OXTR)gene. We found that five variants, V45L, P108A, L206V, V281M, andE339K, significantly altered oxytocin-induced Ca2+ signalingor β-arrestin recruitment and proceeded to assess the effectsof these variants on OXTR trafficking to the cell membrane, desensitization,and internalization. The variants P108A and L206V increased OXTR localizationto the cell membrane, whereas V281M and E339K caused OXTR to be retainedinside the cell. We examined how the variants altered the balancebetween OXTR activation and desensitization, which is critical forappropriate oxytocin dosing. The E339K variant impaired OXTR activation,internalization, and desensitization to roughly equal extents. Incontrast, V281M decreased OXTR activation but had no effect on internalizationand desensitization. V45L and P108A did not alter OXTR activationbut did impair β-arrestin recruitment, internalization, anddesensitization. Molecular dynamics simulations predicted that V45Land P108A prevent extension of the first intracellular loop of OXTR,thus inhibiting β-arrestin binding. Overall, our data suggestmechanisms by which OXTR genetic variants could alterclinical response to oxytocin.The hormone oxytocinis commonly administered during childbirthto initiate and strengthen uterine contractions and prevent postpartumhemorrhage. However, patients have wide variation in the oxytocindose required for a clinical response. To begin to uncover the mechanismsunderlying this variability, we screened the 11 most prevalent missensegenetic variants in the oxytocin receptor ( To bind to oxytocin, OXTR must first trafficto the myometrial smooth muscle cell surface. Upon oxytocin binding,OXTR activates Gq, leading to CaOXTR gene affect the response to exogenousoxytocin. For example, Reinl et al. and Grotegut et al. identifiedsingle nucleotide OXTR variants in patients who requiredhigh or low doses of oxytocin to induce labor, but these studies werenot powered to detect significant associations.16 In an ex vivo study, one coding and one noncoding OXTR variant altered the oxytocin-induced contractions ofuterine tissue strips isolated from pregnant individuals.17 Although exome sequencing studies have shownthat missense variants in the OXTR gene are prevalentin the global human population,18 the functionaleffects of most of these variants have not been determined. However,prevalent missense variants in other GPCRs genes lead to aberrantdrug responses.19 Here, we assessed theeffects of genetic variants of unknown significance in OXTR on oxytocin response in cells.Several investigatorshave tested the hypothesis that variantsin the 2.HEK293T cells were maintained in Dulbecco’smodified Eagle’s medium/Ham’s F12 medium without phenolred and supplemented with 10% fetal bovine serum and 25 μg/mLgentamicin. Cells were kept in a humidified cell culture incubatorat 37 °C with 5% COThe wild-type (WT) OXTR and P108A OXTRconstructs in pcDNA3.1(+) vector were a kind gift from Dr. JeffreyMurray (University of Iowa). Other missense single nucleotide variantswere introduced by site-directed mutagenesis . The WT OXTR sequence was identical to the coding region of theNational Center for Biotechnology Information reference sequence NM_000916.3.www.cdna.org). A plasmid containingthe Rluc8 cDNA was a kind gift from Dr. Brian Finck (Washington Universityin St. Louis).The β-arrestin-1-Rluc8 fusion construct in the vector pcDNA3.1(+)encoded β-arrestin-1 with a C-terminal linker SGGSTSA followedby Rluc8. The β-arrestin-2-Rluc8 fusion construct in the vectorpcDNA3.1(+) encoded β-arrestin-2 with a C-terminal linker GGGSEFfollowed by Rluc8. The template cDNA clones for β-arrestin-1(ARRB100002) and β-arrestin-2 (ARRB200001) were obtained fromthe cDNA Resource Center  encodedOXTR with a C-terminal linker SGGKL followed by GFP10. A plasmid containingthe GFP10 cDNA was a kind gift Dr. Céline Gales .http://n2t.net/addgene:67848; RRID: Addgene_67848).20 Note that this plasmid includes the missensesingle nucleotide variant A218T, which was corrected before introducingthe variants of interest. An N-terminal HA tag was added (linker GPT)to generate the HA-OXTR-GFP construct.The plasmid encoding OXTR-GFP was a gift from Christian Gruber stock solutions dilutedto 500 μM in water were stored at −80 °C until justbefore use.4) were plated in each well of 96-well black-walled,clear-bottompolystyrene microplates coated with poly-d-lysine. The followingday, cells were transfected with a construct encoding WT or variantOXTR. Each variant was tested alongside WT controls on the same plate.For transfections, 50 ng of DNA and 0.5 μL of TransIT-293 reagent diluted in Opti-MEM reduced-serum media  were added to each well. After 24h, media was removed and replaced with 100 μL of Brilliant Calciumindicator solution , which was preparedby diluting Brilliant Calcium indicator, DrySolv, and TRS reagentin assay buffer. After incubation for 1 h, a Synergy2 plate reader was used to add 100 μL of oxytocin ofthe appropriate concentration and record the fluorescence intensity every0.14 s for 20 s/well. Fluorescence increase (increase in intracellularCa2+) was calculated as the average of fluorescence intensityreadings from 10 to 20 s after oxytocin addition minus the minimumfluorescence intensity averaged over five points from 0 to 10 s.HEK293T cells were plated in each wellof 96-well white-walled, clear-bottom polystyrene microplates coatedwith poly-d-lysine. The following day, cells were transfectedwith WT or variant OXTR-GFP10 and β-arrestin-1-Rluc8 or β-arrestin-2-Rluc8at a ratio of 15:1 (w/w). For transfections, 50 ng of DNA and 0.5μL of Lipofectamine 2000 reagent (Thermo Fisher Scientific),both diluted in Opti-MEM reduced-serum media, were added to each well.After 24 h, media was removed and replaced with 100 μL of Hank’sbuffered salt solution (HBSS) supplemented with 20 mM HEPES. A Synergy2plate reader was used to add 100 μL of assay buffer containing10 μM coelenterazine 400a  and the indicatedconcentrations of oxytocin to 10 wells at a time. Luminescence at520 and 400 nm was read every 26 s for a total of 182 s. The BRETratio was calculated as the average ratios of emission at 520 nm/400nm at the five time points from 78 to 182 s. WT controls were testedon each plate in parallel with variants.HEK293T cells (4 × 106) were plated in T25 flasks and transfected the nextday with HA-OXTR-GFP, OXTR-GFP, or HA-OXTR. Cells were transfectedwith 300 ng of plasmid DNA and 4 uL of TransIT-LT1 reagent (MirusBio). Cells were detached 24 h later with CellStripper (Corning) andcollected by centrifugation. To measure receptor internalization,cells were incubated with the indicated concentration of oxytocinfor 30 min before and during detachment. Cells were incubated withan empirically determined saturating concentration (8–16 μg/mL)of phycoerythrin (PE)-conjugated anti-HA antibody  in staining buffer (0.5% BSA and 0.1% sodium azidein Ca2+/Mg2+-free PBS) on ice for 40 min andthen washed twice in staining buffer before flow cytometry to quantifycell surface OXTR. For quantification of total OXTR, the PE-labeledliving cells were fixed with 2% paraformaldehyde and permeabilizedwith 0.5% Tween20 in PBS. Cells were washed with 0.1% Tween 20 inPBS, incubated with 16 μg/mL PE anti-HA antibody for 40 minat room temperature, and washed twice before flow cytometry.HEK293T cells . Three technical replicates were performedfor each experimental condition, and data from 5000 transfected cellswere collected from each replicate. Three independent trials wereperformed. SYTOX Blue (Thermo Fisher Scientific) was used to excludedead cells where appropriate. PE Quantibrite beads (BD Biosciences)were used for calibration. Flow cytometry gating was performed asfollows: (1) forward and side scatter were used to exclude debris;(2) forward scatter width vs height was used to exclude doublets;(3) SYTOX blue staining was used to identify dead cells; (4) GFP fluorescencewas used to gate transfected cells (GFP+ population). The GFP+ thresholdwas determined relative to the GFP signal in the GFP-negative control(cells transfected with HA-OXTR).21 Values fromnonspecific binding of PE-HA antibody to HA-negative cells (cellstransfected with OXTR-GFP) were subtracted from all samples.The number of receptors ontransfected cells was calculated fromthe geometric mean of PE fluorescence intensity calibrated to PE standardsas previously described.2+ and BRET assays,responses were normalized by subtracting the average basal responsefrom all samples and then dividing by the average WT response at thehighest oxytocin concentration for each trial. For desensitizationand internalization experiments, responses were normalized by dividingvalues from all samples by the average response from the correspondingnonpretreated sample(s). Normalization was performed separately foreach replicate experiment.For CaEmax, EC50, and IC50 values(GraphPad Prism 8). The three-parameter regression method, which wasused to fit the BRET data and internalization data, used the model: Y = Bottom + (Top – Bottom)/(1 + 1050) or IC50–X)(log(EC). The four-parameter regression method, which was used to fit theCa2+ activation and desensitization data, used the equation Y = Bottom + (Top – Bottom)/(1 + 1050) or IC50–X)×HillSlope)), and no constraints were placed onany values. Buffer controls were assigned a nominal concentrationvalue of 10–9 M for BRET assays or 10–12 M for all other assays.Nonlinear regression with least-squaresfitting was used to generate dose–response curves and calculate P-values were determined as previously described.23P-values shown reflect comparisons of log EC50 values or Top values (see equations above), as indicated.All experiments were performed intriplicate, with WT controlstested alongside each variant on the same plate to control for day-to-dayvariation in assay response. Average values from three biologicalreplicates were used to construct dose–response curves foreach variant and the matched WT controls, which were compared by performingnested extra sum-of-squares F tests. F statistics were calculatedand 24 This model was then preparedfor simulation by the CHARMM-GUI membrane protein input generator.28 Mutations  and palmitate lipid tails on C346 and C347were introduced by the CHARMM-GUI PDB manipulator.29 All proteins were simulated in 0.15 M KCl (111 K+ ions and 92 Cl– ions) in a rectangular box ofsize 99.5 × 99.5 × 171.2 Å with a membrane consistingof 121 (upper leaflet) or 120 (lower leaflet) POPC molecules and 12cholesterol molecules (upper and lower leaflet). All systems contained∼100 000 TIP3P30 water molecules.Systems were minimized in the default manner supplied by CHARMM-GUI.Briefly, using the CHARMM36m force field,31 each system’s energy was minimized by using gradient descent,then simulated NVT with progressively weaker and fewer restraintson positions of atoms and membrane components.The initial homologymodel of WT OXTR was provided by the I-TASSER GPCR homology modeldatabase.32 Hydrogen bondswere constrained with the LINCS algorithm.33 Cutoffs of 1.2 nm were used for the neighbor list, Coulomb interactions,and van der Waals interactions. The force-switch modifier was usedto smoothly switch forces from van der Waals interactions to zerobetween 1.0 and 1.2 nm. The Verlet cutoff scheme was used for theneighbor list. The Nosé–Hoover thermostat was used tohold the temperature at 300 K.34 The semi-isotropicParrinello–Rahman barostat was used to maintain constant pressureof 1 bar as is standard in protein–membrane simulations.35 Conformations were stored every 20 ps.Production runswere performed in GROMACS.37 was used to enhance conformationalsampling for each OXTR sequence . FiveFAST simulation rounds were conducted with 10 simulations per round.Each simulation was 50 ns in length (2.5 μs aggregate simulation).To explore away from the starting structure, the FAST ranking functionfavored restarting simulations from states that had the fewest numberof preserved native contacts. Additionally, a similarity penalty wasadded to the ranking to promote conformational diversity in startingstructures, as described previously.38The FAST algorithm39 Two DiffNets wereindependently trained to learn about impairment of β-arrestinand Gq signaling. All DiffNet training and analysis was conductedunder the assumption that the regions of Gq and β-arrestin bindingwere most likely to contain differences that explained impaired Gqor β-arrestin signaling. Therefore, the DiffNet analysis onlyconsidered atoms in the binding region . All simulation data (2.5 μs per variant)was converted to DiffNet input as described previously.39 Briefly, XYZ atom coordinates from simulationswere mean-shifted to zero and then multiplied by the inverse of thesquare root of a covariance matrix, which was calculated from simulations.To learn about β-arrestin impairment, a DiffNet was trainedto classify all structures from V45L and P108A as β-arrestinimpaired  and WT and V281M simulationsas normal . To learn about Gq impairment,a DiffNet was trained to classify structures from V281M simulationsas potentially Gq impaired and WT, V45L, and P108A simulations asnormal. In both cases, the labels were iteratively updated in a self-supervisedmanner described previously39 in whichexpectation maximization bounds of [0.1–0.4] were chosen fornormal variants and [0.6–0.9] for impaired variants. Both trainingsessions used 10 latent variables, 10 training epochs in which thedata were subsampled by a factor of 10 in each epoch, a batch sizeof 32, and a learning rate of 0.0001.DiffNets can perform dimensionalityreduction in a way that highlights biochemically relevant differencesbetween data sets.42 To quantify differences between variants, severalmeasurements were made that relied on MSMs, each built with 2.5 μsof simulation data for each variant. All MSMs were constructed withEnspara,43 a python library for clusteringand building MSMs from molecular simulation data. In this work, Ensparawas used to cluster OXTR structures, count transitions between clusters,and derive equilibrium probabilities of structural states exploredduring simulation. A separate MSM was built for each variant, usingthe same methodology for each variant. Namely, simulation frames wereconverted from XYZ atom coordinates to a vector containing a valueindicating the amount of solvent-accessible surface area (SASA) ofeach residue side chain . SASAcalculations were computed by using the Shrake–Rupley algorithm44 (with a solvent probe radius of 0.28 nm) asimplemented in the python package MDTraj.45 SASA featurization was used for subsequent clustering because, unlikeother clustering schemes , SASA emphasizes the conformationalchanges of surface residues over internal residues, which should bemost useful for understanding signaling of a transmembrane receptorthat has a surface for binding ligands. Next, the SASA-featurizeddata were clustered with a hybrid clustering algorithm. First, a k-centersalgorithm46 was used to cluster the datainto 1000 clusters. Next, three sweeps of k-medoids update steps wereapplied to refine the cluster centers to be in the densest regionsof conformational space. Then, transition probability matrices wereproduced by counting transitions between states  usinga 2 ns lag time, adding a prior count of 47 Equilibrium populations werecalculated as theeigenvector of the transition probability matrix with an eigenvalueof one. For the distance histograms in Figure S2).A MarkovState Model (MSM) is a statistical framework for analyzing moleculardynamics simulations and provides a network representation of a freeenergy landscape.18 whichincludes 141,456 exomes, to identify the most prevalentsingle nucleotide missense variants in OXTR. We identified11 OXTR variants , onein the first extracellular loop (ECL1), two in the third intracellularloop (ICL3), and two in the C-terminal tail  OXTR or the 11 variants into HEK293Tcells and then performed high-throughput assays to measure effectson these pathways. First, to measure increases in intracellular Ca2+ in response to oxytocin, we used a fluorescent Ca2+ indicator dye. Second, to measure β-arrestin recruitment inresponse to oxytocin, we performed bioluminescence resonance energytransfer assays in HEK293T cells transfected with green fluorescentprotein (GFP)-tagged OXTR and luciferase-tagged β-arrestin-1or β-arrestin-2. V45L, P108A, L206V, V281M, and E339K had thelargest statistically significant effects on EC50 or Emax in two or more assays and were thereforeselected for further study (Tables S1–S3).V45L decreased the Emax for β-arrestin-1recruitment and increased the EC50 for β-arrestin-2recruitment . P108A increasedthe EC50 for β-arrestin-1 recruitment and increasedboth the EC50 and the Emax forβ-arrestin-2 recruitment. L206V increased the Emax for β-arrestin-1 and β-arrestin-2 recruitment.V281M increased the EC50 for Ca2+ signalingand decreased the Emax for Ca2+ signaling and β-arrestin-2 recruitment. Finally, E339K increasedthe EC50 for Ca2+ signaling and decreased the Emax for Ca2+ signaling, β-arrestin-1recruitment, and β-arrestin-2 recruitment  -conjugated anti-HA antibody to quantifythe HA epitope on the extracellular N-terminus of OXTR. To quantifysurface OXTRs, living cells were labeled by PE; to quantify totalOXTRs throughout the cell, an additional PE-labeling step was performedafter fixing and permeabilizing the PE-labeled living cells.P > 0.01 in one-sample t tests, P = 0.0003 and P = 0.0002, one sample t tests). Conversely, two variants (V281M and E339K) decreased thenumber of cell surface OXTRs by 49 ± 0.7% and 36 ± 2%, respectively. Conversely, V281M and E339K decreased OXTR surface localizationto 12 ± 1% and 17 ± 1%, respectively (adjusted P = 0.01 for both).When we graphed cell surface OXTRsas a percentage of total OXTRs2C, we fo13 Thus, to assess the potentialclinical implication of variants, we aimed to define their effectson OXTR desensitization and internalization. As expected, for allfive variants, relative differences in the number of cell surfacereceptors  of oxytocin. In contrast, V45L, P108A, andE339K caused a rightward shift in the dose–response curve andincreased the IC50 for desensitization .V45L and P108A caused a similar rightward shift in internalizationassays . Although E339Kdid not cause a statistically significant increase in EC50 for internalization (P > 0.05), it preventedmaximalinternalization, with 44% of E339K OXTRs versus 24% of WT OXTRs remainingon the cell surface . Of the threevariants that impaired OXTR internalization and desensitization, onlyone, E339K, also altered potency and efficacy for OXTR activation,potentially due to decreased cell surface localization  reduced β-arrestin recruitment,OXTR internalization, and OXTR desensitization compared to WT OXTR.Thus, three lines of evidence suggest that V45L and P108A decreaseOXTR’s ability to activate β-arrestin. To define thestructural basis of β-arrestin impairment, we used moleculardynamics simulations to computationally model the motions of all atomsin WT and variant OXTRs in solution over time  to e39 We first trained a DiffNet to identify differences betweenconformational ensembles of the two β-arrestin-impaired OXTRs(V45L and P108A) and two OXTRs (WT and V281M) with normal desensitizationand internalization. From this training, the DiffNet learned a labelfor each simulation frame  from zero toone that indicated the probability that it was associated with thisclassification. To interpret these labels, we calculated the correlationbetween interatom distances in the OXTR cytosolic region  and changesin the DiffNet label. We then plotted the 100 distances that weremost correlated with the DiffNet label   6A. Thus,48 Second, shortening ICL1 reduces the distancebetween ICL1 and helix 8 (H8), causing a collapsed state (49) onto the OXTR homology model, the model predicted that this shorteneddistance created a steric clash between ICL1 and the β-arrestinfinger loop, but not between ICL1 and the G protein . Taken together,our data suggest that the mechanism underlying reduced β-arrestinfunction was similar in V45L and P108A OXTR.This conformational change has important implications for β-arrestinbinding. First, shortening ICL1 may prevent the interactions betweenICL1 and the bottom loop of β-arrestin 6B previoed state 6C. When  protein 6D. TakenOur resultsin We then used Markov State Models to plot the probabilitythat OXTR adopts a conformation with a given distance between TM3and TM5. V281M OXTR was more likely to adopt conformations with ashorter distance between TM3 and TM5 than were WT, V45L, and P108AOXTR  impairedOXTR desensitization and OXTR internalization upon exposure to oxytocin.Our molecular dynamics simulations predict that both V45L and P108Aintroduce an extra helical turn at the end of TM1, which may explainthe impaired coupling to β-arrestin seen in vitro.Our data indicate that 2+ signaling , whereas V281Mand E339K, which decreased cell surface OXTR, caused lower maximalrecruitment  but had no effect on desensitization orinternalization. Our Markov State Models predicted conformationalchanges in V281M OXTR consistent with steric hindrance of G proteinbinding  explain the differences in OXTR internalization and recycling observedby Ma et al. Second, Kim et al. characterized three missense OXTR variants, including P108A, that they identified inpatients who experienced premature labor.53 These authors reported that P108A decreased oxytocin binding butdid not significantly affect Gq activation as measured by inositolphosphate production, which was consistent with our results. Furthermore,our findings show that P108A impaired OXTR desensitization, meaningthat some OXTR Gq activation occurred unopposed. This could resultin premature initiation of uterine contractions and thus explain anassociation between P108A and premature labor. Future studies areneeded to determine whether P108A—and V45L, which we foundto have similarly impaired desensitization and structural changes—predisposepatients to preterm labor.Our findings add totwo previous OXTR helped us to prioritize the variants most likely toaffect OXTR function for further study. These variants caused EC50 changes in the 2–4-fold range, consistent with effectscaused by other naturally occurring GPCR variants linked to diseaserisk and drug response.57 Additionally, our data indicate that the two mostprevalent missense variants, A218T and A238T, are unlikely to appreciablyaffect OXTR function.Understanding how genetic variantsalter receptor function is animportant step toward personalized drug dosing. Our functional annotationof the 11 most prevalent variants of unknown significance in 2+ signalingpathway play an important role in determining clinical response tooxytocin. Currently, most oxytocin dosing protocols for labor inductioncall for providers to increase the oxytocin infusion rate at steadyintervals, which compensates for a given amount of OXTR desensitizationover time.58 Imbalance between these processes,also known as signaling bias, may therefore have clinical consequences,as shown in other GPCRs.59 In our study, we identified three variants that may cause signalingbias: (1) V45L and P108A impaired OXTR desensitization but not activationand (2) V281M decreased OXTR activation but not desensitization. However,further studies are necessary to determine whether these changes representsignaling bias between β-arrestin and Gq. Our data indicatethat individuals who carry the V281M allele may be less responsiveto oxytocin but still susceptible to the potential adverse effectsthat result from OXTR desensitization during labor . These individuals may require higher dosesof oxytocin to achieve labor induction and thus may have increasedrisk of these adverse events. Furthermore, oxytocin may be less effectivein preventing postpartum hemorrhage in these individuals. In contrast,patients with V45L or P108A variants may be less susceptible to theadverse effects that result from OXTR desensitization but more susceptibleto uterine hyperstimulation as a result of induction with oxytocin.Finally, patients with the E339K variant, which impairs OXTR activationand desensitization to roughly the same extent, may require higheroxytocin doses to achieve clinical effects.Both activation and desensitization ofthe Ca60 Further studies in these populations are necessary to determinethe utility of genetic analyses in developing precision medicine approachesto oxytocin dosing.Our studies indicatethat individuals who carry the V45L, P108A,V281M, or E339K variants may benefit from personalized oxytocin dosingprotocols or alternative methods of labor induction. P108A is foundin 0.3% of the Finnish population, V281M is found in 0.7% of the Swedishpopulation, and E339K is found in 1.5% of the Ashkenazi Jewish population.


Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.The identification of two variants of the canonical pore-forming subunit of the CaThe online version contains supplementary material available at 10.1007/s00018-021-04098-w. The magnitude and pattern of Ca2+ signals depend on the type and concentration of the agonist; thus, low-intensity stimulation leads to oscillatory responses and higher-intensity stimulations generate more sustained responses [2+ entry (SOCE) is a mechanism for Ca2+ influx involved in a variety of cellular functions including gene transcription and the maintenance of Ca2+ oscillations [2+ stores, primarily the endoplasmic reticulum (ER), so that occupation of phospholipase C-coupled plasma membrane agonist receptors leads to the generation of inositol trisphosphate (IP3), which, in turn, induces Ca2+ efflux from the intracellular Ca2+ stores. Store depletion triggers the activation of stromal interaction molecule (STIM) proteins leading to a conformational change that allows the interaction with and activation of Orai channels in the plasma membrane [2+ responses [2+ signals in response to physiological concentrations of agonists [2+-selective Icrac, which involves activation of Orai channels by STIM proteins, and Isoc, described in certain cell types, which involves the transient receptor potential (TRP) family member TRPC1, Orai1 and STIM1 [Icrac, is non-selective for cations and exhibits higher conductance[Agonist-induced changes in cytosolic Caesponses , 2. Storllations –5. SOCE membrane –10. In aesponses . In addiagonists –14. Two nd STIM1 , 16, thanductance.2+ store-independent), store-operated (Orai1-dependent) and store-operated (Orai1-independent) mechanisms [v1.2 channels to mediate vasoconstriction [There is substantial evidence that TRPC1 channels are activated by multiple pathways, including receptor-operated . Complete EDTA-free protease inhibitor cocktail tablets were from Roche Diagnostics GmbH . DharmaFECT kb transfection reagent was obtained from Horizon Discovery . N-Glycosidase F (PNGase F) was purchased from New England Biolabs Inc . Mouse monoclonal Anti-GOK/Stim1 antibody  was purchased from BD Biosciences . Thapsigargin (TG), histamine, protein A agarose beads, HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid), EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), EDTA (ethylenedinitrilotetraacetic acid), bovine serum albumin (BSA), biotin fenol, trolox, sodium azide, sodium ascorbate, biotin, rabbit polyclonal anti-Orai1 antibody , rabbit polyclonal anti-Orai1 (AB-1) antibody  and rabbit polyclonal anti-β-actin antibody  were obtained from Sigma . Horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) antibody and goat anti-rabbit IgG antibody were from Jackson laboratories . CMV-promoter EYFP-Orai1 plasmid (which might give Orai1α and Orai1β) was kindly provided by Christoph Romanin . CMV-promoter Orai1α-EGFP and Orai1β-EGFP plasmids were kindly provided by Mohamed Trebak , been optimized to give essentially 100% Orai1α and 100% Orai1β, respectively. Green fluorescent genetically encoded Ca2+ indicator for optical imaging (version 1.2) (G-GECO1.2)-Orai1 was a gift from Michael Cahalan . TRPC1-flag was a gift from Craig Montell . pRK5-HA-TRPC1-F562A (dnTRPC1) plasmid was a gift from Jessica Sabourin . MO7O-Orai1-E106Q-flag (dnOrai1) was provided by Thierry Capiod . MO91-STIM1-CFP was a gift from Anjana Rao . All other reagents were of an analytical grade.Fura-2 acetoxymethyl ester (fura-2/AM) was from Molecular Probes . High-glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, trypsin, penicillin/streptomycin, rabbit polyclonal anti-TRPC1 antibody , mouse monoclonal anti-PMCA antibody , high-fidelity PCR kit (Platinum™ SuperFi™ DNA Polymerase), Clean-Blot™ IP detection reagent, SuperSignalPlasmids encoding GECO-Orai1 and Orai1β-EGFP were used as template to generate their respective dominant-negative mutants GECO-Orai1-E106Q and Orai1βE43Q-EGFP (corresponding to the Orai1αE106Q-EGFP mutant), using high-fidelity PCR kit  and the following primers; ORAI1-E106Q_F: 5′-GGTGGCAATGGTGCAGGTGCAGCTGGA-3′, ORAI1-E106Q_R: 5′-TCCAGCTGCACCTGCACCATTGCCACC-3′. Sequencing analysis results are depicted in Fig. S1.2 in high-glucose DMEM supplemented with 10% (v/v) fetal bovine serum and 100 U/mL penicillin and streptomycin, as described previously [6) were plated in 100-mm Petri dish and cultured for 48 h, while, for calcium imaging, determination of Mn2+ entry and confocal determination of G-GECO1.2 fluorescence assays, cells (4 × 105) were seeded in a 35-mm six-well multi-dish.HeLa cells were obtained from the American Type Culture Collection  and cultured at 37 °C with a 5% COeviously . For tra6 cells) were stimulated with 3 μM histamine or with vehicle and subsequently lysed with ice-cold NP-40 buffer pH 8 containing 137 mM of NaCl, 20 mM of Tris, 2 mM of EDTA, 10% glycerol, 1% nonidet P-40, 1 mM of Na3VO4, and complete EDTA-free protease inhibitor tablets. Cell lysates (1 mL) were immuno-precipitated by incubation with 2 µg of anti-Orai1, anti-STIM1 or anti-TRPC1 antibody and 50 µL of protein A-agarose overnight at 4 °C on a rotary platform. Orai1 variants expression and detection were analyzed after protein de-glycosilation by treatment of whole-cell lysates with PNGase F according to the protocols provided by the manufacturer. Cell lysates and immuno-precipitates were resolved by 10% or 12% SDS-PAGE and separated proteins were electrophoretically transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (w/v) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein-binding sites. Immuno-detection of Orai1 variants, β-actin, STIM1, PMCA and TRPC1 was achieved by incubation for 1 h with anti-Orai1 antibody diluted 1:1000 in TBST, 1 h with anti-β-actin antibody diluted 1:2000 in TBST, 1 h with anti-STIM1 diluted 1:500 in TBST, 2 h with anti-PMCA diluted 1:1000 in TBST or 2 h with anti-TRPC1 diluted 1:1000 in TSBT. The primary antibody was removed, and blots were washed six times for 5 min each with TBST. To detect the primary antibody, blots were incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG antibody diluted 1:10,000 in TBST, or Clean-Blot™ IP Detection Reagent diluted 1:250 in TBST, and then exposed to enhanced chemiluminescence reagents for 5 min. The antibody binding was assessed with a C-DiGit Chemiluminescent Western Blot Scanner  and the density of bands was measured using ImageJ software v.1.8.0_172 . Data were normalized to the amount of protein recovered by the antibody used for the immunoprecipitation or to β-actin from the same gel.Immunoprecipitation and Western blotting were performed as described previously . Briefly2 for 30 min in cell culture medium supplemented with 2.5 mM biotin–phenol. Cells were stimulated with 3 μM histamine for 5 min or with vehicle, then medium was removed and cells were washed three time with PBS  and incubated for 1 min in biotinylation buffer (PBS supplemented with 1 mM H2O2). Later, biotinylation buffer was aspired and cells were washed three times with STOP/wash buffer . Cells were subsequently lysed with ice-cold lysis (RIPA) buffer pH 8  supplemented with 5 mM Trolox, 10 mM sodium ascorbate, 10 mM sodium azide and then cells were scraped. Cell lysates were transferred to micro-centrifuge tubes and samples were sonicated. Clear cell lysates were obtained by centrifugation at 20,000 × g for 30 min at 4 °C and total protein concentrations were determined using the BCA method. Biotinylated proteins were isolated by incubation with 50 µL of streptavidin beads at 4 °C for 2 h on a rotary platform. Later, beads were washed twice with 1 mL RIPA buffer, once with 1 mL 1 M KCl, once with 1 mL 0.1 M Na2CO3, once with 1 mL 2 M urea in Tris–HCl pH 8.0 and once with 1 mL RIPA buffer. For Western blotting assay, the biotinylated and non-biotinylated fractions were then eluted by boiling the beads at 95 °C for 15 min in Laemmli buffer  supplemented with 100 mM DTT and 1 mM biotin, and Western blotting was performed as described above.The APEX2 proximity labeling assays were performed as described previously . Briefly2, 5 glucose, and 25 HEPES, pH 7.4, supplemented with 0.1% (w/v) BSA. Cells were examined at 40× magnification  and were alternatively excited with light from a xenon lamp passed through a high-speed monochromator Optoscan ELE 450  at 340/380 nm. Fluorescence emission at 505 nm was detected using a cooled digital sCMOS camera Zyla 4.2  and recorded using NIS-Elements AR software . Fluorescence ratio (F340/F380) was calculated pixel by pixel, and the data were presented as ΔF340/F380. Histamine-evoked changes in cytosolic free Ca2+ concentration were estimated as the area under the curve measured as the integral of the rise in fura-2 fluorescence ratio 10 min after the addition of histamine and taking a sample every second. TG-induced Ca2+ entry was estimated as the integral of the rise in fura-2 fluorescence ratio 2.5 min after the addition of Ca2+ and taking a sample every second.Cells were loaded with fura-2 by incubation with 5 μM fura-2/AM for 30 min at 37 °C. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope  with an image acquisition and analysis system for video-microscopy . Cells were continuously super-fused at room temperature with HEPES-buffered saline (HBS) containing (in mM) 125 NaCl, 5 KCl, 1 MgCl2+ oscillations was performed as described previously [2+ traces obtained in the Ca2+-imaging experiments were plotted using GraphPad Prism v.8.4.3 . The numbers of oscillations per 10 min were manually counted. Then, cells were classified in three groups and a percentage of each group of cells was calculated for each individual coverslip. The first group, oscillating cells, includes cells showing regenerative oscillations after histamine stimulation for the duration of the experiment, where each oscillation returns to baseline before the start of the next oscillation. A second group, plateau cells, includes cells showing a sustained a cytosolic Ca2+ signal that was ≥ 25% of the initial peak, for at least 5 min after stimulation. Finally, the last group includes cells that show either no response to histamine stimulation or showed only one initial spike and remained at baseline for the duration of the recording. All manual counts were independently recorded by two individuals to ensure accuracy of counts.The analysis of Caeviously . All Ca22, 0.5 MnCl2, 125 NaCl, 5 KCl, 1 MgCl2, 5 glucose, and 25 HEPES, pH 7.4, supplemented with 0.1% (w/v) BSA. Cells were examined at 40× magnification  and were excited with light from a xenon lamp passed through a high-speed monochromator Optoscan ELE 450  at 360 nm. Fluorescence emission at 505 nm was detected using a cooled digital sCMOS camera Zyla 4.2  and recorded using NIS-Elements AR software . Mn2+ influx was monitored as the quenching of fura-2 fluorescence at the isoemissive wavelength of 360 nm and presented on an arbitrary linear scale. To compare the rate of decay of fura-2 fluorescence when cells were subjected to different experimental procedures, traces were fitted to the equation y = S × eKX− + A, where K is the slope, S is the span and A is the plateau, as described previously [Cells were loaded with fura-2 by incubation with 5 μM fura-2/AM for 30 min at 37 °C. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope  with an image acquisition and analysis system for video-microscopy . Cells were continuously super-fused at room temperature with HEPES-buffered saline (HBS) containing (in mM) 1 CaCleviously .w/v) BSA at room temperature. Cells were examined at 60× magnification and excited using a confocal laser-scanning system  at 488 nm. Fluorescence emission at 515 nm was detected and recorded using NIS-Elements AR software . GECO fluorescence was determined before and after the addition of 3 µM histamine (resting) in presence of extracellular Ca2+ (1 mM) for 10 min. Images were analyzed using ImageJ software v.1.8.0_172 .G-GECO1.2-Orai1 or G-GECO1.2-dnOrai1-transfected Hela cells were seeded on coverslips and mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope  with an image acquisition and analysis system for video-microscopy NIS-Elements Imaging Software v.5.02.00, . Cells were continuously super-fused with HBS supplemented with 0.1% . Later, cells were incubated at 4 °C for 1 h with biotynilation buffer (PBS supplemented with 1 mg/mL EZ-Link sulfo-NHS-LC-biotin) The biotinylation reaction was terminated by addition of Tris base to a final concentration of 50 mM. Following biotinylation, cells were washed twice in PBS, disrupted using Nonidet P-40 buffer and sonicated. Cell lysates were centrifuged  and protein concentration was measured using BCA assay. Samples were incubated with 50 µL streptavidin beads at 4 °C for 2 h and re-suspended in Laemmli buffer for subsequent analysis by Western blotting. The biotinylated and non-biotinylated fractions were separated in 8% SDS-PAGE, TRPC1 surface expression was detected using a specific anti-TRPC1 antibody, while the detection of PMCA was used as control.The labeling and isolation of cell surface proteins were performed by surface biotinylation assay, as described previously . Hela ce2+ determinations) was used to compare the different experimental groups. For comparison between two groups, the Mann–Whitney U test was used. Throughout the manuscript *, **, and *** indicate p values of < 0.05, < 0.01, and < 0.001, respectively. All data with p < 0.05 were deemed significant; “ns” = nonsignificant.All data are presented as the mean ± standard error of mean (SEM). Analysis of statistical significance was performed using GraphPad Prism v.8.4.3 . Kruskal–Wallis test combined with Dunn´s post hoc test , Orai1β (Orai1βE43Q-EGFP corresponding to the E106Q mutant of the long Orai1 variant) and TRPC1 (HA-TRPC1-F562A). Expression of the different plasmids was demonstrated by Western blotting using the appropriate antibodies . Cells were stimulated with 3 µM histamine to elicit Ca2+ oscillations in the presence of 1 mM extracellular Ca2+. Traces from five representative cells are shown in Fig. 2+ oscillations . As expected, the magnitude of Ca2+ mobilization upon treatment with histamine was significantly greater in cells expressing STIM1, Orai1α, Orai1β and TRPC1 .We have analyzed the functional role of Orai1α, Orai1β and TRPC1 on histamine-induced Ca2+ oscillations significantly increased  or Orai1β-E43Q (dnOrai1β) mutants, the percentage of cells that responded with Ca2+ oscillations, we repeated the previously described protocols in the presence of the dominant-negative TRPC1 mutant . As shown in Fig. 2+ oscillations in these cells.To ascertain the possible role TRPC1 in the maintenance or Ca2+ mobilization, co-transfection of dnOrai1α or dnOrai1β, instead of the WT Orai1 variants, with STIM1 and TRPC1 led to a significant attenuation of histamine-evoked Ca2+ mobilization , which affected exclusively to the cells that responded with a sustained plateau . Co-transfection of dnTRPC1 with STIM1, Orai1α and Orai1β (or the corresponding dnOrai1α and dnOrai1β) significantly decreased histamine-evoked Ca2+ mobilization as compared to HeLa cells expressing WT TRPC1 . In the presence of dnTRPC1, agonist-induced Ca2+ mobilization was still smaller when dnOrai1α, dnOrai1β or both were expressed , with an average of 9.2 ± 0.7 and 7.6 ± 0.7 oscillations/10 min, for Orai1α and Orai1β . When the WT Orai1 variants were replaced by their corresponding dominant-negative mutants, the percentage of cells that responded with Ca2+ oscillations significantly decreased to 18 ± 3% and 21 ± 2%, for Orai1α and Orai1β, respectively, and the average number of oscillations per cell was attenuated . As expected, substitution of the Orai1 variants by their respective dominant-negative mutants significantly attenuated the magnitude of histamine-evoked Ca2+ mobilization . These findings confirm that both variants of Orai1 are required for agonist-induce Ca2+ oscillations. We have further evaluated the role of TRPC1 in this triple co-expression system replacing TRPC1 by dnTRPC1. Substitution of WT TRPC1 for dnTRPC1 did not significantly alter the response pattern of the cells to histamine as reported in Fig. 2+ oscillations in these cells. Interestingly, expression of dnTRPC1 instead of WT TRPC1 significantly attenuated the magnitude of agonist-evoked Ca2+ mobilization exclusively in cells expressing functional Orai1α .We have further analyzed the functional role of Orai1α, Orai1β and TRPC1 on histamine-induced Ca2+ stores had been depleted by 1 min treatment with thapsigargin  in the presence of extracellular Ca2+ (1 mM). After immunoprecipitation with the anti-TRPC1 antibody, and protein de-glycosylation with PNGaseF, Western blotting revealed the presence of Orai1α and, in a significantly less amount, Orai1β in samples from resting cells.  without having any effect on the interaction between Orai1β and TRPC1 . Western blotting with anti-STIM1 antibody confirmed a similar amount of protein in all lanes  or a medium containing 1 mM CaCl2 and were stimulated with TG or left untreated and lysed. Orai1α was pulled down by immunoprecipitation of the whole-cell lysates with anti-Orai1 N-terminal antibody (epitope: amino acids 2–61 exclusive to Orai1α). The immuno-precipitates were subjected to SDS/PAGE and Western blotting with anti-TRPC1 antibody to detect the Orai1α-TRPC1 association. On the other hand, Orai1β was pulled down from the supernatant by immunoprecipitation with anti-Orai1  antibody (epitope: amino acids 288–301 present in both Orai1 variants). The resulting pellet was subjected to SDS/PAGE and Western blotting with anti-TRPC1 antibody to detect the Orai1β-TRPC1 association. A detectable association was found between Orai1α and TRPC1 both in the absence and presence of extracellular Ca2+ but we were unable to detect a significant interaction between TRPC1 and Orai1β .We also analyzed the interaction between TRPC1 and Orai1 variants by immunoprecipitation using an alternative experimental maneuver. As shown in Fig. S5, HeLa cells were suspended in a Ca2+ (1 mM). As shown in Fig. We have further tested the interaction between Orai1 variants and TRPC1 by proximity-dependent biotinylation using APEX2. HeLa cells were co-transfected with STIM1-CFP, TRPC1 and either Orai1α-EGFP or Orai1β-EGFP. Experiments were performed in resting cells and cells stimulated with 3 µM histamine for 5 min in the presence of extracellular CaAltogether, either using co-immunoprecipitation or APEX2 proximity labeling assay, our results indicate that there is a detectable interaction of TRPC1 with Orai1α under resting conditions that was significantly enhanced by treatment with TG or physiological agonists. By contrast, there is a small, if any, interaction between Orai1β and TRPC1 that seems constitutive in nature.2+ influx through TRPC1 on the fluorescence of G-GECO1.2 fused to Orai1α (G-GECO-Orai1α). G-GECO–Orai1α was transiently co-transfected with STIM1 and TRPC1 or dnTRPC1 mutant into HeLa cells and imaged using an epifluorescence inverted microscope. We have previously reported that G-GECO–Orai1α exhibited diffuse expression in the plasma membrane [2+ influx (detected as fluctuations) through Orai1α in the presence of 1 mM extracellular Ca2+. Traces from five representative cells are shown in Fig. Alternatively, we have analyzed the proximity between TRPC1 and Orai1α by looking for the influence of Camembrane . Cells w2+ influx detected by G-GECO1.2 was significantly greater than that observed in cells expressing dnTRPC mutant . These findings suggest that either TRPC1 enhances Ca2+ influx through Orai1α or that both channels are very close so that Ca2+ entry through TRPC1 might be detected by Orai1α-fused G-GECO1.2. Concerning the first hypothesis, it is well established that Orai1/STIM1 forms the primary SOCE channel and leads to the initial Ca2+ influx upon depletion of the intracellular Ca2+ stores, while TRPC1, can be recruited to ER–PM junctions and inserted into the PM by a mechanism that requires Orai1-mediated Ca2+ entry [2+ influx via Orai1α, but once cation entry through TRPC1 is initiated, there are several mechanisms that might negatively influence Orai1α function, including changes in the driving force for Ca2+ entry by membrane depolarization, competition for STIM1 units or the facilitation of Ca2+-dependent Orai1α inactivation [2+ and Orai1α serine phosphorylation was estimated 10 s, 1 min and 10 min after the addition of the agonist. As depicted in Fig. S6a, Western blot analysis reveals that Orai1α serine phosphorylation was comparable in cells expressing TRPC1 and dnTRPC1 mutant, which indicates that Ca2+ influx via TRPC1 does not alter serine phosphorylation-dependent inactivation of Orai1α. We have further investigated whether TRPC1 might influence the plasma membrane expression of Orai1α by biotinylation. HeLa cells were co-transfected with STIM1, Orai1α and either TRPC1, dnTRPC1 or shTRPC1. Cells were stimulated with histamine (3 µM) in the presence of 1 mM extracellular Ca2+ and the exposure of Orai1α in the plasma membrane was estimated 10 s, 1 min and 10 min after the addition of the agonist. As depicted in Fig. S6b, our results indicate that the plasma membrane location of Orai1α was similar in cells expressing TRPC1 or the dnTRPC1, as well as in cells transfected with shTRPC1, thus suggesting that TRPC1-mediated cation entry does not influence the plasma membrane expression of Orai1α. Altogether, these findings challenge a positive role for TRPC1 in Ca2+ influx via Orai1α and further support that Orai1α and TRPC1 are in the close proximity.In cells expressing TRPC1, the overall Cativation . Accorditivation , 35. To 2+ entry via TRPC1 can influence G-GECO1.2 fluorescence, we repeated the experiments in cells expressing G-GECO fused to the dominant-negative Orai1α-E106Q mutant (GECO-dnOrai1α), which lacks Ca2+ influx through the channel. It is noteworthy that under these conditions, TRPC1 is not expected to be fully functional as Ca2+ entry through Orai1α is impaired [2+ influx were significantly greater than in cells expressing dnTRPC1 . By contrast, cells co-expressing STIM1, TRPC1, Orai1α and dnOrai1β exhibited a similar response to TG than those co-expressing Orai1β , although the effect of co-expression of both dominant-negative mutants was not found to be significantly different from that induced by dnOrai1α . This response was abolished when Orai1 was substituted by a dominant-negative Orai1 mutant . As depicted in Fig. S9d–f, and in agreement with Desai et al. [2+ equally well, and generated signals comparable to those observed with wild-type Orai1, which confirms that, in HEK-293 cells, Orai1α and Orai1β are equally effective in activating TRPC1. Interestingly, these findings indicate intercellular differences in the functional role of Orai1α and Orai1β.We have noticed that our findings in HeLa cells differ from those reported by Desai et al. in HEK-293 cells, which showed that both Orai1α- and Orai1β-mediated signals could activate TRPC1 . Therefoi et al. , both Orp < 0.001). Consistent with the results presented in Fig. p < 0.01). In cells transfected with shTRPC1, the expression of TRPC1 in the PM was undetectable  mutant  mutant, thus suggesting that STIM1 is required for the activation of Orai1, but then, it does not seem to be further required to gate TRPC1 channels.We have further evaluated the role of Orai1 in TG-induced Mnte TRPC1 , 40), OrM1K684,68E mutant 2+ store depletion or cell stimulation with physiological agonists. First, we have found interaction between Orai1α and TRPC1 in native cells by co-immunoprecipitation using both anti-Orai1 and anti-TRPC1 antibodies. Second, as antibody-based approaches strongly depend on the specificity of the antibodies themselves, we have further demonstrated co-localization of Orai1α and TRPC1 by an independent mechanism using APEX2 proximity labeling assay in cells expressing STIM1-CFP, TRPC1 and Orai1α-GFP. These approaches indicate co-localization of Orai1α and TRPC1 under resting conditions and that this interaction significantly enhances upon Ca2+ store depletion using TG or histamine. Nevertheless, our findings suggest that Orai1β is scarcely associated to TRPC1 under resting conditions but this interaction, if significant, is constitutive and does not depend on Ca2+ store depletion or stimulation with agonists.Our present studies reveal that Orai1α co-localizes with TRPC1 and is required for the location of this channel in the plasma membrane and its activation upon Ca2+ entry by G-GECO–Orai1α. GECO is a channel-fused fluorescent Ca2+ indicator that detects changes in Ca2+ concentration in the close proximity of the channel to which it is associated [2+ concentration near the channel upon discharge of the endoplasmic reticulum using TG in HeLa cells [2+ store depletion or stimulation with physiological agonists.Our results provide further evidence for the co-localization between Orai1α and TRPC1 by revealing cross-detection of TRPC1-mediated Casociated . We haveLa cells , thus su2+ entry to a similar extent to TRPC1 knockdown in HeLa cells. The experiments were performed in the presence of 1 mM extracellular Ca2+ to maintain Orai1 Ca2+ selectivity [2+ entry-dependent responses, such as TRPC1 expression in the plasma membrane [2+ responses was similar either expressing TRPC1 or the dominant-negative TRPC1 mutant , instead of functional Orai1α, significantly impaired both the surface exposure of TRPC1 and divalent cation entry through the channel. In support of these findings, inhibition of Orai1 by synta66 significantly attenuated Mnectivity  and allogh Orai1  and furtgh Orai1 . Caveoligh Orai1 . As TRPCia Orai1  and bothia Orai1 . For insia Orai1 , 47 migh2+ whose frequency and intensity have been reported to play a key role in the development of a number of cellular events, such as gene transcription [2+ oscillations are triggered by IP3-evoked Ca2+ release from the intracellular Ca2+ stores leading to the activation of store-operated channels in the plasma membrane [2+ oscillations, while TRPC1 has a minor role, if any. While TRPC1 does not seem to play a relevant role in Ca2+ oscillations, recruitment of this channel clearly enhances the magnitude of Ca2+ responses . The observation that TRPC1 is not required for Ca2+ oscillations cannot be attributed to a loss of function of transiently expressed TRPC1 due to ectopic location or any other artifact as co-expression of STIM1, Orai1α, Orai1β and TRPC1 in HeLa cells resulted in a robust Mn2+ entry upon stimulation with TG in the presence of extracellular Ca2+.Upon stimulation with physiological concentrations of agonists, cells develop repetitive oscillations in the concentration of cytosolic Cacription . Ca2+ osmembrane , 11. UsiIn conclusion, our results provide multiple evidence for store depletion-dependent co-localization of Orai1α with TRPC1 and the regulation of TRPC1 plasma membrane expression and channel function by Orai1α; by contrast, the short Orai1 variant, Orai1β, is not required for plasma membrane expression and function of TRPC1. The approaches used do not allow sufficient resolution to discriminate if Orai1α and TRPC1 interact directly or they are located in the same cellular nano-domain as near components of a functional protein complex. Our results provide evidence that Orai1α and Orai1β are non-redundant and might display differential functional roles in calcium signaling.Supplementary file1 (DOCX 2895 KB)Below is the link to the electronic supplementary material.


Non-alcoholic fatty liver disease (NAFLD) is associated with the chronic progression of fibrosis. In general, the progression of liver fibrosis is determined by a histopathological assessment with a collagen-stained section; however, the ultra-early stage of liver fibrosis is challenging to identify because of the low sensitivity in the collagen-selective staining method. In the present study, we demonstrate the feasibility of second-harmonic generation (SHG) microscopy in the histopathological diagnosis of the liver of NAFLD patients for the quantitative assessment of the ultra-early stage of fibrosis. We investigated four representative NAFLD patients with early stages of fibrosis. SHG microscopy visualised well-matured fibrotic structures and early fibrosis diffusely involving liver tissues, whereas early fibrosis is challenging to be identified by conventional histopathological methods. Furthermore, the SHG emission directionality analysis revealed the maturation of each collagen fibre of each patient. As a result, SHG microscopy is feasible for assessing liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological method. The assessment method of the ultra-early fibrosis by using SHG microscopy may serve as a crucial means for pathological, clinical, and prognostic diagnosis of NAFLD patients. Non-alcoholic fatty liver disease (NAFLD) is a complex disease associated with the accumulation of excess fats in the liver, which is considered the most common cause of chronic liver disease ,2,3,4. NVarious diagnostic methods are clinically used to assess fibrosis in the NAFLD liver, including blood tests ,12, ultrIn recent years, second-harmonic generation (SHG) microscopy has emerged as a new imaging technique for visualising fibrosis ,27,28,29In the present study, we sought to demonstrate the feasibility of SHG microscopy in the histopathological diagnosis of the livers of NAFLD patients for the quantitative assessment of the ultra-early stage of fibrosis. We examined four representative livers of NAFLD patients, who were clinically diagnosed as ultra-early stages of NAFLD , early and advanced stages of NASH. We confirmed the histopathological assessment capability of SHG microscopy in terms of histology of fibrosis and maturity of each collagen fibre. Our result suggests the feasibility of SHG microscopy for assessing liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological method.We examined four representative patients who were clinically diagnosed with NAFLD. Clinical and histopathological features examined by a routine blood test, ultrasound elastography, and histopathological examination are shown in Representative histopathological images of the livers of NAFLD patients are shown in Moderate fibrosis was apparent in the liver of Patient 4 as visualised by the Sirius Red-stained image. In the liver of Patient 3, the slightly stained structures were observed, but there was no apparent fibrosis. Most areas of the liver of the ultra-early stage of NAFLD (Patient 1 and Patient 2) had no apparent fibrosis. In all the livers of patients, the sinusoidal walls seemed to be slightly stained but at a level that would be diagnosed as no fibrosis or suspected artefacts by routine histopathological diagnosis.We investigated the imaging capability of SHG microscopy in the liver of NAFLD patients. Firstly, we examined the histological assessment capability of SHG microscopy, as shown in Furthermore, SHG images reveal fine structures of fibrous tissues in all livers of the NAFLD patients. Especially in Patient 2, fine fibrous structures appeared to surround the sinusoid throughout the livers. This may indicate the early stage of the development of fibrosis by activated hepatic stellate cells along sinusoids. Interestingly, these fibrous structures were not apparent in the other livers a,c,d. ThHistological assessment capability of SHG microscopy in backward detection configuration was also investigated, as shown in The SHG light intensity strongly depends on the maturity of the fibrous tissue, such as the molecular organisation of collagen molecules. In addition, SHG imaging is highly quantitative because it does not require a staining process that may yield different results depending on the operator. Therefore, SHG light intensity is a suitable candidate as a quantitative index in assessing fibrosis.3 in volume) [As shown in  volume) ,57,58, tThe mean SHG intensities tended to be stronger as patients with more advanced NAFLD, but with no significant difference among patients except the comparison between Patient 1 and Patient 2, as shown in p < 0.01). The area fractions of Patient 2 with an ultra-early stage of NAFLD, Patient 3 with an early stage of NASH, and Patient 4 with an advanced stage of NASH had no differences. In contrast, in the higher SHG intensity region, there was a tendency for the area fraction to be higher in patients with more advanced NAFLD, although there was no significant difference between patients. These results suggest that the SHG microscopy may have the potential to characterise the ultra-early stage of NAFLD that is hard to precise grading by conventional diagnostic methods.We also performed an area fraction analysis based on SHG intensity, as shown in For fibrosis analysis of NAFLD patients in tissue sections, both forward and backward detection configurations can be employed. In such a case, we can perform the SHG emission directionality analysis for a more quantitative maturation assessment of fibrosis. The major SHG light propagates in the forward direction, but some also propagate in the backward direction, as demonstrated in the previous section. Since incoherent backward-propagated SHG light produced by multiple scattering of forward-propagated SHG light is negligible because of thin tissue sections (ca. 2 µm), the relative SHG intensity between forward- and backward-propagated SHG light depends on fibre diameter, packing density, and molecular order of collagen molecules due to the coherence in generating SHG light ,60.We performed the SHG emission directionality analysis using forward- to backward-detected SHG intensity ratio images, as shown in Statistical analysis of the mean SHG intensity ratios of fine (less than 2 µm) and thick (2 µm or thicker) fibrous tissues of each patient was shown in However, in the thick fibrous tissues, the mean SHG intensity ratio of the advanced NASH liver (Patient 4) was significantly lower than that of the early NASH liver (Patient 3). As shown in inset A of In the present study, we demonstrated the feasibility of SHG microscopy in assessing liver fibrosis of NAFLD patients at the ultra-early stage. Four representative livers of NAFLD patients, who are clinically diagnosed as an ultra-early stage of NAFLD without hepatitis, early stage of NASH, and advanced stage of NASH were successfully examined in terms of histology and fibrosis maturation by using SHG images, SHG intensity, and SHG emission directionality. Importantly, we revealed that the SHG assessment of liver fibrosis enabled to characterise of the fibrous structures in the livers at not only the advanced stage of NASH but also the ultra-early stage of NAFLD patients that were difficult to diagnose by the general histopathological methods such as using Sirius Red-stained sections.The progression of liver fibrosis is an essential indicator for assessing NAFLD, whether the liver will progress to cirrhosis and liver cancer. If liver fibrosis is significantly advanced, irreversible fatal damages may occur to the liver, and few effective treatment options are available. In contrast, it has been suggested that the ultra-early stage of liver fibrosis may be reversible. However, liver fibrosis at the ultra-early stage is challenging to detect by general diagnostic methods, such as routine histopathological assessment using stained tissue sections, because the fibrous structure is not sufficiently matured, as in the case of the ultra-early stage (Patient 1 and Patient 2) in this study. This study suggests that SHG microscopy can detect fibrosis with high sensitivity, which is often missed by the conventional diagnostic method. The further development of this study will lead to the diagnosis of liver fibrosis at a reversible stage and provide effective treatment to patients with NAFLD/MAFLD.For the assessment of liver fibrosis, it is necessary to detect the spatial distribution and the molecular maturity of fibrous tissue with high sensitivity. There are several bioimaging methods focusing on liver fibrosis, such as ultrasound elastography, magnetic resonance (MR) elastography, histopathological imaging with stained tissue sections, and fluorescence microscopy. Ultrasound elastography and MR elastography evaluate the fibrosis in terms of liver stiffness. It is well known that liver stiffness could increase following the progression of fibrosis. The liver stiffness is obtained by evaluating the propagation behaviour of elastic waves through the liver, introduced by ultrasound or mechanical vibration, using ultrasound imaging or MR imaging. These techniques are widely used in clinical practice because they are non-invasive techniques and can be performed several times during the initial diagnosis and follow-up of NAFLD patients. However, the liver stiffness does not significantly change until fibrosis is sufficiently advanced, and thus the detection sensitivity of the liver fibrosis at the ultra-early stage is low. Histopathological imaging using stained tissue sections evaluates fibrosis by staining biopsy-derived liver tissue sections with fibrous tissue-selective dyes. This method is well developed and is a gold standard for the definitive diagnosis of liver fibrosis. However, this approach is based on detecting light transmittance of the dye stained at fibrous tissues, which reduces the detection sensitivity of fine fibrous tissues with little change in light transmittance. Furthermore, the definitive diagnosis of NAFLD using histological features of liver tissues requires a highly trained pathologist. Fluorescence microscopy evaluates fibrosis by detecting the fluorescence emitting from the fluorescent dye selectively stained at the fibrous tissues ,62. The By contrast, SHG microscopy can achieve high sensitivity sufficient to assess liver fibrosis, including the ultra-early stage of NAFLD, as demonstrated in this study. Availability of paraffin block tissues sections and no requirement of staining tissues in SHG measurement suggest that the SHG microscopy is suitable for pathological diagnosis where long-term follow-up is required. Furthermore, as shown in this study, the SHG intensity and emission directionality analyses can be easily quantified. If diagnostic indicators of liver fibrosis in NAFLD are obtained with sufficient numbers of clinical trials in further study, SHG microscopy may have the potential to assess liver fibrosis even in an environment where no skilled pathologist is available in the future. In addition, SHG microscopy offers further unique possibilities for assessing liver fibrosis of NAFLD patients as follows. Since the light source and detection method of SHG measurement are less invasive to living organisms, SHG microscopy has in vivo diagnosis capability. Actually, many researchers have investigated in vivo evaluation capability of SHG microscopy or endoscopy with various organs, including the liver ,66,67,68The limitation of this study was the small number of enrolled patients. We examined the typical four patients with NAFLD and did not evaluate the variabilities among patients and biopsy locations. Thus, further studies should examine a large number of patients with various stages of NAFLD to confirm the clinical efficacy of SHG microscopy and determine the criteria for assessing liver fibrosis of NAFLD in the future. Although the limitation exists, this study provides a proof-of-principle demonstration of the feasibility of SHG microscopy for evaluating liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological methods.Liver tissues were collected by needle biopsy from representative four patients who were clinically diagnosed as NAFLD in Kagawa Prefectural Central Hospital from October 2017 to March 2018. One biopsy was performed on each patient. All experiments were performed in accordance with the relevant guidelines and regulations. The study was approved by the Ethics Committee of Kagawa Prefectural Central Hospital (permission no. 1059). For all patients, fully informed consent was obtained after a full explanation of the study design and before the surgery.The obtained liver tissues were immediately fixed with 10% neutral buffered formalin and embedded in paraffin after dehydration with alcohol and clearing with xylene. The paraffin-embedded tissue blocks were sliced into 2 µm-thick sections with a microtome. Three serial sections were obtained. A section was subjected to SHG measurement without any staining. Two sections were subjected to histopathological examination with HE staining or fibrosis assessment with Sirius Red staining.All the patients were assessed by clinical examination, including routine blood tests, ultrasound elastography, and histopathological diagnosis. A routine blood test was performed for the evaluation of liver enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and platelet count (PLT). By using the age of the patient, AST, ALT, and PLT, we calculated the FIB-4 index, which is one of the indices used for the prediction of liver fibrosis ,70. The SHG imaging was performed with a home-built laser-scanning SHG microscope with a motorised XY stage, as shown in Quantitative data were expressed as mean and standard deviation. For the quantitative SHG intensity analysis, randomly selected six areas with 500 × 500 µm in each sample were used. The mean SHG intensity of each area was calculated, then the mean and standard deviation among these areas were obtained. The same six areas were also subjected to the area fraction analysis. The area fraction of SHG intensity in each area was calculated, then the mean and standard deviation among these areas were obtained. For the quantitative SHG intensity ratio analysis, the randomly sampled 50 pixels with the backward-detected SHG intensity of 5 counts or stronger for each fine (less than 2 µm) or thick (2 µm or thicker) fibres were analysed. The fine and thick fibres were extracted using an image mask, in which the median filter with 10 × 10 pixels and thresholding was applied to the forward-detected SHG image. The final extraction results of the fine and thick fibres were also confirmed manually.Statistical significance was evaluated using Student’s t-test e,f or MaWe revealed the feasibility of the SHG microscopy for assessing liver fibrosis on NALFD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological methods. Various criteria in the SHG imaging assessment of fibrosis, including histological analysis, SHG intensity, and SHG emission directionality, were effective for a quantitative assessment of liver fibrosis in NAFLD patients. If this method is clinically implemented as a successful diagnosis of liver fibrosis in patients diagnosed as NAFLD/MAFLD in the future, ultra-early diagnosis of the risk of liver fibrosis will be realised before it becomes an advanced NASH for which there has been a lack of effective treatment. Therefore, the assessment method of the ultra-early fibrosis by using SHG microscopy may serve as a crucial means for pathological, clinical, and prognostic diagnosis of NAFLD/MAFLD patients.


The aim of this study was to evaluate the impact of stress due to heat  or handling (human restraining), assessed using infrared thermography, on the performance parameters of rabbits of a Spanish Common breed. Thirty-nine rabbits weaned at the age of 28 days were analyzed during a 38-d fattening period at two times of the year: a cold period and a warm period. The rabbits’ stress due to handling was assessed by the temperature difference taken by infrared thermography in the inner ear of the animals, before and after being handled. In general, the productive results were low, since it was an unimproved rustic breed. The animals were more productive in the cold season as the values obtained for daily feed intake (DFI), average daily gain (ADG), total body weight (TBW), total feed intake (TFI) and total weight gain (TWG) were higher then, while the feed conversion ratio (FCR) was higher in the warm season. The greater the stress due to handling, the less efficient the animals were. It was therefore concluded that changes in animal welfare caused by the rabbits’ reactivity to both climatic and individual factors affect animal productivity.Rabbits often experience stress when they perceive certain stimuli, such as handling. The physiological response of animals to stress and temperament is associated with feeding efficiency, with those with the least capacity to react to stress showing the highest performance. The aim of this study was to evaluate the impact of stress due to heat  or handling, assessed using infrared thermography, on the performance parameters of rabbits of a Spanish Common breed. Thirty-nine rabbits were analyzed during a 38-d fattening period at two times of the year: a cold period and a warm period. The rabbits’ stress due to handling was assessed by the temperature difference taken in the inner ear, before and after being handled. The animals were more productive in the cold season. Temperature-humidity index significantly influenced average daily gain (ADG) and daily feed intake (DFI). Rabbits with higher levels of stress showed higher ADG, DFI and feed conversion ratio (FCR) values. The greater the stress due to handling, the less efficient the animals were. FCR increased with higher THI. It was concluded that changes in animal welfare caused by the rabbits’ reactivity to both climatic and individual factors affect animal productivity. Docility and not being afraid of people are among the preferential characteristics selected when domesticating animals . RabbitsIn addition, the environmental conditions, especially temperature and relative humidity, affect both the physiological state and the productivity of animals . RabbitsPhysiological changes occur in order to cope with this stimulus . In the Animals may have different behavioral responses to handling; this is what is called temperament . AnimalsStress can be estimated through biomarkers and changes in body temperature, and these can be analyzed using different techniques, such as by analyzing the biochemical parameters in the blood  and in eAs has been shown, handling and environmental conditions (temperature and humidity) can be two of the most common stressors in current rabbit production systems. No record has been found of any studies describing how heat stress (measured with a temperature-humidity index) and stress caused by handling  in the post-weaning phase can influence the performance parameters of rabbits. For this reason, the main aim of this work was to evaluate the effect of the levels of heat stress, assessed through the temperature-humidity index (THI) and handling stress, evaluated by using infrared thermography. Infrared thermography was measured in the inner ear of rabbits reared for meat production, on the performance parameters of the fattening period, during the cold and warm seasons.Common Spanish agouti-coated domestic meat-oriented rabbits belonging to a strain kept at the Higher Technical School of Agricultural Engineering Teaching Farm of the University of Seville (Spain) were used. The genetic characterization  and prod3 , located in a conventional closed facility with natural ventilation . The animals were subjected to a natural photoperiod. The trial was carried out in accordance with the Spanish legislation [During the experimental periods, the rabbits were individually housed in polyvalent wire-mesh cages measuring 90 × 40 × 30 cmislation  and Direislation . The expThe rabbits were fed a pelleted commercial balanced diet (15.0% crude protein and 15.5% crude fiber) ad libitum as their only feed. Water was supplied ad libitum.n = 17 rabbits ) and winter ). The animals’ stress levels were assessed with inner ear temperature measurements, following previous research by Jaén-Téllez et al. [® 44550, Waltham, MA, USA) every time an infrared temperature sample was taken, so that each infrared temperature had a corresponding humidity and room temperature reading.A total of 39 weaned rabbits, with an age of 28 days old, were used during a 38-d fattening period in two different seasons: spring  = environmental temperature taken just at the moment when the infrared temperatures were taken in undisturbed rabbits inside the cage.Relative humidity, U (%) = Relative humidity taken just at the moment when the infrared temperatures were taken in undisturbed rabbitsInner ear DIF = Infrared inner ear temperature in the handled rabbit (H)—Infrared inner ear temperature in the undisturbed rabbit inside the cage (U).The 39 weaned rabbits were weighed on the sampling days after the temperature samples were collected. The weight gain by each rabbit and the weight of the feed was recorded twice each day on sample collection: once before being filled (weight of the feed remaining in the feeder) and second, when the feed was added, always after the pictures had been taken. With these measurements, the following productive traits were calculated:DFI = Feed weight differential (g) between two data collection dates/Days elapsed between those two data collection dates.Daily feed intake , calculated as:ADG = Live weight differential (g) between two data collection dates/Days elapsed between those two data collection dates.Average Daily Gain , calculated as:FCR = feed intake (g)/weight gain (g).The Feed Conversion Ratio (FCR) was calculated over two consecutive data collection days as:Total body weight (TBW), total weight gain (TWG) and total feed intake (TFI) during the entire fattening period were also calculated.The temperature-humidity index (THI), an indicator of thermal comfort level for animals in a housing system, was calculated according to Marai et al. , and givThe descriptive statistics ) for each trait: TBW, TWG, TFI, ADG, DFI, FCR and THI are shown in n = 39) as the experimental unit (n = 17): Inner ear DIF ≤ 0 °C; Level 2 : 0 °C < Inner ear DIF ≤ 5 °C; Level 3 : 5 °C < Inner ear DIF ≤ 10 °C and Level 4 : Inner ear DIF > 10 °C. Each environmental effect was analyzed one-by-one using one-factor analyses , the root mean squared error (RMSE) and the p value of the analysis were provided for each parameter. Effects were considered as significant when p < 0.05. Statistical analyses were performed using the Statistica v.12.0  and SAS [The quadratic equation with the coefficient of determination . The season (warm or cold) had a statistically significant effect on DFI and FCR (p = 0.007 and p < 0.001), and THI had a significant effect on ADG (p = 0.018) and DFI (p < 0.039).The environmental effects that could most influence the fattening performance were studied . Sex wasThe results of the Tukey post hoc are shown in The parameters depend on the season, warm or cold, with a DFI of 76.81 g/d in summer and 80.37 g/d in winter, which leads to a higher feed consumption in the cold season, and an ADG of 24.96 g/d in summer and 27.39 g/d in winter, which increases the rabbits’ weight in the cold season. The FCR is 3.29 in summer and 3.22 in winter, which therefore makes it a better indicator in the cold season.Depending on the level of stress, there are also changes in the productive parameters. The DFI is 44.71 g/d in non-stressed animals, but as high as 90.41 g/d in highly-stressed animals, producing a higher feed consumption the more stressed the rabbits are. The ADG ranges from 22.45 g/d in non-stressed animals to 29.14 g/d in highly-stressed animals, which results in a greater weight increase in rabbits with higher stress levels. FCR increases with the stress levels of rabbits, with 2.53 in non-stressed animals and up to 3.24 in highly-stressed animals.p < 0.001), the best adjustments (R2 = 0.17) have been produced with the DIF and FCR variables.The evolution of the fattening performance parameters depending on the THI and stress levels  is shown in Daily feed intake, ADG and FCR are among the most essential performance parameters when evaluating productive efficiency during fattening on a rabbit farm : they afAverage Daily Gain showed a lower mean value than that found by González-Redondo  for thisDaily feed intake showed an average value (77.80 ± 1.34 g/day) lower than that of selected lines, which usually present values of around 105 g/day .The low ADG and DFI values observed in the animals in this study are due to the fact that it is an unimproved rustic breed, like the Ibizan rabbit , and a bConsistent with Gupta et al. , we founThe peripheral thermal receptors are triggered to induce the center of appetite in the hypothalamus to cause a decrease in DFI  and ADG,The sex of the animals had no significant effect on the productive parameters, which is normal in rabbits which have not reached sexual maturity , at whicThe handling of the animals during the fattening period had a significant influence on the productive parameters: with the increase in stress levels, there was a tendency towards increased DFI, ADG and FCR. Acute stress can induce hyperphagia with increased feed consumption , which tBoth during and just after the action of the stressful agent, there is a suppression of feed intake induced by the action of the hormones, noradrenaline  and cortEating in response to stress is a common means of relief and gratification in animals . As a reThe stress level of the rabbit examined jointly with THI exerts a greater influence on DFI, ADG and FCR than THI alone. According to Oseni and Poopola , the oveIn conclusion, it has been shown that the performance parameters were altered by the action of weather conditions and the level of individual reactivity to a stressful effect, such as handling. As a result, the productive efficiency of the rabbits was higher in the cold season than in the warm season, when the lowest levels of individual reactivity were recorded. There were an increase in FCR and a decrease in ADG with increasing THI. With the increase in the ability to react to stress, there was a tendency to increase in DFI, ADG and FCR, which implies an overall decrease in the fattening performance. In addition, humidity, temperature and individual reactivity are suitable indicators which can be used to improve the rabbits’ welfare and production.The use of rabbits with low reactivity and controlled climatic conditions could benefit rabbit welfare and productivity. These results could serve as a useful guide for productive management in fattening rabbits and the use of rustic breeds  to benefit animal welfare in rabbit farming.


ABSTRACT IMPACT: The Wake Forest School of Medicine Mentor Academy has adapted to provide continued effective and relevant formal mentoring training to translational researchers in a virtual format, to improve mentoring and provide effective mentor-mentee communication tools. OBJECTIVES/GOALS: To determine whether the WFSM Mentor Academy (MA), an effective long-standing mentoring program for research faculty, is compromised after moving from an in-person to an online format as a result of COVID-19 restrictions. METHODS/STUDY POPULATION: A vetted National Research Mentoring Network (NMRN) implemented at WFSM addresses 6 major competencies  over 6 months with 10 sessions (20 contact hrs). COVID-19 required that the MA (13 participants) move to an online format after 3 (out of 10) in-person sessions. We survey 26 self-rated mentoring competencies pre- and post MA, based on a numerical 7-point scale (abstract published for ACTS 2020) and, in 2020, included additional assessments of online versus in-person MA satisfaction/effectiveness and perceived impact on abilities of MA participants to mentor in an exclusively virtual format. RESULTS/ANTICIPATED RESULTS: All 13 participants responded to the survey and rated the online format as effective (9) or somewhat effective (4) for learning content. However, for participant interactions, only 4 found it effective and 9 somewhat effective. When assessing ability to mentor in a virtual format, most negatively affected competencies were ‘helping your mentee network effectively’ (7 of 13), ‘motivating your mentee’ (7), and ‘identifying and accommodating different communication styles’ (6). Goal setting  was rated easier under COVID-19 restrictions by 3 mentors. Increases in Pre-Post self-expressed mentoring effectiveness  are similar to historical values, and 12 of the 13 mentors changed mentoring practices based on MA experiences. DISCUSSION/SIGNIFICANCE OF FINDINGS: While 2020 ratings for increased effectiveness are similar to prior years, since the 2021 MA will remain online, we will adjust content to address challenges identified in training mentors and in mentoring trainees in virtual settings by strategies to keep MA participants engaged online and sharing new resources for virtual/hybrid format mentoring.


Significant progress has been made in the decoding of Ca2+ signatures into downstream responses to modulate differential developmental and physiological responses in the whole plant. Ca2+ sensor proteins, mainly calmodulins (CaMs), calmodulin-like proteins (CMLs), and others, such as Ca2+-dependent protein kinases (CDPKs), calcineurin B-like proteins (CBLs), and calmodulin-binding transcription activators (CAMTAs) have played critical roles in coupling the specific stress stimulus with an appropriate response. This review summarizes the current understanding of the Ca2+ influx and efflux system in plant cells and various Ca2+ binding protein-mediated signal transduction pathways that are delicately orchestrated to mitigate abiotic and biotic stresses. The probable interactions of different components of Ca2+ sensor relays and Ca2+ sensor responders in response to various external stimuli have been described diagrammatically focusing on established pathways and latest developments. Present comprehensive insight into key components of the Ca2+ signaling toolkit in plants can provide an innovative framework for biotechnological manipulations toward crop improvability in near future.Plant growth, development, and ultimately crop productivity are largely impacted by the interaction of plants with different abiotic and biotic factors throughout their life cycle. Perception of different abiotic stresses, such as salt, cold, drought, heat, and heavy metals, and interaction with beneficial and harmful biotic agents by plants lead to transient, sustained, or oscillatory changes of [calcium ion, Ca Spatio-temporal changes in Ca2+ concentration inside cellular compartments and the whole cell itself have an important function in the signal transduction networks of all eukaryotes. The presence of Ca2+ pumps in the most primitive bacteria suggests that from the very beginning of life, sensing and transporting of Ca2+ have emerged as an absolute homeostatic necessity for cells to combat cytotoxicity , of around 100 nM compared to extracellular surroundings and intracellular storage compartments where Ca2+ concentration ranges in the mM scale , depolarization-activated calcium channels (DACCs), and voltage-independent calcium channels (VICCs) , ionotropic glutamate receptors (GLRs), two-pore channel 1 (TPC1), annexins, mechanosensitive-like channels (MSLs), mid1-complementing activity channels (MCA), and hyperosmolarity-induced [Ca2+]cyt increase channels (OSCAs)  and CNGCs. Moreover, the channels for inositol-1,4,5-trisphosphate (IP3) and cyclic-ADP ribose (cADPR) are expected to be involved in determining the concentration of [Ca2+]cyt in plants, but the molecular elucidation is not yet clear  in a temporal manner , calmodulin-like proteins (CMLs), and calcineurin B-like proteins (CBLs) are the components of sensor relays that do not possess any enzymatic functions but they possess one or more E and F helices (EF) hands for binding with Ca2+ ions and subsequent modulation in their protein conformation  in the plant and among the reported TFs, calmodulin-binding transcription activators (CAMTAs) are the largest well-characterized CaM-binding TF in all multicellular organisms, more or less conserved from plants to human  SOS3 through myristoylation, a post-translational modification of SOS3, to mediate subsequent binding and activation of a serine/threonine kinase protein, SOS2  acts as a SOS3 protein and CBL-interacting protein kinase 24 (CIPK24), a serine/threonine protein kinase, performs as SOS2 , H+-ATPase transporter, and Na+/H+ transporter (NHX1) in Arabidopsis  pathway is a well-established CaArabidopsis and other plants to combat salt stress through phosphatidic acid (PA)-mediated signaling cascade , a CDPK (McCDPK1) has been isolated containing a nuclear localization signal (NLS) for translocation of McCDPK1 into the nucleus to mitigate salt stress , have been detected for decoding of Ca2+ signaling in both cytosol and the nucleus , and IP3  interacts with a protein kinase, PKS3 (CIPK15), to regulate ABA-dependent seed germination, stomatal opening, and subsequent gene expression  containing two C-terminal domains where domain I work in an ABA-independent manner and domain II is activated in presence of ABA  acts as a downstream component of CIPK3  show relatively less water loss from leaves and more drought tolerance  acts as a positive regulator of drought stress in Arabidopsis  reveals negative regulation of gibberellic acid biosynthesis while it activates a 14-3-3 protein (GF14c) and improves drought tolerance in transgenic seedlings during constitutive expression  plants are highly susceptible to drought stress, and CAMTA1 further regulates the expression of several stress-responsive genes and TFs, such as RD26, ERD7, RAB18, LTPs, COR78, CBF1, heat shock proteins (HSPs), and AP2-EREBP  establishes that ZmCCaMK  phosphorylates a NAC TF (ZmNAC84) and induces antioxidant defense by activating downstream genes to enhance drought tolerance  plants expressing aequorin, a luminescent protein, exhibit an increase in [Ca2+]cyt upon heat treatment. Additionally, exogenous application of Ca2+ results in improved thermo-tolerance, whereas the effect is diminished in the presence of Ca2+ chelators and inhibitors  possesses ATPase, holdase, and ATP-dependent foldase activity along with a CaM-binding domain (CaMBD). Moreover, binding to AtCaM2 in the presence of Ca2+ affects the ATPase and foldase activities of DgHsp70 protein  which eventually controls HS TFs (AtHSFA1a) and HS protein genes through phosphorylation or dephosphorylation modulating heat stress response in Arabidopsis  signaling resulting in the activation of heat shock factors (HSFs), accumulation of HSPs, and development of thermotolerance  is a wild species of grapevine having high resistance to cold and disease. Overexpression of VaCPK29 improves heat tolerance in Arabidopsis , and expressions of many BrCMLs, particularly BrCML21–1, are upregulated under heat stress  response in plants. Similar to other stresses, the increase in [Ca2+]cyt which then combines with CaM and activates L-glutamate decarboxylase (GAD) for subsequent decarboxylation of glutamate (Glu) into γ-amino butyrate (GABA). However, calcium and CaM inhibitors block the GABA accumulation in Arabidopsis seedlings , a novel CML is strongly upregulated under various abiotic stresses, such as cold, heat, and drought stress , followed by the activation of protein kinase kinase 2 (MKK2) and MAPK signaling cascade leading to the activation of cold response genes , CaM-binding membrane transporter-like proteins (MCamb1 and MCamb2) in Physcomitrella patens, OsCPK24 in rice and TCH, and a CaM-related protein, in Arabidopsis, show upregulation upon cold stress , the Ca2+-CaM-dependent NAD kinase (NADK) activity has increased after 60 min and 120 min of cold-shock , chromium (Cr), iron (Fe), arsenite (AsIII), arsenate (AsV), zinc (Zn), cadmium (Cd), copper (Cu), mercury (Hg), aluminum (Al), and nickel (Ni)  are capable of binding to the Ca-binding sites of CaM, eventually gaining entry into plant cells through Ca2+ permeable channels , and an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor effectively reduce the Pb2+-induced cell death and MAPK activity depicting the interplay between CDPK and ROS signaling under Pb2+ stress  and Arabidopsis seedlings ]. Further, transcriptome analysis of rice roots upon As(V) exposure reveals overexpression of various calcium-related proteins and kinases, such as CaMs, CBLs, CDPKs, and CIPKs, coupled with several TFs, MAPKs, NADPH oxidases along with genes involved in abiotic stresses  upon Ni stress  affirms that multiple CIPKs are playing a pivotal role to mitigate heavy metal toxicity  and other climatic vagaries such as salt, drought, ABA, cold, and heat stress , a rare earth element, is reported to trigger an increase in Ca2+ levels in the cell, besides interacting with CaM, consequently affecting the expression and conformation of CaM  and beneficial symbiont subsequently exerting differential response either by limiting invasion or by promoting association is the key to survival.Lotus japonicus and Medicago truncatula. Besides rhizobia and mycorrhiza, beneficial interactions also occur with endophytic fungi and plant-growth-promoting bacteria which colonize at the root of the host and result in better plant performance  factors in the case of rhizobial symbiosis, and mycorrhizal (Myc) factors in the case of arbuscular mycorrhizal fungi symbiosis /DMI2 (in M. truncatula), POLLUX/DMI1, CASTOR, NENA, NUP85, NUP133, CCaMK, and CYCLOPS/IPD3  and ERN1  gene is controlled by the same signaling complexes with a substitution of NSP1 by RAM1  and NSP2, are critical for rhizobium-specific gene expression as it interacts with the promoters of rhizobium-induced genes, such as 2+ influx at the plasma membrane  but become more susceptible to insect attack (Trichoplusia ni)  with several elicitors, such as pathogen-associated molecular patterns (PAMPs), microbe-associated molecular patterns (MAMPs), endogenous damage-associated molecular patterns (DAMPs), or compounds produced from cell damage due to pathogenic activity  CML1 and CML2  in tobacco, Arabidopsis, and soybean causing increased resistivity against a wide range of virulent and avirulent pathogens  as important transcriptional regulators of plant defensing gene PDF1.2. HSF HsfB2a promotes transcriptional activation of PDF1.2, after being phosphorylated by CPK3-or CPK13 in response to wounding by herbivores, such as Spodoptera littoralis  which in turn stimulates respiratory burst oxidase homolog protein B (RBOHB)  has been identified as an important modulator of several signal transductions -mediated gene-silencing strategies would be sufficient to knockdown the effect of respective isoforms. In spite of overexpressing a single Ca2+/CaM-player to mitigate stress, gene stacking or gene pyramiding of multiple positive alleles would be more relevant to cope up with the situation. Furthermore, simultaneous editing  of multiple genes could alleviate certain stresses which would eventually improve crop fitness. Although stress is a complex phenomenon, and multiple stresses are encountered by the plant at the same time, Ca2+/CaM signaling pathways are ubiquitous in every stress signaling. Hence, overexpression and genome editing might be the most suitable approach to regulate the master regulator (CaM KMT or CAMTAs) for stress mitigation in the climate-resilient condition to fetch better yield in agricultural crops.The agriculture sector is facing an unnerving challenge due to rapid climate change. In crop breeding programs, researchers are more focused on developing improved stress resilient varieties for better sustenance of mankind. Notable progress has been made in recent decades in the field of both basic and applied research studies highlighting the role of CaNP, AD, and JB conceptualized the review. NP and SH prepared the draft manuscript. AD, MM, and JB reviewed and edited the manuscript. HG made the figure after revision and corrected the manuscript. All authors contributed to the article and approved the submitted version.This publication was supported by SERB, DST, Govt. of India (File no. ECR/2018/000328) and Indian Institute of Technology Kharagpur as well as SRIC, IIT Kharagpur.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.