Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.     

Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma

Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian behavioral rhythm that occurs once every 45-50 s and is mediated in part by posterior body wall muscle contraction (pBoc). pBoc is not regulated by neural input but instead is likely controlled by rhythmic Ca(2+) oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca(2+) signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca(2+) oscillations with a period of approximately 50 s. Oscillations were only detected in the apical cell pole of the intestinal epithelium and occur as a posterior-to-anterior moving intercellular Ca(2+) wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) wave velocity. In contrast, gain-of-function mutations in the IP(3) binding and regulatory domains of ITR-1 have no effect on pBoc or Ca(2+) oscillation frequency but dramatically increase the speed of the intercellular Ca(2+) wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)-encoding genes demonstrated that pBoc and Ca(2+) oscillations require the combined function of PLC-gamma and PLC-beta homologues. Disruption of PLC-gamma and PLC-beta activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca(2+) oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC-gamma functions primarily to generate IP(3) that controls ITR-1 activity. In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation. PLC-beta may function instead to control PIP(2) levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca(2+) signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca(2+) oscillations and intercellular Ca(2+) waves in nonexcitable cells.     

Highly Ca2+-selective TRPM channels regulate IP3-dependent oscillatory Ca2+ signaling in the C. elegans intestine

Posterior body wall muscle contraction (pBoc) in the nematode Caenorhabditis elegans occurs rhythmically every 45-50 s and mediates defecation. pBoc is controlled by inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in the intestine. The intestinal epithelium can be studied by patch clamp electrophysiology, Ca2+ imaging, genome-wide reverse genetic analysis, forward genetics, and molecular biology and thus provides a powerful model to develop an integrated systems level understanding of a nonexcitable cell oscillatory Ca2+ signaling pathway. Intestinal cells express an outwardly rectifying Ca2+ (ORCa) current with biophysical properties resembling those of TRPM channels. Two TRPM homologues, GON-2 and GTL-1, are expressed in the intestine. Using deletion and severe loss-of-function alleles of the gtl-1 and gon-2 genes, we demonstrate here that GON-2 and GTL-1 are both required for maintaining rhythmic pBoc and intestinal Ca2+ oscillations. Loss of GTL-l and GON-2 function inhibits I(ORCa) approximately 70% and approximately 90%, respectively. I(ORCa) is undetectable in gon-2;gtl-1 double mutant cells. These results demonstrate that (a) both gon-2 and gtl-1 are required for ORCa channel function, and (b) GON-2 and GTL-1 can function independently as ion channels, but that their functions in mediating I(ORCa) are interdependent. I(ORCa), I(GON-2), and I(GTL-1) have nearly identical biophysical properties. Importantly, all three channels are at least 60-fold more permeable to Ca2+ than Na+. Epistasis analysis suggests that GON-2 and GTL-1 function in the IP3 signaling pathway to regulate intestinal Ca2+ oscillations. We postulate that GON-2 and GTL-1 form heteromeric ORCa channels that mediate selective Ca2+ influx and function to regulate IP3 receptor activity and possibly to refill ER Ca2+ stores.     

Mapping the interacting domains of STIM1 and Orai1 in Ca2+ release-activated Ca2+ channel activation

STIM1 and Orai1 are essential components of Ca(2+) release-activated Ca(2+) channels (CRACs). After endoplasmic reticulum Ca(2+) store depletion, STIM1 in the endoplasmic reticulum aggregates and migrates toward the cell periphery to co-localize with Orai1 on the opposing plasma membrane. Little is known about the roles of different domains of STIM1 and Orai1 in protein clustering, migration, interaction, and, ultimately, opening CRAC channels. Here we demonstrate that the coiled-coil domain in the C terminus of STIM1 is crucial for its aggregation. Amino acids 425-671 of STIM1, which contain a serine-proline-rich region, are important for the correct targeting of the STIM1 cluster to the cell periphery after calcium store depletion. The polycationic region in the C-terminal tail of STIM1 also helps STIM1 targeting but is not essential for CRAC channel activation. The cytoplasmic C terminus but not the N terminus of Orai1 is required for its interaction with STIM1. We further identify a highly conserved region in the N terminus of Orai1 (amino acids 74-90) that is necessary for CRAC channel opening. Finally, we show that the transmembrane domain of Orai1 participates in Orai1-Orai1 interactions.     

New insights into the molecular mechanisms of store-operated Ca2+ signaling in T cells

The activation of Ca(2+) entry through store-operated channels by agonists that deplete Ca(2+) from the endoplasmic reticulum (ER) is an ubiquitous signaling mechanism, the molecular basis of which has remained elusive for the past 20 years. In T lymphocytes, store-operated Ca(2+)-release-activated Ca(2+) (CRAC) channels constitute the sole pathway for Ca(2+) entry following antigen-receptor engagement, and their function is essential for driving the program of gene expression that underlies T-cell activation by antigen. The first molecular components of this pathway have recently been identified: stromal interaction molecule 1 (STIM1), the ER Ca(2+) sensor, and Orai1, a pore-forming subunit of the CRAC channel. Recent work shows that CRAC channels are activated in a complex fashion that involves the co-clustering of STIM1 in junctional ER directly opposite Orai1 in the plasma membrane. These studies reveal an abundance of sites where Ca(2+) signaling might be controlled to modulate the activity of T cells during the immune response.     

Mitochondrial dynamics and their impact on T cell function

Energy supply is the most prominent function of mitochondria, but in addition, mitochondria are indispensable for a multitude of other important cellular functions including calcium (Ca(2+)) signaling and buffering, the supply of metabolites and the sequestration of apoptotic factors. The efficiency of those functions highly depends on the proper positioning of mitochondria within the cytosol. In lymphocytes, mitochondria preferentially localize into the vicinity (～200nm) of the immune synapse (IS). This localization is regulated by motor-based cytoskeleton-mediated transport, the fusion/fission dynamics of mitochondria, and probably also through tethering with the ER. IS formation also induces the accumulation of CRAC/ORAI1 Ca(2+) channels, the CRAC/ORAI channel activator STIM1, K(+) channels and plasma membrane Ca(2+) ATPase (PMCA) within the IS. Such a large agglomeration of Ca(2+) binding organelles and proteins highlights the IS as a critical cellular compartment for Ca(2+) dependent lymphocyte activation. At the IS, Ca(2+) microdomains generated beneath open CRAC/ORAI channels provide a rapid, robust and reliable mechanism for driving cellular responses in mast cells and T cells. Here, we discuss the relevance of motor-based mitochondrial transport, fusion, fission and tethering for mitochondrial localization in T cells and the importance of subplasmalemmal mitochondria to control local CRAC/ORAI1-dependent Ca(2+) microdomains at the IS for efficient T lymphocyte activation.     

Mitochondrial regulation of store-operated CRAC channels

In eukaryotic cells, one major route for Ca(2+) influx is through store-operated CRAC channels, which are activated following a fall in Ca(2+) content within the endoplasmic reticulum. Mitochondria are key regulators of this ubiquitous Ca(2+) influx pathway. Respiring mitochondria rapidly take up some of the Ca(2+) released from the stores, resulting in more extensive store depletion and thus robust activation of CRAC channels. As CRAC channels open, the ensuing rise in cytoplasmic Ca(2+) feeds back to inactivate the channels. By buffering some of the incoming Ca(2+) mitochondria reduce Ca(2+)-dependent inactivation of the CRAC channels, resulting in more prolonged Ca(2+) influx. However, mitochondria can release Ca(2+) close to the endoplasmic reticulum, accelerating store refilling and thus promoting deactivation of the CRAC channels. Mitochondria thus regulate all major transitions in CRAC channel gating, revealing remarkable versatility in how this organelle impacts upon Ca(2+) influx. Recent evidence suggests that mitochondria also control CRAC channels through mechanisms that are independent of their Ca(2+)-buffering actions and ability to generate ATP. Furthermore, pyruvic acid, a key intermediary metabolite and precursor substrate for the Krebs cycle, reduces the extent of Ca(2+)-dependent inactivation of CRAC channels. Hence mitochondrial metabolism impacts upon Ca(2+) influx through CRAC channels and thus on a range of key downstream cellular responses.     

Some assembly required: constructing the elementary units of store-operated Ca2+ entry

The means by which Ca(2+) store depletion evokes the opening of store-operated Ca(2+) channels (SOCs) in the plasma membrane of excitable and non-excitable cells has been a longstanding mystery. Indirect evidence has supported local interactions between the ER and SOCs as well as long-range interactions mediated through a diffusible activator. The recent molecular identification of the ER Ca(2+) sensor (STIM1) and a subunit of the CRAC channel (Orai1), a prototypic SOC, has now made it possible to visualize directly the sequence of events that links store depletion to CRAC channel opening. Following store depletion, STIM1 moves from locations throughout the ER to accumulate in ER subregions positioned within 10-25nm of the plasma membrane. Simultaneously, Orai1 gathers at discrete sites in the plasma membrane directly opposite STIM1, resulting in local CRAC channel activation. These new studies define the elementary units of store-operated Ca(2+) entry, and reveal an unprecedented mechanism for channel activation in which the stimulus brings a channel and its activator/sensor together for interaction across apposed membrane compartments. We discuss the implications of this choreographic mechanism with regard to Ca(2+) dynamics, specificity of Ca(2+) signaling, and the existence of a specialized ER subset dedicated to the control of the CRAC channel.     

Mechanism of different spatial distributions of Caenorhabditis elegans and human STIM1 at resting state

Recent studies have identified STIM1 and Orai1 as essential and conserved components of the Ca2+ release-activated Ca2+ (CRAC) channel. However, the reason STIM1 exhibits different distributions in nematode Caenorhabditis elegans and in human cells before endoplasmic reticulum (ER) calcium store depletion has not been clarified. Compared to the diffuse ER distribution of human STIM1 (H.STIM1), we found that C. elegans STIM1 (C.STIM1) was pre-oligomerized in puncta at the cell periphery before Ca2+ store depletion when expressed in HEK293 cells. Interestingly, these C.STIM1 puncta failed to induce aggregation of C. elegans Orai1 (C.Orai1), and no CRAC current was detected in quiescent cells. However, upon store depletion, C.Orai1 and C.STIM1 functioned as a pair to locally sense the store depletion signal and to activate the CRAC channel. We substituted the N-terminus of H.STIM1 for the N-terminus of C.STIM1 (H_C.STIM1), which resulted in pre-puncta resting localization. In contrast, by replacing the C-terminus of C.STIM1 with that of H.STIM1, we made a chimeric protein (C.STIM1_H) that exhibited two distribution profiles at resting state, a diffuse ER pattern like H.STIM1, and large aggregates. Taken together, our results suggest that (1) despite highly conserved functional domains, C. elegans STIM1 and human STIM1 display different spatial distributions in HEK293 cells before store depletion; (2) the C.STIM1 puncta at peripheral sites are not sufficient for the aggregation and activation of C.Orai1 in the absence of store depletion; (3) the distinct distributions of C.STIM1 and H.STIM1 at resting state are determined by the cytoplasmic region of STIM1.     

Inositol 1,4,5-trisphosphate signaling regulates rhythmic contractile activity of myoepithelial sheath cells in Caenorhabditis elegans

Intercellular communication between germ cells and neighboring somatic cells is essential for reproduction. Caenorhabditis elegans oocytes are surrounded by and coupled via gap junctions to smooth muscle-like myoepithelial sheath cells. Rhythmic sheath cell contraction drives ovulation and is triggered by a factor secreted from oocytes undergoing meiotic maturation. We demonstrate for the first time that signaling through the epidermal growth factor-like ligand LIN-3 and the LET-23 tyrosine kinase receptor induces ovulatory contractions of sheath cells. Reduction-of-function mutations in the inositol 1,4,5-trisphosphate (IP(3)) receptor gene itr-1 and knockdown of itr-1 expression by RNA interference inhibit sheath contractile activity. itr-1 gain-of-function mutations increase the rate and force of basal contractions and induce tonic sheath contraction during ovulation. Sheath contractile activity is disrupted by RNAi of plc-3, one of six phospholipase C-encoding genes in the C. elegans genome. PLC-3 is a PLC-gamma homolog and is expressed in contractile sheath cells of the proximal gonad. Maintenance of sheath contractile activity requires plasma membrane Ca(2+) entry. We conclude that IP(3) generated by LET-23 mediated activation of PLC-gamma induces repetitive intracellular Ca(2+) release that drives rhythmic sheath cell contraction. Calcium entry may function to trigger Ca(2+) release via IP(3) receptors and/or refill intracellular Ca(2+) stores.     

Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels

Although store-operated calcium release-activated Ca(2+) (CRAC) channels are highly Ca(2+)-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg(2+)-inhibited cation (MIC) channels that open as Mg(2+) is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg(2+) to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca(2+) to divalent-free extracellular solution evokes Na(+) current through open CRAC channels (Na(+)-I(CRAC)) that is initially eightfold larger than the preceding Ca(2+) current and declines by approximately 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs(+) (P(Cs)/P(Na) = 0.13 vs. 1.2 for MIC). Neither the decline in Na(+)-I(CRAC) nor its low Cs(+) permeability are affected by intracellular Mg(2+) (90 microM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca(2+) channels.     

Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells

The nematode Caenorhabditis elegans offers significant experimental advantages for defining the genetic basis of diverse biological processes. Genetic and physiological analyses have demonstrated that inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in intestinal epithelial cells play a central role in regulating the nematode defecation cycle, an ultradian rhythm with a periodicity of 45-50 s. Patch clamp studies combined with behavioral assays and forward and reverse genetic screening would provide a powerful approach for defining the molecular details of oscillatory Ca2+ signaling. However, electrophysiological characterization of the intestinal epithelium has not been possible because of its relative inaccessibility. We developed primary intestinal epithelial cell cultures that circumvent this problem. Intestinal cells express two highly Ca2+-selective, voltage-independent conductances. One conductance, IORCa, is constitutively active, exhibits strong outward rectification, is 60-70-fold more selective for Ca2+ than Na+, is inhibited by intracellular Mg2+ with a K1/2 of 692 microM, and is insensitive to Ca2+ store depletion. Inhibition of IORCa with high intracellular Mg2+ concentrations revealed the presence of a small amplitude conductance that was activated by passive depletion of intracellular Ca2+ stores. Active depletion of Ca2+ stores with IP3 or ionomycin increased the rate of current activation approximately 8- and approximately 22-fold compared with passive store depletion. The store-operated conductance, ISOC, exhibits strong inward rectification, and the channel is highly selective for Ca2+ over monovalent cations with a divalent cation selectivity sequence of Ca2+ > Ba2+ approximately Sr2+. Reversal potentials for ISOC could not be detected accurately between 0 and +80 mV, suggesting that PCa/PNa of the channel may exceed 1,000:1. Lanthanum, SKF 96365, and 2-APB inhibit both IORCa and ISOC reversibly. Our studies provide the first detailed electrophysiological characterization of voltage-independent Ca2+ conductances in C. elegans and form the foundation for ongoing genetic and molecular studies aimed at identifying the genes that encode the intestinal cell channels, for defining mechanisms of channel regulation and for defining their roles in oscillatory Ca2+ signaling.     

Low cytoplasmic [Ca(2+)] activates I(CRAC) independently of global Ca(2+) store depletion in RBL-1 cells

Release of Ca(2+) from inositol (1,4,5)-trisphosphate-sensitive Ca(2+) stores causes "capacitative calcium entry," which is mediated by the so-called "Ca(2+) release-activated Ca(2+) current" (I(CRAC)) in RBL-1 cells. Refilling of the Ca(2+) stores or high cytoplasmic [Ca(2+)] ([Ca(2+)](cyt)) inactivate I(CRAC). Here we address the question if also [Ca(2+)](cyt) lower than the resting [Ca(2+)](cyt) influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both I(CRAC) and [Ca(2+)] within Ca(2+) stores of RBL-1 cells ([Ca(2+)](store)). We found that low [Ca(2+)](cyt) in the range of 30-50 nM activates I(CRAC) and Ca(2+) influx spontaneously and independently of global Ca(2+) store depletion, while elevation of [Ca(2+)](cyt) to the resting [Ca(2+)](cyt) (100 nM) resulted in store dependence of I(CRAC) activation. We conclude that spontaneous activation of I(CRAC) by low [Ca(2+)](cyt) could serve as a feedback mechanism keeping the resting [Ca(2+)](cyt) constant.     

Ca(2+)/Calmodulin-binding proteins from the C. elegans proteome

Calmodulin (CaM) is the primary Ca(2+)-sensor that regulates a wide variety of cellular processes in eukaryotes. Although many Ca(2+)/CaM-binding proteins have been identified, very few such proteins could be found from the genome-wide protein-protein interaction maps of Caenorhabditis elegans constructed by yeast two-hybrid screening. Using a genotype-phenotype conjugation method called mRNA-display, we performed a selection for Ca(2+)/CaM-binding proteins from a proteome library of C. elegans. The method allowed the identification of 9 known and 47 previously uncharacterized Ca(2+)-dependent CaM-binding proteins from the adult worm proteome. The Ca(2+)/CaM-binding properties of these proteins were characterized and their binding motifs were identified. The availability of such information could facilitate our understanding of the signaling pathways mediated by Ca(2+)/CaM in C. elegans. Due to its simplicity and efficiency, the method could be readily applied to examine the Ca(2+)-dependent binding partners of numerous other Ca(2+)-binding proteins, which may play important roles in many signaling pathways in C. elegans.     

Store depletion-activated CaT1 currents in rat basophilic leukemia mast cells are inhibited by 2-aminoethoxydiphenyl borate. Evidence for a regulatory component that controls activation of both CaT1 and CRAC (Ca(2+) release-activated Ca(2+) channel) channels

The intestinal Ca(2+) transport protein CaT1 encoded by TRPV6 has been reported (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705-709) to be all or a part of the Ca(2+) release-activated Ca(2+) channel (CRAC). The major characteristic of CRAC is its activation following store depletion. We expressed CaT1 in HEK293 cells and rat basophilic leukemia (RBL) mast cells and measured whole-cell currents by the patch clamp technique. In HEK293 cells, the expression of CaT1 consistently yielded a constitutively active current, the size of which was strongly dependent on the holding potential and duration of voltage ramps. In CaT1-expressing RBL cells, the current was either activated by store depletion or was constitutively active at a higher current density. CaT1 currents could be clearly distinguished from endogenous CRAC by their typical current-voltage relationship in divalent free solution. 2-aminoethoxydiphenyl borate (2-APB), which is considered a blocker of CRAC, was tested for its inhibitory effect on both cell types expressing CaT1. Endogenous CRAC as well as store-dependent CaT1-derived currents of RBL cells were largely blocked by 75 microm 2-APB, whereas constitutively active CaT1 currents in both RBL and HEK293 cells were slightly potentiated. These results indicate that despite the difference in the permeation properties of CRAC and CaT1 channels, the latter are similarly able to form store depletion-activated conductances in RBL mast cells that are inhibited by 2-APB.     

Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.     

Molecular basis of the CRAC channel

Ca(2+) release-activated Ca(2+) (CRAC) channels, located in the plasma membrane, are opened upon release of Ca(2+) from intracellular stores, permitting Ca(2+) entry and sustained [Ca(2+)](i) signaling that replenishes the store in numerous cell types. This mechanism is particularly important in T lymphocytes of the immune system, providing the missing link in the signal transduction cascade that is initiated by T cell receptor engagement and leads to altered expression of genes that results ultimately in the production of cytokines and cell proliferation. In the past three years, RNA interference screens together with over-expression and site-directed mutagenesis have identified the triggering molecule (Stim) that links store depletion to CRAC channel-mediated Ca(2+) influx and the pore subunit (Orai) of the CRAC channel that allows highly selective entry of Ca(2+) ions into cells.     

Calcium release in HSY cells conforms to a steady-state mechanism involving regulation of the inositol 1,4,5-trisphosphate receptor Ca2+ channel by luminal [Ca2+]

In many cell types, low concentrations of inositol 1,4,5-trisphosphate (IP3) release only a portion of the intracellular IP3-sensitive Ca2+ store, a phenomenon known as "quantal" Ca2+ release. It has been suggested that this effect is a result of reduced activity of the IP3- dependent Ca2+ channel with decreasing calcium concentration within the IP3-sensitive store ([Ca2+]s). To test this hypothesis, the properties of IP3-dependent Ca2+ release in single saponin-permeabilized HSY cells were studied by monitoring [Ca2+]s using the Ca(2+)-sensitive fluorescent dye mag-fura-2. In permeabilized cells, blockade of the sarco/ER Ca(2+)-ATPase pump in stores partially depleted by IP3 induced further Ca2+ release via an IP3-dependent route, indicating that Ca2+ entry via the sarco/ER Ca(2+)-ATPase pump had been balanced by Ca2+ loss via the IP3-sensitive channel before pump inhibition. IP3- dependent Mn2+ entry, monitored via quenching of luminal mag-fura-2 fluorescence, was readily apparent in filled stores but undetectable in Ca(2+)-depleted stores, indicating markedly reduced IP3-sensitive channel activity in the latter. Also consistent with reduced responsiveness of Ca(2+)-depleted stores to IP3, the initial rate of refilling of these stores was unaffected by the presence of 0.3 microM IP3, a concentration that was clearly effective in eliciting Ca2+ release from filled stores. Analysis of the rate of Ca2+ release at various IP3 concentrations indicated a significant shift of the IP3 dose response toward higher [IP3] with decreasing [Ca2+]s. We conclude that IP3-dependent Ca2+ release in HSY cells is a steady-state process wherein Ca2+ efflux via the IP3 receptor Ca2+ channel is regulated by [Ca2+]s, apparently via changes in the sensitivity of the channel to IP3.