Calcium Homeostasis and Cone Signaling Are Regulated by Interactions between Calcium Stores and Plasma Membrane Ion Channels

Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca²⁺ entry (SOCE) to Ca²⁺ homeostasis and its role in regulation of neurotransmission at cone synapses. Mn²⁺ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca²⁺ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca²⁺ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca²⁺ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca²⁺ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca²⁺ channels. Exposure to MRS 1845 resulted in ∼40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca²⁺ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca²⁺ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse.     

Hair loss and defective T- and B-cell function in mice lacking ORAI1

ORAI1 is a pore subunit of the store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1^−/− mice. Orai1^−/− mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1^−/− mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1^−/− mice, but B cells showed a substantial decrease in Ca²⁺ influx and cell proliferation in response to B-cell receptor stimulation. Naïve and differentiated Orai1^−/− T cells showed substantial reductions in store-operated Ca²⁺ entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null.     

Maintenance of Calcium Homeostasis in the Endoplasmic Reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca²⁺) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca²⁺ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca²⁺-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca²⁺ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca²⁺ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca²⁺ pool in untreated cells when extracellular Ca²⁺ is low. However, low extracellular Ca²⁺ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca²⁺ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca²⁺ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.     

Orai1 mutations alter ion permeation and Ca2+-dependent fast inactivation of CRAC channels: evidence for coupling of permeation and gating

Ca²⁺ entry through store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca²⁺ selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I–II loop (E106, E190, D110, D112, D114) are essential for the CRAC channel's high Ca²⁺ selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca²⁺ block, and channel regulation by Ca²⁺. We find that alteration of the acidic residues lowers Ca²⁺ selectivity and results in striking increases in Cs⁺ permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs⁺ permeation. Ca²⁺ binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca²⁺ binding. Unexpectedly, the mutations diminish Ca²⁺-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca²⁺ selectivity, increase in Cs⁺ permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels.     

A Store-Operated Nonselective Cation Channel in Human Lymphocytes

1. Agonist interaction with phospholipase C-linked receptors at the plasma membrane can elicit both Ca²⁺ and Na⁺ influxes in lymphocytes. While Ca²⁺ influx is mediated by Ca²⁺ release-activated Ca²⁺ (CRAC) channels, the pathway responsible for Na⁺ influx is largely unknown.2. We show that thapsigargin, ionomycin, ADP-ribose and IP₃ activated a nonselective cation channel in lymphocytes that had a slightly outwardly rectifying I–V relationship, and a single channel conductance of 23.1 pS. We termed this channel a Ca²⁺ release-activated nonselective cation (CRANC) channel.3. On activation in cell-attached configuration, switching to an inside-out configuration abolished CRANC channel activity.4. Transfection of Jurkat T cells with antisense oligonucleotides for LTRPC2 reduced capacitative Ca²⁺ entry.5. These results suggest that CRANC channels are responsible for the Na⁺ influx as well as a portion of the Ca²⁺ influx in lymphocytes induced by store depletion, that sustained activation of CRANC channels requires some property of the environment of a cell depleted of its Ca²⁺ stores; and that LTRPC2 protein is a likely component of the CRANC channel.     

Mitochondrial Participation in the Intracellular Ca2+ Network

Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca²⁺ uptake and extrusion modulate free cytosolic [Ca²⁺] (Ca_c) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca²⁺ entry, and to monitor Ca²⁺-activated currents that report near-surface [Ca²⁺]. We show that rapid recovery from elevations of Ca_c requires both the mitochondrial Ca²⁺ uniporter and the mitochondrial energization that drives Ca²⁺ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca²⁺] (Ca_m). The indicated resting Ca_m of 100–200 nM is similar to the resting Ca_c reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Ca_m and Ca_c at high temporal resolution shows that, although Ca_m increases less than Ca_c, mitochondrial sequestration of Ca²⁺ is fast and has high capacity. We find that mitochondrial Ca²⁺ uptake limits the rise and underlies the rapid decay of Ca_c excursions produced by Ca²⁺ entry or by mobilization of reticular stores. We also find that subsequent export of Ca²⁺ from mitochondria, seen as declining Ca_m, prolongs complete Ca_c recovery and that suppressing export of Ca²⁺, by inhibition of the mitochondrial Na⁺/ Ca²⁺ exchanger, reversibly hastens final recovery of Ca_c. We conclude that mitochondria are active participants in cellular Ca²⁺ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca²⁺.     

Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion

TRPC3 (or Htrp3) is a human member of the trp family of Ca²⁺-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca²⁺ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca²⁺ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca²⁺ over Na⁺. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca²⁺ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca²⁺. Likewise, infusion of Ca²⁺ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca²⁺-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca²⁺ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca²⁺-permeable channel that supports Ca²⁺-induced Ca²⁺-entry but should not be considered store operated.     

Orai1-NFAT signalling pathway triggered by T cell receptor stimulation

T cell receptor (TCR) stimulation plays a crucial role in development, homeostasis, proliferation, cell death, cytokine production, and differentiation of T cells. Thus, in depth understanding of TCR signalling is crucial for development of therapy targeting inflammatory diseases, improvement of vaccination efficiency, and cancer therapy utilizing T cell-based strategies. TCR activation turns on various signalling pathways, one of the important one being the Ca²⁺-calcineurin-nuclear factor of activated T cells (NFAT) signalling pathway. Stimulation of TCRs triggers depletion of intracellular Ca²⁺ store and in turn, initiates store-operated Ca²⁺ entry (SOCE), one of the major mechanisms to raise the intracellular Ca²⁺ concentrations in T cells. Ca²⁺-release-activated-Ca²⁺ (CRAC) channels are a prototype of store-operated Ca²⁺ (SOC) channels in immune cells that are very well characterized. Recent identification of STIM1 as the endoplasmic reticulum (ER) Ca²⁺ sensor and Orai1 as the pore subunit has dramatically advanced the understanding of CRAC channels and provides a molecular tool to investigate the physiological outcomes of Ca²⁺ signalling during immune responses. In this review, we focus on our current understanding of CRAC channel activation, regulation, and downstream calcineurin-NFAT signaling pathway.     

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells

Anoxia induces a rapid elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca²⁺ release is important for gene activation and survival in O₂-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca²⁺ to the anoxic [Ca²⁺]_cyt elevation in maize cells. Imaging of intramitochondrial Ca²⁺ levels showed that a majority of mitochondria released their Ca²⁺ in response to anoxia and took up Ca²⁺ upon reoxygenation. We also investigated whether the mitochondrial Ca²⁺ release contributed to the increase in [Ca²⁺]_cyt under anoxia. Analysis of the spatial association between anoxic [Ca²⁺]_cyt changes and the distribution of mitochondrial and other intracellular Ca²⁺ stores revealed that the largest [Ca²⁺]_cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca²⁺]_cyt under aerobic conditions but prevented a [Ca²⁺]_cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca²⁺]_cyt and participate in the signaling of O₂ deprivation.     

Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle

Activation of store-operated Ca²⁺ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca²⁺ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca²⁺ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca²⁺ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca²⁺ into the sarcoplasmic reticulum (SR). A novel Ca²⁺-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca²⁺. Our data suggest that cytosolic Ca²⁺ can facilitate SOCE whereas SR luminal Ca²⁺ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca²⁺ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca²⁺ entry in response to physiological demand. Such Ca²⁺ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.     

Mitochondrial Ca2+ Overload Underlies Aβ Oligomers Neurotoxicity Providing an Unexpected Mechanism of Neuroprotection by NSAIDs

Dysregulation of intracellular Ca²⁺ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer''s Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ_1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca²⁺ in neurons and promote mitochondrial Ca²⁺ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca²⁺ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca²⁺ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca²⁺ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca²⁺ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD.     

Extramitochondrial Ca2+ in the Nanomolar Range Regulates Glutamate-Dependent Oxidative Phosphorylation on Demand

We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca²⁺ in physiological concentration ranges (S_0.5 = 360 nM Ca²⁺). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca²⁺ uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca²⁺-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca²⁺. At micromolar concentrations, Ca²⁺ can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca²⁺ regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca²⁺.     

Calcium signals regulate the functional differentiation of thymic iNKT cells

How natural or innate‐like lymphocytes generate the capacity to produce IL‐4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4⁺ T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca²⁺) signals and continuing activity of the calcium release‐activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca²⁺ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca²⁺ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca²⁺ buffering in NKT2 cells. In addition, the low Ca²⁺ extrusion rate may also contribute to the higher Ca²⁺ level in NKT2 cells. Altogether, we identified ORAI‐dependent Ca²⁺ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells.     

Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca²⁺. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer''s and Parkinson diseases. One key regulator that underlies cell survival and Ca²⁺ homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca²⁺ dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca²⁺ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca²⁺ through the InsP3 receptor (InsP3R). The Ca²⁺ efflux in IRE1α-deficient cells correlates with dissociation of the Ca²⁺-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca²⁺ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca²⁺ dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca²⁺ influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca²⁺ homeostasis and cell survival during ER stress and reveal a previously unknown Ca²⁺-mediated cell death signaling between the IRE1α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.     

Characterization of a Membrane Calcium Pathway Induced by Rotavirus Infection in Cultured Cells

Some viruses induce changes in membrane permeability during infection. We have shown previously that the porcine strain of rotavirus, OSU, induced an increase in the permeability to Na⁺, K⁺, and Ca²⁺ during replication in MA104 cells. In this work, we have characterized the divalent cation entry pathway by measuring intracellular Ca²⁺ in fura-2-loaded MA104 and HT29 cells in suspension. The permeability to Ca²⁺ and other cations was evaluated by the change of the intracellular concentration following an extracellular cation pulse. Rotavirus infection induced an increase in permeability to Ca²⁺, Ba²⁺, Sr²⁺, Mn²⁺, and Co²⁺. The rate of cation entry decreased over time as the intracellular concentration increased during the first 20 s. This indicates that regulatory mechanisms, including channel inactivation, are triggered. La³⁺ did not enter the cell and blocked the entry of the divalent cations in a dose-dependent manner. Metoxyverapamil (D600), a blocker of L-type voltage-gated channels, partially inhibited the entry of Ca²⁺ in virus-infected MA104 and HT29 cells. The results suggest that rotavirus infection of cultured cells activates a cation channel rather than nonspecific permeation through the plasma membrane. This activation involves the synthesis of viral proteins through mechanisms yet unknown. The increase in intracellular Ca²⁺ induced by the activation of this channel may be related to the increase in cytoplasmic and endoplasmic reticulum Ca²⁺ pools required for virus maturation and cell death.     

The Skeletal L-type Ca2+ Current Is a Major Contributor to Excitation-coupled Ca2+ entry

The term excitation-coupled Ca²⁺ entry (ECCE) designates the entry of extracellular Ca²⁺ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca²⁺ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn²⁺. Here, we have examined the effects of these agents on the L-type Ca²⁺ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La³⁺, and Gd³⁺) and dantrolene all inhibited the L-type Ca²⁺ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca²⁺ channel displays permeability to Mn²⁺ in the absence of external Ca²⁺ and produces a Ca²⁺ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca²⁺ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca²⁺; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α_1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca²⁺-free solution and were unaffected by Ca²⁺ removal; and (3) after block of SR Ca²⁺ release by 200 μM ryanodine, normal myotubes still displayed a large Ca²⁺ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca²⁺ entry attributed to ECCE.     

Essential Role for the CRAC Activation Domain in Store-dependent Oligomerization of STIM1

Oligomerization of the ER Ca²⁺ sensor STIM1 is an essential step in store-operated Ca²⁺ entry. The lumenal EF-hand and SAM domains of STIM1 are believed to initiate oligomerization after Ca²⁺ store depletion, but the contributions of STIM1 cytosolic domains (coiled-coil 1, CC1; coiled-coil 2, CC2; CRAC activation domain, CAD) to this process are not well understood. By applying coimmunoprecipitation and fluorescence photobleaching and energy transfer techniques to truncated and mutant STIM1 proteins, we find that STIM1 cytosolic domains play distinct roles in forming both “resting” oligomers in cells with replete Ca²⁺ stores and higher-order oligomers in store-depleted cells. CC1 supports the formation of resting STIM1 oligomers and appears to interact with cytosolic components to slow STIM1 diffusion. On store depletion, STIM1 lacking all cytosolic domains (STIM1-ΔC) oligomerizes through EF-SAM interactions alone, but these oligomers are unstable. Addition of CC1 + CAD, but not CC1 alone, enables the formation of stable store-dependent oligomers. Within the CAD, both CC2 and C-terminal residues contribute to oligomer formation. Our results reveal a new function for the CAD: in addition to binding and activating Orai1, it is directly involved in STIM1 oligomerization, the initial event triggering store-operated Ca²⁺ entry.     

Exposure to GSM RF Fields Does Not Affect Calcium Homeostasis in Human Endothelial Cells,Rat Pheocromocytoma Cells or Rat Hippocampal Neurons

In the course of modern daily life, individuals are exposed to numerous sources of electromagnetic radiation that are not present in the natural environment. The strength of the electromagnetic fields from sources such as hairdryers, computer display units and other electrical devices is modest. However, in many home and office environments, individuals can experience perpetual exposure to an “electromagnetic smog”, with occasional peaks of relatively high electromagnetic field intensity. This has led to concerns that such radiation can affect health. In particular, emissions from mobile phones or mobile phone masts have been invoked as a potential source of pathological electromagnetic radiation. Previous reports have suggested that cellular calcium (Ca²⁺) homeostasis is affected by the types of radiofrequency fields emitted by mobile phones. In the present study, we used a high-throughput imaging platform to monitor putative changes in cellular Ca²⁺ during exposure of cells to 900 MHz GSM fields of differing power (specific absorption rate 0.012–2 W/Kg), thus mimicking the type of radiation emitted by current mobile phone handsets. Data from cells experiencing the 900 Mhz GSM fields were compared with data obtained from paired experiments using continuous wave fields or no field. We employed three cell types (human endothelial cells, PC-12 neuroblastoma and primary hippocampal neurons) that have previously been suggested to be sensitive to radiofrequency fields. Experiments were designed to examine putative effects of radiofrequency fields on resting Ca²⁺, in addition to Ca²⁺ signals evoked by an InsP₃-generating agonist. Furthermore, we examined putative effects of radiofrequency field exposure on Ca²⁺ store emptying and store-operated Ca²⁺ entry following application of the Ca²⁺ATPase inhibitor thapsigargin. Multiple parameters (e.g., peak amplitude, integrated Ca²⁺ signal, recovery rates) were analysed to explore potential impact of radiofrequency field exposure on Ca²⁺ signals. Our data indicate that 900 MHz GSM fields do not affect either basal Ca²⁺ homeostasis or provoked Ca²⁺ signals. Even at the highest field strengths applied, which exceed typical phone exposure levels, we did not observe any changes in cellular Ca²⁺ signals. We conclude that under the conditions employed in our experiments, and using a highly-sensitive assay, we could not detect any consequence of RF exposure.