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Key Points: Ca 2+ entry through Ca 2+  release-activated Ca 2+  channels activates numerous cellular responses. Under physiological conditions of weak intracellular Ca 2+ buffering, mitochondrial Ca 2+ uptake regulates CRAC channel activity. Knockdown of the mitochondrial Ca 2+ uniporter channel prevented the development of I CRAC in weak buffer but not when strong buffer was used instead. Removal of either extracellular or intra-pipette Na + had no effect on the selectivity, kinetics, amplitude, rectification or reversal potential of whole-cell CRAC current. Knockdown of the mitochondrial Na + -Ca 2+ exchanger did not prevent the development of I CRAC in strong or weak Ca 2+ buffer. Whole cell CRAC current is Ca 2+ -selective. Mitochondrial Ca 2+ channels, and not Na + -dependent transport, regulate CRAC channels under physiological conditions., Abstract: Ca 2+ entry through store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels plays a central role in activation of a range of cellular responses over broad spatial and temporal bandwidths. Mitochondria, through their ability to take up cytosolic Ca 2+ , are important regulators of CRAC channel activity under physiological conditions of weak intracellular Ca 2+ buffering. The mitochondrial Ca 2+ transporter(s) that regulates CRAC channels is unclear and could involve the 40 kDa mitochondrial Ca 2+ uniporter (MCU) channel or the Na + -Ca 2+ -Li + exchanger (NCLX). Here, we have investigated the involvement of these mitochondrial Ca 2+ transporters in supporting the CRAC current (I CRAC ) under a range of conditions in RBL mast cells. Knockdown of the MCU channel impaired the activation of I CRAC under physiological conditions of weak intracellular Ca 2+ buffering. In strong Ca 2+ buffer, knockdown of the MCU channel did not inhibit I CRAC development demonstrating that mitochondria regulate CRAC channels under physiological conditions by buffering of cytosolic Ca 2+ via the MCU channel. Surprisingly, manipulations that altered extracellular Na + , cytosolic Na + or both failed to inhibit the development of I CRAC in either strong or weak intracellular Ca 2+ buffer. Knockdown of NCLX also did not affect I CRAC . Prolonged removal of external Na + also had no significant effect on store-operated Ca 2+ entry, on cytosolic Ca 2+ oscillations generated by receptor stimulation or on CRAC channel-driven gene expression. In the RBL mast cell, Ca 2+ flux through the MCU but not NCLX is indispensable for activation of I CRAC ., (© 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The epithelial cells of the lung are at the interface of a host and its environment and are therefore directly exposed to the inhaled air-borne particles. Rather than serving as a simple physical barrier, airway epithelia detect allergens and other irritants and then help organize the subsequent immune response through release of a plethora of secreted signals. Many of these signals are generated in response to opening of store-operated Ca(2+) channels in the plasma membrane. In this review, we describe the properties of airway store-operated channels and their role in regulating airway epithelial cell function.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'., (© 2016 The Authors.)

In polarized cells or cells with complex geometry, clustering of plasma-membrane (PM) ion channels is an effective mechanism for eliciting spatially restricted signals. However, channel clustering is also seen in cells with relatively simple topology, suggesting it fulfills a more fundamental role in cell biology than simply orchestrating compartmentalized responses. Here, we have compared the ability of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels confined to PM microdomains with a similar number of dispersed CRAC channels to activate transcription factors, which subsequently increase nuclear gene expression. For similar levels of channel activity, we find that channel confinement is considerably more effective in stimulating gene expression. Our results identify a long-range signaling advantage to the tight evolutionary conservation of channel clustering and reveal that CRAC channel aggregation increases the strength, fidelity, and reliability of the general process of excitation-transcription coupling., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Ca2+ entry into airway epithelia is important for activation of the NFAT family of transcription factors and expression of genes including epidermal growth factor that help orchestrate local inflammatory responses. However, the identity of epithelial Ca2+ channel that activates these transcriptional responses is unclear. In many other non-excitable cells, store-operated Ca2+ entry is a major route for Ca2+ influx and is mediated by STIM1 and Orai1 proteins. This study was performed to determine if store-operated Ca2+ channels were expressed in human bronchial epithelial cells and, if so, whether they coupled Ca2+ entry to gene expression. Cytoplasmic Ca2+ measurements, patch clamp recordings, RNAi knockdown and functional assays were used to identify and then investigate the role of these Ca2+ channels in activating the NFAT and c-fos pathways and EGF expression. STIM1 and Orai1 mRNA transcripts as well as proteins were robustly in epithelial cells and formed functional Ca2+ channels. Ca2+ entry through the channels activated expression of c-fos and EGF as well as an NFAT-dependent reporter gene. Store-operated Ca2+ entry was also important for epithelial cell migration in a scrape wound assay. These findings indicate that store-operated Ca2+ channels play an important role in stimulating airway epithelial cell gene expression and therefore comprise a novel potential therapeutic target for the treatment of chronic asthma and related airway disorders.

Ca2+ flux into mitochondria is an important regulator of cytoplasmic Ca2+ signals, energy production and cell death pathways. Ca2+ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca2+ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC4 evokes a series of cytoplasmic Ca2+ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca2+ entry into the matrix, prevents the mitochondrial Ca2+ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca2+ oscillations appeared to be a consequence of enhanced Ca2+-dependent inactivation of InsP3 receptors, which arose from the loss of mitochondrial Ca2+ buffering. Ca2+ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca2+ release, mitochondria also sequestrated Ca2+ entry through store-operated Ca2+ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca2+.

NFAT-dependent gene expression is essential for the development and function of the nervous, immune, and cardiovascular systems and kidney, bone, and skeletal muscle. Most NFAT protein resides in the cytoplasm because of extensive phosphorylation, which masks a nuclear localization sequence. Dephosphorylation by the Ca(2+)-calmodulin-activated protein phosphatase calcineurin triggers NFAT migration into the nucleus. In some cell types, NFAT can be activated by Ca(2+) nanodomains near open store-operated Orai1 and voltage-gated Ca(2+) channels in the plasma membrane. How local Ca(2+) near Orai1 is detected and whether other Orai channels utilize a similar mechanism remain unclear. Here, we report that the paralog Orai3 fails to activate NFAT. Orai1 is effective in activating gene expression via Ca(2+) nanodomains because it participates in a membrane-delimited signaling complex that forms after store depletion and brings calcineurin, via the scaffolding protein AKAP79, to calmodulin tethered to Orai1. By contrast, Orai3 interacts less well with AKAP79 after store depletion, rendering it ineffective in activating NFAT. A channel chimera of Orai3 with the N terminus of Orai1 was able to couple local Ca(2+) entry to NFAT activation, identifying the N-terminal domain of Orai1 as central to Ca(2+) nanodomain-transcription coupling. The formation of a store-dependent signaling complex at the plasma membrane provides for selective activation of a fundamental downstream response by Orai1., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)