Depletion Of Calcium Stores Research Articles

STIMs and Orai1 regulate cytokine production in spinal astrocytes.

BackgroundOur previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes.MethodsPrimary cultured astrocytes were prepared from neonatal (P2–P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay.ResultsWe found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability.ConclusionsThis study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

THE ROLE OF TRPV4 CATION CHANNELS IN THE REGULATION OF PHENYLEPHRINE-INDUCED CONTRACTION OF RAT PULMONARY ARTER.

The aim of our study was to investigate the role of mechanosensitive TRPV4 channels in the regulation of rat pulmonary artery smooth muscle (PASM) contractile activity induced by the activation of α-adrenoceptors and the possibility of their use as novel pharmacological targets in pulmonary hypertension. TRPV4 selective agonist, GSK1016790A, in the presence of the agonist of α-adrenoceptors phenylephrine (PhE) evoked biphasic contractile reaction with initial relaxation (63,5% ± 7,1) followed by significant vasoconstriction (142% ± 17,9). GSK1016790A evoked similar effects in PASM rings with and without endothelium, indicating that its main site of action was TRPV4 expressed in smooth muscle cells. TRPV4 selective blocker, HC-067047, completely inhibited the effects of GSK1016790A confirming the specific role of TRPV4 in these vascular responses. Application of Ca2+-free external solution reduced the relaxation phase and completely abolished the sustained contractile response to GSK1016790A (from 43,9 % to 0,3 %). The biphasic reaction could be explained as an initial calcium store depletion by PhE and further calciuminduced calcium release activated by TRPV4 that causes BKCa activation, membrane hyperpolarisation and vasorelaxation, followed by Ca2+ entry via TRPV4 and contraction. We conclude that TRPV4 channels play an important role in the regulation of the adrenergic vascular tone of PASM cells, but TRPV4 activation mechanism(s) and signaling pathways remain unclear.

Protection of Human Pancreatic Islets from Lipotoxicity by Modulation of the Translocon.

Type 2 diabetes is characterized by peripheral insulin resistance and pancreatic beta cell dysfunction. Elevated free fatty acids (FFAs) may impair beta cell function and mass (lipotoxicity). Altered calcium homeostasis may be involved in defective insulin release. The endoplasmic reticulum (ER) is the major intracellular calcium store. Lipotoxicity induces ER stress and in parallel an ER calcium depletion through unknown ER calcium leak channels. The main purposes of this study is first to identify one of these channels and secondly, to check the opportunity to restore beta cells function (i.e., insulin secretion) after pharmacological inhibition of ER calcium store depletion. We investigated the functionality of translocon, an ER calcium leak channel and its involvement on FFAs-induced alterations in MIN6B1 cells and in human pancreatic islets. We evidenced that translocon acts as a functional ER calcium leak channel in human beta cells using anisomycin and puromycin (antibiotics), respectively blocker and opener of this channel. Puromycin induced a significant ER calcium release, inhibited by anisomycin pretreatment. Palmitate treatment was used as FFA model to induce a mild lipotoxic effect: ER calcium content was reduced, ER stress but not apoptosis were induced and glucose induced insulin secretion was decreased in our beta cells. Interestingly, translocon inhibition by chronic anisomycin treatment prevented dysfunctions induced by palmitate, avoiding reticular calcium depletion, ER stress and restoring insulin secretion. Our results provide for the first time compelling evidence that translocon actively participates to the palmitate-induced ER calcium leak and insulin secretion decrease in beta cells. Its inhibition reduces these lipotoxic effects. Taken together, our data indicate that TLC may be a new potential target for the treatment of type 2 diabetes.

Nucleotide modulates odor response through activation of purinergic receptor in olfactory sensory neuron

Extracellular nucleotides are important neurotransmitters, neuromodulators and paracrine factors in the neural sensory system [16]. Most of purines and pyrimidines act on the associated purinergic cell-surface receptors to mediate sensory transduction and modulation. Previously, we reported a subgroup of heptaldehyde (H)/2-hepatanone (Ho)-responsive olfactory sensory neurons (H/Ho-OSNs) in the ventral endoturbinates [31]. Through the calcium image recording, we characterized that ATP elicited [Ca2+]i increase in the presence of extracellular calcium, while depletion of intracellular calcium stores blocked UTP-evoked [Ca2+]i increase. Pharmacological studies indicated that P2X3 was expressed in the H/Ho-OSNs, modulating both heptaldehyde (H) and 2-hepatanone (Ho)-induced responses. These data indicated that activation of purinergic receptor negatively modulated odor response, providing the evidence to support the possible protective effect of purinergic receptor in OSNs.

Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores

Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca2+ signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca2+ signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca2+ signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca2+ stores, thereby leaving less Ca2+ available for release upon IP3- and ryanodine-receptor activation. The Ca2+-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca2+ release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression.

Calcium-induced calcium release and gap junctions mediate large-scale calcium waves in olfactory ensheathing cells in situ

Olfactory ensheathing cells (OECs) are a specialised type of glial cells, supporting axon growth and guidance during development and regeneration of the olfactory nerve and the nerve layer of the olfactory bulb. We measured calcium signalling in OECs in olfactory bulb in-toto preparations using confocal and epifluorescence microscopy and the calcium indicator Fluo-4. We identified two subpopulations of olfactory bulb OECs: OECs in the outer sublamina of the nerve layer responded to purinergic neurotransmitters such as adenosine triphosphate with calcium transients, while OECs in the inner sublamina of the nerve layer did not respond to neurotransmitters. However, the latter generated spontaneous calcium waves that covered hundreds of cells. These calcium waves persisted in the presence of tetrodotoxin and in calcium-free saline, but were abolished after calcium store depletion with cyclopiazonic acid or inositol trisphosphate receptor blockage with 2-APB. Calcium waves could be triggered by laser photolysis of caged inositol trisphosphate. Blocking purinoceptors with PPADS had no effect on calcium wave propagation, whereas blocking gap junctions with carbenoxolone or meclofenamic acid entirely suppressed calcium waves. Increasing calcium buffer capacity in OECs with NP-EGTA (“caged” Ca2+) prevented calcium wave generation, and laser photolysis of NP-EGTA in a small group of OECs resulted in a calcium increase in the irradiated cells followed by a calcium wave. We conclude that calcium waves in OECs can be initiated by calcium-induced calcium release via InsP3 receptors and propagate through gap junctions, while purinergic signalling is not involved.

Store-operated calcium entry contributes to abnormal Ca2+ signalling in dystrophic mdx mouse myoblasts

Sarcolemma damage and activation of various calcium channels are implicated in altered Ca2+ homeostasis in muscle fibres of both Duchenne muscular dystrophy (DMD) sufferers and in the mdx mouse model of DMD. Previously we have demonstrated that also in mdx myoblasts extracellular nucleotides trigger elevated cytoplasmic Ca2+ concentrations due to alterations of both ionotropic and metabotropic purinergic receptors. Here we extend these findings to show that the mdx mutation is associated with enhanced store-operated calcium entry (SOCE). Substantially increased rate of SOCE in mdx myoblasts in comparison to that in control cells correlated with significantly elevated STIM1 protein levels. These results reveal that mutation in the dystrophin-encoding Dmd gene may significantly impact cellular calcium response to metabotropic stimulation involving depletion of the intracellular calcium stores followed by activation of the store-operated calcium entry, as early as in undifferentiated myoblasts. These data are in agreement with the increasing number of reports showing that the dystrophic pathology resulting from dystrophin mutations may be developmentally regulated. Moreover, our results showing that aberrant responses to extracellular stimuli may contribute to DMD pathogenesis suggest that treatments inhibiting such responses might alter progression of this lethal disease.

Dysbalance of astrocyte calcium under hyperammonemic conditions.

Increased brain ammonium (NH4 +/NH3) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4 +/NH3 on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4 +/NH3-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4 +/NH3 for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼90%) experienced a persistent calcium increase by ∼50 nM. This persistent increase was already evoked at concentrations of 1–2 mM NH4 +/NH3, developed within 10–20 minutes and was maintained as long as the NH4 +/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH4 +/NH3, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH4 +/NH3-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH4 +/NH3 causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.

Native store-operated calcium channels are functionally expressed in mouse spinal cord dorsal horn neurons and regulate resting calcium homeostasis.

Store-operated calcium channels (SOCs) are calcium-selective cation channels that mediate calcium entry in many different cell types. Store-operated calcium entry (SOCE) is involved in various cellular functions. Increasing evidence suggests that impairment of SOCE is responsible for numerous disorders. A previous study demonstrated that YM-58483, a potent SOC inhibitor, strongly attenuates chronic pain by systemic or intrathecal injection and completely blocks the second phase of formalin-induced spontaneous nocifensive behaviour, suggesting a potential role of SOCs in central sensitization. However, the expression of SOCs, their molecular identity and function in spinal cord dorsal horn neurons remain elusive. Here, we demonstrate that SOCs are expressed in dorsal horn neurons. Depletion of calcium stores from the endoplasmic reticulum (ER) induced large sustained calcium entry, which was blocked by SOC inhibitors, but not by voltage-gated calcium channel blockers. Depletion of ER calcium stores activated inward calcium-selective currents, which was reduced by replacing Ca(2+) with Ba(2+) and reversed by SOC inhibitors. Using the small inhibitory RNA knockdown approach, we identified both STIM1 and STIM2 as important mediators of SOCE and SOC current, and Orai1 as a key component of the Ca(2+) release-activated Ca(2+) channels in dorsal horn neurons. Knockdown of STIM1, STIM2 or Orai1 decreased resting Ca(2+) levels. We also found that activation of neurokinin 1 receptors led to SOCE and activation of SOCs produced an excitatory action in dorsal horn neurons. Our findings reveal that a novel SOC signal is present in dorsal horn neurons and may play an important role in pain transmission.

Thapsigargin Induces Apoptosis by Impairing Cytoskeleton Dynamics in Human Lung Adenocarcinoma Cells

The objective of this study was performed to investigate the effects of thapsigargin on apoptosis, actin cytoskeletal dynamics, and actin cytoskeletal proteins in human lung adenocarcinoma cell. Thapsigargin is a specific irreversible inhibitor of ER calcium-ATPase, which may promote ER stress by depletion of lumenal calcium stores and show potential to induce cell death. The effects of thapsigargin on the apoptosis in A549 cells were assayed by Hoechst staining. Moreover, the F-actin staining by Rhodamine-phalloidin and RhoA antibody for cytoskeleton organizations were applied to A549 cells. To confirm the impairment of cytoskeletal dynamics treated with thapsigargin, western blots were applied to analyze the protein levels of p-Cofilin-1 (Ser3), Cofilin-1, and pPaxillin (Tyr118), as well as RhoA and pS6 (S240/244). Results suggest that thapsigargin may induce cell death in A549 cells with a time- and dose-dependent manner. The F-actin fibers and RhoA signals are also reduced with a time- and dose-dependent manner by thapsigargin treatment. The phosphorylation forms of Cofilin-1 and paxillin are attenuated by 1 μM thapsigargin treatment for 24 h. These alternations may be caused by the inhibition of of mTORC1 activities (indicated by pS6 (Ser240/244)) and RhoA pathways after thapsigargin treatment. The present findings highlight important roles of calcium entry in cytoskeleton organization and apoptosis in human lung adenocarcinoma cells and will help to set a stage to the clinical treatment of cancer cell metastasis.

Mechanism of Activation of Calcium Channel Orai1 by its Regulatory Partner Stim1

Store-operated calcium entry in T lymphocytes depends on the sensing by STIM1 of cellular calcium store depletion, leading to an interaction of STIM1 with ORAI1 and culminating in calcium flux through the ORAI1 channel. Some important features of the STIM1-ORAI1 interaction have been inferred from studies of mutated channels. A recently published structure of the Drosophila Orai channel, which has high sequence homology with human ORAI1, revealed the resting state pore architecture of the channel and confirmed evidence from biochemical and electrophysiological studies that transmembrane helices 1 line the pore. However, the critical steps of STIM1 binding and the resulting transition of the channel to its active calcium-conducting state are yet to be delineated. Here we demonstrate a STIM1-mediated conformational change in the purified wildtype ORAI1 channel that correlates with calcium flux through the channel reconstituted into liposomes. Introduction of the known disabling mutation R91W blocks the conformational change as well as calcium flux through the reconstituted channel. Mutations in the N-terminal cytoplasmic region of ORAI1 that disrupt STIM1 interaction with this part of ORAI1 are sufficient to abrogate the gating signal, although these mutations do not affect STIM1 interaction with the C-terminal region of ORAI1. Our study offers key insights into the STIM1-dependent gating of the ORAI1 channel.

Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors.

BackgroundProlonged intracellular calcium elevation contributes to sensitization of nociceptors and chronic pain in inflammatory conditions. The underlying molecular mechanisms remain unknown but store-operated calcium entry (SOCE) components participate in calcium homeostasis, potentially playing a significant role in chronic pain pathologies. Most G protein-coupled receptors activated by inflammatory mediators trigger calcium-dependent signaling pathways and stimulate SOCE in primary afferents. The aim of the present study was to investigate the role of TRPC3, a calcium-permeable non-selective cation channel coupled to phospholipase C and highly expressed in DRG, as a link between activation of pro-inflammatory metabotropic receptors and SOCE in nociceptive pathways.ResultsUsing in situ hybridization, we determined that TRPC3 and TRPC1 constitute the major TRPC subunits expressed in adult rat DRG. TRPC3 was found localized exclusively in small and medium diameter sensory neurons. Heterologous overexpression of TRPC3 channel subunits in cultured primary DRG neurons evoked a significant increase of Gd3+-sensitive SOCE following thapsigargin-induced calcium store depletion. Conversely, using the same calcium add-back protocol, knockdown of endogenous TRPC3 with shRNA-mediated interference or pharmacological inhibition with the selective TRPC3 antagonist Pyr10 induced a substantial decrease of SOCE, indicating a significant role of TRPC3 in SOCE in DRG nociceptors. Activation of P2Y2 purinoceptors or PAR2 protease receptors triggered a strong increase in intracellular calcium in conditions of TRPC3 overexpression. Additionally, knockdown of native TRPC3 or its selective pharmacological blockade suppressed UTP- or PAR2 agonist-evoked calcium responses as well as sensitization of DRG neurons. These data show a robust link between activation of pro-inflammatory receptors and calcium homeostasis through TRPC3-containing channels operating both in receptor- and store-operated mode.ConclusionsOur findings highlight a major contribution of TRPC3 to neuronal calcium homeostasis in somatosensory pathways based on the unique ability of these cation channels to engage in both SOCE and receptor-operated calcium influx. This is the first evidence for TRPC3 as a SOCE component in DRG neurons. The flexible role of TRPC3 in calcium signaling as well as its functional coupling to pro-inflammatory metabotropic receptors involved in peripheral sensitization makes it a potential target for therapeutic strategies in chronic pain conditions.

G protein-coupled receptor signalling potentiates the osmo-mechanical activation of TRPC5 channels

TRPC5 is an ion channel permeable to monovalent and divalent cations that is widely expressed in different tissues. Although implicated in the control of neurite extension and in the growth cone morphology of hippocampal neurons, as well as in fear-related behaviour, the mechanisms by which TRPC5 is activated remain poorly understood. TRPC5 is known to be activated downstream of Gq-coupled receptors and by membrane stretch, and since there is evidence that mechanical stress may directly activate Gq-coupled receptors, we examined the relationship between the activation of TRPC5 by the type 1 histamine receptor and osmotic stress. Using calcium imaging and patch clamp recordings, we found that a higher proportion of cells expressing TRPC5 respond to hypoosmotic solution when they co-express H1R. This response is associated with a phospholipase C-dependent increase in the cells internal calcium concentration, which is abolished on depletion of calcium stores. We also found that the hypoosmotic stimulus that provokes mechanical stress drives the translocation of TRPC5 to the plasma membrane by a mechanism dependent on PI3K. This increase in TRPC5 at the plasma membrane augments the proportion of cells that respond to hypoosmotic stimulation. Together, these results suggest that hypoosmotic cell-swelling activates Gq-coupled receptors, which in turn enhance the activation of TRPC5 by regulating this channel membrane trafficking. Gq-coupled receptors and TPRC5 are co-expressed in several tissues such as those of the vascular system and in somatosensory neurons, suggesting that this mechanism of TRPC5 activation may have interesting and important implications in arterial pressure sensing and mechanotransduction.

Short- and long-term plasticity in CA1 neurons from mice lacking h-channel auxiliary subunit TRIP8b.

Hyperpolarization-activated cyclic nucleotide-gated nonselective cation channels (HCN or h-channels) are important regulators of neuronal physiology contributing to passive membrane properties, such as resting membrane potential and input resistance (R(N)), and to intrinsic oscillatory activity and synaptic integration. The correct membrane targeting of h-channels is regulated in part by the auxiliary h-channel protein TRIP8b. The genetic deletion of TRIP8b results in a loss of functional h-channels, which affects the postsynaptic integrative properties of neurons. We investigated the impact of TRIP8b deletion on long-term potentiation (LTP) at the two major excitatory inputs to CA1 pyramidal neurons: Schaffer collateral (SC) and perforant path (PP). We found that SC LTP was not significantly different between neurons from wild-type and TRIP8b-knockout mice. There was, however, significantly more short-term potentiation in knockout neurons. We also found that the persistent increase in h-current (I(h)) that normally occurs after LTP induction was absent in knockout neurons. The lack of I(h) plasticity was not restricted to activity-dependent induction, because the depletion of intracellular calcium stores also failed to produce the expected increase in I(h). Interestingly, pairing of SC and PP inputs resulted in a form of LTP in knockout neurons that did not occur in wild-type neurons. These results suggest that the physiological impact of TRIP8b deletion is not restricted to the integrative properties of neurons but also includes both synaptic and intrinsic plasticity.

Abstract 198: Stim/orai Channel Complex In Normal And Hypertrophied Adult Cardiac Myocytes

Cardiac myocytes use Ca2+ not only in excitation-contraction coupling but also as a signaling molecule for cardiac growth. It is however unclear how Ca2+ triggers signaling in cardiac myocytes in the presence of the rapid and large Ca2+ fluctuations that occur during excitation-contraction coupling. We have recently reported that stromal interaction molecule 1 (STIM1), a calcium sensor in the sarcoplasmic reticulum, is a critical element in promoting cardiomyocyte growth through the control of a previously unrecognized sarcolemmal voltage-independent current. Our goal was to characterize the plasma membrane partners that contribute to STIM1-dependent currents in normal and hypertrophied adult cardiac myocytes. We firstly characterized the expression profile of TRPCs and ORAIs proteins in both normal and hypertrophied ventricular myocytes from abdominal aortic banded rats. The expression of TRPC1, 3, 4, 5 and 6 and of ORAI1 - 3 was identified with an up-regulation of TRPC1 in hypertrophied cells. We then used a non-viral method to deliver cy3-tagged siRNAs to ventricular myocytes to knock-down the candidate channels. The cardiac myocytes were isolated and whole cell patch-clamp technique as well as Fura-2 imaging were performed to measure the currents in cy3-labelled and in control cardiac myocytes. Our data demonstrated that the drug-inducible STIM1-dependent store-operated calcium entry was marginal in normal myocytes but was enhanced in hypertrophied myocytes. In addition, a basal inwardly rectifying current was observed in hypertrophied but not in control myocytes. This spontaneous current was recorded in the absence of calcium store depletion, required STIM1 expression and meets the characteristics of Arachidonate-Regulated Calcium selective (ARC) channels. ORAI silencing completely inhibited both store-dependent and store-independent currents suggesting the critical implication of ORAIs whereas TRPCs were only marginally involved. In conclusion, ORAI channels are involved in STIM1-dependent currents observed in hypertrophied adult cardiac myocytes. In addition to store-operated calcium entry, the STIM1/ORAI complex provides an alternative, store-independent pathway for agonist-activated Ca2+ entry.

How Many Orai's Does It Take to Make a CRAC Channel?

CRAC (Calcium Release-Activated Calcium) channels represent the primary pathway for so-called “store-operated calcium entry” – the cellular entry of calcium induced by depletion of intracellular calcium stores. These channels play a key role in diverse cellular activities, most noticeably in the differentiation and activation of Tcells, and in the response of mast cells to inflammatory signals. CRAC channels are formed by members of the recently discovered Orai protein family, with previous studies indicating that the functional channel is formed by a tetramer of Orai subunits. However, a recent report has shown that crystals obtained from the purified Drosophila Orai protein display a hexameric channel structure. Here, by comparing the biophysical properties of concatenated hexameric and tetrameric human Orai1 channels expressed in HEK293 cells, we show that the tetrameric channel displays the highly calcium-selective conductance properties consistent with endogenous CRAC channels, whilst the hexameric construct forms an essentially non-selective cation channel.

The C‐ and N‐terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels

Ca(2+) release-activated Ca(2+) (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, and Orai1, the CRAC channel protein, at overlapping sites in the ER and plasma membranes (PMs). The redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, an important event that not only controls gating, but also regulates Orai1 ion selectivity. Orai1 harbours two STIM1 binding sites, one each on the intracellular C- and N-termini. Previous studies have proposed modular functions for these sites, with the C-terminal site thought to regulate STIM1-Orai1 binding and trapping of Orai1 at the ER-PM junctions, and the N-terminal site mediating gating. However, here we find that a variety of mutations in the N-terminal site impair the binding of Orai1 to STIM1 and to the soluble CRAC activation domain (CAD). Gating could be restored in several N- and C-terminal point mutants by directly tethering the minimal STIM1 activation domain (S) to Orai1 (Orai1-SS channels), indicating that loss of gating in these mutants by full-length STIM1 results from insufficient ligand binding. By contrast, gating could not be restored in mutant Orai1-SS channels carrying more drastic deletions that removed the STIM1 binding sites (1-85, 73-85, or 272-279 Orai1), suggesting that STIM1 binding to both sites is essential for channel activation. Moreover, analysis of ion selectivity indicated that the molecular requirements for gating and modulation of ion selectivity are similar, yet substantively different from those for Orai1 puncta formation, suggesting that ion selectivity and gating are mechanistically coupled in CRAC channels. Our results indicate that the C- and N-terminal STIM1 binding sites are both essential for multiple aspects of Orai1 function including STIM1-Orai1 association, Orai1 trapping, and channel activation.

Store-operated calcium entry and calcium influx via voltage-operated calcium channels regulate intracellular calcium oscillations in chondrogenic cells

Chondrogenesis is known to be regulated by calcium-dependent signalling pathways in which temporal aspects of calcium homeostasis are of key importance. We aimed to better characterise calcium influx and release functions with respect to rapid calcium oscillations in cells of chondrifying chicken high density cultures. We found that differentiating chondrocytes express the α1 subunit of voltage-operated calcium channels (VOCCs) at both mRNA and protein levels, and that these ion channels play important roles in generating Ca2+ influx for oscillations as nifedipine interfered with repetitive calcium transients. Furthermore, VOCC blockade abrogated chondrogenesis and almost completely blocked cell proliferation. The contribution of internal Ca2+ stores via store-operated Ca2+ entry (SOCE) seems to be indispensable to both Ca2+ oscillations and chondrogenesis. Moreover, this is the first study to show the functional expression of STIM1/STIM2 and Orai1, molecules that orchestrate SOCE, in chondrogenic cells. Inhibition of SOCE combined with ER calcium store depletion abolished differentiation and severely diminished proliferation, suggesting the important role of internal pools in calcium homeostasis of differentiating chondrocytes. Finally, we present an integrated model for the regulation of calcium oscillations of differentiating chondrocytes that may have important implications for studies of chondrogenesis induced in various stem cell populations.

Honokiol blocks store operated calcium entry in CHO cells expressing the M3 muscarinic receptor: honokiol and muscarinic signaling

BackgroundHonokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay.ResultsHonokiol had a potent (EC50 ≈ 5 μmol/l) inhibitory effect on store operated calcium entry (SOCE) that was induced by activation of the M3 receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated calcium release, depletion of ER calcium stores, or disruption of M3 receptor binding.ConclusionsIt is likely that an inhibition of SOCE contributes to honokiol disruption of parasympathetic motor functions, as well as many of its beneficial pharmacological properties.

Distinct Functional Roles of the N- and C-Terminal STIM1 Binding Sites in Orai1 for Trapping and Gating of CRAC Channels

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of the ER Ca2+ sensor, STIM1, and the CRAC channel protein, Orai1, to overlapping sites in the ER- and plasma-membranes. Redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, a critical step that not only controls CRAC channel gating, but also regulates its pore properties including calcium selectivity. Previous findings indicate that Orai1 harbors two STIM1 binding sites, located in the intracellular N- and C-termini. Here, we compared the functional contributions of these sites for puncta formation, gating, and modulation of CRAC channel ion selectivity. Consistent with past studies, C-terminal mutations that impaired STIM1-Orai1 association and puncta formation also abrogated channel gating. However, gating was fully restored in some of these mutants by directly tethering a STIM1 fragment containing the minimal CRAC activation domain (CAD) in duplicate to Orai1 (Orai1-SS channels). By contrast, mutants lacking the N-terminal CAD binding site displayed no channel activity despite retaining significant STIM1-Orai1 binding, and this defect could not be restored in Orai1-SS channels. Analysis of ion selectivity indicated that the N-terminal site is essential for conferring high calcium selectivity to Orai1 while the C-terminal site is dispensable if CAD is directly linked to Orai1. Thus, the structural requirements for gating and modulation of ion selectivity are similar but substantively different from those for Orai1 puncta formation. Collectively, these results are broadly consistent with a two-step model for Orai channel activation, in which the C-terminal binding site tethers and retains CAD to Orai1, thereby allowing functionally critical interactions at the Orai1 N-terminus to gate the channel and modulate its pore properties.