Store-operated Ca 2+ channels, which are activated by the emptying of intracellular Ca 2+ stores, provide one major route for Ca 2+ in¯ux. Under physiological conditions of weak intracellular Ca 2+ buffering, the ubiquitous Ca 2+ releasing messenger InsP 3 usually fails to activate any store-operated Ca 2+ entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca 2+ that has been released by InsP 3 , enabling stores to empty suf®ciently for store-operated channels to activate. Here, we report a novel role for mitochondria in regulating store-operated channels under physiological conditions. Mitochondrial depolarization suppresses storeoperated Ca 2+ in¯ux independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP 3 -sensitive store depletion, can be distinguished from Ca 2+ -dependent inactivation of the store-operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidi®cation, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca 2+ uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store-operated in¯ux and raise the possibility of bidirectional Ca 2+ -dependent crosstalk between mitochondria and store-operated Ca 2+ channels.

In many non-excitable cells, a Ca 2+ influx pathway is activated following the process of store emptying, and has been called store-operated or capacitative Ca 2+ entry (Putney, 1986 1. One popular model for the activation of store-operated Ca 2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP 3 ) receptors on the stores to bind to, and thus activate, store-operated Ca 2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I CRAC , the most widely distributed and best characterised store-operated Ca 2+ current, in a model system, the RBL-1 rat basophilic cell line.2. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I CRAC . Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment.3. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution.4. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I CRAC was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores.5. Pre-treatment with the membrane-permeable InsP 3 receptor antagonist 2-APB blocked the activation of I CRAC . However, intracellular dialysis with 2-APB failed to prevent I CRAC from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I CRAC had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La 3+ .6. Studies with the more conventional InsP 3 receptor antagonist heparin revealed that occupation of the intracellular InsP 3 -sensitive receptors was not necessary for the activation or maintenance of I CRAC . Similarly, the InsP 3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I CRAC . Exposure to Li + , which reduces InsP 3 levels by interfering with inositol monophosphatase, also failed to alter I CRAC . Caffeine and Li + did not affect the size of the intracellular Ca 2+ signal that arose when external Ca 2+ was re-admitted to cells which had been pre-exposed to thapsigargin/ionomycin in Ca 2+ -free external solution.7. Our findings demonstrate that the cytoskeleton does not seem to regulate calcium influx and that functional InsP 3 receptors are not required for activation of I CRAC . If the secretion-like coupling model indeed accounts for the activation of I CRAC in RBL-1 cells, ...

In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca 2+ concentration: an initial transient phase re¯ecting Ca 2+ release from intracellular stores, followed by a plateau phase due to Ca 2+ in¯ux. A major component of this Ca 2+ in¯ux is store-dependent and often can be measured directly as the Ca 2+ release-activated Ca 2+ current (I CRAC ). Under physiological conditions of weak intracellular Ca 2+ buffering, respiring mitochondria play a central role in store-operated Ca 2+ in¯ux. They determine whether macroscopic I CRAC activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP 3 ) that is required to activate I CRAC . By increasing the sensitivity of store-operated in¯ux to InsP 3 , respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca 2+ entry or not. Mitochondrial Ca 2+ buffering therefore increases the dynamic range of concentrations over which the InsP 3 is able to function as the physiological messenger that triggers the activation of store-operated Ca 2+ in¯ux.

SummaryNFAT-dependent gene expression is essential for the development and function of the nervous, immune, and cardiovascular systems and kidney, bone, and skeletal muscle [1]. Most NFAT protein resides in the cytoplasm because of extensive phosphorylation, which masks a nuclear localization sequence. Dephosphorylation by the Ca2+-calmodulin-activated protein phosphatase calcineurin triggers NFAT migration into the nucleus [2, 3]. In some cell types, NFAT can be activated by Ca2+ nanodomains near open store-operated Orai1 and voltage-gated Ca2+ channels in the plasma membrane [4, 5]. How local Ca2+ 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 Ca2+ 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 Ca2+ entry to NFAT activation, identifying the N-terminal domain of Orai1 as central to Ca2+ 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.

Stimulation of cells with physiological concentrations of calciummobilizing agonists often results in the generation of repetitive cytoplasmic Ca 2+ oscillations. Although oscillations arise from regenerative Ca 2+ release, they are sustained by store-operated Ca 2+ entry through Ca 2+ release-activated Ca 2+ (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca 2+ oscillations, CRAC channel activity, and downstream Ca 2+ -dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca 2+ sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca 2+ oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca 2+ signals and downstream responses.excitation-transcription coupling | transcription S timulation of cell-surface receptors that couple to the phospholipase C pathway with physiological concentrations of agonist generally evokes repetitive cytoplasmic Ca 2+ oscillations (1). Oscillatory Ca 2+ signals enable cytoplasmic Ca 2+ to reach high levels transiently, thereby avoiding the deleterious effects of a prolonged, elevated Ca 2+ rise. Information is encoded in the oscillatory amplitude and frequency (2) and the spatial profile of the Ca 2+ signal (3), each of which can be deciphered by cells to drive selective downstream responses.Ca 2+ oscillations are triggered by inositol trisphosphate (InsP 3 )-mediated Ca 2+ release from intracellular Ca 2+ stores, primarily the endoplasmic reticulum (ER) (2). The resulting fall in Ca 2+ within the stores opens store-operated CRAC channels in the plasma membrane (4, 5). Ca 2+ entry through these channels refills the stores and, thus, sustains InsP 3 -dependent Ca 2+ oscillations (6). In addition to this supportive role, local Ca 2+ entry through Ca 2+ release-activated Ca 2+ (CRAC) channels during oscillatory responses in mast cells, and not the oscillations per se, signals to the nucleus to regulate Ca 2+ -dependent gene expression (7).The two main molecular components of store-operated Ca 2+ entry are the stromal interaction molecule (STIM) and Orai proteins (reviewed in refs. 8-10). The transmembrane ER proteins STIM1 and STIM2 detect ER Ca 2+ content through an EF-hand domain in their respective N-termini, which face the lumen of the store. Loss of luminal Ca 2+ leads to STIM aggregation within the ER followed by migration to ER-plasma membrane junctions located just below the plasma membrane. Here, they bind to and activate Orai1, a four transmembrane domain spanning plasma membrane protein, which forms the CRAC channel.Despite significant homology between STIM1 and STIM2, there are some important differences between them. First, they differ in their respective abilities to activate Orai1. STIM2 activates Ca 2+ ...