Activation of endoplasmic reticulum calcium leak by 2-APB depends on the luminal calcium concentration

It has been shown that 2-APB is a nonspecific modulator of ion channel activity, while most of the channels are inhibited by this compound, there are few examples of channels that are activated by 2-APB. Additionally, it has been shown that, 2-APB leads to a reduction in the luminal endoplasmic reticulum Ca²⁺ level ([Ca²⁺]_ER) and we have carried out simultaneous recordings of both [Ca²⁺]_i and the [Ca²⁺]_ER in HeLa cell suspensions to assess the mechanism involved in this effect. This approach allowed us to determine that 2-APB induces a reduction in the [Ca²⁺]_ER by activating an ER-resident Ca²⁺ permeable channel more than by inhibiting the activity of SERCA pumps. Interestingly, this effect of 2-APB of reducing the [Ca²⁺]_ER is auto-limited because depends on a replete ER Ca²⁺ store; a condition that thapsigargin does not require to decrease the [Ca²⁺]_ER. Additionally, our data indicate that the ER Ca²⁺ permeable channel activated by 2-APB does not seem to participate in the ER Ca²⁺ leak revealed by inhibiting SERCA pump with thapsigargin. This work suggests that, prolonged incubations with even low concentrations of 2-APB (5 μM) would lead to the reduction in the [Ca²⁺]_ER that might explain the inhibitory effect of this compound on those signals that require Ca²⁺ release from the ER store.     

Molecular mechanism of 2-APB-induced Ca influx in external acidification in PC12

2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca²⁺ (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca²⁺]_i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca²⁺ influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca²⁺]_i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca²⁺]_i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca²⁺]_i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca²⁺ and SOC channel blockers such as SK&F96365, La³⁺ and Gd³⁺. PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca²⁺]_i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca²⁺]_i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling.     

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.     

STIM1 Regulates Platelet-Derived Growth Factor-Induced Migration and Ca2+ Influx in Human Airway Smooth Muscle Cells

It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca²⁺ sensor that activates Orai1, the Ca²⁺ channel responsible for store-operated Ca²⁺ entry (SOCE). We investigated the role of STIM1 in [Ca²⁺]_i and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca²⁺-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca²⁺]_i followed by sustained [Ca²⁺]_i elevation. Sustained increases in [Ca²⁺]_i due to PDGF-BB were significantly inhibited by a Ca²⁺ chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca²⁺]_i or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca²⁺ influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling.     

Orai3 TM3 point mutation G158C alters kinetics of 2-APB–induced gating by disulfide bridge formation with TM2 C101

After endoplasmic reticulum (ER) Ca²⁺ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident STIM proteins to form the Ca²⁺-selective Ca²⁺ release–activated Ca²⁺ (CRAC) channel. However, in the absence of Ca²⁺ store depletion and STIM interaction, the mammalian homologue Orai3 can be activated by 2-aminoethyl diphenylborinate (2-APB), resulting in a nonselective cation conductance characterized by biphasic inward and outward rectification. Here, we use site-directed mutagenesis and patch-clamp analysis to better understand the mechanism by which 2-APB activates Orai3. We find that point mutation of glycine 158 in the third transmembrane (TM) segment to cysteine, but not alanine, slows the kinetics of 2-APB activation and prevents complete channel closure upon 2-APB washout. The “slow” phenotype exhibited by Orai3 mutant G158C reveals distinct open states, characterized by variable reversal potentials. The slow phenotype can be reversed by application of the reducing reagent bis(2-mercaptoethylsulfone) (BMS), but in a state-dependent manner, only during 2-APB activation. Moreover, the double mutant C101G/G158C, in which an endogenous TM2 cysteine is changed to glycine, does not exhibit altered kinetics of 2-APB activation. We suggest that a disulfide bridge, formed between the introduced cysteine at TM3 position 158 and the endogenous cysteine at TM2 position 101, hinders transitions between Orai3 open and closed states. Our data provide functional confirmation of the proximity of these two residues and suggest a location within the Orai3 protein that is sensitive to the actions of 2-APB.     

Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

The presence and physiological role of Ca²⁺-induced Ca²⁺ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca²⁺] ([Ca²⁺]_c). Using targeted aequorin, we have directly monitored [Ca²⁺] changes inside the ER ([Ca²⁺]_ER) in bovine adrenal chromaffin cells. Ca²⁺ entry induced by cell depolarization triggered a transient Ca²⁺ release from the ER that was highly dependent on [Ca²⁺]_ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca²⁺ release was quantal in nature due to modulation by [Ca²⁺]_ER. Whereas caffeine released essentially all the Ca²⁺ from the ER, inositol 1,4,5-trisphosphate (InsP₃)- producing agonists released only 60–80%. Both InsP₃ and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca²⁺ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca²⁺]_c measurements showed that the wave of [Ca²⁺]_c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca²⁺ pool that can release Ca²⁺ both via InsP₃ receptors or CICR.     

Potent functional uncoupling between STIM1 and Orai1 by dimeric 2-aminodiphenyl borinate analogs

The coupling of ER Ca²⁺-sensing STIM proteins and PM Orai Ca²⁺ entry channels generates “store-operated” Ca²⁺ signals crucial in controlling responses in many cell types. The dimeric derivative of 2-aminoethoxydiphenyl borinate (2-APB), DPB162-AE, blocks functional coupling between STIM1 and Orai1 with an IC₅₀ (200 nM) 100-fold lower than 2-APB. Unlike 2-APB, DPB162-AE does not affect L-type or TRPC channels or Ca²⁺ pumps at maximal STIM1–Orai1 blocking levels. DPB162-AE blocks STIM1-induced Orai1 or Orai2, but does not block Orai3 or STIM2-mediated effects. We narrowed the DPB162-AE site of action to the STIM–Orai activating region (SOAR) of STIM1. DPB162-AE does not prevent the SOAR–Orai1 interaction but potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores SOAR–Orai binding but only slowly restores Orai1 channel-mediated Ca²⁺ entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1–Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.     

State-dependent block of Orai3 TM1 and TM3 cysteine mutants: Insights into 2-APB activation

After endoplasmic reticulum (ER) Ca²⁺ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca²⁺-selective Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1–Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB–activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd²⁺). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB–activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd²⁺-induced block that is state dependent: Cd²⁺ only blocks 2-APB–activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB–activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.     

Tuning store-operated calcium entry to modulate Ca2+-dependent physiological processes

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca²⁺ channels that are activated when luminal [Ca²⁺] of the endoplasmic reticulum ([Ca²⁺]_ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca²⁺]_ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca²⁺ store depletion. Another family of plasma membrane Ca²⁺ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca²⁺]_i elevation. Although Ca²⁺ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca²⁺ signal generated by Ca²⁺ entry pathways in response to depletion of ER-Ca²⁺ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

Impact of Geometrical Characteristics of the Organellar Store and Organelle-Free Cytosol on Intracellular Calcium Dynamics in the Dendrite: a Simulation Study

The dependence of intracellular calcium dynamics on geometrical size relations between calcium-exchanging parts of the intracellular space was studied in mathematical models corresponding to a thin fragment of the Purkinje neuron spiny dendrite. The plasma membrane contained ion channels typical of this cell type, including channels that conduct an excitatory synaptic current, and ion pumps. The model equations took into account calcium exchange between the cytosol, extracellular medium, intracellular store (a cistern of the endoplasmic reticulum, ER), endogenous calcium buffers, and an exogenous buffer (fluorescent dye used in the experiments). The ER membrane contained the calcium pump and channels of calcium-dependent and inositol-3-phosphate-dependent calcium release, as well as leakage channels. With the compartment size fixed, the ER cistern diameter was varied so that the proportion of the organelle in the total volume changed from 1 to 36%. Under these conditions, identical synaptic excitation caused similar electrical reactions (calcium spikes) but different concentration responses. Equal increments in the ER diameter led to unequal, more pronounced at thicker diameters, increments of the peak cytosolic concentrations of Са²⁺ ([Ca²⁺] _i ) and of a Са²⁺-fluorescent dye complex [CaD], as well as those of the Са²⁺ concentration in the dendrite ER (characterized by a shift from the basal level, Δ[Ca²⁺]_ER). The changes in [Ca²⁺] _i and [CaD] followed more adequately those in the volume of the organelle-free cytosol, while Δ[Ca²⁺]_ER changes were more similar to those in the ER membrane area. Therefore, the relative occupancy of the intracellular volume by organellar calcium stores and their sizes in a dendritic compartment are important structural factors that essentially modulate the calcium dynamics, and this structural dependence can be adequately reflected in the experiments using fluorophores. Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 19–31, January–February, 2009.     

内质网上的蛋白质RCN2与STIM1-Orai1复合体相互作用

膜蛋白质Orail组成了一类被称为钙释放激活钙通道（CRAC）的离子通道,并且由相互作用的蛋白质STIM1作为其在内质网上的钙感受器．但是这类通道的调节机制还未研究透彻．通过串连亲和纯化STIM1-Orai1复合体,发现与之相互作用的内质网蛋白质RCN2．共聚焦显微术显示RCN2与STIM1在钙库排空前后完全共定位．对RCN2的EFhands结构突变体所作单细胞测钙,结果显示其对钙库操控通道电流特性有微弱影响．全内反射荧光显微术显示,RCN2以花环状围绕包围STIM1聚集堆,这提示RCN2在STIM1聚集中起到一种结构约束作用．     

Synergistically regulated spontaneous calcium signaling is attributed to cartilaginous extracellular matrix metabolism

Ca²⁺ has been recognized as a key molecule for chondrocytes, however, the role and mechanism of spontaneous [Ca ²⁺] _i signaling in cartilaginous extracellular matrix (ECM) metabolism regulation are unclear. Here we found that spontaneous Ca ²⁺ signal of in-situ porcine chondrocytes was [Ca ²⁺] _o dependent, and mediated by [Ca ²⁺] _i store release. T-type voltage-dependent calcium channel (T-VDCC) mediated [Ca ²⁺] _o influx was associated with decreased cell viability and expression levels of ECM deposition genes. Further analysis revealed that chondrocytes expressed both inositol 1,4,5-trisphosphate receptor (InsP3R) and Orai isoforms. Inhibition of endoplasmic reticulum (ER) Ca ²⁺ release and store-operated calcium entry significantly abolished spontaneous [Ca ²⁺] _i signaling of in-situ chondrocytes. Moreover, blocking ER Ca ²⁺ release with InsP3R inhibitors significantly upregulated ECM degradation enzymes production, and was accompanied by decreased proteoglycan and collagen type II intensity. Taken together, our data provided evidence that spontaneous [Ca ²⁺] _i signaling of in-situ porcine chondrocytes was tightly regulated by [Ca ²⁺] _o influx, InsP3Rs mediated [Ca ²⁺] _i store release, and Orais mediated calcium release-activated calcium channels activation. Both T-VDCC mediated [Ca ²⁺] _o influx and InsP3Rs mediated ER Ca ²⁺ release were found crucial to cartilaginous ECM metabolism through distinct regulatory mechanisms.     

Lysosomal exocytosis of ATP is coupled to P2Y2 receptor in marginal cells in the stria vascular in neonatal rats

Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague–Dawley rats were established. P2Y₂ receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immunolabelled in marginal cells of the stria vascular. We found that 30 μM ATP and 30 μM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Ca²⁺, whereas the response was suppressed by 100 μM suramin, 10 μM 1-(6-(17β-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 μM 2-aminoethoxydiphenyl borate (2-APB) and 5 μM thapsigargin (TG), thus indicating that ATP coupled with the P2Y₂R-PLC-IP3 pathway to evoke Ca²⁺ release from the endoplasmic reticulum (ER). Incubation with 200 μM Gly-Phe-β-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca²⁺]_I; this effect was partly inhibited by P2Y₂R-PLC-IP3 pathway antagonists. After pre-treatment with 5 μM TG, [Ca²⁺]_i was significantly lower than that after treatment with P2Y₂R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca²⁺ triggers Ca²⁺ release from ER Ca²⁺ stores. Baseline [Ca²⁺]_i declined after treatment with the Ca²⁺ chelator 50 μM bis-(aminophenolxy) ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 μM ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 μM vacuolin-1. Release of ATP and β-hexosaminidase both increased after treatment with 200 μM GPN and 5 μM TG, but decreased after incubation with 50 μM BAPTA-AM, 4 IU/ml apyrase and 5 μM vacuolin-1. We suggest that ATP triggers Ca²⁺ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca²⁺ triggers Ca²⁺ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca²⁺ signals indirectly via the P2Y₂R-PLC-IP3 pathway.     

Post-conditioning protects cardiomyocytes from apoptosis via PKCε-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum–mitochondria crosstalk

The intracellular Ca²⁺ concentration ([Ca²⁺]_i) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca²⁺]_i, results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca²⁺]_i via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP₃) to increase the release of intracellular Ca²⁺ from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca²⁺]_i through an interaction of PKC with CaR to alleviate [Ca²⁺]_ER depletion and [Ca²⁺]_m elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC_ε translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca²⁺]_i, the [Ca²⁺]_ER was not reduced and [Ca²⁺]_m was not increased in the PC and GdCl₃–PC groups. Furthermore, there was no evident ?ψ_m collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl₃–PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC_ε interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the apoptotic pathway.     

Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells

Mutations in the cation channel TRPC6 result in a renal-specific phenotype of familial nephrotic syndrome, affecting intracellular calcium ([Ca²⁺]_i) signalling in the glomerular podocyte. Tools to study native TRPC6 activity are scarce, although there has been recent success with flufenamic acid (FFA). We confirm the specificity of FFA for TRPC6 both in an artificial expression system and in a human conditionally immortalised podocyte cell line (ciPod).Cells were loaded with fura-2AM and changes in intracellular calcium ([Ca²⁺]_i) were calculated. 200 μM FFA induced an increase in [Ca²⁺]_i in HEK293 cells with native TRPC6 expression, which was enhanced by overexpression of TRPC6 and completely blocked in the absence of extracellular calcium. Expressed TRPC7 did not significantly affect the response to FFA whereas expressed TRPC3 reduced it. FFA also induced an increase ciPod in [Ca²⁺]_i, which was inhibited using SKF96365 and 2-APB, but not indomethacin. In ciPod, adenovirus (Ad-v) wild type (WT) TRPC6 increased [Ca²⁺]_i activity to FFA compared to native TRPC6, whereas activity was significantly reduced with Ad-v dominant negative (DN) TRPC6. The niflumic acid (NFA) induced increase in [Ca²⁺]_i in ciPod was not affected by Ad-v TRPC6 DN, and in HEK293 cells was not affected by WT TRPC6.In conclusion, FFA activates TRPC6 [Ca²⁺]_i signalling in both ciPod and HEK293 cells independently of TRPC3 and TRPC7, and independently of properties of the fenamate family.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.     

Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca2+]i Signals Determine Specificity of Ca2+-Dependent Gene Expression

Regulation of critical cellular functions, including Ca²⁺-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca²⁺ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca²⁺]_i due to intracellular Ca²⁺ release and Ca²⁺ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca²⁺]_i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca²⁺]_i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca²⁺ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca²⁺ store depletion in these cells, it is not clear how the cells decode individual Ca²⁺ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca²⁺]_i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca²⁺ entry generates [Ca²⁺]_i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca²⁺ entry generates sustained [Ca²⁺]_i elevation at high [CCh] and contributes to frequency of [Ca²⁺]_i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca²⁺ dependent gene expression pathways, consistent with the different patterns of [Ca²⁺]_i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca²⁺]_i generated by Orai1, independent of global [Ca²⁺]_i changes or TRPC1-mediated Ca²⁺ entry. In contrast, Ca²⁺ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca²⁺ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca²⁺-dependent gene expression pathways.     

Calcium-activated chloride fluxes in cultured NCL-SG3 sweat gland cells

The dependence of chloride permeability of the human sweat gland cell line NCL-SG3 cell line on cytosolic free calcium ([Ca²⁺]_i) was investigated. X-ray microanalysis, fura-2 fluorescence and patch clamp methodology were used. Carbachol and A23187 decreased cellular Cl and K for cells grown on permeable supports, but carbachol had no effect on cells grown on impermeable supports. In perforated patch experiments with impermeable supports, ATP and calcium ionophores increased the inward current (ic) whereas carbachol had no effect. ic was unaffected by cation channel blockers or removal of extracellular Na⁺ but was blocked by chloride channel blockers. Lowering bath Ca²⁺ decreased ic. On raising bath Ca²⁺ ic and [Ca²⁺]_i responded with a transient rise which was not blocked by La³⁺ or D-600. La³⁺, but not D-600, blocked the entry of Mn²⁺. K⁺-depolarization and Bay-K-8644 had little effect on [Ca²⁺]_i. The rise in [Ca²⁺]_i may be mediated primarily via depletion operated Ca²⁺-channels. Irrespective of substrate NCL-SG3 cells have a chloride permeability which depends on [Ca²⁺]_i.     

Mechanisms Underlying Leakage of Calcium from the Endoplasmic Reticulum of Acinar Cells of the Submandibular Salivary Gland

In the resting state, the Ca²⁺ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca²⁺, which is mediated by Ca²⁺-ATPase (SERCA), and passive leakage of Ca²⁺. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments, we examined possible pathways of passive leakage of Ca²⁺ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements of the concentration of Ca²⁺ in the ER ([Ca²⁺]_ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with the help of β-escin (40 μg/ml); the Ca²⁺ concentration in the cytoplasm ([Ca²⁺] _i ) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca²⁺ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin, resulted in complete inhibition of the uptake of Ca²⁺ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding solutions and was accompanied by the leakage of Ca²⁺ from the ER; this is confirmed by a gradual drop in the [Ca₂₊]_ER. Such a leakage of Ca²⁺ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol 1,4,5-trisphosphate-sensitive receptors (InsP₃R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca²⁺ from the ER. This effect did not depend on the functioning of SERCA, InsP₃R, or RyR. Therefore, passive leakage of Ca²⁺ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the ER membrane. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 339–346, July–August, 2005.     

Increased Response to High KCl-Induced Elevation in the Intracellular-Ca2+ Concentration in Differentiated NG108-15 Cell and the Inhibitory Effect of the L-Type Ca2+ Channel Blocker, Calciseptine

Characteristics of the increasing effect for the concentration of intracellular calcium ions ([Ca²⁺]_i) by high-KCl application were investigated in the neuroblastoma×glioma hybrid NG108-15 cell line (NG108-15 cells). The present study confirmed that the increasing effect of [Ca²⁺]_i by high-KCl application in single NG108-15 cells, differentiated with dibutyryl cAMP (Bt₂cAMP), was significantly enhanced, compared to undifferentiated cells. The following observations were made at first: (1) The response to high-KCl application, in both undifferentiated and differentiated cells, was significantly inhibited by calciseptine (CaS), an L-type Ca²⁺ channel blocker, but not by N-, P- and R-type Ca²⁺ channel blockers. The IC₅₀ values for CaS in both undifferentiated and differentiated cell was almost identical. (2) The inhibitory effect of CaS was irreversible. (3) The increasing effect for [Ca²⁺]_i by high-KCl application was completely dependent on the presence of extracellular calcium ions. (4) The increased [Ca²⁺]_i by high-KCl application under a plateau concentration was quickly decreased to basal levels when the high-KCl solution was exchanged for a high-KCl solution containing EGTA (without CaCl₂). Together, these results suggest that the enhancement of the response effect of [Ca²⁺]_i by high-KCl application in differentiated single NG108-15 cells was mainly due to the quantitative increase of L-type voltage-sensitive calcium channels (VSCCs), which were irreversibly inhibited by CaS.