Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) serves as an intracellular second messenger for several neurotransmitters, hormones and growth factors by initiating calcium release from intracellular stores. A cerebellar Ins(1,4,5)P3 receptor has been characterized biochemically and shown by immunocytochemistry to be present in intracellular membranes in Purkinje cells. We show that a previously described Purkinje-cell messenger RNA encodes a protein of relative molecular mass 260,000 (260 K) with the same properties as the cerebellar Ins(1,4,5)P3 receptor. Its sequence is partially homologous to the skeletal muscle ryanodine receptor. By immunocytochemistry and electron microscopy the protein is shown to be present in all parts of the endoplasmic reticulum, including those that extend into axon terminals and dendritic spines. Our results indicate that gated calcium release from intracellular stores in muscle and Purkinje cells uses similar calcium-channel proteins localized in analogous intracellular compartments. This implies that the intracellular calcium stores in the endoplasmic reticulum of neurons extend into presynaptic terminals and dendritic spines where they may play a direct role in regulating the efficacy of neurotransmission.     

Inositol 1,4,5-trisphosphate induces calcium release from sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum of skeletal muscle is a specialized form of endoplasmic reticulum that controls myoplasmic calcium concentration and, therefore, the contraction-relaxation cycle. Ultrastructural studies have shown that the sarcoplasmic reticulum is a continuous but heterogeneous membranous network composed of longitudinal tubules that surround myofibrils and terminal cisternae. These cisternae are junctionally associated, via bridging structures called 'feet', with sarcolemmal invaginations (the transverse tubules) to form the triadic junction. Following transverse tubule depolarization, a signal, transmitted along the triadic junction, triggers Ca2+ release from terminal cisternae, but the mechanism of this coupling is still unknown. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) has recently been shown to mobilize Ca2+ from intracellular stores, referable to endoplasmic reticulum, in a variety of cell types (see ref. 8 for review), including smooth muscle cells of the porcine coronary artery and canine cardiac muscle cells. Here we show that Ins(1,4,5)P3 releases Ca2+ from isolated, purified sarcoplasmic reticulum fractions of rabbit fast-twitch skeletal muscle, the effect being more pronounced on a fraction of terminal cisternae that contains morphologically intact feet structures; and elicits isometric force development in chemically skinned muscle fibres.     

Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes

Hydrolysis of membrane-associated phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)-P2) to water soluble inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is a common response by many different kinds of cells to a wide variety of external stimuli (see refs 1 and 2 for review). Ins (1,4,5)P3 is a putative second messenger which increases intracellular Ca2+ by mobilizing internal Ca2+ stores, a hypothesis which has been substantiated by studies with chemically permeabilized cells and with isolated microsomal membrane fractions. But the possibility that Ins(1,4,5)P3 could induce in intact cells an influx of external Ca2+ through transmembrane channels, originally hypothesized by Michell in 1975, has never been directly tested. We report here single-channel recordings of an Ins(1,4,5)P3-activated conductance in excised patches of T-lymphocyte plasma membrane. The Ins(1,4,5)P3-activated transmembrane channel appears to be identical to the recently described mitogen-regulated, voltage-insensitive Ca2+ permeable channel involved in T-cell activation. We suggest that Ins(1,4,5)P3 acts as the second messenger mediating transmembrane Ca2+ influx through specific Ca2+-permeable channels in mitogen-stimulated T-cell activation.     

A saturable receptor for 32P-inositol-1,4,5-triphosphate in hepatocytes and neutrophils

Several receptors for neurotransmitters, hormones and growth factors cause accelerated phosphodiesteratic breakdown of polyphosphoinositides when activated. One of the soluble products of this reaction, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) is thought to act as a second messenger signalling the release of Ca2+ from intracellular stores. In support of this hypothesis, several studies have shown that Ins(1,4,5)P3 releases sequestered Ca2+ from permeable cells and microsomes. On the basis of certain structural requirements for Ca2+-releasing activity by inositol phosphates, it has been postulated that Ins(1,4,5)P3 acts by binding to a specific intracellular receptor, probably on a component of the endoplasmic reticulum. Here we report that 32P-Ins(1,4,5)P3 binds to a specific saturable site in permeabilized guinea pig hepatocytes and rabbit neutrophils, and that the properties of this binding site suggest that it is the physiological receptor for Ins(1,4,5)P3.     

Purified inositol 1,4,5-trisphosphate receptor mediates calcium flux in reconstituted lipid vesicles

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), a second messenger molecule involved in actions of neurotransmitters, hormones and growth factors, releases calcium from vesicular non-mitochondrial intracellular stores. An Ins(1,4,5)P3 binding protein, purified from brain membranes, has been shown to be phosphorylated by cyclic-AMP-dependent protein kinase and localized by immunohistochemical techniques to intracellular particles associated with the endoplasmic reticulum. Although the specificity of the Ins(1,4,5)P3 binding protein for inositol phosphates and the high affinity of the protein for Ins(1,4,5)P3 indicate that it is a physiological Ins(1,4,5)P3 receptor mediating calcium release, direct evidence for this has been difficult to obtain. Also, it is unclear whether a single protein mediates both the recognition of Ins(1,4,5)P3 and calcium transport or whether these two functions involve two or more distinct proteins. In the present study we report reconstitution of the purified Ins(1,4,5)P3 binding protein into lipid vesicles. We show that Ins(1,4,5)P3 and other inositol phosphates stimulate calcium flux in the reconstituted vesicles with potencies and specificities that match the calcium releasing actions of Ins(1,4,5)P3. These results indicate that the purified Ins(1,4,5)P3 binding protein is a physiological receptor responsible for calcium release.     

Synergism of inositol trisphosphate and tetrakisphosphate in activating Ca2+-dependent K+ channels

Inositol 1,4,5-trisphosphate (Ins P3) is a second messenger releasing intracellular Ca2+ into the cytosol. It has recently been proposed that inositol 1,3,4,5-tetrakisphosphate (Ins P4), which is formed from Ins P3 by Ins P3-3-kinase, acts with Ins P3 as a second messenger by promoting extracellular Ca2+ entry. It has been suggested that Ins P3 itself can act to stimulate Ca2+ uptake from the extracellular fluid, although a physiological function for Ins P4 was not excluded. Transmembrane currents can now be measured in single cells by voltage clamping under conditions where the intracellular perfusion fluid can be changed several times during individual experiments. We have used this method to test the effects of Ins P3 and Ins P4 on the Ca2+-activated K+ current, and now show that neither Ins P3 alone nor Ins P4 alone can activate a sustained current, whereas Ins P3 and Ins P4 in combination evoke a sustained increase in Ca2+-activated K+ current which is dependent on external Ca2+.     

Rapid mobilization of Ca2+ from rat insulinoma microsomes by inositol-1,4,5-trisphosphate

Several hormones and neurotransmitters raise the cytosolic free Ca2+ concentration by stimulating the influx of Ca2+ and/or by mobilizing stored Ca2+. However, the link between the agonist receptor on the cell surface and the organelle(s) from which Ca2+ is mobilized is unknown. One feature of the agonists that increase cytosolic Ca2+ is their rapid induction of phosphatidylinositol turnover and polyphosphoinositide hydrolysis; in some tissues this leads, within seconds, to a marked accumulation of the water-soluble products, inositol 1,4-bisphosphate ( Ins1 , 4P2 ) and inositol-1,4,5- trisphosphate ( Ins1 ,4, 5P3 ), suggesting that these might mediate Ca2+ mobilization from internal pools. Such an action of Ins1 ,4, 5P3 has recently been inferred from studies with permeabilized pancreatic acinar cells and hepatocytes. Here we show directly that Ins1 ,4, 5P3 rapidly releases Ca2+ from a microsomal fraction of rat insulinoma but not from mitochondria or secretory granules. Moreover, this response is transient and desensitizes the microsomes to subsequent Ins1 ,4, 5P3 additions. These results suggest that Ins1 ,4, 5P3 functions as a cellular messenger inducing early mobilization of Ca2+ from the endoplasmic reticulum.     

Ca2+ signals induced from calcium stores in pancreatic islet β cells

In single rat pancreatic β cells,using fura-2 microfluorometry to measure [Ca2+]i response upon different stimuli,the ways of calcium regulation have been studied.When the extracellular calcium concentration was 2.5 mmol/L,either 60 mmol/L KCl,20 mmol/L D-glucose or 0.1 mmol/L tolbutamide induced increase in [Ca2+]i.Such increase in [Ca2+]i was absent when the same stimuli were applied under zero extracellular calcium.These results indicate that the increase of [Ca2+]i is induced by the activation of voltage-dependent calcium channels in β cells.The manifold forms of [Ca2+]i change induced by glucose imply that the effects of glucose are complex.5 mmol/L caffeine or 5 mmol/L MCh increase the [Ca2+]i ,which is independent of the external calcium,suggesting that [Ca2+]i can be regulated by Ca2+ release from not only the IP3-sensitive but also the ryanodine sensitive calcium stores in β cells.The latency of Ca responses for IP3 pathway (5 s) is faster than that for ryanodine pathway (30 s).It is concluded that there are multiple calcium stores in rat pancreatic β cells.     

Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons

Inositol 1,4,5-trisphosphate (InsP3) mediates the effects of several neurotransmitters, hormones and growth factors by mobilizing Ca2+ from a vesicular, non-mitochondrial intracellular store. Many studies have indirectly suggested the endoplasmic reticulum (ER) to be the site of InsP3 action, though some have implicated the plasma membrane or a newly described smooth surfaced structure, termed the calciosome. Using antibodies directed against a purified InsP3-receptor glycoprotein, of relative molecular mass 260,000, in electron microscope immunocytochemical studies of rat cerebellar Purkinje cells, we have now localized the InsP3 receptor to ER, including portions of the rough endoplasmic reticulum, a population of smooth-membrane-bound organelles (smooth ER), a portion of subplasmalemmal cisternae and the nuclear membrane, but not to mitochondria or the cell membrane. These results suggest that in cerebellar Purkinje cells, InsP3-induced intracellular calcium release is not the property of a single organelle, but is effected by specialized portions of both rough and smooth ER, and possibly by other smooth surfaced structures. The present findings are the first immunocytochemical demonstration of an InsP3 receptor within a cell.     

Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.

Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.     

Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca(2+)-permeable channel.

Receptor-mediated increases in the cytosolic free calcium ion concentration in most mammalian cells result from mobilization of Ca2+ from intracellular stores as well as transmembrane Ca2+ influx. Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores by opening a Ca(2+)-permeable channel in the endoplasmic reticulum. But the mechanism and regulation of Ca2+ entry into nonexcitable cells has remained elusive because the entry pathway has not been defined. Here we characterize a novel inositol 1,3,4,5-tetrakisphosphate (InsP4) and Ca(2+)-sensitive Ca(2+)-permeable channel in endothelial cells. We find that InsP4, which induces Ca2+ influx into acinar cells, enhances the activity of the Ca(2+)-permeable channel when exposed to the intracellular surface of endothelial cell inside-out patches. Our results suggest a molecular mechanism which is likely to be important for receptor-mediated Ca2+ entry.     

Heterotrimeric G protein participated in modulation of cytoplasmic calcium concentration in pollen cells

Cytoplasmic free calcium concentration([Ca2+]c) in pollen cells of Lilium daviddi is measured with confocal laser scanning microscopy to investigate the effect of heterotrimeric G protein (G protein) on [Ca2+]c and the possible signal transduction pathway of G protein triggering cellular calcium signal. After application, cholera toxin (CTX), an agonist of G protein, triggers a transient increase of [Ca2+]c in pollen cells, and evokes a spatial-temporal characteristic calcium dynamics; while pertussis toxin (PTX), a G protein antagonist, leads to the decrease of [Ca2+]c. Both L-type Ca2+ channel blocker verapamil and inhibitor of IP3 receptor heparin inhibit CTX-induced [Ca2+]c increase. The results show that G protein may play a role in the modulation of [Ca2+]c through enhancing the extracellular Ca2+ influx and releasing of Ca2+ from intracellular stores.     

Stepwise enzymatic dephosphorylation of inositol 1,4,5-trisphosphate to inositol in liver

Many receptors for hormones, neurotransmitters and other signals cause hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and effect a rise in cytosolic Ca2+ concentration. The inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) liberated during PtdIns(4,5)P2 breakdown seems to serve as a second messenger that activates the release of Ca2+ from a nonmitochondrial intracellular compartment. As expected if it is an important intracellular messenger, Ins(1,4,5)P3 is relatively rapidly degraded, both within stimulated cells and when added to homogenates of blowfly salivary gland or to permeabilized, but not intact, hepatocytes. Here we report that the dephosphorylation reactions responsible for the conversion of Ins(1,4,5)P3 to free inositol in rat liver are catalysed by two or more enzymes, and that these reactions are distributed between the plasma membrane and cytosol. The Ins(1,4,5)P3 5-phosphatase and inositol 1-phosphate (Ins(1)P) phosphatase of liver appear similar to enzymes described previously in erythrocytes and brain.     

Bidirectional control of cytosolic free calcium by thyrotropin-releasing hormone in pituitary cells

It is now established that a key step in the action of calcium-mobilizing agonists is stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). The latter substance acts as a second messenger, controlling the release of calcium from intracellular stores (see ref. 3 for review). The bifurcating nature of the signalling system is exemplified by the fact that the other product of PtdIns(4,5)P2 hydrolysis, 1,2-diacylglycerol, can alter cellular function by activating protein kinase C, the cellular target for several tumour-promoting agents such as the phorbol esters. In various tissues, including GH3 pituitary tumour cells, a synergistic interaction between calcium ions and protein kinase C underlies agonist-induced changes in cell activity. The data presented here suggest that when GH3 cells are stimulated by thyrotropin-releasing hormone (TRH), an agonist inducing PtdIns(4,5)P2 hydrolysis, the two limbs of the inositol lipid signalling system interact to control free cytosolic calcium levels [( Ca2+]i). At low levels of TRH receptor occupancy, [Ca2+]i increases rapidly, then declines relatively slowly. As receptor occupancy increases, the calcium signal becomes more short-lived due to the appearance of a second, inhibitory, component. This latter component, which is enhanced when [Ca2+]i is elevated by high potassium depolarization, is mimicked by active phorbol esters and by bacterial phospholipase C. It seems likely that protein kinase C subserves a negative feedback role in agonist-induced calcium mobilization.     

Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration

A rapid, transient rise in the free cytosolic Ca2+ concentration ([Ca2+]i) is one of the earliest events in neutrophil activation and is assumed to be involved in many of the subsequent cellular reactions. Both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space contribute to the rise in [Ca2+]i. In an attempt to assess the relative importance of these pools and the sequences leading to the rise in [Ca2+]i, we have studied the time course of changes in [Ca2+]i after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet-activating factor (PAF) using the Ca2+ indicators quin-2 and fura-2. We observed a time lag of 1-3 s between stimulation and rise in [Ca2+]i. This lag depends on the agonist concentration but is independent of extracellular Ca2+. Thus Ca2+ release from intracellular stores is rate limiting for the rise in [Ca2+]i. After this, cation channels in the plasma membrane (measured with the patch clamp method) are opened. These non-selective channels, which also pass Ca2+, are activated by the initial rise in [Ca2+]i, but by neither fMLP nor inositol 1,4,5-trisphosphate (IP3) directly.     

Inositol trisphosphate receptor localization in brain: variable stoichiometry with protein kinase C

Many neurotransmitters, hormones and growth factors act at membrane receptors to stimulate the phosphodiesteratic hydrolysis of phosphatidyl-inositol 4,5-bisphosphate generating the comessengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. Diacylglycerol stimulates protein kinase C3 while Ins(1,4,5)P3 is postulated to activate specific receptors leading to release of intracellular calcium, probably from the endoplasmic reticulum. In recent preliminary reports, Rubin and associates detected 32P-Ins(1,4,5)P3 binding to liver and adrenal microsomes and to permeabilized neutrophils and liver cells. We now report the biochemical and autoradiographic demonstration in brain of high affinity, selective binding sites for 3H- and 32P-labelled Ins(1,4,5)P3 at levels 100-300 times higher than those observed in peripheral tissues. The potencies of various myoinositol analogues at the Ins(1,4,5)P3 binding site correspond to their potencies in releasing calcium from microsomes, supporting the physiological relevance of this receptor. Brain autoradiograms demonstrate discrete, heterogeneous localization of Ins(1,4,5)P3 receptors. In some regions localizations of Ins(1,4,5)P3 receptors resemble those of protein kinase C14, while in others they differ markedly, suggesting a novel mechanism whereby the relative activity of the two limbs of the PI cycle can be differently regulated.     

Irreversible inhibition of Ca2+ release in saponin-treated macrophages by the photoaffinity derivative of inositol-1, 4, 5-trisphosphate

D-myo-inositol-1,4,5-trisphosphate (InsP3) is a putative intracellular second messenger for the mobilization of Ca2+ from intracellular stores, in particular, the endoplasmic reticulum. Specific binding sites on the endoplasmic reticulum may participate in the InsP3-induced release of Ca2+ from the Ca2+ pool. To examine the specific binding sites on the endoplasmic reticulum, we synthesized an arylazide derivative of InsP3 for photoaffinity labelling; InsP3 coupled to p-azidobenzoic acid (InsP3-pAB) using N,N'-carbonyldiimidazole (CDI) was obtained at a 9-11% yield. Here, we report that InsP3-pAB, but not an arylazide derivative of inositol-1,4-bisphophate (Ins(1,4)P2), causes the irreversible inhibition of InsP3-induced release of Ca2+ in saponin-permeabilized photo-irradiated macrophages. The irreversible inhibition by InsP3-pAB after photo-irradiation was prevented by a 10-fold excess of unmodified InsP3.     

InsP4 facilitates store-operated calcium influx by inhibition of InsP3 5-phosphatase

Receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) initiates Ca2+ release from intracellular stores and the subsequent activation of store-operated calcium influx. InsP3 is metabolized within seconds by 5-phosphatase and 3-kinase, yielding Ins(1,4)P2 and inositol 1,3,4,5-tetrakisphosphate (InsP4), respectively. Some studies have suggested that InsP4 controls Ca2+ influx in combination with InsP3 (refs 3 and 4), but another study did not find the same result. Some of the apparent conflicts between these previous studies have been resolved; however, the physiological function of InsP4 remains elusive. Here we have investigated the function of InsP4 in Ca2+ influx in the mast cell line RBL-2H3, and we show that InsP4 inhibits InsP3 metabolism through InsP3 5-phosphatase, thereby facilitating the activation of the store-operated Ca2+ current I(CRAC) (ref. 9). Physiologically, this mechanism opens a discriminatory time window for coincidence detection that enables selective facilitation of Ca2+ influx by appropriately timed low-level receptor stimulation. At higher concentrations, InsP4 acts as an inhibitor of InsP3 receptors, enabling InsP4 to act as a potent bi-modal regulator of cellular sensitivity to InsP3, which provides both facilitatory and inhibitory feedback on Ca2+ signalling.     

Inositol 1,4,5-trisphosphate mimics muscarinic response in Xenopus oocytes

The enhanced metabolism of phosphoinositides, which is associated with a wide variety of stimuli and physiological responses, has been studied intensively. Berridge and his collaborators demonstrated that the first measurable reaction following cell membrane receptor activation is a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and that the product of this reaction, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), could cause a release of non-mitochondrial calcium. These findings have been verified in other systems. Although the relationship between the hydrolysis of PtdIns(4,5)P2 and the mobilization of intracellular calcium was clearly demonstrated, the direct link between Ins(1,4,5)P3 production and the physiological response was only implied. We have investigated the possibility that the intracellular release of Ins(1,4,5)P3 mediates the muscarinic-cholinergic response is Xenopus oocytes, and we show here that intracellularly injected Ins(1,4,5)P3 mimics the muscarinic depolarizing chloride current in Xenopus oocytes. This is the first demonstration of a direct link between phosphoinositides metabolism and a neuro-transmitter-induced physiological response.