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.     

Soft substrate up-regulates the interaction of STIM1 with store-operated Ca2+ channels that lead to normal epithelial cell apoptosis

We have demonstrated that soft substrate induced apoptosis in polarized cells, but not in transformed cells by disturbance of Ca²⁺ homeostasis. This study aims to further investigate the regulatory mechanisms underlying the disruption of Ca²⁺-signaling integrity in soft substrate–induced epithelial apoptosis. Soft substrate up-regulated the store-operated Ca²⁺ (SOC) entry across the plasma membrane of normal cervical epithelial cells, which resulted in increased cytosolic Ca²⁺ levels. Concomitantly, soft substrate induced the aggregation and translocation of stromal interacting molecule 1 (STIM1) toward the cell periphery to colocalize with Orai1, an essential pore subunit of SOC channel, detected by fluorescence resonance energy transfer approach and confocal image analyses. The disturbed Ca²⁺ homeostasis resulted in the activation of μ-calpain, which cleaved α-spectrin, induced actin disorganization, and caused apoptosis. In contrast, soft substrate did not disturb Ca²⁺ homeostasis or induce apoptosis in cervical cancer cells. Chelating extracellular Ca²⁺ by EGTA and down-regulated SOC entry by small interfering RNA targeting STIM1 or inhibitors targeting Ca²⁺-binding site of calpain significantly inhibited soft substrate–induced activation of μ-calpain and epithelial cell apoptosis. Thus, soft substrate up-regulates the interaction of STIM1 with SOC channels, which results in the activation of μ-calpain and subsequently induces normal epithelial cell apoptosis.     

N-terminal myristoylation alters the calcium binding pathways in neuronal calcium sensor-1

Neuronal calcium sensor-1 (NCS-1) interacts with many membranes and cytosolic proteins, both in a Ca²⁺-dependent and in a Ca₂₊-independent manner, and its physiological role is governed by its N-terminal myristoylation. To understand the role of myristoylation in altering Ca²⁺ response and other basic biophysical properties, we have characterized the Ca²⁺ filling pathways in both myristoylated (myr) and non-myristoylated (non-myr) forms of NCS-1. We have observed that Ca²⁺ binds simultaneously to all three active EF-hands in non-myr NCS-1, whereas in the case of myr NCS-1, the process is sequential, where the second EF-hand is filled first, followed by the third and fourth EF-hands. In the case of myr NCS-1, the observed sequential Ca²⁺ binding process becomes more prominent in the presence of Mg²⁺. Besides, the analysis of ¹⁵N-relaxation data reveals that non-myr NCS-1 is more dynamic than myr NCS-1. The overall molecular tumbling correlation time increases by approximately 20% upon myristoylation. Comparing the apo forms of non-myr NCS-1 and myr NCS-1, we found the possibility of existence of some substates, which are structurally closer to the holo form of the protein. There are more such substates in the case of non-myr NCS-1 than in the case of the myr NCS-1, suggesting that the former accesses larger volumes of conformational substates compared with the latter. Further, the study reveals that the possibility of Ca²⁺ binding simultaneously to different parts of the protein is more favourable in non-myr NCS-1 than in myr NCS-1.     

Involvement of Ca<Superscript>2+</Superscript> signaling in tachykinin-mediated contractile responses in swine trachea

Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca²⁺ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca²⁺]_i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca²⁺ channel blocker verapamil and withdrawal of external Ca²⁺. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP₃) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca²⁺ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca²⁺-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP₃ receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca²⁺ mobilization. This potentiation was virtually abolished by removal of external Ca²⁺, suggesting native Ca²⁺ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca²⁺ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca²⁺ influx and release of Ca²⁺ from InsP₃- and ryanodine-sensitive Ca²⁺ stores. Blockade of both InsP₃ receptor and RyR enhances the Ca²⁺ influx through native Ca²⁺ channels in plasma membrane, which is crucial to Ca²⁺ signaling in response to NK1 receptor activation.     

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.     

A re-evaluation of the apparent effects of luminal Ca on inositol 1,4,5-trisphosphate-induced Ca release

It has been suggested that the release of Ca²⁺ from intracellular stores by inositol 1,4,5-trisphosphate (InsP₃) is modulated by the luminal Ca²⁺ content of the stores and that such an effect could underlie the apparent ‘quantal’ nature of InsP3-induced release. Although initial studies failed to find evidence in support of such a modulation, several subsequent reports have indicated luminal Ca²⁺ effects that become apparent only after a greater than 70–75% depletion of Ca²⁺ stores. In these studies, Ca²⁺ release was expressed as a percentage of an A23187-releasable pool which comprised both InsP₃-sensi-tive and InsP₃-insensitive components. In model calculations we have found that the presence of even a minor InsP₃-insensitive component in the total Ca²⁺ pool significantly distorts interpretation of the data. We show that the published results can be accurately duplicated without any requirement for a shift in the true InsP3 sensitivity of Ca²⁺ release if either: (a) the InsP₃-insensitive component does not remain a constant proportion of the total pool during depletion (i.e. depletion disproportionally affects the InsP₃-sensitive component); or (b) during generation of InsP₃-response curves, additional Cal ²⁺ is released from the InsP₃-insensitive component as the InsP₃-sensitive component is progressively emptied. Examination indicates that either, or both, of these conditions apply in the published reports and we conclude that the demonstrated effects of luminal Ca²⁺ may be artifacts.     

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca²⁺-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca²⁺/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca²⁺/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca²⁺/NCS-1·D2R peptide complex, the C-terminal region adopts a 3₁₀ helix-turn-3₁₀ helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1.     

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.     

Neuronal Calcium Sensor-1 Regulation of Calcium Channels,Secretion, and Neuronal Outgrowth

Calcium (Ca²⁺) is an important intracellular messenger underlying cell physiology. Ca²⁺ channels are the main entry route for Ca²⁺ into excitable cells, and regulate processes such as neurotransmitter release and neuronal outgrowth. Neuronal Calcium Sensor-1 (NCS-1) is a member of the Calmodulin superfamily of EF-hand Ca²⁺ sensing proteins residing in the subfamily of NCS proteins. NCS-1 was originally discovered in Drosophila as an overexpression mutant (Frequenin), having an increased frequency of Ca²⁺-evoked neurotransmission. NCS-1 is N-terminally myristoylated, can bind intracellular membranes, and has a Ca²⁺ affinity of 0.3 μM. Over 10 years ago it was discovered that NCS-1 overexpression enhances Ca²⁺-evoked secretion in bovine adrenal chromaffin cells. The mechanism was unclear, but there was no apparent direct effect on the exocytotic machinery. It was revealed, again in chromaffin cells, that NCS-1 regulates voltage-gated Ca²⁺ channels (Cavs) in G-Protein Coupled Receptor (GPCR) signaling pathways. This work in chromaffin cells highlighted NCS-1 as an important modulator of neurotransmission. NCS-1 has since been shown to regulate and/or directly interact with many proteins including Cavs (P/Q, N, and L), TRPC1/5 channels, GPCRs, IP3R, and PI4 kinase type IIIβ. NCS-1 also affects neuronal outgrowth having roles in learning and memory affecting both short- and long-term synaptic plasticity. It is not known if NCS-1 affects neurotransmission and synaptic plasticity via its effect on PIP2 levels, and/or via a direct interaction with Ca²⁺ channels or their signaling complexes. This review gives a historical account of NCS-1 function, examining contributions from chromaffin cells, PC12 cells and other models, to describe how NCS-1’s regulation of Ca²⁺ channels allows it to exert its physiological effects.     

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.     

Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca²⁺ signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca²⁺-specific) and structural (Ca²⁺- or Mg²⁺-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca²⁺ binding using NMR and EF-hand mutants. Ca²⁺ titration, as monitored by [¹⁵N,¹H] heteronuclear single quantum coherence, suggests that Ca²⁺ binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg²⁺ binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca²⁺-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca²⁺ and not Mg²⁺. Ca²⁺ binding induces conformational rearrangements in the protein by reversing Mg²⁺-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg²⁺ with Ca²⁺ reduces the Ca²⁺-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca²⁺-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg²⁺-bound NCS-1 are similar, the early unfolding transitions of Ca²⁺-bound NCS-1 are partially influenced in the presence of Mg²⁺. This study demonstrates the importance of Mg²⁺ as a modulator of calcium homeostasis and active-state behavior of NCS-1.     

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.     

Spatially Defined InsP3-Mediated Signaling in Embryonic Stem Cell-Derived Cardiomyocytes

The functional role of inositol 1,4,5-trisphosphate (InsP₃) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP₃ receptors can translate Ca²⁺ release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP₃-mediated Ca²⁺ release. [InsP₃]_i was monitored with the FRET-based InsP₃-biosensor FIRE-1 (Fluorescent InsP₃ Responsive Element) and heterogeneity in sub-cellular [InsP₃]_i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP₃ 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca²⁺ transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca²⁺]_i and spontaneous activity was modulated by [InsP₃]_i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP₃ by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP₃ signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca²⁺ released from InsP₃Rs is more effective than Ca²⁺ released from RyRs to enhance I_NCX. The results support the hypothesis that in ESdCs InsP₃Rs form a functional signaling domain with NCX that translates Ca²⁺ release efficiently into a depolarization of the membrane potential.     

Mechanisms involved in α6β1-integrin-mediated Ca signalling

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting α6β1-integrin-mediated Ca²⁺ signalling. Both Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and capacitative Ca²⁺ entry via Ca²⁺ channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of α6β1-integrin-mediated Ca²⁺ signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca²⁺-releasing second messengers d-myo-inositol 1,4,5-trisphosphate (InsP₃) and cADPR.     

Redox modulation of calcium entry and release of intracellular calcium by thimerosal in GH4C1 pituitary cells

In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in GH₄C₁ pituitary cells. In intact Fura-2 loaded cells, low concentrations of thimerosal potentiated the spike phase of the TRH-induced (thyrotropin-releasing hormone) rise in [Ca²⁺]_i, whereas high thimerosal concentrations inhibited it. The effect of thimerosal on the plateau phase was always inhibitory.The effect of thimerosal on the IP₃-induced calcium release (IICR) was studied in permeabilized cells using the Ca²⁺ indicator Fluo-3. A low concentration of thimerosal (10 μM) stimulated IICR: the Ca²⁺ release induced by 300 nM inositol-1,4,5-trisphosphate (IP₃) was enhanced in cells treated with thimerosal for 1 or 6 min (67 ± 11 nM and 34 ± 5 nM, respectively) as compared to control cells (17 ± 2 nM). On the other hand, a high concentration of thimerosal (100 μ inhibited IICR: when IP₃ (10 μM) was added after a 5 min preincubation with thimerosal, the IP₃-induced rise in [Ca²⁺]_i (46 ± 14 nM) was 57% smaller as compared with that seen in control cells (106 ± 10 nM).The effect of thimerosal on the voltage-operated Ca ²⁺ channels (VOCCs) was studied by depolarizing intact Fura-2 loaded cells by addition of 20 mM K⁺ to the cuvette. The depolarization-evoked increase in [Ca²⁺]_i was inhibited in a dose-dependent manner by thimerosal. Direct evidence for an inhibitory effect of thimerosal on VOCCs was obtained by using the whole-cell configuration of the patch-clamp technique: thimerosal (100 μM) potently inhibited the Ba²⁺ currents through VOCCs.In addition, our results indicated that thimerosal inhibited the caffeine-induced increase in [Ca²⁺]_i, and activated a capacitative Ca²⁺ entry pathway. The actions of thimerosal were apparently due to its oxidizing activity because the effects were mostly reversed by the thiol-reducing agent dithiothreitol (DTT).We conclude that, in GH₄C₁ pituitary cells, the mobilization of intracellular calcium and the different Ca²⁺ entry pathways are sensitive to redox modulation.     

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.     

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     

Calcium-dependent Clustering of Inositol 1,4,5-Trisphosphate Receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP₃), which operates as an InsP₃-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcεR1) leads to activation of phospholipase C γ isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP₃-sensitive intracellular Ca²⁺ stores, and a sustained phase of Ca²⁺ influx. These events are accompanied by a redistribution of type II InsP₃ receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP₃ receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP₃ receptor clustering occurs within 5–10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 μM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP₃ receptor aggregation may be a characteristic cellular response to Ca²⁺-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4–2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36^M3R cells. Stimulation of these three cell types leads to a reduction in InsP₃ receptor levels only in AR4–2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP₃ receptors may contribute to the previously observed changes in affinity for InsP₃ in the presence of elevated Ca²⁺ and/or may establish discrete regions within refilled stores with varying capacity to release Ca²⁺ when a subsequent stimulus results in production of InsP₃.     

Ca-stimulated, Mg-independent ATP hydrolysis and the high affinity Ca-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m = 0.25 μM, V_max = 24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.