Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Background information. Interconnections between the Ca²⁺ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase‐anchoring proteins). In many cell types, the activation of InsP₃R (inositol 1,4,5‐trisphosphate receptor), an endoplasmic reticulum Ca²⁺ channel, is a key event of Ca²⁺ signalling. The phosphorylation of InsP₃R1 by PKA stimulates Ca²⁺ mobilization. This control is thought to be tight, involving the association of PKA with InsP₃R1. The InsP₃R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP₃R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP₃R1 associated with PKA type IIβ and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co‐purification of AKAP450 with InsP₃R1 on heparin‐agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP₃R1 and RIIβ (regulatory subunit of PKA IIβ) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP₃R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP₃R1 forms a complex with AKAP450 and PKAIIβ, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP₃R1 with PKA in Purkinje cells would require a different macromolecular complex.     

Protein Kinase A Increases Type-2 Inositol 1,4,5-Trisphosphate Receptor Activity by Phosphorylation of Serine 937

Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP₃Rs) represents a mechanism for shaping intracellular Ca²⁺ signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca²⁺ release in cells that express predominantly InsP₃R2. PKA is known to phosphorylate InsP₃R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP₃R2 in DT40-3KO cells that are devoid of endogenous InsP₃R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca²⁺ signals and augmented the single channel open probability of InsP₃R2. A PKA phosphorylation site unique to the InsP₃R2 was identified at Ser⁹³⁷. The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser⁹³⁷, since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca²⁺ signaling following PKA activation in cells that express predominantly InsP₃R2.Hormones, neurotransmitters, and growth factors stimulate the production of InsP₃³ and Ca²⁺ signals in virtually all cell types (1). The ubiquitous nature of this mode of signaling dictates that this pathway does not exist in isolation; indeed, a multitude of additional signaling pathways can be activated simultaneously. A prime example of this type of “cross-talk” between independently activated signaling systems results from the parallel activation of cAMP and Ca²⁺ signaling pathways (2, 3). Interactions between these two systems occur in numerous distinct cell types with various physiological consequences (3–6). Given the central role of InsP₃R in Ca²⁺ signaling, a major route of modulating the spatial and temporal features of Ca²⁺ signals following cAMP production is potentially through PKA phosphorylation of the InsP₃R isoform(s) expressed in a particular cell type.There are three InsP₃R isoforms (InsP₃R1, InsP₃R2, and InsP₃R3) expressed to varying degrees in mammalian cells (7, 8). InsP₃R1 is the major isoform expressed in the nervous system, but it is less abundant compared with other subtypes in non-neuronal tissues (8). Ca²⁺ release via InsP₃R2 and InsP₃R3 predominate in these tissues. InsP₃R2 is the major InsP₃R isoform in many cell types, including hepatocytes (7, 8), astrocytes (9, 10), cardiac myocytes (11), and exocrine acinar cells (8, 12). Activation of PKA has been demonstrated to enhance InsP₃-induced Ca²⁺ signaling in hepatocytes (13) and parotid acinar cells (4, 14). Although PKA phosphorylation of InsP₃R2 is a likely causal mechanism underlying these effects, the functional effects of phosphorylation have not been determined in cells unambiguously expressing InsP₃R2 in isolation. Furthermore, the molecular determinants of PKA phosphorylation of this isoform are not known.PKA-mediated phosphorylation is an efficient means of transiently and reversibly regulating the activity of the InsP₃R. InsP₃R1 was identified as a major substrate of PKA in the brain prior to its identification as the InsP₃R (15, 16). However, until recently, the functional consequences of phosphorylation were unresolved. Initial conflicting results were reported indicating that phosphoregulation of InsP₃R1 could result in either inhibition or stimulation of receptor activity (16, 17). Mutagenic strategies were employed by our laboratory to clarify this discrepancy. These studies unequivocally assigned phosphorylation-dependent enhanced Ca²⁺ release and InsP₃R1 activity at the single channel level, through phosphorylation at canonical PKA consensus motifs at Ser¹⁵⁸⁹ and Ser¹⁷⁵⁵. The sites responsible were also shown to be specific to the particular InsP₃R1 splice variant (18). These data were also corroborated by replacing the relevant serines with glutamates in a strategy designed to construct “phosphomimetic” InsP₃R1 by mimicking the negative charge added by phosphorylation (19, 20). Of particular note, however, although all three isoforms are substrates for PKA, neither of the sites phosphorylated by PKA in InsP₃R1 are conserved in the other two isoforms (21). Recently, three distinct PKA phosphorylation sites were identified in InsP₃R3 that were in different regions of the protein when compared with InsP₃R1 (22). To date, no PKA phosphorylation sites have been identified in InsP₃R2.Interactions between Ca²⁺ and cAMP signaling pathways are evident in exocrine acinar cells of the parotid salivary gland. In these cells, both signals are important mediators of fluid and protein secretion (23). Multiple components of the [Ca²⁺]_i signaling pathway in these cells are potential substrates for modulation by PKA. Previous work from this laboratory established that activation of PKA potentiates muscarinic acetylcholine receptor-induced [Ca²⁺]_i signaling in mouse and human parotid acinar cells (4, 24, 25). A likely mechanism to explain this effect is that PKA phosphorylation increases the activity of InsP₃R expressed in these cells. Consistent with this idea, activation of PKA enhanced InsP₃-induced Ca²⁺ release in permeabilized mouse parotid acinar cells and also resulted in the phosphorylation of InsP₃R2 (4).Invariably, prior work examining the functional effects of PKA phosphorylation on InsP₃R2 has been performed using cell types expressing multiple InsP₃R isoforms. For example, AR4-2J cells are the preferred cell type for examining InsP₃R2 in relative isolation, because this isoform constitutes more than 85% of the total InsP₃R population (8). InsP₃R1, however, contributes up to ∼12% of the total InsP₃R in AR4-2J cells. An initial report using InsP₃-mediated ⁴⁵Ca²⁺ flux suggested that PKA activation increased InsP₃R activity in AR4-2J cells (21). A similar conclusion was made in a later study, which documented the effects of PKA activation on agonist stimulated Ca²⁺ signals in AR4-2J cells (26). Any effects of phosphorylation observed in these experiments could plausibly have resulted from phosphorylation of the residual InsP₃R1.Although PKA enhances InsP₃-induced calcium release in cells expressing predominantly InsP₃R2, including hepatocytes, parotid acinar cells, and AR4-2J cells (4, 13, 21, 26, 27), InsP₃R2 is not phosphorylated at stoichiometric levels by PKA (21). This observation has called into question the physiological significance of PKA phosphorylation of InsP₃R2 (28). The apparent low levels of InsP₃R2 phosphorylation are clearly at odds with the augmented Ca²⁺ release observed in cells expressing predominantly this isoform. The equivocal nature of these findings probably stems from the fact that, to date, all of the studies demonstrating positive effects of PKA activation on Ca²⁺ release were conducted in cells that also express InsP₃R1. The purpose of the current experiments was to analyze the functional effects of phosphorylation on InsP₃R2 expressed in isolation on a null background. We report that InsP₃R2 activity is increased by PKA phosphorylation under these conditions, and furthermore, we have identified a unique phosphorylation site in InsP₃R2 at Ser⁹³⁷. In total, these results provide a direct mechanism for the cAMP-induced activation of InsP₃R2 via PKA phosphorylation of InsP₃R2.     

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

In hepatocytes, as in other cell types, Ca²⁺ signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP₃R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca²⁺ signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP₃ receptors (InsP₃R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP₃R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP₃-induced Ca²⁺ release in hepatocytes. This can be explained by the rather low expression level expression of InsP₃R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca²⁺ signaling via an inhibitory effect on InsP₃ synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP₃ synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.     

Protein kinase A increases the binding affinity and the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

Background information. In endocrine cells, IP₃R (inositol 1,4,5‐trisphosphate receptor), a ligand‐gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺ concentration. There are three subtypes of IP₃R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP₃R‐3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP₃R‐3 in RINm5F cells. Results. We show that immunoprecipitated IP₃R‐3 is a good substrate for PKA. Using a back‐phosphorylation approach, we show that endogenous PKA phosphorylates IP₃R‐3 in intact RINm5F cells. [³H]IP₃ (inositol 1,4,5‐trisphosphate) binding affinity and IP₃‐induced Ca²⁺ release activity were enhanced in permeabilized cells that were pre‐treated with forskolin or PKA. The PKA‐induced enhancement of IP₃R‐3 activity was also observed in intact RINm5F cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. Conclusion. The results of the present study reveal a converging step where the cAMP and the Ca²⁺ signalling systems act co‐operatively in endocrine cell responses to external stimuli.     

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca²⁺ channel inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3^−/−/Nrl^−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP₃R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca²⁺ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.     

Atypical properties of a conventional calcium channel β subunit from the platyhelminth Schistosoma mansoni

Background

Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP₃/Ca²⁺ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca²⁺ signaling and InsP₃-induced Ca²⁺ release from the endoplasmic reticulum (ER).

Results

Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca²⁺]_i changes into a sustained increase. Intraluminal Ca²⁺ measurements in the ER of β-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP₃-induced Ca²⁺ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP₃ receptor Ca²⁺ channel for InsP₃. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP₃-induced Ca²⁺ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP₃/Ca²⁺ signaling pathway for 5-HT, because IBMX potentiated Ca²⁺-dependent Cl^- transport activated by a threshold 5-HT concentration.

Conclusion

This report shows, for the first time for an insect system, that cAMP can potentiate InsP₃-induced Ca²⁺ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP₃/Ca²⁺ signaling pathways enhances transepithelial electrolyte transport.     

Calcineurin is part of a negative feedback loop in the InsP3/Ca signalling pathway in blowfly salivary glands

The ubiquitous InsP₃/Ca²⁺ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca²⁺ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca²⁺] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP₃-induced Ca²⁺ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP₃/Ca²⁺ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca²⁺ signals; (2) cyclosporin A and FK506, inhibitors of Ca²⁺/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca²⁺ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca²⁺ responses does not involve Ca²⁺ entry into the cells; (4) cyclosporin A increases InsP₃-dependent Ca²⁺ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca²⁺ responses, indicating that PKA and calcineurin act antagonistically on the InsP₃/Ca²⁺ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP₃/Ca²⁺ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations.     

Elevated InsP3R expression underlies enhanced calcium fluxes and spontaneous extra-systolic calcium release events in hypertrophic cardiac myocytes

Cardiac hypertrophy is associated with profound remodelling of Ca²⁺ signalling pathways. During the early, compensated stages of hypertrophy, Ca²⁺ fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca²⁺ homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca²⁺ release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca²⁺ store are primarily responsible for the Ca²⁺ flux that induces myocyte contraction, a role for Ca²⁺ release via the inositol 1,4,5-trisphosphate receptor (InsP₃R) in cardiac physiology has also emerged. Specifically, InsP₃-induced Ca²⁺ signals generated following myocyte stimulation with an InsP₃-generating agonist (e.g. endothelin, ET-1), lead to modulation of Ca²⁺ signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca²⁺ release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP₃R (InsP₃R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca²⁺ release via InsP₃Rs serves to sensitise neighbouring RyRs causing an augmentation of Ca²⁺ fluxes during ECC as well as an increase in non-triggered Ca²⁺ release events. Although the sensitization of RyRs may be a beneficial consequence of elevated InsP₃R expression during hypertrophy, the spontaneous Ca²⁺ release events are potentially of pathological significance giving rise to cardiac arrhythmia. InsP₃R2 expression was also increased in hypertrophic hearts from patients with ischemic dilated cardiomyopathy and aortically-banded mice demonstrating that increased InsP₃R expression may be a general phenomenon that underlies Ca²⁺ changes during hypertrophy.     

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.     

Modulation of Inositol 1,4,5-Trisphosphate Receptor Type 2 Channel Activity by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Phosphorylation

InsP₃-mediated calcium release through the type 2 inositol 1,4,5-trisphosphate receptor (InsP₃R2) in cardiac myocytes results in the activation of associated CaMKII, thus enabling the kinase to act on downstream targets, such as histone deacetylases 4 and 5 (HDAC4 and HDAC5). The CaMKII activity also feedback modulates InsP₃R2 function by direct phosphorylation and results in a dramatic decrease in the receptor-channel open probability (P_o). We have identified S150 in the InsP₃R2 core suppressor domain (amino acids 1–225) as the specific residue that is phosphorylated by CaMKII. Site-directed mutagenesis reveals that S150 is the CaMKII phosphorylation site responsible for modulation of channel activity. Nonphosphorylatable (S150A) and phosphomimetic (S150E) mutations were studied in planar lipid bilayers. The InsP₃R2 S150A channel showed no decrease in activity when treated with CaMKII. Conversely, the phosphomimetic (S150E) channel displayed a very low P_o under normal recording conditions in the absence of CaMKII (2 μm InsP₃ and 250 nm [Ca²⁺]_FREE) and mimicked a WT channel that has been phosphorylated by CaMKII. Phopho-specific antibodies demonstrate that InsP₃R2 Ser-150 is phosphorylated in vivo by CaMKIIδ. The results of this study show that serine 150 of the InsP₃R2 is phosphorylated by CaMKII and results in a decrease in the channel open probability.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Ca2+ Mobilization

Many physiological processes are controlled by a great diversity of Ca^{2 +} signals that depend on Ca^{2 +} entry into the cell and/or Ca^{2 +} release from internal Ca^{2 +} stores. Ca^{2 +} mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP₃), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca^{2 +} release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP₃ and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca^{2 +} stores. Activation of the NAADP-sensitive Ca^{2 +} channels evokes complex changes in cytoplasmic Ca^{2 +} levels by means of channel chatter with other intracellular Ca^{2 +} channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca^{2 +} signaling.     

Identification of possible signal transduction components mediating light induction of the Gsa gene for an early chlorophyll biosynthetic step in Chlamydomonas reinhardtii

 Light-induced expression of the Gsa gene encoding the heme and chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase in Chlamydomonas reinhardtii was previously shown to involve Ca²⁺ and calmodulin (CaM) (C. lm et al. 1996, Plant Cell 8: 2245–2253). To further analyze the signal transduction pathway for light-induced Gsa expression, the effects of several pharmacological agents were examined. Treatment of light-dark synchronized cells with the heterotrimeric G-protein agonist Mas-7 caused partial induction of Gsa in the dark. The phospholipase C inhibitor U73122 inhibited light induction of Gsa. Exposure of cells to light caused a sustained 3-fold increase in cellular d-inositol 1,4,5-trisphosphate (InsP₃) concentration. KN-93, a specific inhibitor of Ca²⁺/CaM-dependent protein kinase II, inhibited light induction of Gsa. In contrast, cyclosporin A, a specific inhibitor of the Ca²⁺/CaM-dependent phosphoprotein phosphatase calcineurin, did not affect light induction of Gsa. These results, together with the earlier results, suggest the involvement of a canonical signal transduction pathway for light-regulated Gsa expression that involves a heterotrimeric G-protein activation, phospholipase C-catalyzed InsP₃ formation, InsP₃-dependent Ca²⁺ release, and activation of a downstream signaling pathway through a Ca²⁺/CaM-dependent protein kinase. Received: 21 October 1999 / Accepted: 3 December 1999     

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP₃)-mediated increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). Local Ca²⁺ release from an InsP₃ receptor (InsP₃R) cluster present on the sarcoplasmic reticulum is termed a Ca²⁺ puff. Ca²⁺ released via InsP₃R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca²⁺ rise. In smooth muscle, mitochondrial Ca²⁺ uptake maintains global InsP₃-mediated Ca²⁺ release by preventing a negative feedback effect of high [Ca²⁺] on InsP₃R. Mitochondria may regulate InsP₃-mediated Ca²⁺ signals by operating between or within InsP₃R clusters. In the former mitochondria could regulate only global Ca²⁺ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP₃-mediated Ca²⁺ release by operating within (local) or between (global) InsP₃R clusters has been addressed. Ca²⁺ puffs evoked by localized photolysis of InsP₃ in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F₀, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP₃R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨ_M to prevent mitochondrial Ca²⁺ uptake and attenuated Ca²⁺ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca²⁺ puffs by ∼62%. The “fast” Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP₃-mediated Ca²⁺ release suggesting that mitochondrial influence is via their Ca²⁺ uptake facility. These results indicate Ca²⁺ uptake occurs quickly enough to influence InsP₃R communication at the intra-cluster level and that mitochondria regulate both local and global InsP₃-mediated Ca²⁺ signals.     

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca binding proteins in RBLm2H3 rat basophilic leukemia cells

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca ²⁺ ATPase (SERCA) isoforms (2b and 3) as well as four Ca ²⁺ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP₃ binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca ²⁺ binding proteins in fraction F3 and the sucrose density gradient fractions. Ins P₃R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP₃R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP₃R-1 and InsP₃R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP₃R-1 and InsP₃R-2 and the SERCs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP₃R isoforms, the two SERCA isoforms and the four Ca²⁺ binding proteins investigated. This heterogeneity may underlie specialization of the Ca²⁺ stores and the subsequent initiation of intracellular Ca²⁺ signals.     

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

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

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

The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP₃R3) is apically localized and triggers Ca²⁺ waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP₃R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP₃R3 in cholangiocyte Ca²⁺ signaling and secretion is well established and because loss of InsP₃R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3′-UTR of human InsP₃R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP₃R3–3′UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP₃R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP₃R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP₃-mediated Ca²⁺ signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP₃R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP₃R3 expression and InsP₃R3-mediated Ca²⁺ signaling and secretion.     

Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating

The ubiquitous inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP₃R) Ca²⁺ release channel plays a central role in the generation and modulation of intracellular Ca²⁺ signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP₃, free Ca²⁺, free ATP⁴⁻) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP₃R channel activity by free Ca²⁺ in the ER lumen ([Ca²⁺]_ER) remains poorly understood because of limitations of Ca²⁺ flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca²⁺]_ER on homotetrameric rat type 3 InsP₃R channel activity. In optimal [Ca²⁺]_i and subsaturating [InsP₃], jumps of [Ca²⁺]_ER from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP₃ but restored when [Ca²⁺]_ER was raised to 1.1 mM. In suboptimal [Ca²⁺]_i, jumps of [Ca²⁺]_ER (70 nM to 300 µM) enhanced channel activity. Thus, [Ca²⁺]_ER effects on channel activity exhibited a biphasic dependence on [Ca²⁺]_i. In addition, the effect of high [Ca²⁺]_ER was attenuated when a voltage was applied to oppose Ca²⁺ flux through the channel. These observations can be accounted for by Ca²⁺ flux driven through the open InsP₃R channel by [Ca²⁺]_ER, raising local [Ca²⁺]_i around the channel to regulate its activity through its cytoplasmic regulatory Ca²⁺-binding sites. Importantly, [Ca²⁺]_ER regulation of InsP₃R channel activity depended on cytoplasmic Ca²⁺-buffering conditions: it was more pronounced when [Ca²⁺]_i was weakly buffered but completely abolished in strong Ca²⁺-buffering conditions. With strong cytoplasmic buffering and Ca²⁺ flux sufficiently reduced by applied voltage, both activation and inhibition of InsP₃R channel gating by physiological levels of [Ca²⁺]_ER were completely abolished. Collectively, these results rule out Ca²⁺ regulation of channel activity by direct binding to the luminal aspect of the channel.     

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

In addition to its well established function in activating Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca²⁺ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca²⁺ signals via changes in ER Ca²⁺ store content, by imaging Ca²⁺ liberation through inositol trisphosphate receptors (IP₃R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca²⁺] was transiently elevated by applying voltage-clamp pulses to induce Ca²⁺ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca²⁺ signals evoked by strong photorelease of IP₃, and increased numbers of local Ca²⁺ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca²⁺ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca²⁺ on the IP₃R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca²⁺ may modulate IP₃R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.     

Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.