Ankyrin B modulates the function of Na,K-ATPase/inositol 1,4,5-trisphosphate receptor signaling microdomain

Na,K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappaB activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na,K-ATPase/IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na,K-ATPase and IP3R. We identify the N terminus tail of the Na,K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na,K-ATPase and IP3R, reduced the number of cells responding to pm doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappaB. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,K-ATPase in the plasma membrane.     

Functional coupling of chromogranin with the inositol 1,4,5-trisphosphate receptor shapes calcium signaling

Chromogranins A and B are high capacity, low affinity calcium (Ca(2+)) storage proteins that bind to the inositol 1,4,5-trisphosphate-gated receptor (InsP(3) R). Although most commonly associated with secretory granules of neuroendocrine cells, chromogranins have also been found in the lumen of the endoplasmic reticulum (ER) of many cell types. To investigate the functional consequences of the interaction between the InsP(3) R and the chromogranins, we disrupted the interaction between the two proteins by adding a chromogranin fragment, which competed with chromogranin for its binding site on the InsP(3)R. Responses were monitored at the single channel level and in intact cells. When using InsP(3) R type I incorporated into planar lipid bilayers and activated by cytoplasmic InsP(3) and luminal chromogranin, the addition of the fragment reversed the enhancing effect of chromogranin. Moreover, the expression of the fragment in the ER of neuronally differentiated PC12 cells attenuated agonist-induced intracellular Ca(2+) signaling. These results show that the InsP(3)R/chromogranin interaction amplifies Ca(2+) release from the ER and that chromogranin is an essential component of this intracellular channel complex.     

Three-dimensional rearrangements within inositol 1,4,5-trisphosphate receptor by calcium

Allosteric binding of calcium ion (Ca2+) to inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) controls channel gating within IP3R. Here, we present biochemical and electron microscopic evidence of Ca2+-sensitive structural changes in the three-dimensional structure of type 1 IP3R (IP3R1). Low concentrations of Ca2+ and high concentrations of Sr2+ and Ba2+ were shown to be effective for the limited proteolysis of IP3R1, but Mg2+ had no effect on the proteolysis. The electron microscopy and the limited proteolysis consistently demonstrated that the effective concentration of Ca2+ for conformational changes in IP3R1 was <10(-7) m and that the IP3 scarcely affected the conformational states. The structure of IP3R1 without Ca2+, as reconstructed by three-dimensional electron microscopy, had a "mushroom-like" appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The projection image of the "head-to-head" assembly comprising two particles confirmed the mushroom-like side view. The "windmill-like" form of IP3R1 with Ca2+ also contains the four bridges connecting from the IP3-binding domain toward the channel domain. These data suggest that the Ca2+-specific conformational change structurally regulates the IP3-triggered channel opening within IP3R1.     

Regulation of inositol 1,4,5-trisphosphate receptor-mediated calcium release by the Na/K-ATPase in cultured renal epithelial cells

It is known that the Na/K-ATPase alpha1 subunit interacts directly with inositol 1,4,5-triphosphate (IP(3)) receptors. In this study we tested whether this interaction is required for extracellular stimuli to efficiently regulate endoplasmic reticulum (ER) Ca(2+) release. Using cultured pig kidney LLC-PK1 cells as a model, we demonstrated that graded knockdown of the cellular Na/K-ATPase alpha1 subunit resulted in a parallel attenuation of ATP-induced ER Ca(2+) release. When the knockdown cells were rescued by knocking in a rat alpha1, the expression of rat alpha1 restored not only the cellular Na/K-ATPase but also ATP-induced ER Ca(2+) release. Mechanistically, this defect in ATP-induced ER Ca(2+) release was neither due to the changes in the amount or the function of cellular IP(3) and P2Y receptors nor the ER Ca(2+) content. However, the alpha1 knockdown did redistribute cellular IP(3) receptors. The pool of IP(3) receptors that resided close to the plasma membrane was abolished. Because changes in the plasma membrane proximity could reduce the efficiency of signal transmission from P2Y receptors to the ER, we further determined the dose-dependent effects of ATP on protein kinase Cepsilon activation and ER Ca(2+) release. The data showed that the alpha1 knockdown de-sensitized the ATP-induced ER Ca(2+) release but not PKCepsilon activation. Moreover, expression of the N terminus of Na/K-ATPase alpha1 subunit not only disrupted the formation of the Na/K-ATPase-IP(3) receptor complex but also abolished the ATP-induced Ca(2+) release. Finally, we observed that the alpha1 knockdown was also effective in attenuating ER Ca(2+) release provoked by angiotensin II and epidermal growth factor.     

Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+ -induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.     

Differential inositol 1,4,5-trisphosphate receptor signaling in a neuronal cell line

Differential intracellular distribution of the three pharmacologically and biophysically distinct types of IP3Rs can lead to different subcellular Ca2+ transients each coupled to discrete intracellular functions. Here, we report the functional localization of differentially distributed IP3 receptor types in the commonly-used hippocampal cell line HT22. The distinct subcellular localization and Ca2+ signaling properties of these receptors determine the potential role of specific IP3 receptor types in cellular function. By utilizing immunochemistry, we conclude that HT22 cells express all three IP3 receptors with types 1 and 3 being expressed predominantly in the endoplasmic reticulum and perinuclear regions and type 2 being expressed predominantly in the nuclear envelope. Optical imaging studies using the Ca2+-sensitive indicator dye fluo-3 show that nuclear IP3 responses have greater amplitude and faster kinetics than cytosolic IP3 responses corresponding to the biophysical characteristics of the differentially distributed receptor types. These results support the hypothesis that differentially distributed IP3R isotypes mediate distinct cellular functions through differential, organelle-specific Ca2+ signaling.     

Two-state conformational changes in inositol 1,4,5-trisphosphate receptor regulated by calcium

Inositol 1,4,5-trisphosphate receptor (IP3R) is a highly controlled calcium (Ca2+) channel gated by inositol 1,4,5-trisphosphate (IP3). Multiple regulators modulate IP3-triggered pore opening by binding to discrete allosteric sites within IP3R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca2+, the most pivotal regulator, induced marked structural changes in the tetrameric IP3R purified from mouse cerebella. Electron microscopy of the IP3R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca2+ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP3-binding domains, assigned by binding to heparin-gold. Ca2+-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca2+ appeared to regulate IP3 gating activity through the rearrangement of functional domains.     

Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium release through the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1.

Our previous studies have demonstrated that calmodulin binds to IP3R type I (IP3R1) in a Ca2+ dependent manner, which suggests that calmodulin regulates the IP3R1 channel. In the present study, we investigated real-time kinetics of interactions between calmodulin and IP3R1 as well as effects of calmodulin on IP3-induced Ca2+ release by purified and reconstituted IP3R1. Kinetic analysis revealed that calmodulin binds to IP3R1 in a Ca2+ dependent manner and that both association and dissociation phase consist of two components with time constants of k(a) = 4.46 x 10(2) and > 10(4) M(-1) s(-1) k(d) = 1.44 x 10(-2) and 1.17 x 10(-1) s(-1). The apparent dissociation constant was calculated to be 27.3 microM. The IP3-induced Ca2+ release through the purified and reconstituted IP3R1 was inhibited by Ca2+/calmodulin, in a dose dependent manner. We interpret our findings to mean that calmodulin binds to IP3R1 in a Ca2+ dependent manner to exert inhibitory effect on IP3R channel activity. This event may be one of the mechanisms governing the negative feedback regulation of IP3-induced Ca2+ release by Ca2+.     

Atrial local Ca signaling and inositol 1,4,5-trisphosphate receptors

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca²⁺ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca²⁺ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in atrial myocytes, and show our new findings on the role of IP₃R subtype in the regulation of spontaneous focal Ca²⁺ releases in the compartmentalized areas of atrial myocytes. The Ca²⁺ sparks, representing focal Ca²⁺ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca²⁺ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP₃Rs compared with ventricular cells and possess significant levels of type 1 IP₃R (IP₃R1) and type 2 IP₃R (IP₃R2). Over the last decade the roles of atrial IP₃R on the enhancement of Ca²⁺-induced Ca²⁺ release and arrhythmic Ca²⁺ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca²⁺ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP₃R1 in the maintenance of peri-nuclear and non-junctional Ca²⁺ sparks via stimulating a posttranslational organization of RyR clusters.     

Solubilization of rat liver inositol 1,4,5-trisphosphate receptor

High affinity Ins(1,4,5)P₃-binding sites of permeabilized hepatocytes are probably the ligand recognition sites of the receptors that mediate the effects of Ins91,4,5)P₃ on intracellular Ca²⁺ mobilization. We have now solubilized these sites from rat liver membranes in the zwitterionic detergent, CHAPS, and shown that the solubilized bind Ins(1,4,5)P₃ with an affinity (K_d = 7.26 ± 0.52 nM, Hill coefficient H = 1.05 ± 0.06) similar to that of the sites in native membranes (K_d = 6.02 ± 0.02). ATP and a range of inositol phosphates (Ins(2,4,5)P₃ Ins(4,5)P₂, and inositol 1,4,5-trisphosphorothioate) also bound with similar affinities to the native and solubilized sites. Solubilization of the liver InsP₃ receptor will allow its further characterization, purification, and comparison of its properties with those of InsP₃ receptors already purified from cerebellum and smooth muscle.     

Effects of inositol 1,4,5-trisphosphate receptor-mediated intracellular stochastic calcium oscillations on activation of glycogen phosphorylase

In various cell types cytosolic calcium (Ca(2+)) is an important regulator. The possible role of Ca(2+) release from the inositol 1,4,5-trisphosphate (IP(3)) receptor channel in the regulation of the phosphorylation-dephosphorylation cycle process involved in glycogen degradation by glycogen phosphorylase have theoretically investigated by using the Li-Rinzel model for cytosolic Ca(2+) oscillations. For the case of deterministic cytosolic Ca(2+) oscillations, there exists an optimal frequency of cytosolic Ca(2+) oscillations at which the average fraction of active glycogen phosphorylase reaches a maximum value, and a mutation for the average fraction of active glycogen phosphorylase occurs at the higher bifurcation point of Ca(2+) oscillations. For the case of stochastic cytosolic Ca(2+) oscillations, the fraction of active phosphorylase is strongly affected by the number of IP(3) receptor channels and the level of IP(3) concentration. Small number of IP(3) receptor channels can potentiate the sensitivity of the activity of glycogen phosphorylase. The average frequency and amplitude of active phosphorylase stochastic oscillations are increased with the level of increasing IP(3) stimuli. The various distributions for the amplitude of active glycogen phosphorylase oscillations in parameters plane are discussed.     

Type 3 inositol 1,4,5-trisphosphate receptor: A calcium channel for all seasons

Inositol 1,4,5 trisphosphate receptors (ITPRs) are a family of endoplasmic reticulum Ca²⁺ channels essential for the control of intracellular Ca²⁺ levels in virtually every mammalian cell type. The three isoforms (ITPR1, ITPR2 and ITPR3) are highly homologous in amino acid sequence, but they differ considerably in terms of biophysical properties, subcellular localization, and tissue distribution. Such differences underscore the variety of cellular responses triggered by each isoform and suggest that the expression/activity of specific isoforms might be linked to particular pathophysiological states. Indeed, recent findings demonstrate that changes in expression of ITPR isoforms are associated with a number of human diseases ranging from fatty liver disease to cancer. ITPR3 is emerging as the isoform that is particularly important in the pathogenesis of various human diseases. Here we review the physiological and pathophysiological roles of ITPR3 in various tissues and the mechanisms by which the expression of this isoform is modulated in health and disease.     

Studying isoform-specific inositol 1,4,5-trisphosphate receptor function and regulation

Inositol 1,4,5-trisphosphate receptors (InsP3R) are a family of ubiquitously expressed intracellular Ca2+ channels. Isoform-specific properties of the three family members may play a prominent role in defining the rich diversity of the spatial and temporal characteristics of intracellular Ca2+ signals. Studying the properties of the particular family members is complicated because individual receptor isoforms are typically never expressed in isolation. In this article, we discuss strategies for studying Ca2+ release through individual InsP3R family members with particular reference to methods applicable following expression of recombinant InsP3R and mutant constructs in the DT40-3KO cell line, an unambiguously null InsP3R expression system.     

Novel regulation of calcium inhibition of the inositol 1,4,5-trisphosphate receptor calcium-release channel

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), a Ca2+-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca2+ signals in many cell types. Three InsP3R isoforms are expressed in different subcellular locations, at variable relative levels with heteromultimer formation in different cell types. A proposed reason for this diversity of InsP3R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca2+ concentrations ([Ca2+]i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca2+] in mediating high [Ca2+]i inhibition of InsP3R gating by studying single endogenous type 1 InsP3R channels through patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentrations of a calmodulin antagonist nor overexpression of a dominant-negative Ca2+-insensitive mutant calmodulin affected inhibition of gating by high [Ca2+]i. However, a novel, calmodulin-independent regulation of [Ca2+]i inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca2+] approximately 400 nM were inhibited by 290 muM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (<5 nM, for approximately 300 s) before the patch-clamp experiments reversibly relieved Ca2+ inhibition, with channel activities observed in [Ca2+]i up to 1.5 mM. Although InsP3 activates gating by relieving high [Ca2+]i inhibition, it was nevertheless still required to activate channels that lacked high [Ca2+]i inhibition. Our observations suggest that high [Ca2+]i inhibition of InsP3R channel gating is not regulated by calmodulin, whereas it can be disrupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca2+]i inhibition may not be an immutable property of different InsP3R isoforms. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the InsP3R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP3.     

Metabotropic receptor activation,desensitization and sequestration-I: modelling calcium and inositol 1,4,5-trisphosphate dynamics following receptor activation

A mathematical account is given of the processes governing the time courses of calcium ions (Ca2+), inositol 1,4,5-trisphosphate (IP(3)) and phosphatidylinositol 4,5-bisphosphate (PIP(2)) in single cells following the application of external agonist to metabotropic receptors. A model is constructed that incorporates the regulation of metabotropic receptor activity, the G-protein cascade and the Ca2+ dynamics in the cytosol. It is subsequently used to reproduce observations on the extent of desensitization and sequestration of the P(2)Y(2) receptor following its activation by uridine triphosphate (UTP). The theory predicts the dependence on agonist concentration of the change in the number of receptors in the membrane as well as the time course of disappearance of receptors from the plasmalemma, upon exposure to agonist. In addition, the extent of activation and desensitization of the receptor, using the calcium transients in cells initiated by exposure to agonist, is also predicted. Model predictions show the significance of membrane PIP(2) depletion and resupply on the time course of IP(3) and Ca2+ levels. Results of the modelling also reveal the importance of receptor recycling and PIP(2) resupply for maintaining Ca2+ and IP(3) levels during sustained application of agonist.     

Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.     

The inositol 1,4,5-trisphosphate (InsP3) receptor

Conclusion In this review, we have described the functional properties and regulation of the InsP₃R. Not all aspects of InsP₃R function and regulation were covered, the main focus was on the most recent and physiologically important data. Information about the structure, heterogeneity, functional properties, and regulation of the InsP₃R is useful for understanding the spatiotemporal aspects of Ca signaling. The combination of biochemical, biophysical and molecular biological techniques has revealed the intricacies of the InsP₃R over the past decade. However, questions about the functional differences between various isoforms and splice variants of the InsP₃R, the structural determinants responsible for regulation of InsP₃R by Ca and ATP, the functional effects of InsP₃R phosphorylation and many others remain to be elucidated. Future investigations can be expected to provide answers to these important questions.We thank S. Bezprozvannaya for expert technical assistance. This work was supported by National Institutes of Health grants HL 33026 and GM 39029, and a Grant-in-Aid from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.     

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 50 mM 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 7-8 ms, the mean closed dwell time is approximately 10 ms; 4), InsP3R2 has the highest apparent affinity for InsP(3) (0.10 microM), followed by InsP3R1 (0.27 microM), and then by InsP3R3 (0.40 microM); 5), InsP3R1 has a high-affinity (0.13 mM) ATP modulatory site, InsP3R2 gating is ATP independent, and InsP3R3 has a low-affinity (2 mM) ATP modulatory site; 6), ATP modulates InsP3R1 gating in a noncooperative manner (n(Hill) = 1.3); 7), ATP modulates InsP3R3 gating in a highly cooperative manner (n(Hill) = 4.1). Obtained results provide novel information about functional properties of mammalian InsP3R isoforms.     

Kinetics of calcium channel opening by inositol 1,4,5-trisphosphate.

The subsecond mobilization of intracellular Ca2+ by IP3 was measured with rapid mixing techniques to determine how cells achieve rapid rises in cytosolic [Ca2+] during receptor-triggered calcium spiking. In permeabilized rat basophilic leukemia cells at 11 degrees C, more than 80% of the 0.7 fmol of Ca2+/cell sequestered by the ATP-driven pump could be released by IP3. Half of the stored Ca2+ was released within 200 ms after addition of saturating (1 microM) IP3. The flux rate was half-maximal at 120 nM IP3. Ca2+ release from fully loaded stores was highly cooperative; the Hill coefficient over the 2-40 nM range was greater than 3. The delay time of channel opening was inversely proportional to [IP3], increasing from 150 ms at 100 nM IP3 to 1 s at 15 nM, indicating that the rate-limiting step in channel opening is IP3 binding. Multiple binding steps are required to account for the observed delay and nonexponential character of channel opening. A simple model is proposed in which the binding of four IP3 molecules to identical and independent sites leads to channel opening. The model agrees well with the data for KD = 18 nM, kon = 1.2 X 10(8) M-1 s-1, and koff = 2.2 s-1. The approximately 1-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having approximately 100-s exchange times that were previously found with radiolabeled IP3. The approximately 1-1s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes.     

Activation of the inositol 1,4,5-trisphosphate receptor by the calcium storage protein chromogranin A

Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) stores in which the Ca(2+) storage protein, chromogranin A (CGA), couples with InsP(3)-gated Ca(2+) channels (InsP(3)R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP(3)R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP(3)Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP(3)R channel activity by CGA appears to be an essential component in the control of intracellular Ca(2+) concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis.