All effects were also independent of Dap1, the yeast orthologue of the progesterone receptor. Altogether, our observations provide new insights into the cytoprotective effects of progesterone.The steroid hormone progesterone is not only a crucial sex hormone, but also serves as a neurosteroid, thus playing an important role in brain function. Epidemiological data suggest that progesterone improves the recovery of patients after traumatic brain injury. Brain injuries are often connected to elevated calcium spikes, reactive oxygen species (ROS) and programmed cell death affecting neurons. Here, we establish a yeast model to study progesterone-mediated cytoprotection. External supply of progesterone protected yeast cells from apoptosis-inducing stress stimuli and resulted in elevated mitochondrial oxygen uptake accompanied by a drop in ROS generation and ATP levels during chronological aging. In addition, cellular Ca Progesterone is a sterol-derived hormone that is crucial for female reproductive capacity and plays major regulatory roles in the monthly menstrual cycle and upon conception as well as during pregnancy and embryogenesis. In addition, it also serves as a neurosteroid, thus playing an important role in brain function in both sexes 345681011Saccharomyces cerevisiae, knowing that this organism has repeatedly been shown to be suitable for mechanistic studies of programmed cell death (PCD) 13141516171820212223242526DAP1 - a heme-binding protein related to the mammalian membrane progesterone receptor - did not alter susceptibility towards progesterone treatment. Altogether, we reveal that progesterone exerts potent cytoprotective effects in yeast.In order to investigate the cytoprotective potential of progesterone, we took advantage of 2O2 or acetate, which are both well-known PCD inducers in yeast 1430313233Traumatic brain injury is connected to elevated PCD and ROS accumulation in the brain tissue 282 consumption was significantly enhanced during progesterone treatment, ATP levels were reduced . Altogether, this indicates an uncoupling phenotype with diminished oxidative phosphorylation. Accordingly, we observed reduced growth of wild type yeast upon progesterone treatment on a non-fermentable carbon source (glycerol), while no changes were detected on a fermentable carbon source (glucose) . Importantly, this effect was also observed in a mutant strain lacking the heme-binding protein Dap1, which is the sole yeast orthologue of the human progesterone receptor  0 cells . Altogether, it appears that progesterone impacts yeast mitochondrial respiration in a receptor-independent fashion.To further explore the mechanisms underlying progesterone cytoprotection, we next examined the physiology of mitochondria, since these organelles constitute one of the main sources of ROS 3536372+ homeostasis, knowing that mitochondria are one of the organelles responsible for buffering cytosolic Ca2+ under normal conditions 2+ accumulation in the cytosol of neurons followed by cell death and neurodegeneration 2+ uptake under the influence of progesterone. Specifically, wild type yeast cell cultures were challenged with 150 mM CaCl2 and transient concentrations of cytoplasmic Ca2+ levels ([Ca2+]cyt) / responses were monitored. Progesterone caused a significantly reduced Ca2+ uptake capacity alongside with a faster reduction of cytoplasmic Ca2+ levels . Of note, basal Ca2+ levels before and after the Ca2+ pulse were already lowered when cells were treated with progesterone . However, mitochondrial respiration was not involved in this phenotype, since progesterone treatment continued to affect basic cytosolic Ca2+ levels in respiration-deficient rho0 cells .Next we investigated progesterone effects on Ca2+ channels/transporters in yeast, including the cytoplasmic membrane transporters Cch1 and Mid 1, the organelle transporters Vcx1, Pmr1, Cod1, Yvc1, and Pmc1 as well as Emc7, an ER protein associated to Ca2+ homeostasis. Although Ca2+ uptake and clearance was influenced by some of these gene deletion, all mutants continued to exhibit significantly reduced Ca2+ uptake when treated with progesterone . Thus, the effects observed in wild type cells could not be reversed by single gene deletions in any of these transporters. Similarly, the effects of progesterone treatment on Ca2+ homeostasis/uptake were independent of the mammalian membrane progesterone receptor homolog Dap1 . Taken together, progesterone seems to influence Ca2+ homeostasis/uptake in a general manner, independently from known Ca2+ transporters and respiration capacity.To further investigate the observed phenotypes, we tested single-gene deletion mutants of all currently known CaS. cerevisiae as a model to investigate cytoprotection by progesterone. We observed that progesterone increased stress tolerance of yeast to the well-known PCD inducers H2O2 and acetate 14303132332 consumption (+50%) but lower ATP levels (-50%), arguing for a mitochondrial uncoupling effect. Indeed, growth on the non-fermentable carbon source glycerol was diminished in the presence of progesterone. Notably, mild uncoupling induced by chemical substances (such as dinitrophenol), caloric restriction or ectopic expression of mammalian uncoupling proteins in yeast - S. cerevisiae does not possess any known uncoupling proteins 43442 consumption with decreased 32P uptake (as a parameter for ATP production) has been reported for isolated rat mitochondria treated with progesterone 0 strain was not influenced by progesterone treatment.Here, we establish 2+ homeostasis and, in particular, on Ca2+ susceptibility/uptake. However, we could not identify any single Ca2+ channel in yeast that would influence these effects. However, we cannot exclude that yet unidentified Ca2+ channels or a combinations of known Ca2+ channels mediate these effects 2+ homeostasis could reside in its direct interaction with biological membranes. Since the chemical structure of progesterone shows four-ring as well as hydrophobic backbone and polar groups at both ends of the molecule, it could directly interact with cellular and mitochondrial membranes 2+, H+). This mode of action could connect our observations of mitochondrial uncoupling and modulation of Ca2+ homeostasis. Of note, a progesterone-treated rho0 strain still showed Ca2+ effects but no enhanced stress tolerance, suggesting that altered Ca2+ homeostasis may lie upstream of mitochondrial uncoupling. However, these mechanistic hypotheses remain to be empirically tested.Progesterone had major effects on Ca2+512+2+ uptake in the rabbit myometrial smooth muscle cells, it increased the accumulation of Ca2+ in mitochondria 2+ levels as well as the capacity of these cells to accumulate Ca2+in different cellular compartments. Similar effects were reported for other types of smooth muscles 562+ channels 562+ signaling and the following inhibition of excitotoxic neuron death seem to be the neuroprotective mechanism induced by acute administration of progesterone after various neuronal injuries 596062Certainly, the putative relevance of the herein described progesterone effects for TBI pathology remains to be explored. In some mammalian cell types, progesterone leads to a significant increase of intracellular CaThe role of progesterone in the pathological development of TBI has been well described in recent years. It has been shown that progesterone improves the function of the blood-brain-barrier after TBI 2+ levels, uncoupled mitochondria and ROS reduction - were not lost when the sole possible yeast orthologue of the human progesterone receptor was removed from the system. This suggests that progesterone mediates its broad cytoprotective effects through other proteins than steroid receptors or perhaps with cellular membrane lipids. We surmise that yeast constitutes an ideal platform for exploring these effects in further detail.Interestingly, the intracellular effects exerted by progesterone in our model - reduced intracellular CaS. cerevisiae strains (Table 1) were inoculated to 5*105 (for growth curve OD600 of 0.05) cells in SC medium containing 0.17% yeast nitrogen base , 0.5% (NH4)2SO4 , 30 mg/L of all amino acids (except 80 mg/L histidine and 200 mg/L leucine), 30 mg/L adenine, and 320 mg/L uracil with 2% glucose (SCD) or alternatively with 3% glycerol (SCGly), w/o treatment with progesterone . Controls were treated with respective solvent (EtOH). Where indicated, stress (acetate or H2O2) was inflicted as described previously in mid-log phase . Due to the inherent reduced respiration-rate of BY4741 strains, TB50a strains were used for respiration-related experiments. DAP1 deletion was carried out by classical homologous recombination 67600 of 0.05 in SCD media and SCGly media with or without 10 µg/ml progesterone addition. Untreated cultures were supplemented with 0.1% EtOH for solvent control. To obtain growth curves, 300 µl of respective cultures per well were transferred into Honeycomb® plates, and measured with Bioscreen C MBR system (Oy Growth Curves Ab Ltd.) for a period of 48 hours at 28°C, using continuous shaking and OD600 measurements every 30 minutes.Cells from ONC in SCD media were inoculated to an ODOxygen consumption was measured using a FireSting oxygen electrode (Pyro-Science) under constant stirring at a temperature of 28.0 ± 0.2°C in sealed 2 ml bottles. The corresponding cell counts were measured using a CASY Cell Counter, whereas percentage of living cells in the sample were established by flow cytometry with propidium iodide (PI: 100 ng/ml) stained samples. The slope of the oxygen concentration as the function of time in its linear region was calculated and normalized to the number of living cells in the sample.6 cells from each sample were collected, washed and incubated with DHE solution (2.5 µg/ml in PBS) for 10 min in the dark. After washing samples were re-suspended in PBS buffer and measured using flow cytometry. The relative mean fluorescence measured for the cell population was used for analysis Oxidation of non-fluorescent di-hydroethidium (DHE) to fluorescent ethidium was used to measure ROS accumulation in yeast cells ATP extraction was done with flash-frozen cells by adding 0.5 ml preheated (90°C) BES buffer and incubation at 90°C for 3 minutes. After centrifugation, supernatants were stored at -80°C until the measurement. ATP levels were determined by using the ATP detection kit from Invitrogen in a Luminoskan (Thermo Scientific).2+]cyt were measured using yeast strains carrying the vector pYX212 encoding the bioluminescent protein aequorin under the control of a TPI promoter. For analysis of the cellular response to high doses of external Ca2+, an equivalent of 6*106 cells was harvested, resuspended in 200 μl SCD containing 4 μM coelenterazine and incubated for 1 h in the dark. After washing cells were measured in a Luminoskan for 10 s and then challenged with high dose of Ca2+ (pump injection of 150 mM Ca2+). Kinetics were recorded over 120 s. The luminescence signal was normalized to the OD600 of each well and reported in relative luminescence units, normalized to the global maximum value of the ethanol treated control of the respective run for better comparability.[CaClick here for supplemental data file."http://microbialcell.com/researcharticles/the-neuroprotective-steroid-progesterone-promotes-mitochondrial-uncoupling-reduces-cytosolic-calcium-and-augments-stress-resistance-in-yeast-cells/".All supplemental data for this article are also available online at


The channel-blocking activity is most apparent in ASIC3, as removing Ca2+ results in channel opening, with the site’s location remaining unresolved. Here we show that a ring of rat ASIC3 (rASIC3) glutamates (Glu435), located above the channel gate, modulates proton sensitivity and contributes to the formation of the elusive Ca2+ block site. Mutation of this residue to glycine, the equivalent residue in chicken ASIC1, diminished the rASIC3 Ca2+ block effect. Atomistic molecular dynamic simulations corroborate the involvement of this acidic residue in forming a high-affinity Ca2+ site atop the channel pore. Furthermore, the reported observations provide clarity for past controversies regarding ASIC channel gating. Our findings enhance understanding of ASIC gating mechanisms and provide structural and energetic insights into this unique calcium-binding site.Acid-sensing ion channels (ASICs) evolved to sense changes in extracellular acidity with the divalent cation calcium  sense changes in extracellular acidity with Ca In disease and human health, ASICs play a role in neurodegeneration following ischemia and inflammatory pain6. Four genes encode six ASIC subtypes , each exhibiting additional physiological functions12. In the central nervous system, ASIC1 acts as the primary proton receptor in the brain and has been implicated in stroke13, while the peripherally located ASIC3 primarily mediates the perception of pain15.Acid-sensing ion channels (ASICs) are trimeric proton-activated sodium channels characterized by protein subunits comprised of intracellular amino-termini and carboxyl-termini, two transmembrane domains, and a large extracellular domain. These channels are part of the epithelial sodium channel (ENaC)/degenerin (DEG) family of ion channels+) and calcium (Ca2+) concentrations. This interplay between cations has resulted in two distinct activation models: the relief of Ca2+ block and the allosteric gating mechanisms20. The Ca2+ block model19 proposes a high-affinity Ca2+-binding site sits at the extracellular side of the channel pore, and argues that proton-catalyzed Ca2+ release from this block site is sufficient to induce channel opening, accompanying no conformational change. This model was considered controversial at the time, but intriguing with the data shown. In particular, the model appears to work well for ASIC3 but cannot explain why disruption of the single Ca2+ block site does not constitutively open the channels18, suggesting an underdetermined gating manner independent of Ca2+ block. In the allosteric mechanism20, additional Ca2+/H+-binding sites are presumed to exist within multiple extracellular protein domains where conformational changes are caused by proton binding and Ca2+ unbinding, leading to channel opening. In support of the allosteric hypothesis, studies with ASIC1a have predicted a second Ca2+ binding/modulating site that is located near the wrist region and was shown to mediate Ca2+ regulation of proton gating18. Typically, the Ca2+ sensitivity is most unique in ASIC3 among the five ASIC subtypes, as simple removal of extracellular Ca2+ alone results in channel activation19, while the same removal does not result in ASIC1 opening in most cases18. Most likely, the Ca2+ block and allosteric effects contribute differentially to gating regulation among the ASIC family members. Noticeably, several conserved residues at the channel pore entrance have been implicated in Ca2+ binding23, however, little is known regarding the exact location of the putative site and its chemical principles for Ca2+ block action. Still, whether protons and Ca2+ compete for binding at one site or whether Ca2+ unbinding allosterically affects proton-induced activation remains to be further resolved.The gating behavior of many ASICs is intricately linked to the changes of both extracellular protons (H2+ sensitivity of rASIC3 is influenced by a ring of glutamate residues (rASIC3 Glu435) located above the channel gate. Mutation of this residue to glycine, the equivalent amino acid in cASIC1, diminishes the Ca2+ block effect in rASIC3. Correspondingly, the reverse substitution at the equivalent position in cASIC1, i.e., G429E, results in increased proton sensitivity and channel activation in the absence of Ca2+. Furthermore, our molecular dynamics simulations unveil the exact location of the Ca2+ block site and provide the structural and energetic characterization at this site. Our data provide strong evidence supporting the competition between H+ and Ca2+ at the gating site, and suggest that a pronounced local conformational change is necessary for channel opening accompanying proton-aided Ca2+ release.Here, we investigate the gating mechanisms in ASICs by combining the electrophysiological and computational approaches, focusing on the chicken ASIC1 (cASIC1) and rat ASIC3 (rASIC3). We show that the unique Ca2+ block activation mechanism was behind the gating characteristics of ASIC319, the residues near the channel gate may differ between the ASIC subtypes. We compared the primary sequences between cASIC1 and rASIC3 in the transmembrane segments to pinpoint the residues that were responsible for ASIC distinct Ca2+ sensitivities. Interestingly, we identified a residue positioned approximately one alpha-helical turn from the aspartate gate (Asp433 in cASIC1), which corresponds to glycine in cASIC1 (Gly429) but is glutamate in rASIC3 (Glu435)  and subsequently to pH 7.4 environment with no Ca2+ , resulting in a channel with remarkably reduced currents compared to its wild type and other channels studied , indicating an ASIC3-like Ca2+ sensitivity afforded by the glutamate substitution.To test our hypothesis that the glutamates that were an alpha-helical turn away from the gate were responsible for a Caied Fig. . On the 2+ and the corresponding substitution in cASIC1 (G429E) conferred rASIC3-like characteristics. Next, we investigated the influence of this residue on the proton sensitivity. We generated the pH response profiles for the wild-type cASIC1 and rASIC3, along with the cASIC1 G429E mutant. In these experiments, both conditioning and test solutions either had 1 mM CaCl2 or no additional CaCl2. The cASIC1 pH sensitivity was influenced by the presence of 1 mM Ca2+ . When cASIC1 was activated in the absence of Ca2+ , the pH50 was 7.01 ± 0.03, a more alkaline value, with a Hill coefficient of 2.05 ± 0.23 (Table n = 5). Both the low calcium pH50 and Hill value were significantly different than the corresponding values in the presence of Ca2+ (p < 0.001). rASIC3 has an established sensitivity to extracellular Ca2+19. Here, we confirmed that rASIC3 became more sensitive to alkaline pH in the absence of Ca2+ by showing a pH50 value of 7.32 ± 0.04 with a Hill coefficient of 1.48 ± 0.20 . The value represents a significant difference (p < 0.01) compared to that observed in the presence of Ca2+: pH50 of 6.86 ± 0.02 with a Hill value of 3.54 ± 1.08 .The glutamate residue, rASIC3 Glu435, influenced channel opening following the removal of Caa2+ Fig. , display50 values are significantly different between the wild-type cASIC1 and G429E mutant channels in the presence of Ca2+ . In the absence of Ca2+, pH50 in the mutant G429E was shifted to more alkaline (7.42) than that in wild type (7.01)  of GFP-positive cells had noticeable currents elicited by pH 6.6. The average current is 134 ± 41 pA in rASIC3 E435G compared to 5397 ± 706 pA in wild-type rASIC3. These data suggest that the single mutation at ASIC3 may impair channel insertion into the membrane or the gating mechanism. Poor functional expression prohibited us from achieving stable recordings required for pH response studies.The pH2+ block (binding) site at the beginning of the transmembrane segments in rASIC3 that subtly defines its unique Ca2+ sensitivity. To shed additional light on this mechanism, we performed all-atom molecular dynamics (MD) simulations on the cASIC1 G429E mutant and wild-type channels in an explicit solvent and membrane environment with and without Ca2+ at the channel pore mouth  upon removing Ca2+  or an octahedral coordination (involving 6 ligands) with the conserved gating residue Asp433 on each subunit, Glu429 on one subunit, and two water molecules  channel TRPV626 that also exploit a cluster of four carboxylate groups for binding Ca2+ 28. The use of non-polarizable force fields also tends to overestimate the binding affinity to Ca2+; however, the results are reasonable as a relative measure31.Using two distinct ion models representing the Caems Fig. . The Ca22+ impeded water flux through the gate . Yet, the Na+ inside the extracellular vestibule failed to diffuse into the intracellular vestibule through the channel gate . The same inter-subunit interactions are disfavorable in the desensitized state due to steric clashes  channels under various protonation conditions to probe the impact of protonation at the Asp433 ring on Ca2+ affinity (Supplementary Table 33 (Methods section). For the mutant system, single and double protonation at the Asp433 cluster resulted in negligible changes in Ca2+-binding affinity  would effectively destabilize Ca2+ and eventually eliminate Ca2+ blockade. The wild-type system exhibited a similar trend, but required less protons to destabilize Ca2+ binding  protocol35 could be a desirable choice for further studying pH-dependent effect on channel gating. Yet, it would not qualitatively influence the overall trend herein.In the above simulations, the gating residue Asp433 was assumed to be in a deprotonated state . To explore the binding completion between Ha2+ Fig. , as compa2+ Fig. . Hence, 2+-binding site above the channel gate in rat ASIC3 and engineer this site within cASIC1 G429E mutant receptor. This site implicates four acidic residues, including three symmetry-related aspartate residues and one glutamate residue from either subunit  mutant channels. The pH sensitivities of the rASIC3 and the mutant channel were remarkably similar, with pH sensitivity shifting to more alkaline pH values in the absence of Ca2+ . Based on the data presented here, we conclude that the mutation has introduced proton-binding site cooperativity that does not change when Ca2+ is removed from external solution. Further study is needed to demonstrate how this site affects specific ASIC gating properties, such as activation and deactivation kinetics.Introducing the cASIC1 G429E mutation has a pH2+-binding site at the extracellular mouth of the channel. Conversely, we have provided evidence to suggest that a weak Ca2+-binding site is present at the pore gate in other ASICs. The gating aspartate residue (cASIC1 Asp433) is highly conserved across all of ASICs 21. One of the proposed cesium densities appeared to coordinate with the carboxylates of cASIC1 D433. What was not seen in the protein electron density map were other additional contributors to this coordination, unlike the coordination observed with the second cesium electron density. The second cesium ion coordinated with the carboxylates of D433 and the backbone oxygen atoms of G432, in a trigonal antiprism pattern. Coordinating with cesium provides evidence that these residues coordinate with cations, in at least a weak-binding site, and suggests that other cations  may bind here. Furthermore, the presence of a weak Ca2+-binding site at the pore gate may provide an explanation for potentiation of the currents in mouse ASIC1a38 and fish ASIC116 by decreasing the concentrations of external Zn2+ and Ca2+. Interestingly, the glutamate residue (Glu435 in rASIC3) is only conserved among the ASIC3 subtype, whereas the equivalent position is occupied by glycine or alanine for the other ASIC subtypes  and always 1 for Ca2+. However, the Immke–McCleskey model suggested Ca2+/H+ site catalysis fails to trigger a conformational change; in other words, these channels should be constitutively active (open) upon removal of Ca2+19. In contrast, our simulations of Ca2+-bound and Ca2+-free channels demonstrated that considerable conformational changes occurred  in rat ASIC1a (rASIC1a) by single (E425G and D432C) or combined substitutions did not open the channels constitutively. In light of the crystallographic and computational data, we contend that a re-interpretation of the previous experimental data is warranted. The rASIC1a Glu425 (Glu426 in cASIC1) is very unlikely to comprise an additional Ca2+ binding/modulating site in that: (i) it is exposed to the outside of the extracellular vestibule in our simulations and the reported cASIC1 structures40; (ii) the Glu426 ring diameter is large (22~30 Å) in each structure; and (iii) Glu426 lacks partners to coordinate/stabilize Ca2+. Instead, the rASIC1a E425G mutation may result in disengagement with Arg64 (Arg65 in cASIC1) in the intermediate state as indicated, thereby facilitating pore opening and destabilization of Ca2+ binding, especially alongside D432C replacement. Additionally, the D432C mutation is not able to completely destroy the Ca2+ block site as the cysteine itself can contribute a ligand via the cysteine sulfhydryl24. In view of the spatial proximity of the trapped Ca2+ to the Asp433 side chains 18. Furthermore, the observation that the rASIC1a double mutant retains the principal gating characteristics of the wild type 18 suggests the possibility for ASIC1 to have Ca2+/H+ binding sites in the extracellular domain that allosterically mediate functional conformational changes.Though simple, the model of a single Cains Fig. , a low-a40 have revealed a prominent acidic pocket located in the extracellular domain, where there are several pairs of close carboxyl–carboxylate interactions between aspartate and glutamate residues . This acidic pocket was proposed to be engaged in the H+/Ca2+ sensing and the associated allosteric control of channel activation40. Sequence analysis shows that the equivalent residue to cASIC1 Asp346 is a serine in rASIC3, the only difference in the acidic pocket composition between the two channels. To test the hypothesis that the residues in the acidic pocket function as the H+/Ca2+ allosteric site, we additionally performed two sets of simulations .The crystal structures and the experiments of cASIC12+ is strengthened by the presence of additional acidic residues near the channel mouth. The inclusion of the Gly to Glu mutation at the cASIC1 position 429 and the converse in rASIC3 may be key for the Ca2+ block site in the ASICs studied. A similar Ca2+ sensitivity was observed in marine ASIC, where a reduction in activating solution pH and Ca2+ concentration resulted in a shift towards more alkaline pH in the activation profile16. From this study, there was a significant finding in support of our proposal that there are weak Ca2+ block sites in non-ASIC3 subtypes. In the marine ASIC study, Ca2+ blocked low pH generated current in a voltage-dependent manner at higher concentrations that what are needed for gating, which suggests that there is a weak Ca2+ block site within the electric field, or channel pore16. The marine ASIC study highlights the need to keep under consideration that differences exists between the ASIC subtypes.The channel-binding site for Ca2+-block site at the entry of the ASIC3 channel pore is the result of a single acidic residue one alpha-helical turn away from the canonical ASIC gate. The data here confirm the presence of a high-affinity Ca2+-binding site and reveal for the first time the Ca2+-binding mode at this block site. Future studies, and development of novel therapeutics targeting ASICs, will be needed to account for the unique differences in ASIC subtype gating and calcium sensitivity.In summary, the combined electrophysiological and computational results suggest that the CaN-methyl-d-glucamine.All reagents were purchased from Sigma-Aldrich. The external pH was adjusted using 41. Site-directed mutagenesis for cASIC1 G429E and rASIC3 E435G were made using the QuikChange Lightning Site-Directed Mutagenesis kit, according to manufacturer’s instructions (Agilent Technologies). Each ASIC construct was transiently transfected into Chinese hamster ovarian  cells for electrophysiological study using Lipofectamine LTX , as previously reported41. Briefly, cells were plated on 9×9 mm glass coverslips in 35 mm cell culture dishes in completed growth media . After 24 h of incubation, transfection reactions were generated using serum-free media (500 μL) and was placed in sterile microcentrifuge tubes under a laminar flow hood. To each tube, 1 μg ASIC plasmid DNA was added followed by 4 μL of Lipofectamine LTX reagent. Reactions were tapped gently to mix and allowed to incubate for 20 min at room temperature. Next, media was removed from the cell culture dishes and replaced with fresh complete growth media (1.5 mL). Each transfection reaction mix was applied to separate culture dishes, bringing the final concentration to 2 mL. Transfected cells were allowed to grow in a 37 °C/5% CO2 incubator for 18–24 prior to electrophysiological assay. Only cells that exhibited fluorescence were chosen for electrophysiological study.Full-length chicken ASIC1 and rat ASIC3 were subcloned into a vector that attached an enhanced green fluorescent protein to the amino terminusN-methyl-d-glucamine (NMDG). Additionally, intracellular solutions contained (in mM): KCl (100), MgCl2 (5), EGTA (10), HEPES (40), and NaCl (5) at pH 7.35. The extracellular base solution was used for all other solutions and adjusted to the specified pH. Calcium was added at a concentration of 1 mM in calcium-rich solutions and omitted from those mimicking calcium-depleted solutions . Solution exchange was obtained using arrays of microcapillary tubes arranged perpendicularly to the patch cell using digitally controlled PTFE solenoid or pinch valves with a ValveLink8.2 controller (AutoMate Scientific) or a gravity driven Y-tube solution delivery system.Patch pipettes were pulled from borosilicate glass and fire-polished with a final resistance 5–7 MΩ . Whole-cell patch-clamp electrophysiology was performed on an inverted fluorescent microscope fitted with a FITC fluorescence filter (Nikon) that was equipped with an Axopatch 200B patch-clamp amplifier or an upright microscope fitted with a FITC fluorescence filter (Nikon) equipped with a Axopatch 200 B patch-clamp amplifier (Molecular Devices) using pClamp10 data acquisition software. External solutions were applied at room temperature and mimicked the extracellular and intracellular cellular compartment. Extracellular bath and test solutions consisted of (in mM): NaCl (150), KCl (5), MES (5), HEPES (5), with pH adjusted using HCl or 42 using SWISS-MODEL webservers43. Trimeric arrangements for both cASIC1 and resulting rASIC3 models were generated using Pymol molecular visualization system44 with a symmetry command.A three-dimensional model was generated using the cASIC1 desensitized crystal structure (PDB code 4NYK)t-test or F-test, where appropriate. pH response profiles were generated for wild-type cASIC1, rASIC3, and cASIC1 G429E mutant receptors in the presence and absence of calcium to determine the pH that elicits half of the maximal response, or pH50 by fitting with a logarithmic equation. For pH response profiles, pH50 and Hill values, significance was determined using the sum of least-square F test (GraphPad Prism 6). For response to removal of calcium at pH 7.4, upaired Student’s t-test was used for comparison.Peak current amplitudes of test solutions were normalized against a control pH response in each experiment. Statistical significance was determined using unpaired Student’s 21) as a starting point for all the simulations herein. The channel pore of this desensitized structure is closed and assumes considerable conformational similarity to that of the resting, closed state channel46. Given that this work is mainly focused on the transmembrane domain, the used structure is suitable for the simulation aim, though the major difference between the two states in the extracellular domain46. To test our experimental hypothesis, four simulation systems, i.e., the cASIC1 G429E mutant with/without Ca2+ and the wild type with/without Ca2+, were built based on the same procedures. The bound Cl− and water molecules in the crystal structure were retained. To generate the Ca2+-containing systems, 1 Ca2+ ion was placed at the mouth of channel pore  by reference to the position of Cs+ ion at site 2 in the Cs2+-soaked crystals (PDB code: 3IJ4)21. The principal axis of the trimeric cASIC1 was pre-aligned along the z-axis  through the orient plug-in included with VMD47. The construction of the channel/membrane systems was then facilitated using the on-line tool CHARMM-GUI Membrane Builder48. The disulfide bridges rich in the extracellular domain were explicitly taken into account. The protonation states of the ionizable residues outside of the transmembrane domain were predicted using the web server H++ 49, followed by visual inspection. For the symmetry-related Asp433 ring inside the channel pore, four different protonation conditions were considered as described below. The replacement scheme was employed to pack the 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) lipid bilayer around the transmembrane zone of the channels. The dimensions of the membrane plane (x–y) were set to be 118×118 Å, and the water layer thickness from either of the solute boundaries along the z-axis be 20 Å. Na+ and Cl− were added to yield a physiological salt solution of 150 mM. The above settings resulted in a simulation box of ~118×118×153 Å3, a total of 207,000 atoms and a Ca2+ concentration of ~0.8 mM for each system.Since no rASIC3 structure is available, we chose the minimally functional construct (residues 46–451) of the chicken ASIC1 (cASIC1) determined in a low pH desensitized state  and this model has been widely used in combination with the additive, non-polarizable biomolecular force fields (FF) 53. Despite continuous improvements, the non-polarizable FFs and the non-bonded ion parameter still face limitations in accurately characterizing the ion-ligand interactions due to insufficient inclusion of quantum effects, especially at the enzymatic centers50. Polarizable FFs and quantum mechanical description of the metal centers provide a more rigorous physical framework, however, the high computational cost make them untenable with increasing system size (like the membrane system herein), particularly in free energy calculations that require extensive sampling of conformations54. The alternative strategy, viz., the cationic dummy atom model (a.k.a. multisite ion model)55, provides a promising balance between the computational accuracy and efficiency. In this approach, the metal site is represented by a set of dummy atoms connected around a central atom  FFs were used for the solutes (proteins and lipids) and all ions 57; the latest AMBER ff14SBonlysc58 and lipid17 FFs59 were selected for the proteins and lipids, respectively, and the Joung-Cheatham ion parameter sets60 for Na+ and Cl−. In addition, the water was described by the TIP3P model61.Accurate modeling of metal–ion-containing biological systems has been a nontrivial issue in computational chemistry2+ spherical model) and the AMBER FF (plus Ca2+ dummy model) were performed using the package NAMD (v2.10)62 and the GPU accelerated version of AMBER16 engine PMEMD63, respectively. The format conversion of the CHARMM-GUI generated lipids into the AMBER-style lipids was facilitated via the charmmlipid2amber.py script within the AmberTool1663. The input parameters were kept as consistent as possible between the two simulation programs. The periodic boundary conditions were applied in all of the simulations. The short-range non-bonded interactions were truncated at 12.5 Å, with the smoothing function switched on from 10 Å, and the long-range electrostatics were calculated via the particle mesh Eward summation method64 with a grid spacing of 1 Å. The covalent bonds involving hydrogens were constrained employing the SHAKE algorithm65. The time step of integration was set to 1 fs during the minimization and equilibration stages and 2 fs for the production runs. The Langevin thermostat was used to maintain the temperature at 303.15 K, with a damping coefficient of 1 ps−1. The pressure was kept constant at 1.013 bar by the Nosé-Hoover Langevin piston with an oscillation period of 100 ps (in NAMD), or by the Monte Carlo barostat with a relaxation time of 2 ps (in AMBER). The trajectory snapshots were recorded at 10-ps intervals. The channel/membrane systems underwent a sequence of steps to reach stability following the collective variable-based equilibration strategy implemented in NAMD33. In brief, three different types of restraints were simultaneously imposed and gradually released: the planar restraint is used to keep the lipid head groups on the x–y plane that is perpendicular to the membrane normal; the root-mean-square displacement (RMSD)-based restraint is used to maintain the global conformation of the protein; and the dihedral restraint is applied to restrain the local backbone configuration of the protein. Each system was then further equilibrated 150 ns, following a production run in the isobaric–isothermal (NPT) ensemble that reaches 1000 ns. Moreover, in evaluating the effect of increasing protons on the binding of Ca2+ , a set of MD simulations on the mutant and wild-type channels were carried out under different protonation circumstances, wherein the symmetry-related Asp433 cluster was singly, doubly or triply protonated, as compared to the fully charged state applied above. A summary of all the simulations reported in this study is presented in Supplementary Table The simulations with the CHARMM FF  approach33. Compared to MM-PBSA (molecular mechanics-Poisson Boltzmann surface area), MM-GBSA is computationally more efficient and has shown to gain comparable or even better accuracy in predicting the relative affinities66. Specifically, the five water molecules closest to the Ca2+ in each trajectory snapshot were consistently retained and regarded as part of the receptor, given the maximum number of five water molecules involved in coordination with the ion 47, as described in our recent studies68. Moreover, the water density was calculated with the VolMap tool in VMD47 and the radius scaling in VolMap was increased to 2 to produce a smoother map. A detailed comparison and an extended discussion of the results with the two different Ca2+ models are presented in Supplementary Information.The last 300 ns of the simulation trajectories were extracted for the following analyses. In detail, the binding affinities of Caion Fig. . This scData supporting the findings of this manuscript are available from the corresponding authors upon reasonable request.Supplementary Information


Their fundamental mechanism, based on the periodic Ca2+ exchange between the endoplasmic reticulum and the cytoplasm, has been well characterized. However, how the kinetics of cytosolic Ca2+ changes are related to the extent of a physiological response remains poorly understood. Here, we review data suggesting that the downstream targets of Ca2+ are controlled not only by the frequency of Ca2+ oscillations but also by the detailed characteristics of the oscillations, such as their duration, shape, or baseline level. Involvement of non-endoplasmic reticulum Ca2+ stores, mainly mitochondria and the extracellular medium, participates in this fine tuning of Ca2+ oscillations. The main characteristics of the Ca2+ exchange fluxes with these compartments are also reviewed.Ca In non-excitable cells, these repetitive spikes mainly arise through the periodic exchange of Ca2+ between the endoplasmic reticulum (ER) and the cytosol, through the interplay between inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ channels and SERCA pumps2. This basic mechanism, summarized in2+ oscillations with increasing concentrations of InsP3 accompanying the rise in stimulation. Such a process is referred to asfrequency encoding. It was often hypothesized that oscillations provide a digital signal to downstream effectors that are in turn stimulated in an ON or OFF manner. Indeed, if a process is activated above a threshold Ca2+ concentration, oscillations allow Ca2+ to reach this threshold repetitively even if the average Ca2+ signal remains below the threshold4.Most of the time, the hormone-induced Ca2+ oscillations8. Although intuitively attractive, suchfrequency sensitivity of the downstream targets of Ca2+ has not been well corroborated by data. Besides the beautiful example of Ca2+-dependent calmodulin kinase II regulation by high-frequency Ca2+ spikes9 or of selective gene expression in T-lymphocytes4, there are few clear examples of physiological responses to Ca2+ increases that are quantitatively controlled by the frequency of the Ca2+ spikes. This statement does not mean that frequency encoding does not occur or that the frequency of Ca2+ oscillations does not affect the extent of the Ca2+-mediated physiological response. Indeed, a higher frequency of oscillations implies a larger average Ca2+ level, which may beper se the reason for the larger response. However, modulating the amplitude of the oscillations, their baseline level, or the duration of the spikes also modifies the average level and hence the response. As another example, spikes preceded by an important pacemaker-like Ca2+ increase could activate slower downstream targets characterized by a low threshold of activation. In such cases, frequency cannot be considered as the key characteristic of the oscillatory pattern and the response is not simply frequency sensitive. However, in the numerous studies about Ca2+ oscillations, frequency is the most studied parameter and the most commonly related to the extent of Ca2+-mediated physiological responses.Based on the observed frequency encoding of the extracellular signal, it was also postulated that the physiological response in the form of secretion, gene expression, proliferation, etc., would in turn be sensitive to the frequency of Ca2+ oscillations is not so surprising given that the period of Ca2+ oscillations can be subject to a significant level of randomness  that mediates Ca2+ release from mitochondria slightly affects cytosolic Ca2+ changes and, by doing so, significantly reduces Ca2+-dependent processes, such as glutamate release, wound closure, and proliferation20. Cell survival, death, and adaptation are sensitive to changes in Ca2+ patterns due to the interplay between ER/cytoplasmic Ca2+ exchanges and mitochondria and lysosomes21.Shigella bacteria also fine tune the Ca2+ responses when they invade epithelial cells. While the wild-type strain induces rather smooth and low-amplitude Ca2+ variations in the cytoplasm of the host cell, a less-invasive mutant strain induces more robust Ca2+ responses which, paradoxically, are associated with a higher survival of the host cells during the first hours following invasion22.Also, recent investigations tend to suggest that rather than the frequency alone, the detailed dynamic characteristics of the Ca3 receptors expressed by a given cell – which have been shown to substantially affect the shape of the Ca2+ oscillations25 – are critical for cell death and survival decisions26. Finally, bioinformatics analyses highlighted that in cancer cells and tissues, the main processes associated with Ca2+ dynamics that are perturbed are the mechanisms of store-operated calcium entry (SOCE) and of calcium reuptake into mitochondria27. Both of these processes are related to the fine tuning of Ca2+ oscillations, as discussed below. Altogether, these observations call for a more detailed understanding of oscillation-associated Ca2+ dynamics. Understanding why Ca2+ oscillates and what regulates the frequency of oscillations is not sufficient to understand their physiological impact, but the duration and shape of the peaks, their sustainability, and the baseline Ca2+ level must be carefully taken into account. In the following sections, we elaborate on two key controllers of the InsP3R-based Ca2+ oscillations, both related to Ca2+ stores other than the ER, namely the mitochondria and the extracellular medium. We briefly review and discuss some of the main recent observations about their interplay with the InsP3-induced Ca2+ spikes.On another level, the precise isoforms of InsP2+ entry into mitochondria stimulates metabolism, thereby coupling ATP synthesis with energy demand28. That Ca2+ exchange between the cytosol and the mitochondria in turn affects InsP3-induced cytosolic Ca2+ signals was put forward quite early30, but this concept was somewhat put aside for a decade. The molecular identification of the mitochondrial Ca2+ uniporter (MCU), a voltage, cytosolic, and mitochondrial Ca2+-sensitive transporter34, awakened interest in this question. Ca2+ entry into mitochondria through the MCU is a highly non-linear function of cytosolic Ca2+33. The MCU is in fact the Ca2+ pore-forming component of the uniporter and is part of a large complex of proteins that are required for Ca2+ channel activity or to regulate it under various conditions. For example, MICU1  limits mitochondrial Ca2+ influx at low cytosolic Ca2+ concentration and the interaction between the MCU and MICU1 requires the expression of another component, called EMRE for essential MCU regulator35. Ca2+ efflux back into the cytoplasm occurs through a NCLX. As expected, modifying any of these pathways affects the frequency of the oscillations; interestingly, increasing the activity of the MCU can both increase and decrease the frequency of oscillations36. In addition to its effect on the frequency of the oscillations, the MCU controls the width of the spikes and the sustainability of the oscillations, as knocking-down the MCU broadens Ca2+ oscillations and accelerates the rundown of the oscillations in rat basophilic leukemia (RBL)-1 cells  of poorly identified molecular nature has been reported in studies on isolated mitochondria from cardiac and liver cells46. However, the implication of RyR and RaM in mitochondrial Ca2+ influx remains to be firmly established48. Finally, in a more indirect manner, mitochondrial metabolism also affects cytosolic Ca2+ signals, mainly by modifying the mitochondrial voltage across the internal mitochondrial membrane, which greatly affects the activities of all of the above-mentioned fluxes29. All of the above-cited phenomena are thus potentially implicated in the control of the detailed characteristics of Ca2+ oscillations. Their interplay with the activities of the MCU and the NCLX is regulated by an intricate and complex network of interactions implicating cytosolic and mitochondrial Ca2+ as well as mitochondrial voltage and numerous accessory proteins.The kinetics of the MCU and the NCLX have been fairly well characterized, but much remains to be done to fully identify other fluxes. The permeability transition pore (PTP) in its low conduction mode participates in the Ca2+ oscillations are sustained by SOCE from the extracellular medium49. This mechanism involves the stromal interaction molecule (STIM) and the Orai protein50. The transmembrane ER protein STIM is sensitive to Ca2+ changes in the ER through an EF-hand facing the lumen of the store. Decrease in luminal Ca2+ below ~200 μM (for the STIM1 isoform) leads to STIM aggregation, followed by migration to ER–plasma membrane junctions. Here, STIM oligomers can bind and activate Orai, a four-transmembrane-domain plasma-membrane-spanning protein, thus forming a channel (known as CRAC for Ca2+-release-activated Ca2+ channel) allowing Ca2+ to enter down the chemical gradient. Another STIM isoform, STIM2, has a lower affinity for ER Ca2+, which allows for activation of Ca2+ entry at moderate ER depletion, although at a reduced rate51. Mammalian cells have genes for the three homologs Orai1, Orai2, and Orai3, and it is thought that Orai2 and/or Orai3 act as compensative types for the lack of Orai1. Orai channels are made of multiple subunits, and CRAC channel gating by STIM is best described by a Monod-Wyman-Changeux scheme in which tetramers of Orai have four STIM binding sites52.Cytosolic Ca2+ pools are emptied artificially, studies performed in a variety of cell types demonstrate that STIM expression is essential for an ensemble of physiological processes53. To quote here just one recent example, in airway smooth muscle, altered expression and function of STIM/Orai proteins have been linked to pathologies including restenosis, hypertension, and atopic asthma54.Although the mechanism just described has most of the time been investigated in conditions when the Ca2+ entry displays a hysteretic behavior: STIM-Orai association and dissociation do not occur at similar ER Ca2+ concentrations55. Although the origin and the physiological significance of this unusual behavior remains unknown, it might be related to the inactivation of SOCE-mediated Ca2+ entry by cytosolic Ca2+ itself, a process that has long been thought to be mediated by calmodulin56 but was recently suggested to be due to a calmodulin-independent conformational change within the pore allowed by two specific Orai residues, Y80 and W7658. This Ca2+-induced inactivation (CDI) of SOCE allows for a modulation of Ca2+ entry depending on the level of cytosolic Ca2+, thus shaping the oscillations.The STIM-Orai pathway for Ca2+ pattern and its downstream targets are also much documented. Through the specific ER Ca2+ sensor STIM2 that has a high KM for Ca2+, SOCE determines the basal level of Ca2+ in HeLa cells51. More generally, SOCE-mediated Ca2+ entry has a significant effect on Ca2+ oscillations, as it can in turn affect all Ca2+ exchanges between the cytoplasm and the internal stores. A less straightforward but highly interesting effect was uncovered in RBL-2H3 cells. Because the activity of the plasma membrane phosphatidylinositol 4-phosphate 5 kinase that replenishes the PIP2 pool is Ca2+ sensitive, SOCE is necessary to avoid the rundown of the oscillations. Indeed, in the absence of SOCE, cysteinyl leukotriene type I receptor activation leads to the exhaustion of the PIP2 pool and hence to the disappearance of InsP3-induced Ca2+ release from internal stores59.The key effect of STIM and Orai on the oscillatory Ca2+ oscillating, gene expression is entirely driven by SOCE and proceeds as an all-or-nothing process in individual cells60. During maturation of mouse oocytes, STIM1 and Orai1 control the basal Ca2+ level and the whole Ca2+ homeostasis, thus controlling meiosis resumption61. At fertilization of pig eggs, overexpression of STIM1 and Orai1 substantially decreases the number of Ca2+ spikes induced by sperm binding (62. This observation contrasts with the observed decreased frequency of fertilization-induced Ca2+ oscillations in hamster eggs when decreasing external Ca2+ concentration63. It shows that the control of Ca2+ signaling by SOCE cannot be directly assimilated to the control of Ca2+ signals by the extracellular Ca2+ concentration. Interestingly, if SOCE is inhibited, fertilization is also impaired, as oscillations last for about 1 hour instead of at least 2 hours. In mice, cytoplasmic Ca2+ levels are elevated for ~50% of the time in STIM1+Orai1-overexpressing oocytes in the first 2 hours after fertilization, as compared to only less than 20% of the time in control oocytes. Despite this larger Ca2+ signal, most of the STIM1/Orai1-overexpressing oocytes do not reach the two-cell stage64. However, female mice lacking Orai1 are fertile65, while male mice are sterile due to severe defects in spermatogenesis66.In RBL cells displaying Cabinding . Moreove2+ oscillations does not provide an ON/OFF signal for the Ca2+-mediated response to the stimulus nor is the extent of the response only determined by the frequency of the oscillations. How the exact shape of this Ca2+ signal is controlled, i.e. what we refer to here as its fine tuning, can alter the response qualitatively and quantitatively. Ca2+ exchanges with mitochondria and SOCE play an important role in fine tuning cytosolic Ca2+ oscillations. Interestingly, there is some dynamic interplay between these two Ca2+ sources as, in mesothelial cells in the absence of external Ca2+, mitochondrial Ca2+ takes over to provide a Ca2+ influx pathway during oscillations67. Moreover, other organelles such as the Golgi69 or the acidic organelles71 are also involved. Genetic regulations further complicate the intricate network of Ca2+ fluxes: in lymphocytes, Ca2+-dependent activation of CREB controls the level of expression of the MCU, which explains why the expression of this uniporter is modified in the absence of InsP3 receptors or of the STIM/Orai machinery72. Much remains to be done to understand how diverse factors interact to control the detailed pattern of Ca2+ oscillations and how this pattern can in some cases significantly affect the physiological response. Integration of the Ca2+ signal over long periods of time may explain why small changes in the pattern of the Ca2+ spikes become significant in some cases. By such integration, the extent of activation of the downstream targets of Ca2+ is modified by apparently minor changes in the Ca2+ oscillatory pattern, which are less visible than its frequency. Spatial aspects most certainly also play an important role in this respect, as the Ca2+-sensitive targets are far from being homogeneously distributed within the cell73. Finally, the kinetics and thresholds for Ca2+ activation of these targets are expected to be at least as important, as in other signaling cascades playing a key role in the storage of information74.It is by now clear that in many cases the existence of Ca


Cytogenetic abnormalities play important roles in the diagnosis and prognosis of leukemias . TrisomyChromosome aberrations detected in therapy-related AML (t-AML) and de novo AML cases are identical but their frequencies may differ . In a seAutosomal trisomies have been described in several hematologic malignancy cases. The first case of sole trisomy 6 was reported in aplastic anemia. Other reports showed that trisomy 6 was associated with hypoplastic bone marrow, dyserythropoiesis, and AML . MohamedYu et al. reviewed ten reports in PubMed describing 18 cases of AML presenting with trisomy 6 as the sole karyotypic abnormality along with 3 cases of their own [7]. They concluded that there were no direct correlations between the number of blasts and the percentage of abnormal metaphases. They could not identify any correlation between morphology or prognosis and trisomy 6 [7].Under these circumstances, as in our case, we lack information on the impact of trisomy 6 on prognosis in secondary AML patients.


Notably, the function of TRPA1 depends on Ca2+, with low Ca2+ potentiating and high Ca2+ inactivating TRPA1. However, it remains unknown how Ca2+ exerts such contrasting effects. Here, we show that Ca2+ regulates TRPA1 through calmodulin, which binds to TRPA1 in a Ca2+-dependent manner. Calmodulin binding enhanced TRPA1 sensitivity and Ca2+-evoked potentiation of TRPA1 at low Ca2+, but inhibited TRPA1 sensitivity and promoted TRPA1 desensitization at high Ca2+. Ca2+-dependent potentiation and inactivation of TRPA1 were selectively prevented by disrupting the interaction of the carboxy-lobe of calmodulin with a calmodulin-binding domain in the C-terminus of TRPA1. Calmodulin is thus a critical Ca2+ sensor enabling TRPA1 to respond to diverse Ca2+ signals distinctly.TRPA1 is a Ca The survival of animals depends on their ability to detect damaging signals in the environment. TRPA1 is a polymodal damage sensor detecting a wide array of damaging signals including noxious cold, chemical and mechanical stimuli11345TRPA1 is highly conserved in detecting harmful chemicals across different species810213141516172192+ is a ubiquitous second messenger playing a pivotal role in regulating TRPA121222+ activates and/or potentiates TRPA1 at low concentrations21222+]i after activating phospholipase C (PLC)-coupled receptors924252+]i has also been proposed as a mechanism by which cold activates TRPA1212+ potentiated TRPA1 response to mechanical stimuli2+-dependent activation and/or potentiation of TRPA1 thus further diversifies the function of TRPA1. Secondly, high Ca2+ rapidly inactivates TRPA1 following initial channel activation, a process known as desensitization or tachyphylaxis122229Ca2+-dependent regulation of TRPA1, the underlying mechanisms remain elusive. It has been reported that Ca2+ activates TRPA1 by directly binding to an EF hand domain in the N-terminus of TRPA121222329322+ acts on TRPA1 via Ca2+-sensing calmodulin (CaM). CaM binds to TRPA1 depending on Ca2+, and is essential for the basal sensitivity of TRPA1. Importantly, CaM binding is sufficient to evoke TRPA1 potentiation and inactivation without changes in Ca2+, and this effect can be selectively prevented by disrupting the binding of CaM to TRPA1. Therefore, CaM functions as both a Ca2+ sensor and an effector enabling TRPA1 to sense and respond to diverse Ca2+ signals distinctly.Despite the central importance of Ca2+ concentrations potentiate TRPA1 while high Ca2+ concentrations desensitize TRPA122232+ in the modulation of TRPA1, we recorded TRPA1 currents from HEK293 cells expressing TRPA1 elicited by the non-electrophilic agonist carvacrol. We found that peak TRPA1 currents increased progressively with increased concentrations of carvacrol without appreciable desensitization in the absence of [Ca2+]e  (2+]e and [Ca2+]i were chelated (Ca2+-free + BAPTA-AM), negligible TRPA1 currents were induced across all doses of carvacrol , leading to a marked leftward shift in the dose-response curve . By contrast, the inhibition and/or desensitization of TRPA1 became more and more prominent with further increases in Ca2+ at the high Ca2+ range (i.e. >1 mM), resulting in gradually reduced TRPA1 responses, a process reflecting Ca2+-dependent inactivation (CDI), in addition to CDP , we wondered whether Ca2+ act through an intermediate protein, which is not only sensitive to Ca2+, but also binds to TRPA1, forming a Ca2+-sensitive channel complex. CaM is such a candidate with remarkable Ca2+-sensing capability. Indeed, CaM mediates Ca2+ gating of many ion channels, such as TRP, SK, Na+ and Ca2+ channels3435363738We next investigated how TRPA1 senses and responds to different Ca2+. The binding was, however, absent without either Ca2+ or TRPA1/TRPV1 co-expression (2+ (2+]i (∼100 nM). Furthermore, despite a much lower affinity of CaM for Ba2+ over Ca2+ ions2+ elicited substantial TRPA1-CaM binding, albeit reduced when compared to Ca2+ . However, Mg2+ did not induce significant TRPA1-CaM binding  than N-lobe 2+ to CaM can thus be selectively disrupted by mutating two different lobes2+ binding to N-lobe by mutating the first two Ca2+ binding sites (EF12) on N-lobe (CaM12 mutant) (3) and the fourth Ca2+ binding sites (EF4) on C-lobe  caused a 60% and an 87% reduction in TRPA1-CaM binding, respectively (2+ binding sites on C-lobe (CaM34 mutant), or by Ca2+-insensitive CaM1234 in which all four Ca2+-binding sites are mutated2+ loading of CaM, and different Ca2+-binding sites on CaM play distinct roles in TRPA1 binding: CaM C-lobe is more important in triggering TRPA1 binding, while N-lobe may only play an auxiliary role in the binding process. Even within CaM C-lobe, the role of EF3 and EF4 is not identical, with EF4 having a higher capability of triggering TRPA1 binding than EF3.In support of CaM pull-down assay, TRPA1 was found to bind to CaM in a co-immunoprecipitation experiment . CaM con mutant) . Mutatinectively . TRPA1-C-lobe CaM mutant, 2+ channels4344CaM binds to a variety of molecular targets through several classes of CaMBD such as the “IQ” motif in Cabona fide CaMBD, we synthesized a peptide identical in sequence to the 17AA. As anticipated, the binding of CaM to the C-terminus of TRPA1 was abrogated by incorporating the peptide in CaM pull-down assay , though incompletely , suggestOf note, the CaMBD is rich in hydrophobic residues, consistent with the binding preference of CaM2+. Indeed, CaM over-expression potently increased TRPA1 responses to all doses of carvacrol in nominal 0 [Ca2+]e  is sufficient to trigger enhanced TRPA1-CaM binding in the presence of excess CaM.To test a possible role for CaM in regulating TRPA1, we first investigated whether CaM potentiates TRPA1, as does low Ca [Ca2+]e , suggest binding  and 4A. 2+ range between 10 μM and submaximal 0.5 mM that elicits no appreciable desensitization. TRPA1 current evoked by carvacrol was rapidly potentiated by 10 μM Ca2+  or no binding  to TRPA1 . Indeed in 2 mM Ca2+, TRPA1 currents evoked by all doses of carvacrol were robustly inhibited by over-expressed CaM . TRPA1 CDP was abolished by mutating W996, R999, and P1010 . A prono2+ gating of TRPA1. Taken together, these results demonstrate that TRPA1 CDP and CDI are mediated by overlapping but distinct sets of effector sites on the CaMBD of TRPA1.We also tested whether CDP and CDI are affected in ΔTRPA1 in which CaMBD has been deleted, but found that ΔTRPA1 was non-functional (data not shown). The lack of function in ΔTRPA1 could be due either to impaired CaM binding or to structural disturbance. Of note, tryptophan is a conserved residue targeted by CaM among many other CaM effectors, such as CaMKIIWe finally demonstrated whether CaM binding is disrupted in the TRPA1 mutants with defective CDP/CDI. In silver staining of CaM-peptide complex, enhanced CaM staining due to bound CaMBD peptide was significantly diminished by mutating either W996, or V1008 or P1010 , suggest2+ exerts multiple and opposing effects on TRPA1. However, it has been a mystery how TRPA1 senses different Ca2+ levels (Ca2+ sensor) and translates Ca2+ signals into the gating of the channel (Ca2+ effector), leading to opposing effects. In this study we have found that Ca2+ gating of TRPA1 depends on CaM, which binds to TRPA1, forming a Ca2+-sensing channel complex. In the basal state, CaM either enhances (low Ca2+) or inhibits (high Ca2+) TRPA1 sensitivity. In the activated state, CaM promotes either TRPA1 activation (low Ca2+) or desensitization (high Ca2+). These effects were prevented by either mutating the Ca2+-binding sites on CaM or by mutating the CaM binding sites on TRPA1. CaM thus acts as a Ca2+ sensor and an effector responsible for regulating TRPA1 sensitivity and activation in both the basal state and activation state.Ca2+ effects were mediated by CaM C-lobe, without a significant role for N-lobe in these processes. The two lobes of CaM are thus not the source that drives opposing Ca2+ effects on TRPA1. This is in contrast to CaV2.1 and TMEM16 chloride channels that employ the C-lobe and N-lobe of CaM to transduce channel facilitation and inactivation, respectively453 and EF4 in the C-lobe triggered different degrees of TRPA1 binding  did not pre-associate with TRPA1, in contrast to other ion channels, such as L-type Ca2+ channels431234 had no dominant-negative action on TRPA1, which is the main evidence leading to the exclusion of a possible role for CaM in TRPA1 regulation in a previous studyIt is noteworthy that over-expressed CaM can still exert an additional effect on TRPA1 CDP/CDI, supporting that TRPA1 is far from saturated by endogenous CaM. TRPA1-CaM binding can even be triggered by nanomolar ranges of Ca2+ , suggest2+-dependent regulation of TRPA1. Indeed, all the TRPA1-CaM binding can be eliminated by the C-lobe mutant CaM34  domain in TRPA1 has been implicated as a critical region for mediating TRPA1 desensitization2+ oscillations, agreeing with previous studies demonstrating that [Ca2+]e regulates TRPA1 through binding to a channel site that needs to be very close to the channel pore222+, but also allows the channel to translate Ca2+ signals into channel gating. The revealed accessible CaMBD in TRPA1 could thus be a potential drug targeting region for tuning the channel for therapeutics.The C-terminal CaMBD of TRPA1 is adjacent to the ion permeation pathway allowing CaM to detect rapid CaHEK293 cells and mice DRG neurons were cultured as described previously5354Thermo-TRP ion channels including TRPV1-4, TRPM8 (rat) and TRPA1 (mouse) cDNAs were subcloned into pcDNA3-V5-His-TOPO vector (Life Technology) as described previously2+ or Ba2+ concentrations as indicated in related Figures. For CaM pull down assay in 2+-free, 0.5 mM EGTA was included to deplete background Ca2+ in the solution. Similarly in experiments for studying the effect of Ba2+, corresponding Ba2+ ions were added to the above Ca2+-free solution to exclude the effect of background Ca2+. For CaM pull down in 2+ of 100 nM and 500 nM were obtained by adding Ca2+ (in mM) of 0.28 and 0.422, respectively, to the above Ca2+-free binding solution buffered by 0.5 mM EGTA. Free Ca2+ concentrations were calculated using the programme MaxChelator (University of Stanford). After 4 times of thorough washing (20 mins each) with corresponding free Ca2+, bound proteins were eluted by boiling in sample buffer followed by separation on a 7.5% SDS-PAGE gel and detection with anti-V5 (Life Technologies). A peptide (NH2-LWYLRKVDQRSTIVYPN-COOH) with identical sequence to CaMBD in TRPA1 and corresponding scrambled peptide (NH2-YNQIRVYKVTPRLSLDW-COOH) (Biomatik) were also included in CaM pull down assay to investigate the effect of peptide on TRPA1-CaM binding.CaM pull down assay was performed by incubating CaM-agarose (Sigma) with HEK293 cell lysate expressing TRPA1-V5-His (×6) or other TRP channels in a lysis buffer ) at 4 °C under different CaTo examine the binding of CaM to cytoplasmic tails of TRPA1, GST-coupled TRPA1 cytoplasmic tails expressed from HEK293 cells were purified using GST-agarose (Sigma) followed by elution with Glutathione (10 mM), similarly as previously described552+ or without Ca2+ (5 mM EGTA) at room temperature for 1 h. The formed protein complexes were then resolved on 12% non-denaturing polyacrylamide gels in the presence of 2 mM Ca2+ or without Ca2+ (2 mM EGTA) followed by staining with a silver kit (Sigma) in accordance with the manufacturer’s instructions.Silver staining of CaM-peptide complex was performed as described by others with slight modifications2+) consisted of (in mM): 140 NaCl, 4 KCl, 1.0 MgCl2, 10 HEPES, 5 Glucose, pH 7.4 with NaOH. Ca2+-free solution was prepared by adding 5 mM EGTA to the basic extracellular solution. Solutions with millimolar free Ca2+ or Ba2+ were obtained by adding Ca2+ or Ba2+ directly to the basic extracellular solution. Internal pipette solution contained (in mM): 140 KCl, 2.0 MgCl2, 5.0 EGTA, 10 HEPES, pH 7.4 with KOH. To obtain 10 μM free Ca2+, 1.99 mM Ca2+ was added to a Ca2+-free solution buffered by 2 mM EGTA. To study the effect of the CaM antagonist W-7, cells were perfused with 100 μM W-7 (Tocris) throughout the recording. To study the effect of peptides on TRPA1 currents in DRG neuros, peptides were included in the pipette to dialyse the cells as described previouslyWhole-cell recordings were conducted using Axopatch 200B patch clamp amplifier (Axon) controlled by pClampx 10.2 softwares (Molecular Device) as before55Student’s t test and was considered significant if P < 0.05. Comparisons among multiple groups were assessed by one-way analysis of variance with Bonferroni’s post hoc test.All data were presented as mean ± SEM. Differences between two groups were determined using How to cite this article: Hasan, R. et al. Calmodulin is responsible for Ca2+-dependent regulation of TRPA1 Channels. Sci. Rep.7, 45098; doi: 10.1038/srep45098 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In this study, our data revealed the following: (1) in both rat distal pulmonary arteries and PASMCs, chronic hypoxia exposure upregulated the expression of Orai1 and Orai2, without affecting Orai3 and STIM1; (2) either heterozygous knockout of HIF-1α in mice or knockdown of HIF-1α in PASMCs abolished the hypoxic upregulation of Orai2, but not Orai1, suggesting the hypoxic upregulation of Orai2 depends on HIF-1α; and (3) using small interference RNA knockdown strategies, Orai1, 2, 3 and STIM1 were all shown to mediate SOCE in hypoxic PASMCs. Together, these results suggested that the components of SOCCs, including Orai1, 2, 3 and STIM1, may lead to novel therapeutic targets for the treatment of chronic hypoxia-induced pulmonary hypertension.Previous studies have demonstrated that besides the classic canonical transient receptor potential channel family, Orai family and stromal interaction molecule 1 (STIM1) might also be involved in the regulation of store-operated calcium channels (SOCCs). An increase in cytosolic free Ca PH group 3 is due to lung diseases and/or hypoxia, including chronic obstructive pulmonary disease, sleep-disordered breathing, alveolar hypoventilation disorders, diffuse parenchymal lung diseases, chronic exposure to high altitude and developmental abnormalities.2+]i) is a major trigger for pulmonary vasoconstriction and the proliferation and migration of PASMCs.2 Among the multiple pathways that can lead to increase in [Ca2+]i, the hypoxia-induced enhanced store-operated Ca2+ entry (SOCE) through store-operated calcium channels (SOCCs) largely accounts for the elevated [Ca2+]i in PASMCs.4 SOCCs are mainly constituted by canonical transient receptor potential channel (TRPC) and calcium release-activated calcium modulator .5–8 Orai consists of three members, Orai1, Orai2 and Orai3. Recent studies have revealed that Orai1 might be involved in the constitution of SOCCs, or regulates the function of SOCCs.9 In mesenteric artery smooth muscle cells, the expression level of Orai members are upregulated in proliferating cells.10 Orai1 consists of four transmembrane domains. Under resting conditions, Orai1 exists either as a homodimer or homotetramer; while upon activation, it forms a hexamer and mediates Ca2+ release-activated Ca2+ current (ICRAC), a highly Ca2+-selective and nonvoltage-gated current.11–15 The activation kinetics of Orai1 are relatively slow and determined by the rate of Ca2+ depletion as well as the translocation rate of both STIM1 and Orai1 to endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR)–plasma membrane (PM) junctions. The duration of Orai1 activation can be sustained with prolonged store depletion.16 Among the three Orai homologs, Orai1 contributes the most to mediate SOCE.17 Soon after Orai1 was discovered in 2006, Orai2 was reported as another component of SOCCs.17–19 Similar with Orai1, Orai2 and Orai3 are highly Ca2+ selective corresponding to the characteristic of CRAC (Ca2+ release-activated Ca2+) channels.21 Orai3 was only found in mammals, with a tissue distribution at least as wide as that of Orai1.23 Orai3 combines with Orai1 to form a hexameric CRAC channel, and at least one native Orai1 subunit is contained in the complex.24 Orai1 and Orai3 arrange as pentamer to form the arachidonic acid-regulated calcium (ARC) channels, whose characteristics are similar to CRAC channels, but are store-independent.25 However, whether the Orais contribute to hypoxia-induced enhancement of SOCE remains largely unknown.CHPH is characterized by excessive contraction, proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), which progressively leads to the thickening and remodeling of distal pulmonary arteries (PAs). The increase of intracellular free calcium concentration  overproduction, temperature variation, hypoxic stress and pH changes in the cells, indicating that STIM1 might be a stress sensor sensing a range of cellular stress condition.37–40 In our previous study, we elucidated that knockdown of STIM1 abolishes acute hypoxia -induced enhancement of SOCE.34 Considering SOCE largely accounts for the elevated [Ca2+]i in PASMCs, we hypothesized that STIM1 may also have an important role in prolonged hypoxia-induced SOCE.Stromal interaction molecule 1 (STIM1), a single-pass transmembrane protein, has been well known to predominantly localize in the ER/SR membrane where it acts as a CaTherefore, in this study, we further investigated the regulation and action of Orai family and STIM1 in chronic hypoxia-induced elevation of SOCE in PASMCs.2) for both mRNA and protein assessment. Results showed that hypoxia induced a 64.7±22.7% and 162.2±100.3% increase in Orai1 and Orai2 mRNA level, compared with those of their respective normoxic control, while not affecting the expression of Orai3 and STIM1  and their littermate were subjected to either normoxic or chronic hypoxic exposure for establishment of CHPH mice model. First, in the distal PAs isolated from wild-type α+/+HIF-1 mice, CH  increased Orai1 and Orai2 by 144.7±18.1% and 250.2±105.9% in mRNA levels, and 101.6±8.8% and 35.2±7.8% in protein levels, comparing with their respective normoxic controls  against HIF-1α (siHIF-1α) to evaluate the role of HIF-1α in hypoxic upregulation of Orai1 and Orai2 in cultured PASMCs. First, the expression of HIF-1α protein was decreased by 71.7±7.7% from hypoxic exposed PASMCs treated with siHIF-1α, compared with that of the hypoxic non-targeted siRNA (siNT) control, indicating effective knockdown. Then, in hypoxic PASMCs, knockdown of HIF-1α largely abolished the hypoxic upregulation of Orai2, while not affecting the hypoxic upregulation of Orai1 . Compared with that of the siNT control, the knockdown efficiency of Orai1 was 76.1±2.2% and 54.2±2.9% at mRNA and protein levels, respectively  and then subjected to exposure of prolonged hypoxia . Compared to that of the siNT control, the knockdown efficiency of STIM1 was 83.5±2.1% and 77.4±16.4% at mRNA and protein levels, respectively  and then subjected to exposure of prolonged hypoxia . Compared with that of the siNT control, the knockdown efficiency of was 86.7±3.8% (mRNA) and 54.5±1.2% (protein) for Orai2, and 81.0±1.4% (mRNA) and 58.6±10.0% (protein) for Orai3  or Orai3-specific siRNA (siOrai3), and then subjected to exposure of prolonged hypoxia 41 or the important SOCC-regulated proteins (such as caveolin-1),42 all contributed to the hypoxic-triggered SOCE in PASMCs. However, whether the other SOCE-related proteins STIM1 and Orais also contribute to this process remains unknown. Therefore, in this study, by using comprehensive knockdown of STIM1 or Orais, our results suggested that knockdown of either Orai1, Orai2, Orai3 or STIM1 could significantly reverse prolonged hypoxia-induced increases of SOCE and basal [Ca2+]i in cultured rat distal PASMCs.As is well known, during the PH development, the hypoxic elevation of i in cultured rat distal PASMCs. These results suggest that the hypoxic upregulation of Orai2 contributes to the elevation of SOCE and basal [Ca2+]i via stabilizing the expression of HIF-1α, whereas the hypoxia-elevated pathway of Orai1 expression is independent of HIF-1α. In view of the key role of HIF-1α in the development of CHPH, the Orai2 may be considered a potential target retarding pulmonary vasoconstriction and proliferation. In addition, much work have been done in evaluating the role of Orai1 in the regulation of SOCE. Similar to Orai2, downregulation of Orai1 reversed the increase of SOCE and basal [Ca2+]i caused by CH, suggesting hypoxia-elevated Orai1 enhance SOCE and basal [Ca2+]i. But the transcriptional upregulation mechanisms for Orai1 were not evaluated clearly, what we do is at a beginning of this pathway so that further study need to be developed next. Unlike Orai1 and Orai2, we did not observe an upregulation of Orai3 expression upon hypoxic exposure, while knockdown of Orai3 could also significantly reversed the hypoxic elevation of SOCE and basal [Ca2+]i in rat distal PASMCs, suggesting the basal level of Orai3 is also essential for the elevation of SOCE and basal [Ca2+]i. Notably, we also observed that after knockdown of Orai3, the expression of Orai1 was decreased by ~30%  knockdown of either STIM1 or Orai family proteins  could attenuate the hypoxia-elevated SOCE and basal [Ca2+]i in PASMCs. As elevation of basal [Ca2+]i leads to contraction, proliferation and migration of PASMCs, eventually the pathogenesis of CHPH, blockage of the elevation of basal [Ca2+]i would reverse the process and prevent the genesis of CHPH. Our results added knowledge that the Orais and STIM1 could act as novel therapeutic targets for the treatment against CHPH.An outline was shown in α heterozygous-mutant (HIF-1α+/–) mice were genotyped as described before.49 All mice were housed in specific pathogen-free facilities. Littermate mice from HIF-1α+/+×HIF-1α+/– mating were genotyped at 3–4 weeks old.Animal protocols were approved by the Animal Care and Use Committee of Guangzhou Medical University. Sprague–Dawley rats  were purchased from Guangdong Medical Laboratory Animal Center . HIF-150 The chamber was continuously flushed with a mixture of room air and N2 to maintain 10±0.5% O2 and CO2<0.5%. The chamber O2 concentration was continuously monitored using a PRO-OX unit . Animals were exposed to room air for 10 min twice a week for changing cage and replenishing food and water. Normoxic control animals were kept in room air next to the hypoxic chamber.Rats  or mice  were placed in a hypoxia chamber for 21 days to establish the chronic hypoxia-induced PH animal model, as previously described.51 PASMCs were enzymatically isolated and plated onto 25 mm glass coverslips .52 Rat PASMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1 g/l D-glucose  with 10% fetal bovine serum (Life Technologies) and 1% penicillin–streptomycin  until 60–70% confluence prior each experiment.The distal intrapulmonary arteries (⩾fourth generation) were dissected from normoxic or chronic hypoxic rats and mice, as we previously described.α (siHIF-1α), Orai1 (siOrai1), Orai2 (siOrai2), Orai3 (siOrai3), STIM1 (siSTIM1) and siNT used as negative control were designed and synthesized by GenePharma . Rat PASMCs were transfected with 50 nM siHIF-1α, siOrai1, siOrai3, siSTIM1, siNT or 100 nM siOrai2 using HiPerFect Transfection Reagent  at 6 μl/ml as transfection vehicle in 0 serum DMEM. After 6 h transfection, serum was added to a final concentration of 0.3% and PASMCs were exposed to normoxia or hypoxia (4% O2) for 60 h. Knockdown efficiency was evaluated by quantitative real-time PCR (qRT-PCR) and western blotting.siRNAs targeting to rat HIF-1μM fura-2  for 60 min at 37 °C under an atmosphere of 5% CO2–95% air, coverslips with PASMCs were mounted in a closed polycarbonate chamber clamped in a heated aluminum platform  on the stage of a Nikon TSE 100 Ellipse inverted microscope . The chamber was perfused at 1 ml/min with Krebs–Ringer bicarbonate (KRB) solution, which contained (in mM) 118 NaCl, 4.7 KCl, 0.57 MgSO4, 1.18 KH2PO4, 25 NaHCO3, 2.5 CaCl2 and 10 glucose. To assess SOCE, we perfused PASMCs for at least 10 min with Ca2+-free KRB containing 5 μM nifedipine to prevent calcium entry through L-type VDCCs and 10 μM CPA to deplete SR calcium stores. KRB perfusate also contained 0.5 mM EGTA to chelate any residual Ca2+. SOCE was assessed in two ways. First, we measured [Ca2+]i at 12 s intervals before and after restoration of extracellular [Ca2+] to 2.5 mM. SOCE was evaluated from the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] in the continued presence of cyclopiazonic acid (CPA) and nifedipine. Second, we monitored fura-2 fluorescence excited at 360 nm at 30 s intervals before and after addition of MnCl2 (200 μM) to the cell perfusate. SOCE was evaluated from the rate at which fura-2 fluorescence was quenched by Mn2+, which enters the cell as a Ca2+ surrogate and reduces fura-2 fluorescence on binding to the dye. Fluorescence excited at 360 nm is the same for Ca2+-bound and Ca2+-free fura-2; therefore, changes in fluorescence can be assumed to be caused by Mn2+ alone. Quenching was quantified as the change in F360 (ΔF360) measured from 5 to 15 min and expressed as a percentage of F360 at 5 min.After incubation with 7.5 34 Reverse transcription was performed using PrimeScript RT Reagent Kit . The reaction mixture contained 1 μg total RNA in a 20 μl volume. cDNA was quantified by qRT-PCR using QuantiTect SYBR Green PCR Master Mix (Qiagen) in a iCycler IQ real-time PCR detection system  using the following conditions: 95 °C for 15 min and 45 cycles, each at 94 °C for 15 s, 57.5 °C for 20 s and 72 °C for 20 s. The volume of each qRT-PCR reaction mixture was 25 μl containing 300 nM forward and reverse primers and cDNA template from 50 ng RNA. Primer sequences of rat Orai1, Orai2, Orai3, STIM1 and 18 s were designed using Primer3 software (http://simgene.com/Primer3), and are shown as follows, where S is sense and AS is antisense:Total RNA in de-endothelialized distal PA and PASMCs was extracted using TRIzol method.Orai1-S (5′-GATGAGCCTCAACGAGCACT-3′),Orai1-AS (5′-GACTTCCACCATCGCTACCA-3′),Orai2-S (5′-GTGGGTCTCATCTTCGTGGT-3′),Orai2-AS (5′-CCACCTGTAGGCTTCTCTCG-3′),Orai3-S (5′-GCCCAGCTTTAGACTGTTGC-3′),Orai3-AS (5′-CTGAGCAGGAATTTGGCTTC-3′),STIM1-S (5′-ATGCCAATGGTGATGTGGAT-3′),STIM1-AS (5′-CCATGGAAGGTGCTGTGTTT-3′),18s-S (5′-GCAATTATTCCCCATGAACG-3′),18s-AS (5′-GGCCTCACTAAACCATCCAA-3′).T) values were generated by iCycler IQ software. Relative concentration of each transcript was calculated using the Pfaffl method.53 Efficiency for each gene was determined from five-point serial dilutions of positive control cDNA samples.Identity of the qPCR products was confirmed by (1) a single sharp peak in the melting curve performed after cDNA amplification, (2) a single band of the expected size resolved by agarose gel electrophoresis and (3) DNA sequencing. Melting curves were performed at 95 °C for 1 min and 55 °C for 1 min, followed by 80 increments of 0.5 °C at 10 s intervals. qRT-PCR detection threshold cycle . After blocking with 5% non-fat dry milk in TBS-0.1% Tween-20, membranes were blotted with mouse monoclonal antibody for HIF-1α , α-actin (Sigma-Aldrich), β-actin , rabbit polyclonal antibodies for Orai1, Orai2  and Orai3 , or rabbit monoclonal antibodies for STIM1 . The membranes were then washed and probed with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG . The bound antibodies were detected using an enhanced luminol-linked chemiluminescence detection system .Total proteins in tissues or cells were extracted in ice-cold RIPA buffer  containing 1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% sodium deoxycholate and 1% 100 mM PMSF. De-endothelialized distal PA was homogenized using ultrasonication. PASMCs were washed with phosphate buffered saline and homogenized by scraping in lysis buffer on ice and then were span down to remove cell debris. Total protein concentration in homogenate samples were determined by BCA protein assay . Samples were combined with 5× loading buffer, boiled and resolved on SDS-PAGE calibrated with prestained protein molecular weight markers. Separated proteins were transferred to polyvinylidene difluoride membranes  of CPA and nifedipine were made in pluronic dimethyl sulfoxide . Fura-2 AM (Invitrogen) was prepared on the day of the experiment as a 2.5 mM stock solution in DMSO.t-test for two groups and one-way ANOVA for multiple groups of data. Differences were considered significant when P< 0.05. Data are presented as means±S.E.M.; ‘n’ refers to the sample size .Statistical analyses were conducted using Student’s Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.