The volume-regulated anion channel VRAC has now been identified as LRRC8/SWELL1. However, apart from VRAC, the Ca2+-activated Cl– channel (CaCC) TMEM16A and the phospholipid scramblase and ion channel TMEM16F were suggested to contribute to cell swelling-activated whole-cell currents. Cell swelling was shown to induce Ca2+ release from the endoplasmic reticulum and to cause subsequent Ca2+ influx. It is suggested that TMEM16A/F support intracellular Ca2+ signaling and thus Ca2+-dependent activation of VRAC. In the present study, we tried to clarify the contribution of TMEM16A to ICl,swell. In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of ICl,swell by hypotonic bath solution  was Ca2+ dependent. TMEM16A augmented the activation of LRRC8A/C by enhancing swelling-induced local intracellular Ca2+ concentrations. In HT29 cells, knockdown of endogenous TMEM16A attenuated ICl,swell and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of ICl,swell by Hypo was attenuated by blocking receptors for inositol trisphosphate and ryanodine , as well as by inhibiting Ca2+ influx. The data suggest that TMEM16A contributes directly to ICl,swell as it is activated through swelling-induced Ca2+ increase. As activation of VRAC is shown to be Ca2+-dependent, TMEM16A augments VRAC currents by facilitating Hypo-induced Ca2+ increase in submembraneous signaling compartments by means of ER tethering.All vertebrate cells activate Cl VRAC iss (VRAC) . Its hexs (VRAC) . The esss (VRAC)  and posss (VRAC) . Others s (VRAC) . Despites (VRAC) , intracegulation .2+-dependent TMEM16A  and cause Ca2+ influx that activates TMEM16A and supports activation of LRRC8.Previous studies did not answer the question whether LRRC8 and TMEM16A/F are activated in parallel to give rise to Imembrane . For comnd human , which e2. Human embryonic kidney HEK293T cells (All cells were grown at 37°C in a humidified atmosphere with 5% (v/v) CO3T cells  stably e® M-450 Epoxy; Invitrogen, Carlsbad, California, United States). Knockdown of TMEM16A and LRRC8A in HT29 cells was performed through transfection by electroporation of the siRNAs siTMEM16A  and/or siLRRC8A , using a Neon Transfection System  with a program of three pulses, 1,650 V, and 10 ms. Scrambled siRNA  was transfected as negative control. All experiments were performed 48–72 h after transfection.Human LRRC8A (NM_00127244.1) and LRRC8C (NM_032270.5) were sub-cloned into the bicistronic vector pIRES2  using standard methods. The construct was verified by sequencing . Transfection of cDNAs into HEK293T cells was performed using standard protocols for Lipofectamine 3000 . CD8 cDNA was co-transfected to allow for detection of overexpressing cells by binding of anti-CD8 labeled beads , and 1× protease inhibitor mixture . Proteins were then separated by an 8.5% SDS-PAGE and transferred to a PVDF membrane . Membranes were incubated with primary rabbit monoclonal anti-TMEM16A/DOG1 antibody  NFM/TBS-T) or rabbit polyclonal anti-LRRC8A antibody  BSA/TBS-T) for 2.5 h at room temperature. A rabbit polyclonal anti-β-actin antibody  NFM/TBS-T) was used for loading control. Membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat polyclonal anti-rabbit secondary antibody  at RT for 2 h, and immunoreactive signals were visualized using a SuperSignal Chemiluminescent Substrate detection kit .2+ concentration (Ca2+ activity) was 0.1 μM. To adjust pipette Ca2+ concentrations to 0.01 μM, the pipette-filling solution contained (mM) KCl 30, K-gluconate 95, NaH2PO4 1.2, Na2HPO4 4.8, EGTA 1, Ca-gluconate 0.209, MgCl2 1.03, D-glucose 5, and ATP 3; 290 mosm/l; pH 7.2. Free Ca2+ concentrations were calculated according to a program developed at the Max Planck institute for biophysics . Ca2+ activities (free Ca2+ concentrations) were originally validated by potentiometric determination using Ca2+-sensitive electrodes. In some experiments, patch pipettes were filled with CsCl buffer containing (mM) CsCl 125, NaH2PO4 1.2, Na2HPO4 4.8, EGTA 1, Ca-gluconate 0.209, MgCl2 1.03, D-glucose 5, and ATP 3; pH 7.2. In these experiments, a CsCl buffer was used as bath solution, containing 145 CsCl, 0.4 KH2PO4, 1.6 K2HPO, 4.6 D-glucose, 1 MgCl2, and 1.3 Ca2+ gluconate, 290 mosm/l; pH 7.4.Cells were patch clamped on glass cover slips. If not indicated otherwise, patch pipettes were filled with a cytosolic-like solution containing (mM) KCl 30, K-gluconate 95, NaH2PO4 1.2, Na2HPO4 4.8, EGTA 1, Ca-gluconate 0.758, MgCl2 1.03, D-glucose 5, ATP 3; 290 mosm/l; pH 7.2. The free Ca– currents, isotonic Ringer bath solution  was changed to hypotonic bath solution  by removing 50 mM NaCl from Ringer solution. Patch-clamp experiments were performed in the fast whole-cell configuration. Patch pipettes had an input resistance of 4–6 MΩ when filled with the cytosolic-like  solution. Currents were corrected for serial resistance. The access conductance was measured continuously and was 60–140 nS. Currents (voltage clamp) and voltages (current clamp) were recorded using a patch-clamp amplifier , the LIH1600 interface, and PULSE software  as well as Chart software . Data were stored continuously on a computer hard disc and analyzed using PULSE software. In regular intervals, membrane voltage (Vc) was clamped in steps of 20 mV from −100 to +100 mV from a holding voltage of −100 mV. Current density was calculated by dividing whole-cell currents by cell capacitance.Coverslips were mounted in a perfused bath chamber on the stage of an inverted microscope  and kept at 37°C. The bath was perfused continuously with Ringer solution at a rate of 8 ml/min. For activation of volume-dependent Cl–)-sensitive enhanced yellow fluorescent protein (eYFP-I152L) was used to measure anion conductance. YFP fluorescence was excited at 490 nm using a high-speed polychromatic illumination system for microscopic fluorescence measurements , and the emitted light at 535 ± 25 nm was detected with a CoolSnap HQ CCD camera . Cells were grown on coverslips and mounted in a thermostatically controlled imaging chamber adapted to an inverted microscope , maintained at 37°C, and perfused at a rate of 5 mL/min. After a brief period under standard Ringer solution , cells were stimulated with hypotonic (200 mosm/l) solution with the following composition (in mM): NaCl 45, KH2PO4 0.4, K2HPO4 ⋅ 3 H20 1.6, glucose 5, MgCl2 ⋅ 6 H20 1, Ca-gluconate ⋅ 1 H20 1.3, Na-gluconate 50) or isotonic solution as control . Quenching of YFP-fluorescence by I– influx was induced by replacing 50 mM extracellular gluconate by I–. Control of experiment, imaging acquisition, and data analysis were done with the software package MetaFluor . Autofluorescence was negligible. For quantitative analysis, cells with low or excessively high fluorescence were discarded. Changes in fluorescence induced by I– uptake are expressed as initial rates of maximal fluorescence decrease (au/s).Quenching of the intracellular fluorescence generated by stable transfection of the iodide  added by automated injection through a syringe pump, or isotonic solution  as control. For ATP stimulation, cells were incubated with or without test compounds in a gluconate-substituted Ringer solution  and ATP was added in a symmetrical I–-substituted Ringer solution . The final I– concentration on each well was 20 mM for every experiment. Total intracellular YFP-fluorescence intensity in each well was measured continuously. Background fluorescence was subtracted, and data was normalized to the initial fluorescence. The initial rate of maximal fluorescence decay caused by I– influx was then calculated as a measure of anion conductance.Alternatively, YFP-quenching measurements were performed on a fluorescence microplate reader  kept at 37°C, using an excitation wavelength of 485 nm and emission detection at 520 nm. Cells were plated in transparent 96-well plates , cultured 48–72 h to 80–90% confluence, and incubated with or without test compounds in standard Ringer solution (300 mosm/l). After a short basal fluorescence reading, cells were stimulated with a hypotonic (200 mosm/l) solution  for 1 h at room temperature. Cells were then mounted in a thermostatically controlled imaging chamber adapted to an inverted microscope , maintained at 37°C and perfused at a rate of 5 mL. Fura-2 was excited at 340/380 nm using a high-speed polychromatic illumination system for microscopic fluorescence measurements , and emission was recorded between 470 and 550 nm using a CoolSnap HQ CCD camera . Cells were stimulated with ATP or hypotonic (200 mosm/l) solution . Intracellular calcium ([Ca2+ ]i) was calculated from the 340/380-nm fluorescence ratio after background subtraction using the formula 2+ ]i = Kd × (R−Rmin)/(Rmax−R) × (Sf2/Sb2)i)  take part in whole-cell current activated through hypotonic cell swelling (Iswelling . Here weCa2+ ]i) . Even atLRRC8A/C . This co earlier .2+, to examine how the presence of TMEM16A would affect volume activation of VRAC. However, we found overexpressed TMEM16A to be active even at this basal intracellular Ca2+ concentration and without any additional Ca2+ increase by hormonal stimulation or by Ca2+ ionophores  and an extracellular CsCl buffer , to exclude any possible contribution of K+ currents. In contrast to mock-transfected cells, TMEM16A-overexpressing cells demonstrated a large basal Cl– inward current that was inhibited by removal of extracellular Cl–, causing a right shift of the reversal potential  or whether TMEM16A supports activation of LRRC8A/C. We therefore performed additional experiments in HT29 cells, which express both ion channels endogenously.Overexpressed TMEM16A was not active under basal conditions with 10 nM intracellular Ca2+ . Overexp29 cells, which produce large endogenous Ca2+-activated Cl– currents and swelling-activated VRAC currents . The bath was perfused with Ringer solution . siRNA knockdown of endogenous TMEM16A attenuated hypo-induced whole-cell currents and changed time-independent ICl,swell to VRAC-typical time-dependent currents . Knockdown of endogenous LRRC8A strongly attenuated ICl,swell, while additional knockout of TMEM16A did not further attenuate ICl,swell. These data suggest that TMEM16A expressed in HT29 cells supports activation of LRRC8/VRAC, probably by facilitating hypo-induced Ca2+ release from ER Ca2+ stores.We reexamined the role of TMEM16A for activation of LRRC8/VRAC in HTcurrents . As showcurrents . Notably2+ concentration.The contribution of TMEM16A to swelling activation of endogenous VRAC was examined in mock-transfected HEK293 cells (-T16A) or HEK293 cells overexpressing TMEM16A (+T16A) at different extracellular hypotonicities. VRAC was activated by extracellular bath solution of different hypotonicities . The data demonstrate an increase in hypotonic activation of VRAC by coexpression of TMEM16A, which was more significant at less severe hypotonicity . These eCl,swell in iodide quenching experiments using halide-sensitive yellow fluorescent protein (YFP). When applying hypotonic bath solution (200 mosm/l), immediate increase in YFP-fluorescence is expected due to rapid uptake of water through aquaporins, dilutions of anions, and de-quenching of YFP fluorescence, which occurs within seconds . ATP induced a typical peak (ER-Ca2+ store release) and plateau  response, which were both inhibited in the absence of TMEM16A , to leave intracellular Ca2+ signaling untouched and to avoid artifacts due to voltage clamping.We examined concentration-dependent quenching by ATP and found pronounced inhibition of quenching by siRNA-TMEM16A . Notably2+-store release, xestospongin C, suramin, and probenecid, blocked ATP-induced activation of halide permeability  did not inhibit activation of VRAC (data not shown).Three different inhibitors of IP3R-mediated ER Caeability . Hypo  extracellular Ca2+, which attenuated the activation of VRAC  and TMEM16A (CaCC) are independent molecular entities and Cl– channels activated by cell swelling or intracellular Ca2+. Although TMEM16 proteins are not VRACs per se, previous data suggest a role in ICl,swell  and regulatory volume decrease (RVD). The contribution of TMEM16A and other TMEM16-proteins to ICl,swell and volume regulation is cell dependent and may be particularly relevant in highly differentiated native (non-cultured) cells. Equally important appear the patch-clamp conditions under which ICl,swell are measured, which may explain some of the earlier controversial findings regarding the role of TMEM16F , which, however, was still somewhat delayed when compared to immediate cell swelling (within seconds)  on activation of VRAC at depolarized clamp voltages. TMEM16A and TMEM16F mainly abolish time-dependent inactivation of VRAC. This has also been described in detail in a previous study . Thus, tseconds) . This suseconds) . Others Cl,swell .2+ increase in an intracellular compartment close to the plasma membrane (2+ was necessary but not sufficient to activate LRRC8A-mediated currents (2+ channels and showed that activation of VRAC was strongly dependent on Ca2+ release through IP3R (2+ microdomains. Similarly, Akita and collaborators suggested that VRAC/VSOR channels can be activated by PLC-coupled GPCRs, which depends on Ca2+ store release in close vicinity of the channel (2+ nanodomain-mediated component of VRAC (This additional mechanism may require Camembrane . Liu et currents . Lemonniugh IP3R . They co channel  and prop of VRAC .2+-dependent activation of VRAC. The role of TMEM16A and other members of the TMEM16-family for ER Ca2+ release was found meanwhile in numerous tissues (2+ release is also essential for activation of CFTR (2+ release from the ER and activation of VRAC is facilitated in the presence of TMEM16 proteins. Because knockdown of TMEM16A but not inhibition of TMEM16A by Ani9 attenuated activation of VRAC, it suggests that ER-tethering by TMEM16A rather than Cl– transport supports activation of VRAC (Our present data may help to clarify the role of TMEM16 proteins for Ca tissues . TMEM16A of CFTR . Thus, c of VRAC .2+ release will activate TMEM16 proteins and CFTR (– channel pharmacology (Along this line, TMC8, a member of the closely related family of transmembrane channel-like TMC proteins, also controlled activation of VRAC and volume regulation . Dependiand CFTR , which pmacology .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.RC, JO, and RS performed the experiments and analyzed the data. RC, RS, and KK wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


The morphology and microflora in the ileum, caecum, and colon were also examined. The results proved that the ultrastructure of the sacculus rotundus and vermiform appendix, as well as the integrity of the intestinal barrier , were impaired after DON was administrated to the rabbits. Compared to the control group, the relative abundance and diversity of the microflora decreased in all three intestinal segments in the DON group, particularly in the ileum and caecum. In conclusion, the toxic effect of DON on weaned rabbits may be performed by destroying the structure of the sacculus rotundus and vermiform appendix, as well as affecting the structure and diversity of the intestinal flora.Deoxynivalenol (DON) is a mycotoxin found in grains that poses a potential threat to human and animal health, and the gastrointestinal tract is the primary target organ. There are few studies focused on the toxicology of DON to rabbits, especially on the relation among DON, microbiota, and the gut-associated lymphoid tissue. A total of 30 weaned rabbits (35 d) were evenly divided into the control group and DON group (1.5 mg/kg bodyweight (BW)) based on their body weight. After a 24-day trial, the ultrastructures of the  Deoxynivalenol (DON), mainly synthesized through toxin-producing fungus of the Fusarium genus, is found in grains all over the world, in particular, in wheat, barley, corn, and their by-products . As one sacculus rotundus (SR) and vermiform appendix (VA) [The intestine is one of the primary target organs attacked by mycotoxins following the intake of DON ,7. Reseadix (VA) ,12, whicdix (VA) ,14. The dix (VA) ,16. At pIntestinal microflora acts as a vital role in protecting host health, such as the energy intake in the diet, the production of key metabolites for the host, the development and maturation of the immunity system, and the response to intestinal diseases . The caeConsidering the points mentioned above, the aim of the present study was to investigate the effect of DON on the ultrastructure of the SR and VA, the intestinal morphology, and intestinal microflora in weaned rabbits.Representative morphologies of the SR by SEM are illustrated in Meanwhile, the SEM photographs of the VA b showed Representative morphologies of the SR of rabbits by TEM are illustrated in Representative morphologies of the VA by TEM are illustrated in The typical morphological photographs of the ileum, caecum, and colon of rabbits via SEM are illustrated in In this experiment with two groups, three rabbits were chosen randomly from each group, and three intestinal segments were sampled in each rabbit, and, therefore, the DNA of 18 samples was extracted and sequenced totally. Then, six group libraries were obtained after 18 raw data were analyzed, and they were coded as CI, CM, CJ, DON-I, DON-M, and DON-J, that is, C and DON representing the control group and DON group respectively, as well as I, M, and J representing the samples from the jejunum, duodenum, and caecum, respectively.In the present study, an average of 89,265 effective sequence reads were acquired from each group library based on 16S rDNA amplicon sequencing, wherein the smallest number of reads was 81,336 and the largest was 92,293 . Alpha dThe rarefaction curve and rank–abundance curve were used to describe the richness and diversity of the samples in a group . RarefacThe rank–abundance curve was drawn based on the relative abundance of OTUs as the ordinate, and the sequence numbers as the abscissa, which can intuitively reflect the abundance and uniformity of the species within the sample. The smoother the rank curve in the vertical direction, the more even distribution of species, while the larger the span of the rank curve in the horizontal direction, the higher the richness of species. The results of the rank–abundance curve showed that almost all samples were close to the saturation platform, which indicates that the present data have a sufficient depth to capture most of the diversity information of the samples.Firmicutes, Bacteroidetes, and Proteobacteria were the main intestinal bacteria at the phylum level for all of the samples of rabbits. The relative abundance of Proteobacteria, Actinobacteria, and Cyanobacteria in both the ileum and caecum of rabbits decreased significantly after DON administration, while that of Firmicutes and Bacteroidetes in the ileum and colon increased significantly. However, when compared to the control group, there was no obvious variation for the abundance of bacteria floras in the colon in the DON group animals at the phylum level. In Ruminococca in the ileum and colon of rabbits was upregulated significantly after DON administration, while the abundance in the colon remained stable.The relative abundance of the dominant bacteria floras in six libraries at the phylum and family levels are displayed in Ruminococcaceae, Bacteriods, and Lachnospiraleaes all increased significantly in the ileum, caecum, and colon of rabbits after DON administration. Ruminococcaceae represented the largest number of bacteria in the three intestinal segments at the genus level. In addition, the relative abundance of the “others” significantly decreased in the ileum, caecum, and colon of DON group rabbits, indicating that the variety and richness of the intestinal microflora were reduced after the addition of DON to rabbits, especially in the ileum and caecum.The top 30 bacteria floras with the highest relative abundance at the genus level are displayed in The diagram of the Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) clustering tree based on the Unifrac weighted distance is illustrated in The gastrointestinal tract is the primary target organ attacked by mycotoxin-contaminated food or feed . After mIn the present study, SEM technology was used to observe the integrity of the intestinal barrier of weaned rabbits, and we found that the mucosal epithelium was exfoliated and lost extensively, with irregular microvilli. The mucosa in the colon had the least destruction compared with in the caecum and ileum, indicating that DON caused intestinal damage in rabbits through destroying the integrity of the physiological barrier in the whole intestines but with different degrees of severity. SEM technology also was used to observe the intestinal morphology of piglets in a previous study, and the results illustrated that the histological structure of the mucosa in different intestinal segments was destroyed after the addition of DON to pigs .Unlike our previous study in pigs, the destruction in the intestinal barrier of the rabbits induced by DON in this experiment was milder and was mainly in the ileum, while the damages to the caecum and colon were less. This may be because the rabbits are a typical post-fermented animal, and their intestinal flora are very rich, particularly in the caecum and colon, which could reduce the toxicity of DON by converting DON to de-epoxidation deoxynivalenol (DOM) . Based oThe SR and VA, as the unique GALT organs of rabbits, are primarily responsible for the development and maturation of B lymphocytes, as well as the transformation of secondary lymphoid tissues for adult rabbits, such as peyer patch (PPs) ,34. StudM cells usually enfold lymphocytes and macrophages and play a key role in transporting antigens from the enteric cavity to mucosa-associated lymphoid tissues, such as SR and VA, and certain pathogens can use M cells to invade the host and initiate infections ,36. The Rabbits are hindgut-fermentation herbivores with hundreds of microorganisms inhabiting the intestines, which constitute the intestinal micro-ecosystem and maintain a dynamic balance under the intervention of the intestinal immune system . The staProteobacteria, Actinobacteria, and Cyanobacteria in both the ileum and caecum of rabbits decreased significantly after DON administration, while that of Firmicutes and Bacteroidetes in the ileum and colon increased significantly. However, compared to the control group, there was no significant change for the abundance of bacteria flora in the colon in the DON group animals at the phylum level. The abundance of Ruminococca in the ileum and colon of rabbits was significantly upregulated at the family levels after DON administration, while those levels in the colon remained stable.In this study, the results of 16s RNA sequencing proved that the relative abundance of Ruminococcaceae, Bacteriods, and Lachnospiraleaes all increased significantly at the genus level, and the relative abundance of the “others” significantly decreased in the ileum, caecum, and colon of the DON group rabbits. In all, the abundance and richness of the intestinal microflora in the ileum and caecum were reduced compared with the three intestinal segments after the addition of DON to the rabbits, while those in the colon were kept relatively stable. These data correspond to the morphological results of the intestinal barrier observed by SEM, that is, DON had the greatest impact on the ultrastructure of the ileum of weaned rabbits, and had the least damage to the colon. DON can affect the intestinal flora of weaned rabbits, and may further affect the absorption of DON in the intestinal tract as well as the integrity of the intestinal barrier.In addition, the relative abundance of The intestinal flora not only participates in the processing of nutrients and regulates the formation of intestinal blood vessels, but also promotes the development of the GALT and mucosal immunity, induces immune tolerance, as well as produces a diverse spectrum of pre-immune antibodies . The intsacculus rotundus and vermiform appendix, as well as by affecting the structure and diversity of the intestinal flora.In conclusion, the toxic effect of DON on weaned rabbits may occur via destruction of the structure of the The protocol concerning animal experiments was audited and approved by the Animal protection committee of Shandong Agricultural University (ACSA-2018-032-September).The complete feed of weaned rabbits was provided by the Liuhe Feed Company , and the ingredient composition, nutrition level, and the concentration of Aflatoxin B1 (AFB1), Zearalenone (ZEA), and DON, are listed in sacculus rotundus, vermiform appendix, and intestinal tract  were collected quickly, rinsed using 0.9% saline, and then fixed by 2.5% glutaraldehyde for later electron microscope examination. The content of the homologous three intestinal segments were collected and immediately stored at −70 °C until the 16S DNA sequencing analysisThirty healthy, weaned Rex Rabbits (35-day) were divided evenly into the control group and DON group based on their initial weight, to provide 15 rabbits in each group. All rabbits in the two groups were fed with basic diets, but those in the DON group had DON standard  added into the drinking water. The additional amount of DON was 1.5 mg/kg BW/d, and detailed addition steps were performed according to the method reported by Yang et al. . All exp2, and then adhered to the sample stage with a conductive adhesive under the stereo microscope. Finally, the samples were observed and photographed under a scanning electron microscope  after metal spraying with an Ion Sputter Coater .After approximately two-centimeter segments of the SR, VA, and intestinal tract were collected respectively and fixed in 2.5% glutaraldehyde-paraformaldehyde overnight at 4 °C, the samples were washed using 0.2M phosphate-buffered saline (PBS) three times, 30 min per time, and then passed through a series of graded alcohols for dehydration, i.e., 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol. After being fixed in isoamyl acetate for 24 h, the samples were dried near the critical point of COAfter about one-cubic-millimeter pieces of the SR and VA were cut and immersed in 2.5% glutaraldehyde-paraformaldehyde over 24 h, the samples were fixed in 1% osmium acid for 1 h, and then washed thoroughly with PBS. Ultra-thin slices were prepared according to the conventional technique and dyed using double staining with uranium acetate and lead citrate. The slices were observed and photographed using a transmission electron microscope .We weighed 200 mg intestinal content samples and added them into 1.4 mL of Argininosuccinate Lysis buffer . Then, the QIAamp DNA Stool Mini kit for feces samples  was used to extract the DNA following the instructions. The DU640 Nucleic Acids and Protein Analyzer  was applied to evaluate the quality of the DNA at a wavelength of 260/280. Finally, after germfree ultrapure water was used to dilute and quantify precisely to 1 ng/uL, all the DNA samples were stored at −20 °C for later use.TMXL sequencing platform .16S rDNA Amplicon Sequencing was applied to sequence a polymerase chain reaction (PCR) product or captured fragment of a specific length. First, the barcoded primer pair 341F/806R set was designed for PCR amplification in this study, and the 341F primer was the following: CCTAYGGGRBGCASCAG, while the 806 primer was the following: GGACTACNNGGGTATCTAAT. The diluted genomic DNA was used as template DNA, and the barcoded primers were applied to amply the V3–V4 fragments of the 16S rDNA gene. The detailed steps of PCR reaction were performed on the basis of the method reported by Caporaso et al. . All theTMXL sequencing platform, and clean reads with high quality were obtained through the cutting and filtering of the initial reads. Uparse software  was used to cluster the clean reads of all samples, and the sequences with 97% identity by default were considered as OTUs. During the data analysis, three replicate samples were aggregated into a total database. Therefore, 18 samples were aggregated into six databases, which were coded as CI, CM, CJ, DONI, DONM, and DONJ.A small fragment library for single-end sequencing was constructed based on the IonS5The Mothur method and SSUr RNA database were used to representatively annotate species for the OTU sequence (set threshold 0.8–1), and the taxonomic information of each sample was generated into the phylum, class, order, family, and genera levels. To explore the differences of community structure between different samples or groups, multiple sequence alignment was performed based on the OTUs, and a phylogenetic tree was constructed through dimensionality reduction analysis, such as principal co-ordinates analysis (PCoA) and principal component analysis (PCA). Finally, all data were applied in the analysis of both the Alpha diversity and Beta diversity, which must be normalized in advance.Several Alpha diversity indexes, including ACE, Simpson, Shannon, Chao, and Observed species, were calculated using QIIME software, while the rarefaction curves and rank–abundance curves were drawn and analyzed using R software. The graphs of PCA, PCoA, and NMDS were also drawn and analyzed using R software . In this study, our analyses of the differences in the Alpha diversity indexes and Beta diversity indexes between groups were performed using the parametric test and non-parametric test, respectively. The T-test and Wilcox test were applied to analyze the differences between the two groups, while the Tukey test and Wilcox test were used to analyze among more than three samples .


Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration ([Ca2+]ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca2+]ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca2+]ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, [Ca2+]ER is a major regulator of InsP3R channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.Modulating cytoplasmic Ca This en15.2 : 1 ) and ord3R channel activity can be modulated by [Ca2+]ER indirectly via feed-through effects of Ca2+ flux driven through an open channel by high [Ca2+]ER that raises the local [Ca2+]i in the channel vicinity to regulate its activity through its cytoplasmic activating and inhibitory Ca2+-binding sites  on the cytoplasmic side in the lum-out excised patch configuration . Thus, as long as [Ca2+]free on the cytoplasmic side was well buffered, switching [Ca2+]ER between 70 nM and 300 μM in lum-out excised membrane patches had no effect on channel activity. This definitively ruled out the possibility that InsP3R activity is modulated directly by an intrinsic Ca2+-regulatory site on the luminal side that is either part of the InsP3R or of a protein that constitutively interacts with the channel  is present in the nuclear envelope (2+ leaks equilibrated [Ca2+]free in the perinuclear space of the isolated nuclei (effectively [Ca2+]ER) with bath [Ca2+] at unphysiologically low levels. Accordingly, possible [Ca2+]ER regulation of channel activity by peripheral proteins in the lumen that interact with the InsP3R only in the presence of high, physiological [Ca2+]ER -mediated [Ca2+]ER regulation of InsP3R channel activity exists. We discovered a strong Ca2+ flux-independent inhibition of InsP3R channel activity mediated by accessory ER-luminal protein(s) only in the presence of high, physiological [Ca2+]ER. We identified the region of the InsP3R that is involved in this [Ca2+]ER-dependent inhibition, and discovered that the widely expressed Ca2+-binding protein annexin A1 (ANXA1) localizes not only to the cytoplasm but also to the perinuclear space inside the nuclear envelope, and possibly in the ER lumen as well, where it plays a critical role in channel inhibition through high [Ca2+]ER-mediated interaction with the InsP3R. Reducing this interaction enhanced agonist-induced InsP3R-mediated Ca2+ release, and increased local elementary Ca2+-release events in intact cells. Thus, [Ca2+]ER is a major regulator of InsP3R-channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a luminal peripheral membrane-associated Ca2+-binding protein, likely ANXA1.Here we specifically assessed whether such a 3R genes were ablated and stably replaced with wild-type rat type-3 InsP3R (DT40-r3 cells) (3R channels (2+]ER in the perinuclear space (2+]bath (70 nM). Linear, ohmic InsP3R-channel current vs. applied potential (ich-Vapp) curves were observed with high single-channel conductance, as expected  block of the measured K+ current due to high [Ca2+]ER. Thus, absence or presence of MgATP in the bath provides a mechanism to alter [Ca2+]ER in intact nuclei. In a previous study examining the Ca2+ permeant-ion effect on K+ conductance through InsP3R channels (3R channel slope conductance over a broad range of [Ca2+]ER (0 to 1.1 mM). This enabled us to use the single-channel conductance, rather than Ca2+ indicator dye fluorescence, to estimate that [Ca2+]ER is ~300 μM in the perinuclear space of intact isolated DT40-r3 nuclei in the presence of bath MgATP. Subsequent excision of the membrane patch into the lum-out patch configuration  , so thatchannels  were recar space  equilibrexpected . In contexpected , SERCA ap curves  observedchannels , we deriguration  exposed  was high with 70 nM Ca2+ER  buffered [Ca2+]i at cytoplasmic Ca2+ regulatory sites, possible feed-through effects were eliminated    inhibition affects endogenous InsPp curves  and redup curves ) isolatensP3R–1) , rat PC-InsP3R-3 , and wilInsP3R-3 . In a ba3R-3  connecting transmembrane helices in the pore-forming domain. We selected human sequences (3R (3R isoforms. We excluded the P-region (orange stripe) and selectivity filter (green stripe) involved in other crucial channel functions (3R that could interact with the PLP.We hypothesized that the PLP interacts directly with the InsPequences  based onnces (3R . Since tunctions . Only founctions  fit the 3R-3 channels in the cytoplasmic-side-out (cyto-out) configuration    or cyto-out patch-clamp experiments with 0-MgATP in the bath (magenta). This indicates that the pL2 region interacts with the PLP to suppress InsP3R channel activity, and that the inhibitory effect of the PLP is mostly or completely mediated by the pL2-PLP interaction.We first investigated whether the conserved region of the L2 loop pL2,  is involguration  with pipat 70 nM , left prachieved , right, excision , left, wmoBAPTA) , channelo (~0.6) , magentao (~0.6) . Alternae InsP3R , left. Bachieved , right. mulation , purple,mulation , left. W μM Ca2+ , right. nal side , brown w2+-binding proteins (UniProt protein database) in the ER lumen , and that are broadly expressed. The annexin (ANX) protein family met these criteria. Of 12 members of the ANX family, five were identified to have statistically-significant preference to bind to the mpL2 peptide over the smpL2 peptide .To identify the PLP, peptides with a modified pL2 sequence (mpL2) or scrambled mpL2 (smpL2) were used for in-vitro pull-downs from soluble fractions of bovine hepatocyte ER microsomes . Eluates2+-dependent phospholipid-binding proteins (2+ binding to ANX (dissociation constant Kd ~25 μM to 1 mM) promotes its association with negatively-charged phospholipid-containing membranes  in [Ca2+]ER between 70 nM and 55 μM  containing 1 μM recombinant full-length human ANXA1 under conditions that prevented permeant Ca effects . ANXA1 wnd 55 μM . In cont[Ca2+]ER . Fittingequation :(1)Po=Pm2+]ER (Kinh) is 260 ± 14 μM with Hill coefficient (Hinh) of 2.3 ± 0.3    and Kd oobserved , ANXA1 iurations .3R was without affect  sequence , ANXA1-inhibited channel Po  and rate of change (1/τ) of Fura-2 fluorescence ratio in populations of cells responding to the muscarinic receptor agonist carbachol  in individual ANXA1 and non-targeting (N-T) siRNA-treated HeLa cells  generated by clusters of InsP3R stimulated by uncaging of a non-metabolizable InsP3 analogue  the two proteins from cell lysates. There are many reasons why co-IP can fail, including low expression of ANXA1 in the ER lumen, particularly in comparison with its strong expression in the cytoplasm; and that the interacting regions in InsP3R and ANXA1 are small, particularly in the case of the InsP3R. Second, whereas we have been able to apply immunofluorescence microscopy to localize ANXA1 to the nuclear envelope in different permeabilized cells with cytoplasmic ANXA1 washed out, localization to the ER has been unconvincing   , the preaffinity . Convers, the PLP and ANXA1 strongly suppressed channel activation even in optimal concentrations of cytoplasmic Ca2+ and InsP3, such that channel Po is limited to ~0.1 (Po elicited by saturating [InsP3] in absence of PLP (ANXA1) (3R activity by high (50 μM) [Ca2+]i  . This po [Ca2+]i . FurtherM – 1 mM . We obse0–400 μM . We obse3R channels (~95%) in cells are unresponsive to experimentally-induced elevations of [InsP3], with responsive channels limited to those in immobile clusters from which Ca2+ blips and puffs originate  could play a role. In this scenario, most InsP3R are normally associated with the PLP, rendering them unresponsive to InsP3. However, channels in some clusters may lose their association with the PLP, possibly because of a unique phospholipid composition of the ER membrane there, or because the L2 sequence is not exposed in InsP3R channels in clusters, or for other reasons. Channels in such clusters would be much more responsive to InsP3 and ‘licensed’ to respond even with the ER fully replete with Ca2+. In addition, the strong inhibition of InsP3R channel activity by ANXA1 could possibly help shape intracellular InsP3-induced Ca2+ signals. PLP (ANXA1) inhibition will reduce the amount of Ca2+ released by shortening the open durations of activated channels  as PLP inhibition of channel activity is alleviated , Anx A2 , and Anx A6 .Peptides used in our study: L2 (GNRGTFIRGYKAMVMDMEFLYHVGYILTSVLGLFAHEL), sL2 (DVVIALHGNAMMYLLVFEHTSTGIGKFLRFYGERLMYG), pL2 (GNRGTFIRGYKAMVMDME), mpL2 , smpL2 (K-Ahx-RDYRGMRMIMGETFNVGA), and ANXA1 N-terminal peptide (MAMVSEFLKQAWFIENEEQEYVQTVKSSKGGPGSAVSPYPT) were custom synthesized by Peptide 2.0 .3R genes have been stably ablated were obtained from Riken Bioresource Center, Japan . Generation of DT40–KO-r–InsP3R–3 cells (DT40-r3 cells) that stably express only recombinant rat type 3 InsP3R in DT40-KO cells was described in 2. When cell density exceeded 2.5 × 106/ml, they were sub-cultured to 0.1–0.2 × 106/ml  from Kerafest (cat. # EUR030) were cultured as adherent cells in DMEM , supplemented with 10% FBS and 1% Gibco antibiotic-antimycotic; at 37°C in 5% CO2. Medium was renewed every two to three days. Cells were sub-cultured following instructions provided in ATCC CRL-1573 product sheet.Wild type HEK293 cells from ATCC (CRL-1573) and InsP3R-3 (HEK–3KO-r–InsP3R–3) were generated from HEK293-3KO cells by transfecting them using Transit LT-1 (Mirus cat. # MIR 2304) following manufacturer’s protocols. Using G418 selection, stable clones of HEK293-3KO-r-InsP3R-3 cells expressing various level of recombinant r-InsP3R-3 were generated by limiting dilution.HEK-3KO cells stably expressing rat InsP2. Cells were sub-cultured following instructions provided in ATCC CCL-131 product sheet.Mouse N2a cells from ATCC (CCL-131) were cultured as adherent cells in 1:1 mixture of DMEM  and Opti-MEM I Reduced Serum Medium (Gibco cat. # 31985–070), supplemented with 5% FBS, 1% penicillin-streptomycin; at 37°C in 5% CO2. Cells were sub-cultured following instructions provided in ATCC CRL-1721 product sheet.Rat PC12 cells from ATCC (CRL-1721) were cultured as adherent cells in F-12K Kaighn’s Mod. Nutrient mixture , supplemented with 15% horse serum, 2.5% FBS, 1% Gibco antibiotic-antimycotic; at 37°C in 5% CO2. 70–80% sub-confluent cells were dislodged using 0.25% trypsin solution and split 1:4 to 1:8 as described in ECACC data sheet for HEKtsA201 cells.Human HEKtsA201 cells derived from HEK293 cells were obtained from Sigma-Aldrich (cat. # 96121229) and cultured as adherent cells in the same medium as for HEK293 cells, at 37°C in 5% COA549 human carcinoma cells from ATCC (CCL-185) were cultured using the same protocol as that for HEKtsA201 and HEK293 cells.2. Cells were sub-cultured following instructions provided in ATCC CCL-1469 product sheet.Panc-1 human pancreatic epithelioid carcinoma cells from ATCC (CRL-1469) were cultivated as adherent cells in DMEM with L-glutamine, glucose and sodium pyruvate (Corning cat. # MT10-013-CM), supplemented with 10% FBS and 1% Gibco antibiotic-antimycotic; at 37°C in 5% CO2+] ([Ca2+]free). [Ca2+]free was buffered either by 0.5 mM 5,5′-dibromo 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid  in experiments in which [Ca2+]free on the luminal side of the membrane ≤300 μM; or by 5 mM diBrBAPTA or 10 mM N-(2-Hydroxyethyl)ethylenediaminetriacetic acid  in experiments with luminal [Ca2+]free ≥300 μM to eliminate possible permeant Ca2+ feed-through effects on InsP3R channel activities  .Isolation of intact nuclei from cells, performing nuclear patch-clamp experiments in on-nucleus, cytoplasmic-side-out and luminal-side-out configurations with rapid exchange of ligand conditions are described in detail in tivities . Cytoplaes (2ATP  and eith2ATP (3]  (Invitroa 72559) . [Ca2+]f3, 2 μM [Ca2+]i and 0.5 mM ATP4- on the cytoplasmic side of the outer nuclear membrane). Our experience suggested that most newly-thawed DT40-r3 cells were consistent after culturing for 5 days. If the mean channel Po of a batch of control cells cultured for any time beyond day five was found to be significantly lower than that of previous control cells, that batch was terminated immediately and a new batch of cells was thawed out for new experiments.The nuclear patch-clamp experiments in various patch-clamp configurations  using DT40-r3 cells were acquired over an extensive period. To ensure the behaviors of these cells were consistent, we regularly used cells, cultured in identical conditions and at most one day apart from data-generating cells, to perform control experiments in which nuclei were patched in on-nuc or cyto-out configurations under optimal stimulatory conditions  were acquired at room temperature (RT) as described (Vapp were measured relative to the reference bath electrode regardless of the patch-clamp configuration used. InsP3R channel gating characteristics—number of active channels observed (NA) and open probability (Po)—were derived from current traces using semi-automatic QuB . 10 g of cut liver pieces and 19 ml of ice-cold DPBS with Mg2+ and Ca2+ (Corning cat # 21–030 CM) were put into each of twenty Stomacher 80 strainer bags (Seward cat # BA6040/STR) and processed individually in a Stomacher 80 Biomaster (Seward cat # 0080/000/AJ) at highest setting for 75 s to release intact hepatocytes. Cell suspension was strained through the strainer bags and collected (~500 ml). 250 ml of ACK lysing buffer  at RT was mixed into the cell suspension to specifically lyse red blood cells. The process was terminated after 5 min by adding 500 ml DMEM (Corning cat. # 10–013-CV) with 10% FBS followed with gentle shaking. Hepatocyte suspension was centrifuged at 9,600 g for 18 min and the supernatant was discarded. Hepatocytes were rinsed twice by resuspending cells in 500 ml HBSS, centrifuging at 9,600 g for 18 min, and discarding the supernatant.Bovine liver was procured from three cows within three hours of slaughter, cut into large pieces of ~200 g each, rinsed with ice-cold PBS and flash frozen in –80°C freezer within one hr of procurement to be used later. The following protocol was used for processing 200 g of bovine liver. Quantities of reagents used were scaled according to amount of extracted proteins required. Bovine liver was thawed in ice-cold water and diced into small pieces of 0.8 cm2, 1 mM CaCl2, 20 mM Tris HCl (pH 7.5), 400 μM phenylmethane sulfonyl fluoride (PMSF), and seven cOmplete protease inhibitor cocktail tablets (Roche cat. # 11697498001). Debris in cell suspension was removed with cell strainer. The hepatocytes were homogenized with nitrogen cavitation in an ice-cold large cell disruption vessel  using pressure of 1000 p.s.i. with constant stirring for 20 min. Cell homogenate was centrifuged at 3,300 g for 15 min to pellet unbroken cells and nuclei. The supernatant was centrifuged at 9,600 g for 15 min to pellet cell debris and mitochondria, and at 105,000 g for 15 min to pellet a crude microsomal fraction. The pellet was resuspended into 40 ml of 1.38 M sucrose solution, placed under a step gradient of 1.0 and 0.86 M sucrose and centrifuged at 300,000 g for 75 min. The smooth and rough ER fractions below the interface between 1.38 M and 1.0 M sucrose solution layers were collected and placed on ice.Hepatocytes were resuspended in 150 ml of homogenizing solution containing 150 mM KCl, 1 mM MgCli)-imidazole HCl (NaPPi-I) solution containing 5 mM NaPPi and 3 mM imidazole-HCl, pH 7.4 was added dropwise to the collected ER fractions with continuous stirring on ice to the final volume of 100 ml. After a further 15 min of stirring, the mixture was centrifuged at 105,000 g for 37 min to re-pellet the microsomes. After discarding the supernatant, the ER microsome pellet was resuspended in 45 ml of sucrose-NaPPi-I solution containing 0.25 M sucrose, 5 mM NaPPi, and 3 mM imidazole HCl, pH 7.4; centrifuged again at 105,000 g for 37 min; and the supernatant was discarded.To remove ribosomes from the rough ER, ice-cold sodium pyrophosphate (NaPP2+ K+ with protease inhibitor (LCaK-PI) solution containing 150 mM KCl, 0.5 mM EGTA, 0.05% Tween-20, 25 mM Tris HCl (pH 7.5), 400 μM PMSF, one cOmplete protease inhibitor cocktail mini-tablet (Roche cat. #11836170001). The microsome suspension was processed with nitrogen cavitation twice using an ice-cold small cell disruption vessel  under 2200 p.s.i. with constant stirring for 20 min each cavitation. The homogenate was centrifuged at 105,000 g for 60 min to pellet the broken ER membrane. The supernatant was collected carefully without disturbing the ER membrane pellet so it contained only peripheral proteins released from the ER lumen in the presence of low [Ca2+]free (buffered by 0.5 mM EGTA in the LCaK-PI solution). Total protein concentration in the hepatocyte peripheral ER luminal protein (HPERLP) sample was determined using Bradford protein assay (~1.4–3.0 mg/ml). The HPERLP sample was aliquoted and stored in –80°C for future use.The smooth ER microsome pellet was resuspended with vigorous vortexing in 8 ml of low-Ca3R that is highly conserved among the three InsP3R isoforms (K-Ahx-GNRGTFIRGYRAMVMDME) were synthesized for pull-down experiments. The Lys residue in pL2 was replaced with Arg (underlined) so that the mpL2 peptides could be coupled to magnetic beads through the only Lys  at the N-terminus. The unnatural 6-aminohexanoate residue (Ahx) was added to reduce possible steric hindrance that may interfere with the interaction between the peptide and ER luminal proteins. For control, peptides with a scrambled modified sequence (smpL2: K-Ahx-RDYRGMRMIMGETFNVGA) were also synthesized. Instructions of the manufacturer were followed to covalently couple the mpL2 and smpL2 peptides to NHS-activated magnetic beads (Pierce cat. # 88826) through the stable covalent link formed by reaction between the primary amine group of the N-terminal Lys of the peptides and the N-hydroxysuccinimide ester group on the magnetic beads. Peptide-coupled beads were stored (10 mg/ml) in storage buffer  at 4°C.Based on the pL2 sequence in the L2 region of the InsPisoforms , peptide2+ K+ (HCaK) solution containing 150 mM KCl, 1% NP–40 detergent (Thermo scientific cat. # 85124), 0.8 mM CaCl2, 25 mM Tris HCl (pH 7.5) was added to each. Effluent from the columns was discarded. 320 μl of peptide-coupled magnetic beads were resuspended in storage buffer, rinsed twice with HCaK solution, and resuspended with 290 μl of HCaK solution. 35 μl of the peptide-coupled magnetic bead suspension was added to each of the MS columns. Effluent was examined for presence of magnetic beads. If beads were detected, effluent was returned to the column till no beads were detected. Each column with magnetic beads were washed with 80 μl of HCaK solution and effluent was discarded. 360 μl of the HPERLP sample was thawed and HCaK solution and 1 M CaCl2 standard solution were added to adjust the total protein concentration in the final HCaK-HPERLP mixture to 1.25 mg/ml, and [CaCl2] to 800 μM ([Ca2+]free = 432 μM). 40 μl of the HCaK-HPERLP mixture was added to each column, followed by 80 μl of HCaK-PI solution—HCaK solution with additional 400 μM PMSF and 1% Halt protease inhibitor cocktail (Thermo Fisher Cat. # 78430). The magnetic beads in each column were rinsed once with 2 ml of HCaK-PI solution, and thrice with 2 ml of HCaK solution. Effluent was discarded. 200 μl of low-Ca2+ eluding (LCaE) solution with 150 mM KCl, 1% NP–40 detergent, 0.5 mM EGTA, 25 mM Tris HCl (pH 7.5) was added to each column. The eluate from all the columns was collected and frozen at –80°C.A total of six pull-down procedures were performed in the cold with all apparatus and reagents at 4°C: one procedure using mpL2 peptide-coupled magnetic beads and one control procedure using smpL2 peptide-coupled magnetic beads for each of the three HPERLP samples from individual cows. The same protocol described below was used for each of the six procedures. To obtain sufficient protein for mass spectrometry, eight MACS separation columns  were used in each procedure. The MS columns were placed on the OctoMACS separator (Miltenyi Biotec cat. # 130-042-109) mounted on the MACS Multistand (Miltenyi Biotec cat. #130-042-303). To prime the columns, 250 μl of high-Ca2+ eluate proteins from above were precipitated with 5 volumes of acetone held overnight at –20°C, decanted then solubilized in 20 µL of 1 × reducing NuPAGE LDS (Thermo Fisher Scientific) and run into a precast 10% Bis-Tris SDS-PAGE (Thermo Fisher Scientific) for 1.6 cm. After fixing overnight  the gel was stained with colloidal Coomassie blue dye. Each sample was excised from the gel in 4 (2 × 9 mm) slices and each cut into (1 mm)3 cubes. The gel pieces were destained in a solution containing 50% methanol and 1.25% acetic acid, reduced with 5 mM DTT (Thermo Fisher Scientific), and alkylated with 40 mM IAA (Sigma-Aldrich). The gel pieces were washed with 20 mM ammonium bicarbonate (Sigma-Aldrich) and dehydrated with acetonitrile (Fisher Scientific) twice. Sequencing-grade trypsin (Promega) (5 ng/µL in 20 mM ammonium bicarbonate) was added to the dehydrated-gel pieces and proteolysis was allowed to proceed overnight at 37°C. Peptides were extracted with 0.3% TFA (Fisher Scientific), followed by 50% acetonitrile. The volume of the combined extracts was reduced by vacuum centrifugation and 25% of the extracted peptides were analyzed per LC-MS/MS run  coupled with a nanoLC Ultra (Eksigent). Peptides were separated by reverse phase (RP)-HPLC on a nanocapillary column, 75 μm id ×15 cm Reprosil-pur 3 μm, 120A  in a Nanoflex chip system (Eksigent). Mobile phase A consisted of 1% methanol (Fisher)/0.1% formic acid (Thermo) and mobile phase B of 1% methanol/0.1% formic acid/80% acetonitrile. Peptides were eluted into the mass spectrometer at 300 nL/min with each RP-LC run comprising a 90 min gradient from 10% to 25% B in 65 min, 25–40% B in 25 min. The mass spectrometer was set to repetitively scan m/z from 300 to 1800  followed by data-dependent MS/MS scans on the twenty most abundant ions, with a minimum signal of 1500, dynamic exclusion with a repeat count of 1, repeat duration of 30 s, exclusion size of 500 and duration of 60 s, isolation width of 2.0, normalized collision energy of 33, and waveform injection and dynamic exclusion enabled. FTMS full scan AGC target value was 1e6, while MSn AGC was 1e4, respectively. FTMS full scan maximum fill time was 500 ms, while ion trap MSn fill time was 50 ms; microscans were set at one. FT preview mode; charge state screening, and monoisotopic precursor selection were all enabled with rejection of unassigned and 1+ charge states.Raw MS files were processed using MaxQuant, version 1.6.1.0 for identification of proteins. The peptide MS/MS spectra were searched against the UniProtKB/Swiss-Prot Bovine Reference Proteome database, UP000009136 , comprising 24,333 entries including isoforms. Fragment ion tolerance was set to 0.5 Da, with full tryptic specificity required and a maximum of two missed tryptic cleavage sites. Precursor ion tolerance was seven ppm. Oxidation of methionine, acetylation of the protein N-terminus and conversion of glutamine to pyroglutamic acid were used as variable modifications. Carbamidomethylation of cysteine was set as a fixed modification. The minimal length required for a peptide was seven amino acids. Target-decoy approach was used to control false discovery rate (FDR). A maximum FDR of 1% at both the peptide and the protein level was allowed. If no unique peptide sequence to a single database entry was identified, the resulting protein identification was reported as an indistinguishable ‘protein group’. Protein groups containing matches to decoy database or contaminant proteins were discarded. The MaxQuant match-between-runs feature was enabled.3R compared to the scrambled, control peptide using the false sign rate paradigm  was applied to identify proteins with strong preference to bind to the L2 region of the InsP3R.The local false sign rates were computed by sampling from the posterior using Markov Chain Monte Carlo. A low LFSRImages were collected on a Leica TCS SP8 X system using a 100x/1.4 NA PL APO CS2 objective. Annexin A1 was labeled with rabbit anti-Annexin A1 pAb  and anti-rabbit AlexaFluor568 conjugated pAb  was excited using a continuously tunable white light laser set to 568 nm to detect fluorescence emission between 600–700 nm. Calnexin was labeled with mouse anti-Calnexin mAb  and anti-mouse AlexaFluor488 conjugated pAb  excited at 488 nm to detect fluorescence emission between 510–550 nm. DAPI was present in the mounting medium used to prepare the slides, excited at 408 nm to detect fluorescence emission between 430–470 nm.6 cells/10 cm tissue culture dish in 10 ml of 5% FBS/DMEM with 1 × Anti Anti, and grown at 37°C in 5% CO2 for 72 hr. Media in the dishes was collected, filtered through a 0.22 µm pore syringe filter, and concentrated 8-fold using Amicon Ultra-4 centrifugal filter devices (3 kDa MW-cutoff) as described in the manufacturer’s protocol. The cells in each dish were detached with Versene (5.4 mM EDTA) solution, pelleted by centrifugation at 1000 RPM for 5 min at 4°C, rinsed in 10 ml of 1 × DPBS, pelleted, and re-suspended in 1.5 ml of cell lysis buffer  supplemented with 1 mM PMSF and Protease Inhibitor Cocktail (Roche). Lysates were incubated for 15 min on ice, centrifuged at 14,000 RPM for 10 min at 4°C to pellet genetic material released from nuclei. Supernatants were collected. Total [protein] was determined using the Pierce BCA Protein Assay kit (Thermo Fisher cat. # 23227).HEKtsA201 cells were seeded at 2 × 102CO3; 44 mM NaHCO3; pH 9.6). Plates were incubated overnight at 4°C. The next day, the capture antibody solution was discarded, wells were rinsed 3 × with 200 µl of wash solution , and 200 µl of blocking solution  was added to each well and incubated for 30 min at RT. The wells were rinsed 3 × again as described before. 100 µl of each concentrated cell-culture media (supernatant) or cell lysate sample were added. Five-fold serial dilutions were made of concentrated supernatants  and cell lysates  in six replicates. Wells containing cell-culture media or cell lysis buffer only were made in triplicate as negative controls. The plate was incubated overnight at 4°C. The next day, samples in the wells were discarded, and the wells rinsed 3×. 100 µl of primary mouse α-ANXA1 antibody  diluted 1:1000; or mouse α-pan actin antibody  diluted 1:100 in antibody diluent  was added to the appropriate wells and incubated for 1 hr at RT. Contents in the wells were again discarded and the wells rinsed 3×. 100 µL of secondary goat α-mouse IgG-HRP antibody  diluted 1:2000 in antibody diluent was added to each well, and incubated for 1 hr at RT. Again, contents in the wells were discarded and the wells rinsed 3×. 100 µL of PICO chemiluminescent HRP substrate (Pierce) was added to each well and incubated for 5 min at RT. Chemiluminescence was measured in a Cytation five plate reader (BioTek).A high-binding, white, 96-well microtiter plate  was coated with 100 µl/well of rabbit α-ANXA1 antibody  diluted 1:500; or rabbit α-β actin antibody  diluted 1:100 in carbonate capture buffer  siRNA or not transfected at all. Four pairs of controls and experiments were done: two pairs were experiments using ANXA1 siRNA-transfected cells versus controls using N-T siRNA-transfected cells, the other two were experiments using ANXA1 siRNA-transfected cells versus controls using non-transfected cells.6 cells/ml were plated into each of four 10 cm culture dishes (two for the experiment and two for the control) and incubated overnight (~18 hr) at 37°C. To transfect two 10 cm dishes of HEKtsA201 cells, 30 μl of 20 μM siRNA (ANXA1 or N-T) was thawed and diluted with 90 μl of RNase-free water. 3.9 ml of Opti-MEM (Gibco cat. # 31985–070) was added to the siRNA, mixed by gentle pipetting. The siRNA mixture was incubated for 7 min. For a non-transfected control, 4 ml of Opti-MEM was used. 160 μl of transfection reagent (Dharmacon cat. # T-2001–02) was diluted with Opti-MEM to a final volume of 8 ml, mixed by gentle pipetting. The resulting transfection solution was incubated for 7 min. 4 ml of transfection solution was added to the 4 ml of siRNA mixture (for experiment ANXA1 or control N-T siRNA transfection) or 4 ml of Opti-MEM solution (for non-transfection control). The resulting mixtures were incubated for another 25 min. Each of the mixtures was then diluted with 32 ml of antibiotic-free complete medium (DMEM (Corning cat. # 10–013-CV) with 10% FBS (HyClone cat. # SH30071.03) to give 40 ml of transfection medium.Following the siRNA transfection protocol from Dharmacon , human ANXA1 siRNA (Dharmacon cat. # M-011161-01-0005) and N-T siRNA (Dharmacon cat. # D-001206-13-05) were suspended in 250 μl of 1 × siRNA buffer (50 μl of 5 × siRNA buffer (Dharmacon cat. # B-002000-UB-100) in 200 μl RNase-free water) to a final siRNA concentration of 20 μM, aliquoted and stored at –20°C. For a pair of control and experiment, 10 ml of HEKtsA201 cell suspension with density of ~0.26 × 102+ release.Culture medium was removed from each of the four dishes of HEKtsA201 cells that had been incubated overnight. 20 ml of ANXA1 siRNA transfection medium was added to each of two dishes for the experiment; and 20 ml of N-T siRNA transfection medium or non-transfection medium was added to each of two dishes for the control. The cells in transfection medium were incubated for 6 hr at 37°C. After the incubation, the transfection medium was replaced with 20 ml of regular HEKtsA201 cell medium for each of the four dishes of cells. Cells were incubated at 37°C for an additional 42 hr before being used for measurements of intracellular CaExpression levels of ANXA1 in HEKtsA201 cells were probed in Western blots using anti-ANXA1 antibody , stripped and re-probed with anti-β-actin antibody .6 cells  were in each 10 cm culture dish. To collect the attached cells, the HEKtsA201 medium was removed, the cells were rinsed once with 10 ml of Ca2+-free DPBS (Corning cat. # 21–031 CM) at RT, incubated in 5 ml of Versene solution (Gibco cat. # 15040–066) for 20 min to detach, dispersed by gently pipetting the cell suspension, and the cell density was determined using a hemocytometer. Four million cells were used to quantify the expression level of ANXA1 in the HEKtsA201 cells by Western blotting. The remaining cells were centrifuged at 350 g for 3 min. The supernatant was discarded, and the cell pellet was resuspended in 10 ml of Fura-2 AM dye-loading solution (1 μM Fura-2 AM dye (Molecular Probes cat. # I-1225) and 0.01% pluronic F127 (Thermo Fisher cat. # P3000MP) in HEPES-buffered saline (HBS) containing 135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 11 mM glucose, and 11.6 mM Na-HEPES (pH 7.4). The cell suspension was kept in darkness with gentle shaking for 20 min at RT. The dye-loaded cells were then centrifuged at 350 g for 3 min. The supernatant was discarded, and the cell pellet was resuspended in 10 ml HBS. The cell suspension was again centrifuged at 350 g for 3 min. The supernatant was discarded, and the cell pellet was resuspended in HBS to a cell density of 3–4 × 106 cells/ml. These cells were kept in darkness. Each 10 cm dish of cells was sufficient for 4 to 7 assays. For proper control, dishes of ANXA1 siRNA-transfected cells and control cells were used alternately. Just before each assay, 0.75 ml of cell suspension was centrifuged in a 1.5 ml eppendorf tube at 400 g for 3 min. The supernatant was discarded and the cells were resuspended in 0.75 ml of HBS and transferred to a cuvette containing 0.75 ml of HBS and a 3–mm magnetic stirrer. The cuvette was transferred to the temperature-controlled (37°C) experimental compartment of a multi-wavelength-excitation dual-wavelength-emission high-speed spectrofluorometer . Fluorescence at 340 nm excitation/515 nm emission and 380 nm excitation/515 nm emission for the Ca2+-bound and Ca2+-free Fura-2 species, respectively, were measured at 5 Hz. The ratio (R) of the fluorescence intensity excited by 340 nm versus 380 nm light was used as relative measurement of intracellular [Ca2+]free. After a stable baseline of R had been observed for 90 s, 15 μl of 10 mM carbachol solution was added to the cell suspension  to stimulate sub-maximal InsP3R-mediated release of Ca2+ from the ER. R was further observed for another 120 s.The same protocol was used to perform fluorimetry for experiment and control HEKtsA201 cells. After incubation,~10–20 × 102. Transfection with siRNA for either human ANXA1 siRNA or non-targeting siRNA control was performed as described for HEKtsA201 cells. ~ 3 × 105 cells were seeded on each 12 mm coverslip. At 48 hr post-transfection, cells were loaded with 2 µM Fura-2 AM for 15–20 min then incubated in 2 Ca Tyrode’s solution for 5–10 min at room temperature prior to imaging. Cells were imaged on a Nikon Ti system using a 20x/0.75 NA objective for fluorescence at 340 nm excitation/515 nm emission (Ca2+-bound Fura2), and 380 nm excitation/515 nm emission (Ca2+-free Fura2). Coverslips were perfused with 2 Ca Tyrode’s solution for 30 s, 10 or 100 µM histamine in 2 Ca Tyrode’s solution for 360 s or 120 s respectively, followed by a washout with 2 Ca Tyrode’s solution for 90 s. 100 ms exposure images for each wavelength were collected every 2 s. For analysis of intracellular Ca2+-signals, cells were mass-selected using a binary mask and the Time Series Analyzer v3 plugin in ImageJ (NIH). The 340 nm/380 nm (Rfura2) fluorescence ratio for each time point was measured for every cell in the field of view and the normalized signal (R/R0) was calculated by dividing Rfura2 for each time point by the mean Rfura2 during the first 30 s of each experiment. A series of logic tests were applied to data to identify signaling cells and separate those cells into oscillating and non-oscillating groups. To exclude cells that did not begin and end at baseline Ca2+ levels, only cells with R/R0 values between 1.25–0.75 during the first 30 s and R/R0 values less than 1.5 during the last 30 s of the final washout were selected. To exclude non-responding cells, only cells with maximal R/R0values ≥ 2.0 following histamine stimulation were selected. Cells with a coefficient of variation (%CV) value greater than 15% measured from the end of the initial Ca2+ signal to the end of the histamine stimulation were considered oscillating cells. Oscillating cells were qualitatively characterized as exhibiting either ‘periodic oscillation’ or ‘random spiking’ pattern.HeLa cells were cultured on 15 cm tissue culture dishes in Dulbecco’s Modification of Eagle’s (DMEM Corning) supplemented with 10% fetal bovine serum (Hyclone) and maintained at 37°C in a humidified incubator gassed with 95% air and 5% CO2. For transfection with siRNA, cells were collected using 0.25% Trypsin-EDTA (Gibco #25200–056) and grown on 60 mm culture dishes. When cells reached 80% confluence they were incubated with 15 nM ANXA1 siRNA or with 15 nM non-targeting CTL siRNA together with 15 µl of DharmaFECT transfection reagent  for 6 hr. In some experiments, cells were additionally incubated with 5 nM siGLO Red Transfection Indicator (Dharmacon #D-001630-02-05) to serve as a fluorescent tracer to visualize transfection efficiency. Following incubation, cells were harvested using 0.25% Trypsin-EDTA, pelleted by low speed centrifugation (~800 x g), resuspended in EMEM and distributed among poly-D-lysine coated  35 mm glass-bottom imaging dishes (MatTek #P35-1.5–14 C) where they were grown for 48 hr prior to imaging, at which time cells were ~50% confluent.WT HEK-293 cells were cultured on plastic 75 cm tissue culture flasks in Eagle’s Minimum Essential Medium  supplemented with 10% fetal bovine serum (Omega Scientific #FB-11), and maintained at 37°C in a humidified incubator gassed with 95% air and 5% CO2+ dye Cal-520/AM  and the caged IP3 analogue ci-IP3/PM   for 1 hr at RT in a Ca2+-containing HEPES buffered salt solution (Ca2+-HBSS). Cells were then washed with Ca2+-HBSS and loaded for an additional hour with EGTA/AM . Cal-520/AM, caged i-IP3/PM, and EGTA/AM were all solubilized with DMSO/20% pluronic F127 (ThermoFisher #P3000MP). Ca2+-HBSS contained (in mM) 135 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH = 7.4 at RT).Immediately before imaging, cells were incubated with membrane-permeable esters of the fluorescent Ca2+ signals was accomplished using a home-built system, based around an Olympus IX50 microscope equipped with an Olympus 60X objective (NA 1.45). Using 488 nm laser fluorescence excitation and a 510 nm long pass emission filter, fluorescence images were captured with an Evolve EMCCD camera (Photometrics), utilizing 2 × 2 pixel binning for a final field of 128 × 128 pixels (one pixel = 0.53 µm) at a rate of ~125 frames s−1. To photo-release i-IP3, UV light from a xenon arc lamp was filtered through a 350–400 nm bandpass filter and introduced by a UV-reflecting dichroic in the light path to uniformly illuminate the field of view. The amount of i-IP3 released was controlled by varying the flash duration, set by an electronically controlled shutter (UniBlitz). All image data were streamed to computer memory using Metamorph v7.7  and stored on hard disc for offline analysis.Total internal reflection fluorescence (TIRF) imaging of Ca2+ levels which hamper measurement of local Ca2+ signals. Raw fluorescence records were first black-level subtracted and then processed to create a ratio image stack (F/F0), where the fluorescence intensity of each pixel in a given frame was replaced by that intensity (F) divided by the mean resting fluorescence intensity at that pixel averaged over 500 frames prior to photo-release of i-IP3 (F0). Next, a custom plug-in (‘detect puffs’) was applied for automated detection and analysis of local Ca2+ signals. All Ca2+ puffs identified by the algorithm were verified by visual inspection prior to further analysis. Measurement of peak puff amplitudes (ΔF/F0) and kinetics were performed by the algorithm on a 5 × 5 pixel region of interest centered over the centroid of each event, and were exported to EXCEL spreadsheets for further analysis. Additional analysis and graphing was performed in Microcal Origin v6.0 (OriginLab) and Igor 6 (WaveMetrics).Image data in MetaMorph stk format were processed using Flika , a freel3R channel open probabilities (Po), normalized probabilities of detection of InsP3R channel activity (Pd), normalized maximal change of fluorescence intensity ratio (ΔRmax) and normalized rate of change of fluorescence intensity (1/τ), number of Ca2+ puffs per second per imaging field and amplitudes of the Ca2+ puffs (ΔF/F0), performed under various experimental conditions were compared using unpaired t-test. For data sets involved in multiple comparisons, p values were determined using the Bonferroni correction for multiple comparisons. Quantified western blot intensities from co-immunoprecipitation experiments were compared by paired t-test.All measurements in this study: InsP In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:2+ concentration [Ca2+] in the endoplasmic reticulum (ER) lumen regulates the activity of inositol 1,4,5-trisphosphate receptors (InsP3Rs) independent of flow-through effects on the cytoplasmic surface of the channel.In this manuscript, Vais et al. use an elegant experimental approach to address the seemingly intractable question of whether the high CaDecision letter after peer review:2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Mark T Nelson as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kenton Swartz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Colin W Taylor (Reviewer #3).Thank you for submitting your article "Profound ER-luminal [CaThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:2+ concentration [Ca2+] in the endoplasmic reticulum (ER) lumen regulates the activity of inositol 1,4,5-trisphosphate receptors (InsP3Rs) independent of flow-through effects on the cytoplasmic surface of the channel. The authors show that, under conditions in which [Ca2+] is controlled so as to produce a physiologically high concentration in the lumen (300 nM) and appropriately low concentration in cytoplasm-equivalent spaces (70 nM), InsP3R open probability in the presence of cytosolic IP3 was profoundly reduced compared with that observed under symmetrically low [Ca2+] (70 nM in ER and cyto) conditions. The absence of an inhibitory effect upon elevating [Ca2+] in the ER-equivalent space to 300 μM while recording in the lumen-out configuration suggested the presence of a loosely associated inhibitory lumen protein, which they termed peripheral luminal-protein (PLP). After demonstrating that this PLP-mediated inhibitory phenomenon was generalizable to other cell types expressing different repertoires of InsP3Rs, they further established that a region of the L2 loop in common among all InsP3R subtype was responsible for PLP binding and that an excess of isolated peptide with this sequence was capable of blocking the inhibitory effect of PLP. Using this peptide to pull down proteins from soluble protein fractions of bovine hepatocyte ER microsomes in conjunction with mass spectrometry, they identified PLP as a member of the annexin (ANX) family of Ca2+-binding proteins. Subsequent experiments performed using recombinant ANXA1, ANXA6 and ANXa11, all of which were detected among ER proteins, and provide evidence that ANXA1 as the isoform responsible for the inhibitory effect and showed that endogenous ANXA1 was capable of inhibiting InsP3R-mediated Ca2+ release in intact cells.In this manuscript, Vais et al. use an elegant experimental approach to address the seemingly intractable question of whether the high CaEssential revisions:+ currents; given the familiar role of InsP3Rs in mediating Ca2+ efflux, these readers would be expecting Ca2+ to be monitored directly. This same demographic will also likely have difficulty orienting to the techniques used to create lumen-out and cytoplasm-out patch configurations. Because such "naïve" readers, which include molecular and cell biologists, and many others, are among those most likely to be interested in this work, manoeuvres used to record K+ conductance (instead of Ca2+ flux) and create lumen-out and cytoplasm-out patches should be described in plain English .1) Readers without a background in electrophysiology may be confused by the off-hand allusion to recording K3R-3 cells used for the bulk of the experiments are well described in the Materials and methods, it would be helpful if they were more clearly described when first introduced in Results .2) Although DT40-KO-r-InsP3Rs is profound (e.g. strong effect on Po) and universal . An impressive combination of advanced electrophysiological techniques and molecular biology approaches allowed the authors to convincingly demonstrate that Ca2+ concentration in the ER lumen has strong modulatory effect on the properties of InsP3Rs. The authors also provided compelling evidence for the involvement of a luminal Ca2+-binding protein in this modulation. There was a consensus amongst the reviewers that this phenomenon is novel, important and well-documented, and could stand on its own. It was felt, however, that the role of ANXA-1 is less strong. The major criticism relates to the suggestion that ANXA1 mediates endogenous inhibition by luminal Ca2+. The evidence  that ANXA1 is expressed in the 'ER lumen' is not clear. There is evidence that ANXA1 can confer an effect of luminal [Ca2+] , but that is not in itself evidence that it is the endogenous mediator. While the relatively modest effects of siRNA-mediated knockdown of ANXA1 on Ca2+ signals in intact cells lend support to its role, there is no evidence that these effects are related to luminal [Ca2+]. Immunostaining for AnxA1 looks like staining for nuclear lamins rather than the expected ER distribution. It would be important to verify the presence of AnxA1 in the ER lumen (e.g. by EM or super-resolution microscopy) and perhaps discuss/explain the retention of AnxA1 in this perinuclear compartment (and is consequences for cell physiology).3) The reported effect of the luminal modulation/inhibition of InsPThe authors should bolster their evidence for ANXA1 (within 2 months) or consider it for a follow-up paper.4) The reviewers agree that phenomenon is specific for the perinuclear ER, but it is unclear if it applies to other ER compartments. The authors should provide evidence for this mechanism in other ER compartments, which is tough, or limit their conclusion to the perinuclear ER and discuss its general relevance.2+] are not due to 'feed-through' to a cytosolic site rest on the argument that diBrBAPTA should buffer such effects . This may be true, but the argument would be stronger if the same effects of ATP were also shown at holding potentials where any Ca2+ flux would be directed into the lumen.5) Figure 2: the argument that the effects of cytosolic ATP  suggests a substantial increase in the frequency of gating and a probable decrease in Po in lumen-out recordings when [Ca2+]lumen was increased to 300μM, whereas the summary data (J) and subsequent conclusions (the need for an accessory protein) suggest no change in Po. Please clarify.6) Figure 2H  Figure 3H: it would be preferable to see this figure presented with the actual recorded Po achieved in the presence of pL2 is because it reverses the inhibition by luminal Ca2+. It needs to be shown that pL2 does not increase Po under conditions with reduced luminal [Ca2+].8) Figure 4J: the results shown do not establish that the increased  Essential revisions:+ currents; given the familiar role of InsP3Rs in mediating Ca2+ efflux, these readers would be expecting Ca2+ to be monitored directly. This same demographic will also likely have difficulty orienting to the techniques used to create lumen-out and cytoplasm-out patch configurations. Because such "naïve" readers, which include molecular and cell biologists, and many others, are among those most likely to be interested in this work, manoeuvres used to record K+ conductance (instead of Ca2+ flux) and create lumen-out and cytoplasm-out patches should be described in plain English .1) Readers without a background in electrophysiology may be confused by the off-hand allusion to recording K3R channel activity (Introduction). Schematic diagrams showing different nuclear patch-clamp configurations  have now been added in a new Figure 1. So all subsequent figure numbers are increased by one. We have also now clarified why the electrical currents through open InsP3R channels observed in our nuclear patch-clamp experiments were carried by K+ and not Ca2+.We have now modified the section on nuclear patch-clamp electrophysiology methods to provide better descriptions of the methods we used to study the InsP3R-3 cells used for the bulk of the experiments are well described in the Materials and methods, it would be helpful if they were more clearly described when first introduced in Results .2) Although DT40-KO-r-InsP3R-3 cell line used in our experiments in the main text .We have now added a description of the DT40-KO-r-InsP3Rs is profound (e.g. strong effect on Po) and universal . An impressive combination of advanced electrophysiological techniques and molecular biology approaches allowed the authors to convincingly demonstrate that Ca2+ concentration in the ER lumen has strong modulatory effect on the properties of InsP3Rs. The authors also provided compelling evidence for the involvement of a luminal Ca2+-binding protein in this modulation. There was a consensus amongst the reviewers that this phenomenon is novel, important and well-documented, and could stand on its own. It was felt, however, that the role of ANXA-1 is less strong. The major criticism relates to the suggestion that ANXA1 mediates endogenous inhibition by luminal Ca2+. The evidence  that ANXA1 is expressed in the 'ER lumen' is not clear. There is evidence that ANXA1 can confer an effect of luminal [Ca2+] , but that is not in itself evidence that it is the endogenous mediator. While the relatively modest effects of siRNA-mediated knockdown of ANXA1 on Ca2+ signals in intact cells lend support to its role, there is no evidence that these effects are related to luminal [Ca2+]. Immunostaining for AnxA1 looks like staining for nuclear lamins rather than the expected ER distribution. It would be important to verify the presence of AnxA1 in the ER lumen (e.g. by EM or super-resolution microscopy) and perhaps discuss/explain the retention of AnxA1 in this perinuclear compartment (and is consequences for cell physiology).3) The reported effect of the luminal modulation/inhibition of InsPThe authors should bolster their evidence for ANXA1 (within 2 months) or consider it for a follow-up paper.the luminal protein: our inability to immunoprecipitate it with the InsP3R, and to convincingly localize it to the ER beyond the nuclear envelope. On the other hand, a substantial body of evidence presented here demonstrates that ANXA1 has all of the properties of the peripheral membrane-associated Ca2+ binding protein (PLP) that, as the reviewer notes, we have convincingly demonstrated exists We discuss this in a separate Results section of the manuscript. Accordingly, we have decided to leave the ANXA1 data in the manuscript while providing much stronger caveats and reservations regarding our ability to conclude that ANXA1 is the ubiquitous PLP that regulates the InsP3R. In the revised manuscript, we do the following. We have re-written sentences to avoid conclusions that ANXA1 is the PLP, by including caveat words such as “likely”, or simply not mentioning ANXA1 but instead referring to PLP. In addition, we have more strongly emphasized the section in the Discussion where we point to the IP and localization failures. We now point that all of our patch-clamp studies were performed on the nuclear envelope, the same compartment where we were able to localize ANXA1 to the nuclear envelope lumen, suggesting that our observation regarding ANXA1 might be relevant for this compartment specifically. And we now add a new section at the end of the manuscript that points out limitations of the study, and suggest experiments that could be done in future studies to more definitively identify the PLP. Together, we feel that the reader now will have the strong sense that ANX1 could be the protein, but establishing it as the PLP remains to be determined.We described in the Discussion of the original version of the manuscript two important caveats regarding the conclusion that ANXA1 was 4) The reviewers agree that phenomenon is specific for the perinuclear ER, but it is unclear if it applies to other ER compartments. The authors should provide evidence for this mechanism in other ER compartments, which is tough, or limit their conclusion to the perinuclear ER and discuss its general relevance.Please see previous response. Because of our inability to localize ANXA1 to the entire ER, we now suggest in the limitations of the current study that the phenomena we document could possibly be applicable specifically to the perinuclear ER.2+] are not due to 'feed-through' to a cytosolic site rest on the argument that diBrBAPTA should buffer such effects . This may be true, but the argument would be stronger if the same effects of ATP were also shown at holding potentials where any Ca2+ flux would be directed into the lumen.5) Figure 2: the argument that the effects of cytosolic ATP  Figure 3 vs. Figure 8. We note that even using voltage to impede Ca2+ flux, a high concentration of Ca2+ chelator on the cytoplasmic side of the channel is still needed to buffer the Ca2+ passing through the channel. That is, the electrical potential needed to abrogate the feed-through effect using only a low concentration of chelator (0.5 mM) is much higher than the membrane patch at the pipette tip can tolerate.To clarify the experiments shown in Figure 3 , we have now added three schematic diagrams . We already previously performed the kinds of experiments that the reviewer mentioned, in which the high [Ca2+]ER of InsP3R Single-Channel Activity”, third paragraph) suggests a substantial increase in the frequency of gating and a probable decrease in Po in lumen-out recordings when [Ca2+]lumen was increased to 300μM, whereas the summary data (J) and subsequent conclusions (the need for an accessory protein) suggest no change in Po. Please clarify.6) Figure 2H . Even so, the Po of individual channel records varied substantially, as shown in Figures 3 L-M , so significant number of current traces (tabulated in the graphs) were collected to obtain meaningful statistics for our experiments.In Figure 3K , only a total 1.2 s of the “typical” current trace was shown to give the readers some idea of the gating of the InsP2+]ER before the perfusion solution switch was significantly lower than that in 300μM [Ca2+]ER after perfusion switch. However, it should be noted that most of the channel closings in both 70 nM and 300 μM [Ca2+]ER had very short durations, so the mean Po of the channel recorded in 70 nM [Ca2+]ER for the ~ 0.5 s before the solution switch is not substantially higher than the mean Po recorded in 300μM for the ~ 0.5 s after the solution switch, as the reviewer suggested.As for the current trace in Figure 3K  that the reviewer pointed out, it is obvious that the frequency of channel closings (seen as upward current spikes) in 70 nM i and maximal [InsP3] in the absence  and presence  of 1 mM MgATP in the bath solution. We have also included the normalized channel Po for the various InsP3R channels in +/– MgATP to show how the relative change in Po of those channels is remarkably similar , despite the large absolute difference among the Po of those channels. We rearranged the averaged data in Figure 4I to avoid giving the impression that different data sets were compared.We agree with the reviewer. We now show the actual recorded InsPo achieved in the presence of pL2 is because it reverses the inhibition by luminal Ca2+. It needs to be shown that pL2 does not increase Po under conditions with reduced luminal [Ca2+].8) Figure 4J: the results shown do not establish that the increased PPo is already very high under conditions of reduced luminal [Ca2+]. We could change the conditions, for example use lower [InsP3] to observe channels with a low Po in conditions of reduced luminal [Ca2+]. Preliminary data from such an experiment suggest that the peptide is not stimulatory on its own. However, the number of experiments is limited due to complete shutdown of our research efforts by the COVID-19 pandemic. We point out in the new “limitations of the current study” the absence of this control experiment.


These results suggest that MRD assessment should be a routine investigation in clinical practice in this AML subset.Measurable residual disease (MRD) response during acute myeloid leukemia (AML) treatment is a gold standard for determining treatment strategy, especially in core-binding factor (CBL) AML. The aim of this study was to critically review the literature on MRD status in the CBF-AML to determine the overall impact of MRD status on clinical outcomes. Published studies in the MEDLINE and EMBASE databases from their inception up to 1 June 2019 were searched. The primary end-point was either overall survival (OS) or recurrence-free survival (RFS) between MRD negative and MRD positive CBF-AML patients. The secondary variable was cumulative incidence of relapse (CIR) between groups. Of the 736 articles, 13 relevant studies were included in this meta-analysis. The MRD negative group displayed more favorable recurrence-free survival (RFS) than those with MRD positivity, with a pooled odds ratio (OR) of 4.5. Moreover, OS was also superior in the MRD negative group, with a pooled OR of 7.88. Corroborating this, the CIR was statistically significantly lower in the MRD negative group, with a pooled OR of 0.06. The most common cutoff MRD level was 1 × 10 However, there was no statistically significant difference in RFS outcome between both groups with this cutoff point  and association with clinical outcomes. Our results show that MRD negative CBF-AML patients have significantly better OS/RFS. In addition, the CIR was found to be significantly lower in the MRD negative group than in the MRD positive group. Subgroup analysis of each fusion gene (RUNX1-RUNX1T1 or CBFB-MYH11) found OS, RFS, and CIR to be significantly better in the MRD negative group.This is the first meta-analysis of qualitative MRD assessment in CBF-AML  recommendation, MRD monitoring in AML is suggested after induction chemotherapy . MRD stanagement . In MRD nagement . ConsistThis study has some limitations. First, the MRD cutoff levels vary among the included studies according to their experience and institutional policy. We were, therefore, unable to identify the optimal cutoff point for MRD assessment in CBF-AML. We were also unable to determine the best time point in the evolution of treatment to evaluate MRD. Nonetheless, a previous study proposes suitable timing for MRD assessment post-induction, post-consolidation, and then tri-monthly during the first 18 months of follow-up . Eight oMRD negative CBF-AML patients had better OS, RFS, and CIR than their MRD positive CBF-AML counterparts. These results suggest that MRD assessment should be a routine investigation in clinical practice in this AML subset, even in strained economies, since it improves clinical decision-making and can result in patients avoiding expensive procedures like stem cell transplantation.