The third transmembrane segment of orai1 protein modulates Ca2+ release-activated Ca2+ (CRAC) channel gating and permeation properties

Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.     

Competitive modulation of Ca2+ release-activated Ca2+ channel gating by STIM1 and 2-aminoethyldiphenyl borate

Activation of Ca(2+) release-activated Ca(2+) channels by depletion of intracellular Ca(2+) stores involves physical interactions between the endoplasmic reticulum Ca(2+) sensor, STIM1, and the channels composed of Orai subunits. Recent studies indicate that the Orai3 subtype, in addition to being store-operated, is also activated in a store-independent manner by 2-aminoethyldiphenyl borate (2-APB), a small molecule with complex pharmacology. However, it is unknown whether the store-dependent and -independent activation modes of Orai3 channels operate independently or whether there is cross-talk between these activation states. Here we report that in addition to causing direct activation, 2-APB also regulates store-operated gating of Orai3 channels, causing potentiation at low doses and inhibition at high doses. Inhibition of store-operated gating by 2-APB was accompanied by the suppression of several modes of Orai3 channel regulation that depend on STIM1, suggesting that high doses of 2-APB interrupt STIM1-Orai3 coupling. Conversely, STIM1-bound Orai3 (and Orai1) channels resisted direct gating by high doses of 2-APB. The rate of direct 2-APB activation of Orai3 channels increased linearly with the degree of STIM1-Orai3 uncoupling, suggesting that 2-APB has to first disengage STIM1 before it can directly gate Orai3 channels. Collectively, our results indicate that the store-dependent and -independent modes of Ca(2+) release-activated Ca(2+) channel activation are mutually exclusive: channels bound to STIM1 resist 2-APB gating, whereas 2-APB antagonizes STIM1 gating.     

MicroRNAs are essential for stretch-induced vascular smooth muscle contractile differentiation via microRNA (miR)-145-dependent expression of L-type calcium channels

Stretch of the vascular wall is an important stimulus to maintain smooth muscle contractile differentiation that is known to depend on L-type calcium influx, Rho-activation, and actin polymerization. The role of microRNAs in this response was investigated using tamoxifen-inducible and smooth muscle-specific Dicer KO mice. In the absence of Dicer, which is required for microRNA maturation, smooth muscle microRNAs were completely ablated. Stretch-induced contractile differentiation and Rho-dependent cofilin-2 phosphorylation were dramatically reduced in Dicer KO vessels. On the other hand, acute stretch-sensitive growth signaling, which is independent of influx through L-type calcium channels, was not affected by Dicer KO. Contractile differentiation induced by the actin polymerizing agent jasplakinolide was not altered by deletion of Dicer, suggesting an effect upstream of actin polymerization. Basal and stretch-induced L-type calcium channel expressions were both decreased in Dicer KO portal veins, and inhibition of L-type channels in control vessels mimicked the effects of Dicer deletion. Furthermore, inhibition of miR-145, a highly expressed microRNA in smooth muscle, resulted in a similar reduction of L-type calcium channel expression. This was abolished by the Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN93, suggesting that Ca(2+)/calmodulin-dependent protein kinase IIδ, a target of miR-145 and up-regulated in Dicer KO, plays a role in the regulation of L-type channel expression. These results show that microRNAs play a crucial role in stretch-induced contractile differentiation in the vascular wall in part via miR-145-dependent regulation of L-type calcium channels.     

Store-operated Ca2+ Entry in Malignant Hyperthermia-susceptible Human Skeletal Muscle

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca²⁺ triggers sarcolemmal Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca²⁺] either within the resealed t-system ([Ca²⁺]_t-sys) or within the cytosol. In normal fibers, halothane (0.5 mm) failed to initiate SR Ca²⁺ release or Ca²⁺_t-sys depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca²⁺ release and Ca²⁺_t-sys depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca²⁺ release took the form of a propagated wave, which was temporally coupled to a wave of Ca²⁺_t-sys depletion. SOCE was potently inhibited by “extracellular” application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca²⁺ depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca²⁺ release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca²⁺ pool may contribute to the maintained rise in cytosolic [Ca²⁺] that underlies MH.     

Yap1 protein regulates vascular smooth muscle cell phenotypic switch by interaction with myocardin

The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs.     

Facilitation and Ca2+-dependent inactivation are modified by mutation of the Ca(v)1.2 channel IQ motif

The heart muscle responds to physiological needs with a short-term modulation of cardiac contractility. This process is determined mainly by properties of the cardiac L-type Ca(2+) channel (Ca(v)1.2), including facilitation and Ca(2+)-dependent inactivation (CDI). Both facilitation and CDI involve the interaction of calmodulin with the IQ motif of the Ca(v)1.2 channel, especially with Ile-1624. To verify this hypothesis, we created a mouse line in which Ile-1624 was mutated to Glu (Ca(v)1.2(I1624E) mice). Homozygous Ca(v)1.2(I1624E) mice were not viable. Therefore, we inactivated the floxed Ca(v)1.2 gene of heterozygous Ca(v)1.2(I1624E) mice by the α-myosin heavy chain-MerCreMer system. The resulting I/E mice were studied at day 10 after treatment with tamoxifen. Electrophysiological recordings in ventricular cardiomyocytes revealed a reduced Ca(v)1.2 current (I(Ca)) density in I/E mice. Steady-state inactivation and recovery from inactivation were modified in I/E versus control mice. In addition, voltage-dependent facilitation was almost abolished in I/E mice. The time course of I(Ca) inactivation in I/E mice was not influenced by the use of Ba(2+) as a charge carrier. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as a chelating agent for intracellular Ca(2+), inactivation of I(Ca) was slowed down in control but not I/E mice. The results show that the I/E mutation abolishes Ca(2+)/calmodulin-dependent regulation of Ca(v)1.2. The Ca(v)1.2(I1624E) mutation transforms the channel to a phenotype mimicking CDI.     

Ca2+ Sparks Act as Potent Regulators of Excitation-Contraction Coupling in Airway Smooth Muscle

Ca²⁺ sparks are short lived and localized Ca²⁺ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca²⁺-activated K⁺ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca²⁺ channels. But in many smooth muscles from a variety of organs, Ca²⁺ sparks can additionally activate Ca²⁺-activated Cl⁻ channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca²⁺ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca²⁺ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca²⁺ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca²⁺ channels, resulting in an increase in global [Ca²⁺]_i and cell contraction. Therefore, Ca²⁺ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.     

A novel RhoA/ROCK-CPI-17-MEF2C signaling pathway regulates vascular smooth muscle cell gene expression

Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1α (PP1α), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1α inhibition resulted in increased expression of myocardin, while ectopic expression of PP1α inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of a PP1α inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1α, and CPI-17.     

Lead-dependent effects on arachidonic acid accumulation and the proliferation of vascular smooth muscle

Lead (Pb(2+)) has been implicated in the development of hypertension and atherosclerosis. The proliferation of vascular smooth muscle cells (VSMC) is a central feature of both conditions and there is evidence that Pb(2+) potentiates serum-dependent cell growth. The aim of this work was to examine the role of phospholipase A(2) in mitogen-dependent VSMC proliferation and determine if Pb(2+) interacts with this system in order to potentiate mitotic events. It was observed that cell proliferation induced by angiotensin II, or fetal bovine serum, required the activation of a Ca(2+)-dependent cytosolic phospholipase A(2) and the subsequent release of unesterified arachidonic acid. This path was affected by Pb(2+) as the metal increased the amount of arachidonic acid accumulation induced by either mitogen. In addition, Pb(2+) potentiated mitogen-induced DNA synthesis when present at lower doses (0.02 or 0.2 mg%), but had no effect on DNA synthesis, or cell numbers, in unstimulated cells. However, a high dose (2 mg%) of Pb(2+) attenuated the DNA synthesis stimulated by angiotensin II, or serum, but induced the accumulation of unesterified arachidonic acid in unstimulated cells. A biphasic effect of Pb(2+) on cell numbers and viability was also observed as 0.02 or 0.2 mg% Pb(2+) did not affect cell numbers or trypan blue exclusion in unstimulated cells, while 2 mg% Pb(2+) reduced cell numbers and viability. It appeared, therefore, that the lower concentrations of Pb(2+) increased arachidonic acid release and DNA synthesis only in stimulated VSMC, perhaps due to further activation of a Ca(2+)-dependent processes. In contrast, the high dose of Pb(2+) reduced DNA synthesis in stimulated cells and reduced cell numbers and viability in unstimulated cells, which may relate to the noted increase in unesterified arachidonic acid.     

Vascular smooth muscle mitochondria at the cross roads of Ca(2+) regulation

Mitochondria play an essential role in the regulation of vascular smooth muscle Ca(2+) signaling being simultaneously integrated in the regulation of ion channels and Ca(2+) transporters, oxygen radical production, metabolite recycling and intracellular redox potential. Mitochondria buffer Ca(2+) from cytoplasmic microdomains to alter the spatio-temporal pattern of Ca(2+) gradients following Ca(2+)-influx and Ca(2+)-release, and thus control site-specific, Ca(2+)-dependent ion channel activation and inactivation. The sub-cellular localization of mitochondria in conjunction with tissue-specific channel expression is fundamental to vascular heterogeneity. The mitochondrial electron transport chain recycles metabolic intermediates that modulate cellular redox potential and produces oxygen radicals in proportion to oxygen tension. Perturbation of specific complexes within the transport chain can affects NADH:NAD and ATP:ADP ratios and radical production, which can in turn influence second messenger metabolism, ion channel gating and Ca(2+)-transporter activity. Mitochondria thus provide the common ground for cross-talk between these regulatory systems that are mutually sensitive to one another. This cross-talk between signaling systems provides a means to render the physiological regulation of vascular tone responsive to complex stimulation by paracrine and endocrine factors, blood pressure and flow, tissue oxygenation and metabolic state.     

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.     

Overexpression of Orai1 and STIM1 proteins alters regulation of store-operated Ca2+ entry by endogenous mediators

Orai1 and STIM1 have been identified as the main determinants of the store-operated Ca²⁺ entry (SOCE). Their specific roles in SOCE and their molecular interactions have been studied extensively following heterologous overexpression or molecular knockdown and extrapolated to the endogenous processes in naïve cells. Using molecular and imaging techniques, we found that variation of expression levels of Orai1 or STIM1 can significantly alter expression and role of some endogenous regulators of SOCE. Although functional inhibition of Ca²⁺-independent phospholipase A₂ β (iPLA₂β or PLA2g6A), or depletion of plasma membrane cholesterol caused a dramatic loss of endogenous SOCE in HEK293 cells, these effects were attenuated significantly when either Orai1 or STIM1 were overexpressed. Molecular knockdown of iPLA₂β impaired SOCE in both control cells and cells overexpressing STIM1. We also discovered important cross-talk between expression of Orai1 and a specific plasma membrane variant of iPLA₂β but not STIM1. These data confirm the role of iPLA₂β as an essential mediator of endogenous SOCE and demonstrate that its physiological role can be obscured by Orai1 and STIM1 overexpression.     

Caveolin-1 Facilitates the Direct Coupling between Large Conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ Channels and Their Clustering to Regulate Membrane Excitability in Vascular Myocytes

L-type voltage-dependent Ca²⁺ channels (LVDCC) and large conductance Ca²⁺-activated K⁺ channels (BK_Ca) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca²⁺ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BK_Ca activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BK_Ca and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BK_Ca by Ca²⁺ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BK_Ca co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BK_Ca can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BK_Ca and Cav1.2 but also the functional coupling between BK_Ca and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K⁺ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BK_Ca-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca²⁺ dynamics including the negative feedback, to control the arterial excitability and contractility.     

MicroRNA-31 regulated by the extracellular regulated kinase is involved in vascular smooth muscle cell growth via large tumor suppressor homolog 2

Aberrant growth of vascular smooth muscle cells (VSMCs) is a major cellular event in the pathogenesis of many proliferative vascular diseases. Recently, microRNA-31 (miR-31) has been found to play a critical role in cancer cell proliferation. However, the biological role of miR-31 in VSMC growth and the mechanisms involved are currently unknown. In the present study, the expression of rat mature miR-31 (rno-miR-31) was determined in cultured VSMCs and in rat carotid arteries. We identified that rno-miR-31 is an abundant miRNA in VSMCs, and its expression was significantly increased in proliferative VSMCs and in vascular walls with neointimal growth. The up-regulation of rno-miR-31 in proliferative VSMCs was inhibited by the inhibitor of mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK). By both gain-of-function and loss-of-function approaches, we demonstrated that rno-miR-31 had a proproliferative effect on VSMCs. We further identified that LATS2 (large tumor suppressor homolog 2) is a downstream target gene product of rno-miR-31 that is involved in rno-miR-31-mediated effect on VSMC proliferation. The LATS2 as a target gene protein of rno-miR-31 is verified in vivo in balloon-injured rat carotid arteries. The results suggest that MAPK/ERK/miR-31/LATS2 may represent a novel signaling pathway in VSMC growth. miR-31 is able to enhance VSMC proliferation via its downstream target gene product, LATS2.     

Bone morphogenetic protein 4 promotes vascular smooth muscle contractility by activating microRNA-21 (miR-21), which down-regulates expression of family of dedicator of cytokinesis (DOCK) proteins

The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins.     

Fine-tuning of store-operated calcium entry by fast and slow Ca2+-dependent inactivation: Involvement of SARAF

Store-operated Ca²⁺ entry (SOCE) is a functionally relevant mechanism for Ca²⁺ influx present in electrically excitable and non-excitable cells. Regulation of Ca²⁺ entry through store-operated channels is essential to maintain an appropriate intracellular Ca²⁺ homeostasis and prevent cell damage. Calcium-release activated channels exhibit Ca²⁺-dependent inactivation mediated by two temporally separated mechanisms: fast Ca²⁺-dependent inactivation takes effect in the order of milliseconds and involves the interaction of Ca²⁺ with residues in the channel pore while slow Ca²⁺-dependent inactivation (SCDI) develops over tens of seconds, requires a global rise in [Ca²⁺]_cyt and is a mechanism regulated by mitochondria. Recent studies have provided evidence that the protein SARAF (SOCE-associated regulatory factor) is involved in the mechanism underlying SCDI of Orai1. SARAF is an endoplasmic reticulum (ER) membrane protein that associates with STIM1 and translocate to plasma membrane-ER junctions in a STIM1-dependent manner upon store depletion to modulate SOCE. SCDI mediated by SARAF depends on the location of the STIM1-Orai1 complex within a PI(4,5)P₂-rich microdomain. SARAF also interacts with Orai1 and TRPC1 in cells endogenously expressing STIM1 and cells with a low STIM1 expression and modulates channel function. This review focuses on the modulation by SARAF of SOCE and other forms of Ca²⁺ influx mediated by Orai1 and TRPC1 in order to provide spatio-temporally regulated Ca²⁺ signals.