Positive regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by mammalian target of rapamycin (mTOR) in RINm5F cells

The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, is the main regulator of intracellular Ca(2+) mobilization in non-excitable cells. An emerging body of evidence suggests that specific regulatory control of the Ca(2+) signaling pathway is modulated by the activation of additional signaling pathways. In the present study, we investigated the influence of the PI3-kinase/mammalian target of rapamycin (mTOR) pathway on the activity of the IP(3)R/Ca(2+) signaling pathway in RINm5F cells. We used a co-immunoprecipitation approach to show that mTOR physically interacts with IP(3)R-3 in an mTOR activity-dependent manner. We also showed that IP(3)R is phosphorylated by mTOR in cellulo. All the conditions known to modulate mTOR activity (IGF-1, wortmannin, rapamycin, PP242, and nutrient starvation) were shown to modify carbachol-induced Ca(2+) signaling in RINm5F cells. Lastly, we used an assay that directly measures the activity of IP(3)R, to show that mTOR increases the apparent affinity of IP(3)R. Given that mTOR controls cell proliferation and cell homeostasis, and that Ca(2+) plays a key role in these two phenomena, it follows that mTOR facilitates IP(3)R-mediated Ca(2+) release when the nutritional status of cells requires it.     

cAMP-dependent protein kinase enhances inositol 1,4,5-trisphosphate-induced Ca2+ release in AR4-2J cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). There are three subtypes of IP(3)R that are differentially distributed among cell types. AR4-2J cells express almost exclusively the IP(3)R-2 subtype. The purpose of this study was to investigate the effect of cAMP-dependent protein kinase (PKA) on the activity of IP(3)R-2 in AR4-2J cells. We showed that immunoprecipitated IP(3)R-2 is a good substrate for PKA. Using a back-phosphorylation approach, we showed that endogenous PKA phosphorylates IP(3)R-2 in intact AR4-2J cells. Pretreatment with PKA enhanced IP(3)-induced Ca(2+) release in permeabilized AR4-2J cells. Pretreatment with the cAMP generating agent's forskolin and vasoactive intestinal peptide (VIP) enhanced carbachol (Cch)-induced and epidermal growth factor (EGF)-induced Ca(2+) responses in intact AR4-2J cells. Our results are consistent with an enhancing effect of PKA on IP(3)R-2 activity. This conclusion supports the emerging concept of crosstalk between Ca(2+) signaling and cAMP pathways and thus provides another way by which Ca(2+) signals are finely encoded within non-excitable cells.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling

Background information. The IP₃R (inositol 1,4,5‐trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca²⁺ release in virtually all cell types. We have previously demonstrated that caspase‐3‐mediated cleavage of IP₃R1 during cell death generates a C‐terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP₃R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase‐3‐cleaved C‐terminus of IP₃R1 increased the rate of thapsigargin‐mediated Ca²⁺ leak and decreased the rate of Ca²⁺ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca²⁺ stores. We detected the truncated IP₃R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP₃R1 blocked sperm factor‐induced Ca²⁺ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase‐3‐mediated truncation of IP₃R1 enhanced the Ca²⁺ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca²⁺ leak is largely compensated by enhanced Ca²⁺‐uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca²⁺ leak acts as a feed‐forward mechanism to divert the cell into apoptosis.     

The activation state of the inositol 1,4,5-trisphosphate receptor regulates the velocity of intracellular Ca2+ waves in bovine aortic endothelial cells

Ca(2+) is a highly versatile second messenger that plays a key role in the regulation of many cell processes. This versatility resides in the fact that different signals can be encoded spatio-temporally by varying the frequency and amplitude of the Ca(2+) response. A typical example of an organized Ca(2+) signal is a Ca(2+) wave initiated in a given area of a cell that propagates throughout the entire cell or within a specific subcellular region. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3) R) is responsible for the release of Ca(2+) from the endoplasmic reticulum. IP(3) R activity can be directly modulated in many ways, including by interacting molecules, proteins, and kinases such as PKA, PKC, and mTOR. In the present study, we used a videomicroscopic approach to measure the velocity of Ca(2+) waves in bovine aortic endothelial cells under various conditions that affect IP(3) R function. The velocity of the Ca(2+) waves increased with the intensity of the stimulus while extracellular Ca(2+) had no significant impact on wave velocity. Forskolin increased the velocity of IP(3) R-dependent Ca(2+) waves whereas PMA and rapamycin decreased the velocity. We used scatter plots and Pearson's correlation test to visualize and quantify the relationship between the Ca(2+) peak amplitude and the velocity of Ca(2+) waves. The velocity of IP(3) R-dependent Ca(2+) waves poorly correlated with the amplitude of the Ca(2+) response elicited by agonists in all the conditions evaluated, indicating that the velocity depended on the activation state of IP(3) R, which can be modulated in many ways.     

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

BACKGROUND INFORMATION: 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+. RESULTS: 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. CONCLUSIONS: 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.     

Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells

This study was made to explain the mechanisms for the effects of exposure to a time varying 1.51 T magnetic field on the intracellular Ca(2+) signaling pathway. The exposure inhibited an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine chromaffin cells induced by addition of bradykinin (BK) to a Ca(2+) free medium. The exposure did not change BK induced production of inositol 1,4,5-trisphosphate (IP(3)). [Ca(2+)](i) was markedly increased in IP(3) loaded cells, and this increase was inhibited by the magnetic field exposure. A similar increase in [Ca(2+)](i) by other drugs, which stimulated Ca(2+) release from intracellular Ca(2+) stores, was again inhibited by the same exposure. However, transmembrane Ca(2+) fluxes caused in the presence of thapsigargin were not inhibited by the magnetic field exposure in a Ca(2+) containing medium. Inhibition of the BK induced increase in [Ca(2+)](i) by the exposure for 30 min was mostly recovered 1 h after exposure ended. Our results reveal that the magnetic field exposure inhibits Ca(2+) release from intracellular Ca(2+) stores, but that BK bindings to BK receptors of the cell membrane and intracellular inositol IP(3) production are not influenced.     

Uncoupled IP3 receptor can function as a Ca2+-leak channel: cell biological and pathological consequences

Ca(2+) release via intracellular release channels, IP(3)Rs (inositol 1,4,5-trisphosphate receptors) and RyRs (ryanodine receptors), is perhaps the most ubiquitous and versatile cellular signalling mechanism, and is involved in a vast number of cellular processes. In addition to this classical release pathway there is limited, but yet persistent, information about less well-defined Ca(2+)-leak pathways that may play an important role in the control of the Ca(2+) load of the endo(sarco)plasmic reticulum. The mechanisms responsible for this 'basal' leak are not known, but recent data suggest that both IP(3)Rs and RyRs may also operate as Ca(2+)-leak channels, particularly in pathological conditions. Proteolytic cleavage or biochemical modification (such as hyperphosphorylation or nitrosylation), for example, occurring during conditions of cell stress or apoptosis, can functionally uncouple the cytoplasmic control domains from the channel domain of the receptor. Highly significant information has been obtained from studies of malfunctioning channels in various disorders; for example, RyRs in cardiac malfunction or genetic muscle diseases and IP(3)Rs in neurodegenerative diseases. In this review we aim to summarize the existing information about functionally uncoupled IP(3)R and RyR channels, and to discuss the concept that those channels can participate in Ca(2+)-leak pathways.     

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor impairs ER Ca(2+) buffering and causes neurodegeneration in primary cortical neurons

Disruption of neuronal Ca(2+) homeostasis plays a well-established role in cell death in a number of neurodegenerative disorders. Recent evidence suggests that proteolysis of the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3) R1), a Ca(2+) release channel on the endoplasmic reticulum, generates a dysregulated channel, which may contribute to aberrant Ca(2+) signaling and neurodegeneration in disease states. However, the specific effects of InsP(3) R1 proteolysis on neuronal Ca(2+) homeostasis are unknown, as are the functional contributions of this pathway to neuronal death. This study evaluates the consequences of calpain-mediated InsP(3) R1 proteolysis on neuronal Ca(2+) signaling and survival using adeno-associated viruses to express a recombinant cleaved form of the channel (capn-InsP(3) R1) in rat primary cortical neurons. Here, we demonstrate that expression of capn-InsP(3) R1 in cortical cultures reduced cellular viability. This effect was associated with increased resting cytoplasmic Ca(2+) concentration ([Ca(2+) ](i) ), increased [Ca(2+) ](i) response to glutamate, and enhanced sensitivity to excitotoxic stimuli. Together, our results demonstrate that InsP(3) R1 proteolysis disrupts neuronal Ca(2+) homeostasis, and potentially acts as a feed-forward pathway to initiate or execute neuronal death.     

Protein kinase C decreases the apparent affinity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel which plays a major role in Ca2+ signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2 and IP3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP3R are poorly known. RINm5F cells who express almost exclusively (approximately 90%) the IP3R-3, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) may influence IP3R-3-mediated Ca2+ release. With an immunoprecipitation approach we confirmed that RINm5F cells express almost exclusively the IP3R-3 isoform. With an in vitro phosphorylation approach, we showed that the immunopurified IP3R-3 was efficiently phosphorylated by exogenous PKC. With a direct in cellulo approach and an indirect in cellulo back-phosphorylation approach we showed that phorbol-12-myristate-13-acetate (PMA) causes the phosphorylation of IP3R-3 in intact RINm5F cells. In saponin-permeabilized RINm5F cells, 3-induced Ca2+ release was reduced after a pre-treatment with PMA. PMA also reduced the Ca2+ response of intact RINm5F cells stimulated with carbachol and EGF, two agonists that use different receptor types to activate phospholipase C. These results suggest the existence of a negative feedback mechanism involving two components of the Ca2+ signalling cascade, whereby activated PKC dampens IP3R-3 activity.     

Microfilament and microtubule assembly is required for the propagation of inositol trisphosphate receptor‐induced Ca2+ waves in bovine aortic endothelial cells

Ca²⁺ is a highly versatile second messenger that plays a key role in the regulation of numerous cell processes. One‐way cells ensure the specificity and reliability of Ca²⁺ signals is by organizing them spatially in the form of waves that propagate throughout the cell or within a specific subcellular region. In non‐excitable cells, the inositol 1,4,5‐trisphosphate receptor (IP₃R) is responsible for the release of Ca²⁺ from the endoplasmic reticulum. The spatial aspect of the Ca²⁺ signal depends on the organization of various elements of the Ca²⁺ signaling toolkit and varies from tissue to tissue. Ca²⁺ is implicated in many of endothelium functions that thus depend on the versatility of Ca²⁺ signaling. In the present study, we showed that the disruption of caveolae microdomains in bovine aortic endothelial cells (BAEC) with methyl‐ß‐cyclodextrin was not sufficient to disorganize the propagation of Ca²⁺ waves when the cells were stimulated with ATP or bradykinin. However, disorganizing microfilaments with latrunculin B and microtubules with colchicine both prevented the formation of Ca²⁺ waves. These results suggest that the organization of the Ca²⁺ waves mediated by IP₃R channels does not depend on the integrity of caveolae in BAEC, but that microtubule and microfilament cytoskeleton assembly is crucial. J. Cell. Biochem. 106: 344–352, 2009. © 2008 Wiley‐Liss, Inc.     

Agonist-dependent phosphorylation of the inositol 1,4,5-trisphosphate receptor: A possible mechanism for agonist-specific calcium oscillations in pancreatic acinar cells.

The properties of inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium oscillations in pancreatic acinar cells depend crucially on the agonist used to stimulate them. Acetylcholine or carbachol (CCh) cause high-frequency (10-12-s period) calcium oscillations that are superimposed on a raised baseline, while cholecystokinin (CCK) causes long-period (>100-s period) baseline spiking. We show that physiological concentrations of CCK induce rapid phosphorylation of the IP3 receptor, which is not true of physiological concentrations of CCh. Based on this and other experimental data, we construct a mathematical model of agonist-specific intracellular calcium oscillations in pancreatic acinar cells. Model simulations agree with previous experimental work on the rates of activation and inactivation of the IP3 receptor by calcium (DuFour, J.-F., I.M. Arias, and T.J. Turner. 1997. J. Biol. Chem. 272:2675-2681), and reproduce both short-period, raised baseline oscillations, and long-period baseline spiking. The steady state open probability curve of the model IP3 receptor is an increasing function of calcium concentration, as found for type-III IP3 receptors by Hagar et al. (Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. 1998. Nature. 396:81-84). We use the model to predict the effect of the removal of external calcium, and this prediction is confirmed experimentally. We also predict that, for type-III IP3 receptors, the steady state open probability curve will shift to lower calcium concentrations as the background IP3 concentration increases. We conclude that the differences between CCh- and CCK-induced calcium oscillations in pancreatic acinar cells can be explained by two principal mechanisms: (a) CCK causes more phosphorylation of the IP3 receptor than does CCh, and the phosphorylated receptor cannot pass calcium current; and (b) the rate of calcium ATPase pumping and the rate of calcium influx from the outside the cell are greater in the presence of CCh than in the presence of CCK.     

Mammalian target of rapamycin (mTOR) phosphorylates inositol 1,4,5-trisphosphate receptor type 2 and increases its Ca release activity

There is substantial evidence that crosstalk between the proliferation and Ca²⁺-signaling pathways plays a critical role in the regulation of normal physiological functions as well as in the pathogenesis of a variety of abnormal processes. In non-excitable cells, intracellular Ca²⁺ is mobilized through inositol 1,4,5-trisphosphate sensitive Ca²⁺ channels (IP₃R) expressed on the endoplasmic reticulum. Here we report that mTOR, a point of convergence for signals from mitogenic growth factors, nutrients and cellular energy levels, phosphorylates the IP₃R-2, the predominant isoform of IP₃R in AR4-2J cells. Pretreatment with the mTOR inhibitor rapamycin, decreased carbachol-induced Ca²⁺ release in AR4-2J cells. Rapamycin also decreased IP₃-induced Ca²⁺ release in permeabilized AR4-2J cells. We also showed that IGF-1 potentiates carbachol-induced Ca²⁺ release in AR4-2J cells, an effect that was prevented by rapamycin. Rapamycin also decreased carbachol-induced Ca²⁺ release in HEK 293A cells in which IP₃R-1 and IP₃R-3 had been knocked down. These results suggest that mTOR potentiates the activity of IP₃R-2 by a phosphorylation mechanism. This conclusion supports the concept of crosstalk between Ca²⁺ signaling and proliferation pathways and thus provides another way by which intracellular Ca²⁺ signals are finely encoded.     

Endothelin-3 stimulates inositol 1,4,5-trisphosphate production and Ca2+ influx to produce biphasic dopamine release from rat striatal slices

Summary 1. Real-time monitoring of dopamine (DA) release from rat striatal slices demonstrated that endothelin (ET)-3 (0.1–10M) produced a biphasic DA release consisting of transient and sustained components. When extracellular Ca²⁺ was removed, the sustained but not transient response remarkably decreased.2. ET-3 (1–10M) stimulated an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), which also consisted of two components. The external Ca²⁺ depletion inhibited primarily the sustained component of the Ca²⁺ response to ET-3.3. ET-3 increased inositol 1,4,5-trisphosphate (IP3) concentrations in striatal slices. This response peaked at 10 to 20 sec and returned to the basal level 2 min after stimulation, an event which was in good accord with a prompt and transient phase of both cytosolic Ca²⁺ activity and DA release evoked by ET-3.4. Thus, ET-3 produces a transient and a sustained release of DA from striatal slices by stimulating intracellular Ca²⁺ mobilization via IP3 formation and extracellular Ca²⁺ influx, respectively.     

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP₃) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully. No effect of Ca²⁺ concentration on [³H]-InsP₃ binding to unreconstituted InsP₃ receptor could be observed either at 4°C or at 25°C, whereas the effect of [Ca²⁺] on reconstituted InsP₃ receptor depended on the temperature. The Ca²⁺ concentration outside the proteolipsome ([Ca²⁺]_o) had no detectable effect on InsP₃ binding to InsP₃ receptor at 4°C. In contrast, with increase of [Ca²⁺]_o from 0 to 100 nmol/L at 25°C, the InsP₃ binding activity increased gradually. Then the InsP₃ binding activity was decreased drastically at higher [Ca²⁺]_o and inhibited entirely at 50 μmol/L [Ca²⁺]_o. Conformational studies on intrinsic fluorescence of the reconstituted InsP₃ receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP₃ receptor could not be affected by [Ca²⁺]_o at 4°C. While at 25°C, the effects of 10 μmol/L [Ca²⁺]_o on global, membrane and cytoplasmic conformation of the reconstituted InsP₃ receptor were different significantly from that of 100 nmol/L [Ca²⁺]_o.     

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP3) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully.No effect of Ca2+ concentration on [3H]-InsP3 binding to unreconstituted InsP3 receptor could be observed either at 4℃ or at 25℃,whereas the effect of [Ca2+] on reconstituted InsP3 receptor depended on the temperature.The Ca2+ concentration outside the proteolipsome ([Ca2+]o) had no detectable effect on InsP3 binding to InsP3 receptor at 4℃.In contrast,with increase of [Ca2+]o from 0 to 100 nmol/L at 25℃,the InsP3 binding activity increased gradually.Then the InsP3 binding activity was decreased drastically at higher [Ca2+]o and inhibited entirely at 50 mol/L [Ca2+]o.Conformational studies on intrinsic fluorescence of the reconstituted InsP3 receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP3 receptor could not be affected by [Ca2+]o at 4℃.While at 25℃,the effects of 10 m mol/L [Ca2+]o on global,membrane and cytoplasmic conformation of the reconstituted InsP3 receptor were different significantly from that of 100 nmol/L [Ca2+]o.     

Immunohistochemical localization of type 2 inositol 1,4,5-trisphosphate receptor to the nucleus of different mammalian cells

The inositol 1,4,5-trisphosphate receptor (InsP3R) is a ligand-gated Ca2+ channel responsible for the release of Ca2+ from intracellular stores in the response of a wide variety of cells to external stimuli. Molecular cloning studies have revealed the existence of three types of InsP3R encoded by distinct genes. In the study presented here, we used selective anti-InsP3R antibodies to determine the intracellular location of each InsP3R subtype in bovine aortic endothelial cells, bovine adrenal glomerulosa cells, and COS-7 cells. InsP3R1 was found to be widely distributed throughout the cytosol and most abundantly in the perinuclear region identified as the endoplasmic reticulum (co-localization with protein disulfide isomerase). The intracellular location of InsP3R3 was similar to that of InsP3R1. Surprisingly, InsP3R2 was found mostly associated to the cell nucleus. This observation was made with two antibodies recognizing different epitopes on InsP3R2. Binding studies revealed the presence of a high affinity-binding site for [3H] InsP3 on purified nuclei from bovine adrenal cortex. Confocal images showed that InsP3R2 was not confined to the nuclear envelope but was distributed relatively uniformly within the nucleus. Our results demonstrate that the three types of InsP3R are not similarly distributed within a specific cell type. Our results also suggest the existence of an intranuclear membrane network on which InsP3R2 is abundantly expressed.     

Quantal Ca2+ release and inactivation in a model of the inositol 1,4,5-trisphosphate receptor involving transformation of the ligand by the receptor

A model explaining quantal Ca²⁺ release as an intrinsic property of the inositol 1,4,5-trisphosphate (IP₃) receptor has been put forward. The model is based on the hypothesis that the IP₃ receptor can catalyze a transformation of the IP, molecule differing from its conventional metabolism. A simple kinetic mechanism is considered, in which IP₃-induced Ca²⁺ channel opening is followed by the step of IP₃ conversion and channel closure. Examination of the resulting mathematical model shows that it can reproduce well both partial release of stored Ca²⁺ and the same responsiveness to subsequent IP3 additions. On incorporation of an additional closed state of the channel, the model describes also a time-dependent channel inactivation at a high IP₃ dose. Temperature sensitivity of the catalytic step accounts for the reported elimination of quantal responses and inactivation at low temperature. The transformation product is surmised to be a positional or stereo isomer of IP₃.     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Single channel function of inositol 1,4,5-trisphosphate receptor type-1 and -2 isoform domain-swap chimeras

The InsP3R proteins have three recognized domains, the InsP3-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893-3898). The InsP3 binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 InsP3R channels have been defined. Here, two type-1/type-2 InsP3R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The InsP3 and Ca2+ regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP3R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define InsP3R structure-function.     

An IRBIT homologue lacks binding activity to inositol 1,4,5‐trisphosphate receptor due to the unique N‐terminal appendage

IRBIT is an inositol 1,4,5‐trisphosphate (IP₃) receptor (IP₃R)‐binding protein that inhibits the activation of IP₃R by competing with IP₃ for the common binding site on IP₃R. In this study, we characterize an IRBIT homologue, termed Long‐IRBIT. Long‐IRBIT is highly homologous to IRBIT (～88%) and heteromerizes with IRBIT. In spite of complete conservation of critical amino acids required for the interaction with IP₃R and comparable phosphorylations on critical four Ser residues for IP₃R‐binding, Long‐IRBIT retains little ability to interact with IP₃R. Deletion mutagenesis analysis revealed that this low affinity to IP₃R is attributable to an inhibitory effect of the Long‐IRBIT specific N‐terminal appendage (LISN domain). Immunohistochemical analysis revealed the distinct distribution of Long‐IRBIT and IRBIT in mouse cerebellar cortex, that is, Long‐IRBIT is mainly expressed in interneurons such as basket cells, while IRBIT is mainly expressed in glial cells. Furthermore, Long‐IRBIT, but not IRBIT, underwent phosphorylation during neuronal differentiation in neuroblastoma cells and this phosphorylation was dependent on the LISN domain. These results suggest that Long‐IRBIT has a different function from IRBIT.