were without effect. The different levels of expression of mRNA for three InsP3R isoforms in islets and insulinoma cells, and the influence of glucose and alpha-ketoisocaproic acid on the expression of type III mRNA, suggests that nutrient metabolism plays a role in the regulation of this gene and that the function of InsP3R subtypes may be unique with each playing a distinct role in beta-cell signal transduction and insulin secretion.

Isoform-Specific Function of Single Inositol 1,4,5-Trisphosphate Receptor Channels

The inositol 1,4,5-trisphosphate receptor (InsP 3R) family of Ca 2+ release channels is central to intracellular Ca 2+ signaling in mammalian cells. The InsP 3R channels release Ca 2+ from intracellular compartments to generate localized Ca 2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315–325; Joseph, 1996. Cell Signal. 8:1–7; Kume et al., 1997. Science. 278:1940–1943; Berridge, 1997. Nature. 368:759–760). Most cells express multiple InsP 3R isoforms, but only the function of the single type 1 InsP 3R channel is known. Here the single-channel function of single type 2 InsP 3R channel is defined for the first time. The type 2 InsP 3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP 3 regulation and Ca 2+ regulation of type 1 and type 2 InsP 3R channels are strikingly different. Both InsP 3 and Ca 2+ are more effective at activating single type 2 InsP 3R, indicating that single type 2 channels mobilize substantially more Ca 2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca 2+ concentrations inactivate type 1, but not type 2, InsP 3R channels. This indicates that type 2 InsP 3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca 2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP 3R identity will help define the spatial and temporal nature of local Ca 2+ signaling events and may contribute to the segregation of parallel InsP 3 signaling cascades in mammalian cells.

Agonist-induced down-regulation of type 1 and type 3 inositol 1,4,5-tris-phosphate receptors in A7r5 and DDT 1 MF-2 smooth muscle cells

Prolonged stimulation of rat A7r5 aortic smooth muscle cells with 3 μM vasopressin, or of hamster DDT 1 MF-2 smooth muscle cells with 10 μM bradykinin or 100 μM histamine led within 4 h to a 40–50% down-regulation of the type 1 InsP 3 receptor (InsP 3R-1) and of the type 3 InsP 3 receptor (InsP 3R-3). InsP 3R down-regulation was a cell- and agonist-specific process, since several other agonists acting on PLC-coupled receptors did not change the expression level of the InsP 3R isoforms in these cell types and since no agonist-induced down-regulation of InsP 3Rs was observed in HeLa cells. Down-regulation of InsP 3Rs was prevented by an inhibitor of proteasomal protease activity, N-acetyl-Leu-Leu-norleucinal (ALLN). The Ca 2+ channel blocker verapamil (2 μM) also induced InsP 3R-1 down-regulation (43%) in A7r5 cells, which was inhibited by ALLN. In A7r5 cells transiently transfected with a cDNA construct, bearing a luciferase coding sequence under control of the rat InsP3R-1 promoter, reduced luciferase activity could be demonstrated upon stimulation of cells with vasopressin or verapamil. Thus, besides enhanced protein degradation, a reduction of InsP 3R promoter activity might contribute to the down-regulation of InsP 3Rs in A7r5 cells. We next investigated the effect of InsP 3R down-regulation on Ca 2+ responses in A7r5 cells. A rightward shift in the dose-response curve for InsP 3 induced Ca 2 release was observed in permeabilized monolayers of vasopressinpretreated A7r5 cells (EC 50 630 nM and 400 nM for pretreated and non-pretreated cells, respectively). The Ca 2+ responses to threshold doses of vasopressin were markedly reduced in intact vasopressin-pretreated cells. We conclude that prolonged agonist-exposure leads to down-regulation of InsP 3Rs in A7r5 and DDT 1 MF-2 smooth muscle cells. The mechanism of down-regulation likely involves proteasomal degradation and reduction of InsP 3R promoter activity. Moreover, down-regulation of InsP 3Rs resulted in desensitization of Ca 2+ release from InsP 3 sensitive stores.

Calcium, calcium release receptors, and meiotic resumption in bovine oocytes.

During maturation, mammalian oocytes undergo a series of changes that prepare them for fertilization. These events are regulated by kinases, most notably histone H1 and mitogen-activated protein kinase. Intracellular calcium ([Ca2+]i) oscillations participate in oocyte signaling, and it has been postulated that they play a role in oocyte maturation. In these studies we investigated the association of Ca2+, Ca2+ channels, and activation of kinases in in vitro-maturating bovine oocytes. BAPTA-AM, a Ca2+ chelator, inhibited oocyte maturation and delayed activation of kinases, although spontaneous [Ca2+]i rises were not observed in control oocytes loaded with fura-2, a Ca2+ indicator. The ability of the 1,4,5-inositol trisphosphate receptor (InsP3R) to release Ca2+, monitored after the addition of thimerosal and myo-inositol 1,4,5-trisphosphate (InsP3), increased as maturation progressed. This may be associated with a similar increase, monitored by Western blotting, in the density of the type I InsP3R isoform during oocyte maturation. Injection of heparin, an InsP3R antagonist, blocked oocyte maturation and activation of kinases. The density of the ryanodine receptor, another Ca2+ channel, may be 30- to 100-fold lower than that of the InsP3R in bovine oocytes. Thus, our results demonstrate that [Ca2+]i participates in the progression of meiosis and that the InsP3R may be responsible for the majority of Ca2+ release during maturation and fertilization.

The InsP 3 receptor and intracellular Ca 2+ signaling

The inositol 1,4,5-trisphosphate receptor (InsP 3R) is a ligand-gated Ca 2+-release channel on intracellular Ca 2+ store sites (such as the endoplasmic reticulum), and plays an important role in intracellular Ca 2+ signaling in a wide variety of cell types. Recent studies have shown that binding of inositol 1,4,5-trisphosphate (InsP 3) to InsP 3R isoforms is differentially regulated by Ca 2+, and that InsP 3R functions are finely regulated by phosphorylation via tyrosine kinases and protein kinase C, by dephosphorylation via calcineurin, and by binding to FKBP (FK506-binding protein). In addition, transient receptor potential (TRP) and TRP-like proteins appear to couple conformationally with the InsP 3R for capacitative Ca 2+ entry. The importance of InsP 3R signaling in neuronal function has been demonstrated by gene targeting in mice and by studies of T-cell receptor signaling, apoptosis, meiotic maturation, and cytokinesis.

Identification and characterization of inositol 1,4,5-trisphosphate receptors in rat testis

PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP 3Rs) in rat testis. PCR analysis also revealed that rat testis express both forms of the S1 splice variant (S1 + and S1 −), but only the S2 − form of the S2 splice variant of the type I InsP 3 receptor. PCR analysis was also used to identify InsP 3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [ 3H]-Ins P 3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a B max of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The K d for InsP 3 binding to its receptor in testicular microsomes was 60 ± 10 nM which was similar to that found for cerebellar microsomes (80 ± 20 nM). InsP 3-induced Ca 2+ release (IICR) in testicular microsomes was found to have an EC 50 (concentration which causes a half-maximal response) of 0.5 ± 0.03 μM, also similar to that seen for cerebellar microsomes (0.3 μM). Maximal IICR occurred at about 20 μM InsP 3, with up to 4% of total intracellular Ca 2+ stores being mobilized as compared to between 10–30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s −1 at 30 μM InsP 3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (∼1 s −1) and taken together with the binding data support the proposal that the receptor density/Ca 2+ store is ∼8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP 3 analogues also confirmed similar behaviour for testicular InsP 3Rs and cerebellar InsP 3Rs.

Rat basophiliv leukemia cells as model system for inositol 1,4,5-trisphosphate receptor IV, a receptor of the type II family: functional comparison and immunological detection

This study concerns the detection and analysis of the highly homologous type II-like inositol 1,4,5-trisphosphate (InsP 3) receptors (InsP 3R-II, -IV and -V). We have particularly investigated RBL-2H3 cells, which at the mRNA level predominantly expressed InsP 3RIV [De Smedt H. Missiaen L. Parys JB. et al. (1994) Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. J. Biol. Chem., 269, 21691–21698]. When measured in identical experimental conditions, microsomes from RBL-2H3 cells were characterized by a much higher InsP 3 binding affinity (Kd 3.8 ± 0.8 nM, B max 0.40 ± 0.08 pmol/mg protein) than microsomes from A7r5 cells (K d 65 ± 7 nM, B max 0.65 ± 0.08 pmol/mg protein) or from cerebellum (K d 135 ± 14 nM, B max 7.35 ± 1.13 pmol/mg protein). An affinity-purified antibody against the C-terminus of type II-like InsP 3Rs detected, after SDS-PAGE and immunoblotting, a 250 kD protein in RBL-2H3 and C 3H10T1/2 cells, but not in other cell types. An isoform-specific antibody against the C-terminus of InsP 3R-I was used to determine the presence of the various InsP 3R-I splice isoforms at the protein level. The 273 kD (brain), 261 kD (peripheral tissues) and 256 kD ( Xenopus oocytes) isoforms were recognized. Expression of InsP 3R-I in RBL-2H3 cells was very low. Taken together, our results support the hypothesis that InsP 3R isoforms may differ to a large extent in their affinity for InsP3 and suggest that RBL-2H3 cells are a useful model for the study of InsP 3R-IV.

Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction.

The relative expression of different inositol 1,4,5-trisphosphate receptor (InsP3R) mRNA was determined in a selection of murine and rat cell types that are commonly used to study InsP3-mediated Ca2+ signaling. Different mRNA species (encoding the known InsP3R isoforms) were co-amplified using common polymerase chain reaction primer pairs that recognized sequences that are totally conserved between the various InsP3R. Specific identification of the co-amplified sequences was done by restriction site analysis. In cerebellum, mRNA encoding InsP3R-I accounted for > 90% of the total InsP3R mRNA. This isoform was also present in all other cell types tested and was often the major isoform. In contrast, the level of expression of the other isoforms was cell type-specific. A new InsP3R isoform (type V) was detected that had 94.5% sequence identity with the InsP3R-II in the amplified region. Interestingly, this isoform was largely expressed in murine but not in rat cells. We functionally characterized InsP3R-V using the mouse fibroblast C3H10T1/2 cells, where mRNA encoding InsP3R-V accounted for 76.4% of the total InsP3R mRNA. InsP3-induced Ca2+ release in permeabilized C3H10T1/2 cells was regulated by luminal and cytosolic Ca2+, stimulated by thimerosal, and inhibited by caffeine.

Presence of Inositol 1,4,5-Trisphosphate Receptor, Calreticulin, and Calsequestrin in Eggs of Sea Urchins and Xenopus laevis

The presence of inositol 1,4,5-trisphosphate receptor (InsP 3R), calreticulin, and calsequestrin was demonstrated in eggs of sea urchins ( Lytechinus pictus, Lytechinus variegatus, and Strongylocentroutus purpuratus) and Xenopus laevis. Binding of inositol 1,4,5-trisphosphate (InsP 3) to microsomes of L. pictus eggs was inhibited by heparin and NaCl. An affinity-purified antibody against the C-terminal of the type I InsP 3R, which recognizes InsP 3R isoforms of rabbit brain (273 kDa) and Xenopus oocytes and eggs (256 kDa), reacted with a 373-kDa protein in sea urchin eggs. The 373-kDa protein was tentatively identified as the sea urchin egg InsP 3R. Observations with fluorescence microscopy indicated that the InsP 3R is present throughout the cytoplasm of sea urchin eggs in a pattern consistent with the distribution of endoplasmic reticulum. Small differences in the relative amount of reaction deposits in cortex vs subcortex were noted among the species of sea urchins examined. Reaction product was also localized to the periphery of female pronuclei in eggs of all three sea urchins. InsP 3R reactivity was present in the perinuclear region, along the periphery of the germinal vesicle, and throughout the animal and vegetal hemispheres of Xenopus oocytes. A similar cytoplasmic staining pattern was also observed in eggs, although islands of reactivity, much larger than those in oocytes, were present in the animal hemisphere of eggs. Calreticulin and calsequestrin in sea urchin eggs had the same molecular mass as in rabbit brain (56 and 60 kDa, respectively), but differed from those present in Xenopus oocytes/eggs (61 and 57 kDa, respectively). The distribution of calreticulin and calsequestrin in both sea urchin and Xenopus oocytes and eggs was similar to that observed for the InsP 3R. These results are discussed in relation to previous studies of Ca 2+ regulation during egg development and fertilization and suggest that in the oocytes and eggs of the species examined, InsP 3-sensitive Ca 2+ stores play an important role in the regulation of cellular Ca 2+.