Despite the recent approval of some FLT3 inhibitors by drug regulatory agencies, treatment guidelines for FLT3-mutated AML still require allogeneic transplantation as a necessary procedure to treat the disease in first or second CR, due to the high relapse incidence related to the use of these drugs. The study of the heterogeneity of leukemogenesis and resistance mechanisms related to the use of FLT3 inhibitors, alone or in combination, represents one of the additional challenges in attempting to achieve the eradication of the mutated FLT3 leukemic clone. The analysis and knowledge of these pathways might drive future approach in this setting.FLT3 ITD and TKD mutations occur in 20% and 10% of Acute Myeloid Leukemia (AML), respectively, and they represent the target of the first approved anti-leukemic therapies in the 2000s. Type I and type II FLT3 inhibitors (FLT3i) are active against FLT3 TKD/ITD and FLT3 ITD mutations alone respectively, but they still fail remissions in 30–40% of patients due to primary and secondary mechanisms of resistance, with variable relapse rate of 30–50%, influenced by NPM status and FLT3 allelic ratio. Mechanisms of resistance to FLT3i have recently been analyzed through NGS and single cell assays that have identified and elucidated the polyclonal nature of relapse in clinical and preclinical studies, summarized here. Knowledge of tumor escape pathways has helped in the identification of new targeted drugs to overcome resistance. Immunotherapy and combination or sequential use of BCL2 inhibitors and experimental drugs including aurora kinases, menin and JAK2 inhibitors will be the goal of present and future clinical trials, especially in patients with FLT3-mutated (FLT3mut) AML who are not eligible for allogeneic transplantation. Twenty-five to 30 percent of AMLs harbor FLT3 receptor mutations: 20–25% at the level of the juxtamembrane (JM) domain, recognized as internal tandem duplications (FLT3ITD) and distinguished, on the basis of the insertion site, in JM (70% of FLT3ITD) and TKD1 (30% of FLT3ITD) ,5.The small amount of mutated FLT3 ITD proteins not hypoglycosilated remaining on the cell surface determines the alternative activation of PIK/AKT and MEK-ERK signaling.FLT3TKD mutations have different downstream effects, resulting in blocking of differentiation, rather than stimulation of proliferation. For instance, FLT3ITD-mutated AML cells specifically determine STAT5 activation and p27 inhibition through the binding of their tyrosines residues 589 and 591 with Src family kinases , which might explain the proliferative advantage of FLT3ITD-mutated AML over FLT3TKD-mutated AML ,7. FigurFLT3i drugs differ in inhibition potency, activity on FLT3-ITD and TKD mutations, and specificity, and for these reasons they could have variable off-target toxicities .Type I FLT3i  are active against both FLT3-ITD and TKD mutations, because they interact with tyrosine kinase receptors in the active and inactive forms, while Type II FLT3i (Quizartinib and Sorafenib) are active only in the FLT3ITD forms because they have the binding site at the level of the hydrophobic region adjacent to the ATP-binding site, which is inaccessible when the receptor is in the active form. The availability of these drugs has dramatically changed the treatment guidelines for AML, supported by evidence of their efficacy with a molecularly guided approach.FLT3i were some of the few target drugs approved in the 2000s; however, FLT3ITD AML still has an unfavorable outcome, especially when it occurs with high FLT3 allelic ratio in NPM wild type patients .Incidence of relapse could be decreased by the use of second generation FLT3i, but NGS technologies and single-cell analysis have already demonstrated resistance pathways in cell cultures in vitro and in patients with FLT3ITDmut AML in vivo, and hematopoietic stem cell transplant (HSCT) is still necessary and recommended for curing the disease .FLT3ITDmut AML, with low allelic ratio with NPM1 comutations, is classified in the 2017 ELN classification as a favorable risk disease; nevertheless, the predictive role of this association is still controversial ,12.The resistance could be driven by acquisition of other mutations with or without loss of FLT3 or by acquisition or switching to different FLT3 mutations. Moreover, bone marrow niche could induce resistance through the release of microenvironmental factors which compete for the same therapeutic target of FLT3i or promote blasts and leukemic stem cell survival. Indeed, upregulation of the target-downstream pathways, such as STAT5 or mTOR, may contribute to blocking FLT3i activity. Overexpression of proteins inhibiting the apoptosis and inhibition of drug metabolism by cytochromes expressed in stroma are some other mechanisms of leukemic escape ,14. DeepMidostaurin and Gilteritinib are AIFA-EMA-FDA approved in naïve FLT3mut AML and relapsed FLT3mut AML, respectively. Other FLT3i such as Quizartinib and Sorafenib were investigated in clinical trials with interesting results but with uncertain evidence of efficacy in terms of survival. The current ELN and NCCN guidelines for FLT3mut AML patients eligible or not to intensive therapy are illustrated in Midostaurin is a multi-targeted kinase inhibitor active against FLT3 ITD and TKD, platelet-derived growth factor receptor (PDGFR), KIT, SRC, and other RTKs .The randomized phase III RATIFY trial analyzed the efficacy of Midostaurin in combination with the standard backbone therapy including Cytarabine and Daunorubicin (3 + 7) induction and high dose Cytarabine consolidation in patients <60 years with untreated FLT3 (ITD and/or TKD) AML.The primary endpoint was reached with an HR of 0.78 for OS in Midostaurin arm vs. placebo.Based on these results, Midostaurin was approved by FDA, EMA and AIFA and now, in combination with intensive chemotherapy, is the new standard of care for the treatment of patients with newly diagnosed FLT3mut AML . In the Gilteritinib is a new, multitarget, second-generation type I FLT3i. The phase I–II CHRYSALIS trial showed 41% composite complete remission (CCR) among patients with R/R FLT3mut AML associated with a good safety profile . The ranThe initial data showed that Gilteritinib decreased the risk of death by 36% and improved both rates of CCR and OS, with an advantage of 3.7 months when compared to salvage chemotherapy .Furthermore, these results were confirmed in a recent study update . Based oThe MORPHO phase III placebo-controlled trial, evaluating post-HSCT maintenance with Gilteritinib in FLT3mut AML, recently completed enrollment and results are keenly awaited (NCT02997202).Quizartinib is a second-generation highly selective type II FLT3i . In a raIn this trial, Quizartinib demonstrated a statistically significant OS improvement of 1.5 months in comparison to salvage chemotherapy.The role of post-HSCT maintenance with Quizartinib (60 mg/d) in FLT3-ITD AML was explored in a phase I study which showed a reduced relapse rate .Given the relatively small OS improvement and the concerns over potential side effects, including cardiac toxicity, Quizartinib was not approved in the US and Europe, but is approved in Japan as a monotherapy in R/R FLT3-ITDmut AML.Sorafenib is a first-generation type II multi-kinase inhibitor active against RAS/RAF, c-KIT, vascular endothelial growth factor (VEGF) receptor, PDGFR kinases and FLT3 . Due to its broad spectrum of action, Sorafenib was combined with standard chemotherapy in the randomized SORAML trial, where 267 patients ≤60 years with newly diagnosed AML, irrespective of FLT3 status (only 17% had FLT3-ITDmut), received 3 + 7 induction and high-dose Cytarabine consolidation with or without Sorafenib [Patients in the Sorafenib arm had a significantly improved EFS and RFS in comparison to standard chemotherapy with similar OS results, although a recent update suggested a trend for longer OS .In another trial enrolling 27 newly diagnosed FLT3-ITDmut AML patients who were not candidates for intensive chemotherapy, Sorafenib combined with 5-Azacytidine reported a 78% overall response rate (ORR), with a median duration of remission of 14.2 months and an acceptable safety profile .Finally, a randomized placebo-controlled multicenter phase II trial, called the SORMAIN trial, evaluated the role of Sorafenib as a maintenance therapy after HSCT in FLT3-ITDmut AML patients . In thisUnfortunately, Sorafenib was also associated with higher rate of graft-versus-host disease (GVHD) and skin toxicity compared to the placebo arm. Sorafenib is currently not yet approved in the United States or Europe for the treatment of AML patients.The vast majority of patients affected by FLT3-ITDmut AML, in remission after first-line or salvage chemotherapy, relapse if they do not receive HSCT, which therefore remains the cornerstone of treatment of the disease . DespiteFLT3-ITD mutations determine the switch from the inactive  to the active conformation (DFG-in) of FLT3 receptor. FLT3-TKD (D835) mutations block the receptor in the active conformation (DFG-in), due to the substitution of Asp at position 835 of the activation loop, leading to the opening of the ATP binding and auto-activation of the receptor. Type I FLT3i bind the FLT3 receptor in the DFG-in conformation much more strongly than the DFG-out conformation, either near the activation loop or the ATP binding pocket, and are active against both FLT3ITD and TKDmut AML. Type II FLT3i target the ATP-binding domain of FLT3 receptor, exclusively in the DFG-out conformation, are selectively active against FLT3-ITDmut AML, and resistant to FLT3-TKD mutations. Amino acid changes around the binding site are some of the structural reasons of resistance to type I FLT3i ,34.The “gatekeeper” mutation F691L showed universal resistance to all the currently available FLT3i . SensitiHere, we describe the scenario of primary and secondary resistance, reported in preclinical study, in real life, and in clinical trials investigating FLT3mut AML treatment.Traer et al. identified fibroblast growth factor 2 (FGF2) and CXCL12/CXCR4 stromal release as a possible mechanism of resistance to FLT3i ,40. The Microenvironment-mediated resistance to Gilteritinib, studied in FLT3mut cell lines, analyzed by integrating WES, unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR-Cas9), metabolomics, proteomics, phosphoproteomics, and small molecule inhibitor screenings, confirmed these data . Early rQuizartinib resistance, reconstructed in the in vitro model of Dumas et al., was also found to be related to AXL activation via the canonical GAS6 ligand, through soluble STAT5-activating factors, and local hypoxic environment .In addition, the in vitro model of resistance to Midostaurin and Sorafenib showed elevated levels of CCL5, with restoration of response after exposure to the CXCR4 receptor antagonist, Plerixafor .Ras-related C3 botulinum toxin substrate 1 (Rac1) is a protein involved in actin-mediated cytoskeleton remodeling. Midostaurin-resistant FLT3mut cell lines showed overexpression of RAC1, resulting in hyperphosphorylation of N-WASP, inducing actin polymerization, and of the anti-apoptotic protein BCL-2 . In vitrLast but not least, the metabolism of FLT3i is affected by cytochrome P450 3A4 expressed by bone marrow stromal cells and might also be dependent on pharmacological interactions with other drugs, metabolized by cytochrome P450 . MechaniThese studies identified MEK, AURKB, CDC7, CCL5, BCL2, RAC1, NRAS and MAPK as possible targets deserving of inhibition for overcoming FLT3i resistance.Alotaibi et al. analyzed bone marrow with NGS-based myeloid panel before and after FLT3i-based therapy  in 67 relapsed and 106 refractory patients in a cohort of 946 FLT3mut AML patients treated at MD Anderson . MechaniOff-target mutations were more frequent after type I FLT3i, whereas on-target FLT3 mutations occurred in 65% of patients relapsed after type II FLT3i, with a 30% incidence of FLT3-D835, which was reduced when these were associated with CCT compared with LIT.DNMT3A and IDH2 mutations were more frequent in responders than nonresponders, while RAS mutations with a VAF > 20% were related to refractoriness, particularly to treatment with type I FLT3i.The authors suggest that this analysis has some bias due to the 1% NGS sensitivity threshold and variability in FLT3i combination therapy administered. Analysis of emerging subclones with droplet digital polymerase chain reaction (PCR) or other ultra-deep sequencing platforms, pre-therapy and at relapse, might help in the future to better understand the mechanisms of relapse.F691 and codon D835 mutations were found to be linked to resistance in two clinical trials with Sorafenib ,50. In aN676K mutation, inducing a single amino acid substitution within the FLT3 kinase domain, was the first to be identified as resistance-leading mutation in a patient with R/R AML treated with Midostaurin .FLT3-ITD clone in 46% of cases treated with Midostaurin compared to in 19% in cases who did not receive it. Switched or gained FLT3ITD clones emerged in the Midostaurin group in 11% of relapsed patients vs. 0% of refractory patients, suggesting that the acquisition of new FLT3 mutations is associated with the duration of Midostaurin exposure. FLT3 mutations were stable at relapse in 32% of patients treated with Midostaurin vs. 48% of naïve patients. In patients with FLT3-ITD persistence, selection of resistant ITD clones was found in 11% as a potential driver of disease. The Midostaurin registrative phase III trial RATIFY showed a 59% CR rate in the experimental arm with 40% resistance and 40% of relapse. Genescan-based testing for FLT3-ITD and whole exome sequencing (WES) were performed at diagnosis and relapse or resistance in a selection of 54 patients receiving Midostaurin and chemotherapy and 21 treated with chemotherapy alone, enrolled in RATIFY or the German–Austrian Acute Myeloid Leukemia Study Group 16-10 trial . RelapseAt the onset of resistance or disease progression, the Midostaurin group presented fewer on target than off target mutations.n = 3), RUNX1 (n = 3), RAS (n = 4), IDH1 (n = 2), chromatin/splicing related genes  were identified in resistant patients at WES analysis, while only two relapsed patients had the N676 mutation, with low VAF (<5%).The pathway enrichment analysis detected activation of RAS and MAPK in resistant patients, losing FLT3 mutation at relapse. Mutations of WT1 . NPM1 expression was found to be doubled in relapsed compared to refractory patients (43% vs. 21%), while WT1 mutations were equally distributed in the two groups.Secondary resistance to Quizartinib is influenced by ineffectiveness against D835 and the gatekeeper residue of kinase mutations, F691L, with consequent selection of related clones . Single The ADMIRAL trial’s authors recently published interesting results concerning patients with FLT3mut AML relapsing after Gilteritinib.Acquisition of new mutations occurred in 40 patients: 18 involved RAS/MAPK pathway, 6 FLT3 (5 F691L), 3 WT1 (1 with F691L), 1 IDH1, and 1 GATA2; 13 patients (32.5%) had no new mutations. The acquisitions of RAS/MAPK pathway gene mutations and FLT3 F691L gate keeper mutations at relapse were mutually exclusive . Not traThe correlation between frequency of emergent FLT3 F691L gatekeeper mutations at relapse and dose of Gilteritinib is unclear. In the Admiral trial, patients who received 120-mg/day Gilteritinib had a similar incidence of FLT3 F691L incidence compared to that observed in relapsed patients who received 20 to 200 mg/day Gilteritinib, while none of the patients receiving >200 mg/day Gilteritinib acquired the mutation at relapse. Nevertheless, patients receiving 120 mg/day had better OS compared to other patients .Another study showed a correlation between Gilteritinib dose and resistance in 22 FLT3mut AML patients analyzed at relapse by NGS and single cell analysis, identifying in those receiving doses below 200 mg, a more likely development of RAS or FLT3 F691L mutations .Crenolanib is a second-generation type I FLT3i active against FLT3/PDGFR at concentrations lower than those reported as safe in humans . Zhang eEvaluation of new FLT3i, the combination of several target agents, and the use of multi-target agents represent possible future approaches to overcoming AML FLT3 resistance. Here, we report the most representative in vitro studies and the enrolling and not yet enrolling clinical trials available.Knowledge of the FLT3 receptor sites susceptible to the most relevant mutations, conferring resistance to I and II generation FLT3i, guided the construction of new inhibitors capable of bypassing these resistances. In vitro studies using cell lines and xenograft models have validated their efficacy. Below, we have selected several studies ,70,71,72FLT3i showed several mechanisms of resistance and single cell and NGS analyses showed the presence of multiple complexity in the leukemic escape suggesting the emergence of multiclonal or oligoclonal resistant AML cells at relapse. Scientists are all converging on attempting to bypass potential FLT3i failures by using the association of different FLT3i or the combination of FLT3i with chemotherapy and target or multitarget agents. The principal pathways explored in preclinical studies are summarized in Bregante et al. identified 2 out of 18 compounds active against FLT3-ITD AML, WS6 and Ispinesib, and combined them with two approved drugs, Ponatinib and Cabozantinib, in in vitro models (AML cell lines and samples) . WS6 hadBET inhibitors play an important role in suppressing leukemogenesis through inhibition of leukemic pro-survival factors such as MYC and BCL2 but insufficient single-agent clinical potential and low specificity and hematological tolerance related to activity in normal bone marrow cells are reasons of concern. Lee et al. showed that the novel 4-azaindole derivative PLX51107 has BET-inhibitory activity in vitro (MYC plasma inhibitory activity assay in OCI-AML3 cells for BET inhibition and FLT3 plasma inhibitory activity assay in MOLM-14 cells) and in vivo (MV4-11 mouse xenograft model) .Quizartinib-PLX51107 compared to mice treated with 5 mg/kg Quizartinib alone. PLX51107 appears to be the ideal BET inhibitor because of its short plasma half-life, resulting in high specificity against leukemic cells, compared with normal bone marrow precursors, permitting a safe combination with continuous FLT3i exposure. The association of Quizartinib-PLX51107 could be further investigated in future clinical trials.Tumor growth was significantly inhibited in mice treated with Quizartinib efficacy is hampered by bone marrow stromal niche through STAT3 and STAT5 activation. Patel et al. attempted to reproduce this leukemic protective environment in an in vitro model of FLT3-ITD+ AML cells cultured in conditioned medium, obtained from bone marrow stromal cells cultures . They coFleischmann et al. studied the two distinct phosphoproteome patterns in human FLT3 mut AML (MOLM13) and murine AML cell lines (Ba/F3), depending on the localization of FLT3ITD . Pretrea→the surface pattern responds better to AKT-mTOR inhibitors Rapamycin→the endotelial reticulum pattern might benefit from chaperones (HSP90) and MEK inhibitors. The authors also advocated a synergistic interaction between Valproic Acid and MEK inhibitors.In conclusion, two different patterns of localization of FLT3 are associated with two specific phosphoproteome and chemosensitivity settings:Huang et al. analyzed in vitro activity of PI3K inhibitor, LY294002, in Sorafenib resistant FLT3 mut AML cell lines (BaF3-ITD-R) . Loss ofMomelotinib is a JAK2 inhibitor also active on FLT3 that has recently shown efficacy in FLT3i-resistant cell lines such as Gilteritinib and Quizartinib, expressing mutations  . A recenA recent study showed that FLT3i can determine activation of histone deacetylase 8 (HDAC8) via FOXO1 and FOXO3, blocking p53 and themselves providing an escape from apoptosis, and thus a mechanism of resistance . InhibitNVP-HDM201 in combination with Midostaurin, demonstrating significantly increased susceptibility to FLT3i in NPM1 and TP53 wild type FLT3mut AML cells with high allelic ratio. The combination NVP-HDM201 and Midostaurin was as effective as chemotherapy + Midostaurin in FLT3-ITD positive TP53 wild type cells, suggesting a possible role in future clinical trials [MDM2 is an oncogenic protein inhibiting normal p53 function. Therefore, MDM2 inhibitors retain their activity only in TP53 wild type AML, because they are unable to interact with deleted or absent p53. Seipel et al. analyzed the in vitro efficacy of MDM2 inhibitor l trials .Among the AXL inhibitors ,84, the Liu et al. selected drug-sensitive and drug-resistant human AML cell lines and FLT3-mut AML blast cells with high AXL antigen expression to analyze the cytotoxic effects of DAXL-88, DAXL-88-MMAE and R428 . Drug-reBased on the previous evidence of the downregulation of MEIS1 and its transcriptional target gene FLT3 by inhibitors of the menin-MLL complex, MI-503 was tested with FLT3i Ponatinib and Gilteritinib, demonstrating synergism in suppressing FLT3 and downstream genes . This syYang et al. studied the effect of Quizartinib and Pexidartinib rotation in AML cell lines (MOLM-14 and MV4-11) by analyzing the onset of resistance using computational studies . They obCabozantinib is an oral multitarget inhibitor of FLT3, AXL, MET, VEGFR, and KIT showing a potent inhibition of FLT3-ITD mut cell lines with D835 resistant mutations . It is aCompound 14J, derived from the pharmacophore assemblage of momelotinib and tandutinib, demonstrated enhanced in vitro inhibition of JAK2 and FLT3 in AML cell lines , enough CCT241736 is an oral dual inhibitor of FLT3 and Aurora kinase that also showed efficacy in leukemic cell lines and xenograft models of FLT3-ITDmut and or FLT3-TKDmut tumors resistant to Quizartinib and Sorafenib ,94. The Wu-5 is a novel inhibitor of USP10 that induces degradation of FLT3-mutated protein and downregulates compound C AMPKα, which was shown to effectively inhibit proliferation of FLT3mut cell lines such as MV4-11, Molm13 and MV4-11R .Wu-5 and Crenolanib showed synergism in the inhibition of FLT3 and AMPKα in FLT3-ITDmut cells, while metformin hampers the efficacy of Crenolanib due to the activation of AMPKα, confirming the interactions between Crenolanib and AMPKα activity.Wang P. et al. identified and analyzed the in vitro efficacy of a dual oral FLT3 and tubulin inhibitor KX2-391, with very interesting effects on resistant FLT3mut AML cell lines (D835 and F691L). It also effectively reduced leukemic growth of FLT3-ITD-F691L, FLT3-ITD and FLT3-ITD-D835Y mut AML cells in a xenograft leukemia model .Xu et al. identified a new FLT3/Hedgehog inhibitor, called Triptonide, with interesting abilities to specifically inhibit FLT3-ITDmut AML cells, sparing normal cells. The small molecule induced cell cycle arrest at G0/G1 and apoptosis of MOLM-13 cell line in in vitro and in vivo murine xenograft model . ProteomCAR-T are the most exciting immunological bullets currently available for R/R acute lymphoblastic lymphoma and R/R Non-Hodgkin Lymphoma. Li K. et al. constructed FLT3scFv/NKG2D-bispecific-CAR T cells, including a new CAR construct comprising the extracellular region of the human NKG2D receptor and the FLT3scFv, that showed cytotoxicity against FLT3mut AML cells and significantly prolonged the survival of the MOLM-13 cells engrafted mice. The bispecific CAR T cells eliminated both primary FLT3mut and FLT3wt AML blasts, although the treatment effect on the FLT3mut AML blasts was more profound. The tumor-killing efficacy of CAR T cells improved dramatically when they were administered in combination with Gilteritinib both in vitro and in vivo .Here, we report results of recent phase I trials with new FLT3i or target agents.A phase I study explored twice-daily administration of 100 mg vs. 200 mg of the oral JAK2/FLT3i Pacritinib in combination with 3+7 in five patients with first-line FLT3 AML (cohort A) or in combination with 10-day cycles of Decitabine in eight patients with R/R FLT3 AML (cohort B). Pacritinib administration schedule was from day 1 to day 4 and from day 8 to day 21 in cohort A (with 3+7 infusion from day 5 to day 11), and from day 1 to day 21 in cohort B (with 20 mg/sm Decitabine infusion from day 5 to day 14). There was one early death, two CRs, and two stable disease responses in cohort A, and one morphological leukemia-free state and five stable disease in cohort B, with two cases of dose-limiting toxicity at the 100 mg dose, due to hemolytic anemia and grade 3 QTc prolongation, respectively. The median OS was 292 days, and two CR patients in cohort A were safely transplanted. A 35% reduction in blasts was observed in two patients after pretreatment with Pacritinib in cohort A, suggesting interesting activity even in monotherapy. The study’s low sample size and preliminary results warrant further investigation .The activity of the oral FLT3i Pexidartinib, with interesting in vitro efficacy against the conferring resistance mutation F691L, was explored in a phase I trial with a dose escalation phase at daily doses ranging from 800 to 5000 mg in 34 patients, without dose limiting toxicities, followed by a dose expansion phase of 3000 mg daily dose in 56 R/R FLT3 AML patients . DiarrheThe strategy of increasing leukemic killing through leukemic FLT3mut cells mobilization was investigated in a phase I study combining oral FLT3i Sorafenib at 400, 600 and 800 mg twice daily, with G-CSF and plerixafor administered every other day for seven doses starting on day 1. The trial enrolled 28 patients with R/R FLT3-ITDmut AML, 36% after HSCT and 39% after failing a previous FLT3i treatment, including one patient refractory to sorafenib. Extrahematological ≥ grade 3 treatment-related events were reported in 20 patients mainly due to skin rash, arrhythmia, elevation of liver enzymes, bone pain, and, less frequently, transient renal failure, pleural effusions and pericardial effusions. The CCR rate was 37%, with a median duration of response of 5.3 months. Two patients (7%) achieved negativity of FLT3, 1 is still in remission at 56 months and 1 relapsed after 16 months after acquisition of D835 mutation. The mobilization of leukemic FLT3mut cells represents an intriguing mechanism of resistance escape deserving further investigation .Protein synthesis could be a target for overcoming FLT3i resistance. Omacetaxine mepesuccinate OME, able to inhibit t-RNA binding to ribosomes and t-RNA transcription, showed in vitro synergy with FLT3 . TreatmeAs previously shown, BCL-2 overexpression is one of the mechanisms of resistance to FLT3i and a potential target of inhibition for overcoming resistance.Konopleva et al. investigated the triplet Venetoclax-10 days Decitabine-FLT3i in 12 young R/R FLT3mut AML and in 13 patients, aged >60 years, in frontline FLT3mut AML in a phase II trial (NCT03404193) . The priDoublet regimens had similar results, but with shorter follow-up and in less unfavorable settings. Venetoclax associated with Gilteritinib showed CRc of 85% in a phase IB trial enrolling R/R FLT3mut AML . MoreoveDespite similar results, authors suggested a potential advantage of triplet vs. doublet FLT3i and LIT combinations, due to deeper responses and prevention of secondary resistance. To reduce the hematological toxicity of the triplet, a bone marrow aspirate at day 14 is recommended to assess blast clearance or aplasia requiring Venetoclax discontinuation. Despite the high FLT3 inhibitory efficacy reported in in vitro studies, second-generation FLT3i, Gilteritinib, Quizartinib and Crenolanib showed primary and secondary resistance in FLT3mut AML treatment. The presence of NRAS mutations at baseline or at relapse after FLT3i therapy, and the identification of F691L mutations, represent the most frequent events in those patients requiring new combination therapies.The antiBCL2 agent Venetoclax, one of the most interesting approved drugs in recent AML treatment scenarios , was assClinicalTrials.gov identifier: NCT02997202, NCT02400255).Randomized phase III studies of CCT in combination with Midostaurin versus Gilteritinib (NCT03836209) and with Midostaurin versus Crenolanib (NCT03258931) are currently ongoing to establish which FLT3i should be used in frontline. Furthermore, phase III study of Gilteritinib versus placebo and phase II Crenolanib trials are ongoing and may help to address the benefit of FLT3 inhibition more definitely as maintenance therapy after HSCT in FLT3mut AML .In vitro and in vivo studies confirmed the polyclonal nature of resistance to FLT3i, which is due to microenvironment factors, alterations of glutamine metabolism, cytoskeleton remodeling and hyperactivation of several downstream pathways of FLT3 receptor. Phase III trials and post hoc analyses of real-life experiences have shown correlations between relapses and mutations of RAS and F691L, and between refractoriness and persistence of FLT3 mutations and acquisition of mutations of IDH, RAS and genes controlling cell cycle and splicing of RNA. RAS mutations were predictive of refractoriness after Crenolanib but they were not after Gilteritinib, while D835 mutations were acquired at relapse after treatment with type II FLT3i.Preclinical studies have investigated several new compounds able to overcome the classical mutations conferring resistance to FLT3i, such as F691L and FLT3 D835. The combinations of FLT3i and other target drugs or multitarget agents inhibiting BCL2, MYC, PTPN11, MEK, MDM2, HDA8, Aurora kinases, JAK2, JAK1 and AXL were tested in in vitro and xenograft models. WS6 showed synergy with Ispinesib and Cabozantinib, as Dasatinib did with Quizartinib, especially in AML with PTPN11 mutations. Valproic Acid, MEK and HSP90 inhibitors showed synergy when FLT3 was localized in the endothelial reticulum, while AKT-mTOR inhibitors, such as Rapamycin, are active when FLT3 is situated on the cell surface. The JAK2 inhibitor Momelotenib and the MDM2 inhibitor NVP-HDM201 confirmed their activity, respectively, in JAK2-mutated AML and NPM1 and TP53 wild type settings, while Menin inhibitors were active in MLL and NPM1 comutated FLT3AML. AXL inhibitors target AKT/ERK downstream signaling with interesting results.Among the multitarget agents, we selected Cabozantinib (multitarget inhibitor), Compound 14J (JAK2i/FLT3i), Wu-5 (AMPKa protein inhibitor/FLT3i), CCT241736 (Aurora kinase inhibitor/FLT3i), KX2-391 (tubulin inhibitor/FLT3i) and Triptonide (Hedgehog inhibitor/FLT3i) as the most interesting drugs, active in in vitro and xenograft models of R/R FLT3mut AML. The bispecific CAR T cells FLT3scFv/NKG2D showed synergy with Gilteritinib, providing a new option of cell therapy, hitherto unexplored in this setting. Clinical phase I trials investigated Pacritinib (JAK2/FLT3i) with HMA and chemotherapy, Pexidartinib, the combinations of Sorafenib with Plerixafor/G-CSF and Omacetaxine mepesuccinate, identifying this latter combination as the one providing the best ORR rate of 72%. Phase II trials, investigating triplet combinations of Venetoclax, HMA and FLT3i showed a low early mortality and very high CR rates in first and second line, burdened by high relapse rates, even after HSCT, in R/R setting. The day +14 bone marrow blast count helped to modulate hematological toxicity by reducing the duration of Venetoclax treatment. The depth and duration of response, especially in newly diagnosed patients, make this approach an attractive option for future phase III trial.New FLT3i have shown interesting activity against mutations which confer resistance, such as Compounds 17, 8r, 5o ,71,72, bWe are waiting for results of ongoing clinical trials investigating combinations of Venetoclax and or HMA, CPX-351 and chemotherapy with FLT3i in newly diagnosed and R/R patients. These data might therefore change the paradigm of the cure of the disease, which still represents an unmet medical need, especially in a second-line setting.Anti-FLT3/CD3 DART and anti-FLT3 CAR-T are some of the current specific ‘immune magic bullets’ available in ongoing clinical trials in the R/R FLT3mut AML treatment scenario. However, future trials could also select and investigate well-tolerated possible specific antibody-based immunotherapies, with the aim of eradicating LSCs or pre-emptively treating molecular relapse. CD123 is frequently expressed in AML and CD99 was recently found to be specifically expressed by FLT3mut LSCs . BispeciPost-HSCT maintenance administration is really an intriguing topic because of the frequency and poor prognosis of post-HSCT relapse. However, due to the uncertainty of Sorafenib safety data, we are still waiting for a better FLT3i in this context . This haThe presence of comutations is another important factor to consider when choosing induction therapy. FLT3 and IDH mutations can be co-expressed at diagnosis, and Shoukier et al., on the basis of a real-life experience, suggested that the value of VAF may guide the choice of the best target therapy between FLT3i and IDH inhibitor . As a maNPM1 and FLT3 comutations represent another intriguing subset with more favorable outcome than NPM1 wild type FLT3mut AML. Menin inhibitors showed a potential activity in NPM1mut AML, related to MLL1 and MLL1-fusion protein inhibition. Menin inhibitors also inactivate MEIS1 transcription factor with the particularly interesting effect of downregulating its transcriptional target gene FLT3, suggesting a possible synergy with FLT3i, especially in NPM1mut-FLT3mut AML, and also in MLL-FLT3mut AML . ResearcInhibition of the antiapoptotic protein BCL2 is another important available therapeutic option for overcoming resistance. Double or triplet regimens can increase the depth of response and avoid persistence or the appearance of leukemic subclones. Administration of oral target agents and hypomethylating agents can spare chemotherapy and improve quality of life and the psychological impact of the disease by reducing extrahematological toxicity and increasing time spent out of the hospital, especially in patients who are not transplant candidates. Careful monitoring of quality-of-life-adjusted costs is necessary in future clinical trials, as it is already known that new regimens come with the burden of high costs .We have seen how the acquisitions of secondary mutations can cause FLT3AML relapse through a linear evolution if they occur in the original FLT3mut clone, or through a branching evolution if they arise in a clone different from the original FLT3mut leukemic clone. Clinical trials and real-world experience using FLT3i have reported high rates of acquisition of RAS and epigenetic modifiers mutations in relapsed FLT3mut AML patients, followed by the acquisition of the TP53 and WT1 mutations, whereas F691L represents the classical mutation conferring resistance to all currently available FLT3i. FLT3-mut AML patient relapse might be due to FLT3 and its activation of downstream pathways . Stromal factors could bypass FLT3 silencing, activating its downstream signaling or stimulating the antiapoptotic protein BCL2 and AXL gene. CXCR4-CCL5 interaction may protect and hide leukemic cells in the bone marrow niche, where cytochromes expressed by stromal cells may also interfere with FLT3i metabolism. Increased fatty acid metabolism in resistant leukemic cells, induced by stromal factors, was also shown to activate cell cycle regulator genes such as CDC7/AURK, with consequent increase of leukemic proliferation.Interpretation of past clinical trials and post hoc analysis of primary and secondary mechanisms of resistance could guide future personalized treatment plans, tailored to patient populations. These approaches could be particularly appealing in patients not eligible for HSCT, but could also be crucial in pursuing cure in the unfavorable context of pre- and post-HSCT R/R disease. Sorafenib maintenance after HSCT has not been approved, but new FLT3i are being investigated in this setting and could likely show greater benefit and safety. Immunotherapy, BCL2, Aurora kinases, Menin, JAK2 inhibitors represent some of the exciting target drugs, investigated in clinical and preclinical trials, which could probably overcome FLT3i resistance and give a breakthrough in the future treatment of FLT3mut AML.Analysis of quality-of-life-adjusted costs should be performed to guide the choice between combinations of multiple target agents and or sequential pre-emptive treatment of relapse, based on MRD data and relative mutations repertoire.


The two-edge carbide diamond-coated drill produced by Seco Company was used in the experiments. Cutting-force components and delamination factor were measured in the experiments, and photos of the holes were taken to determine the delamination. In addition, modeling of cause-and-effect relationships between the technological drilling parameters vc and fz was simulated with the use of artificial neural network modeling. For all tested GFRP materials, an increase in fz led to an increase in the amplitude of cutting-force component Fz. The lowest values of the amplitude of cutting-force component Fz were obtained with the lowest tested feed per tooth value of 0.04 mm/tooth for all tested materials. It was observed that materials produced with the use of the specially designed pressing device were characterized by lower values of the cutting-force component Fz. It was also found that the delamination factor increased with an increase in fz for all tested GFRP materials. A comparison of the lowest and the highest values of fz revealed that the lowest delamination factor increase was archived by the B1 material and amounted to about 12.5%. The error margin of the obtained numerical modeling results does not exceed 15%, so it can be concluded that artificial neural networks are a suitable tool for modeling cutting force amplitudes as a function of vc and fz. The study has shown that the use of the special pressing device during the manufacturing of composite materials has a positive effect on delamination.This paper reports the results of measurements of cutting forces and delamination in drilling of Glass-Fiber-Reinforced Polymer (GFRP) composites. Four different types of GFRP composites were tested, made by a different manufacturing method and had a different fiber type, weight fraction (wf) ratio, number of layers, but the same stacking sequence. GFRP samples were made using two technologies: a novel method based on the use of a specially designed pressing device and hand lay-up and vacuum bag technology process. The study was conducted with variable technological parameters: cutting speed v GFRP is a composite material, whose structure is made of a minimum of two components: Glass Fiber (GF) and polymer  . GFRP chThe aviation industry is one of the industries in which drilling accounts for 40% of all machining operations in components assembly  . AircrafResearch works are carried out on various types of drilling, such as conventional drilling , grindinDelamination affects the structural integrity and reliability of FRP composites; therefore, it has a significant economic impact, especially when considering different stages associated with components assembly . In drilA study on GFRP drilling carried out by K. Siva Prasad  demonstrThe aim and novelty of the study presented in this paper was to assess the influence of the different technological drilling parameters on the cutting forces occurring in this process and the delamination of GFRP composites plates. The GFRP plates were made of two different manufacturing technologies: a traditional one, vacuum bag method and a new innovative one which was patented . The resc and feed per tooth fz) and different GFRP materials on the cutting forces and their influence on delamination. On this basis, it will be possible to define the optimum technological parameters for drilling GFRPs manufactured with the new method. The applied research plan is presented in The purpose of this study was to investigate the effect of selected technological drilling parameters . After that, 10 holes  were drilled in each sample a spacing of of 25 mm between the axis of the holes  on vertisoftware . DelaminThe delamination was quantified using the delamination factor and was defined as ,32:(1)FdThe diameters were measured using the software provided with the Keyence VHX-5000 optical microscope at 20× magnification.z (AFz) were developed. Only the component of the cutting force Fz was an object of prediction models, because it plays the largest role in the drilling process and assumes the largest values. The prediction models were designed using artificial neural networks in the Statistica 13 software. Variable technological parameters, i.e., a cutting speed vc [mm/min] and a feed per tooth fz [mm/tooth] and the type of the analyzed material (M) were adopted as input neurons, and the amplitude of the cutting-force component Fz (AFz) was taken as the output neuron. Based on the obtained experimental results of experimental tests, models of the amplitude of the cutting-force component FA shallow neural network with one hidden layer was used for modeling. The Multilayer Perceptron (MLP) and Radial Basis Function (RBF) neural network were used in the study. Different neuron activation functions were used for MLP: linear, exponential, logistic, tanh and sinus, together with different learning algorithms such as the BFGS gradient (Broyden–Fletcher–Goldfarb–Shanno), steepest descent training algorithm and conjugate gradient. The learning algorithm for the RBF was a RBFT, with a Gaussian hidden neuron activation function and a linear function for the output layer. The number of neurons in the hidden layer (2–10) was selected experimentally. The dataset was split in a proportion of 75% and 25% . The test set was not included due to the small amount of data ,34. Netwz for the i-th observation, and z for the i-th observation obtained as a result of the prediction. Similar analyses and neural networks were used in the drilling process case for other materials  and the prediction made demonstrate that the obtained network is characterized by a satisfactory prediction ability of this parameter, because the correlation coefficient is above 0.98 and the size of errors does not exceed 15%.The modeling results of the amplitude of cutting-force component FThe employed measuring for the determination of delamination factor is shown in z and constant cutting speed vc for different types of GFRP. Regardless of the tested material, for the lowest feed value, the delamination factor values are the lowest and most comparable . The diagram also reveals that feed per tooth has a significant impact on the delamination factor, as the delamination factor increases with an increase in the feed per tooth for all materials, which agrees with the results reported in [orted in . A compaz and the component of the cutting force Fz and delamination factor is presented, demonstrates a certain relation for all GFRP types. With the increase in the feed per tooth fz, both the delamination factor and the value of cutting-force component Fz increase. An increase in delamination and cutting force is caused by the cutting edge. The drill edge is in direct contact with the tested sample layer, which is extruded the fastest in comparison to the cutting. The cutting force causes greater thrust on the tested sample, which leads to the higher bending of the sample and can lead to the crack initiation and delamination development.A comparison of the results presented in c = 91 m/min. The delamination factor value decreased with increases in the cutting speed. The lowest delamination factor values were obtained for A1 = 1.13 (at vc = 91 m/min) and for B1 = 1.16 (at vc = 91 m/min). This is unusual, as study [c = 182 m/min) and 3% (at vc = 273 m/min), respectively.The relationship between the cutting speed and the delamination factor differs from that established for feed per tooth, as shown in as study  showed tz = 0.016 mm/tooth and three different cutting speeds vc . It can be observed that for the same drilling technological parameters of different materials, the influence of fiber type in the composite and the production method on delamination and defects in the form of torn fibers on the machined edges are noticeable. The smallest damage to the edges of the machined holes can be observed for materials A1 and B1, while the largest is observed for B2; these materials were made of the same fiber but differ in the number of layers and the manufacturing method. A similar observation can be made for materials A1 and A2, both of which were made of the same fiber, but had a different number of layers and different manufacturing methods—less delamination is observed for material A1, for which a special pressing device was used.The novelty of this study consists of the preparation of GFRP using a new technique and the study of the machinability of these materials. Since the machinability of these materials is yet to be investigated, our study contributes to the knowledge in the field. The optimal parameters of the drilling process, which leads to low delamination phenomena, were determined.z has the greatest influence on the total cutting force compared to other components;The cutting-force component Fz were obtained for material A1 and the highest for material B2 (an increase of approximately 64%);The lowest values of cutting-force component Fz are obtained for materials consisting of twill woven fibers . In this case, the GF fabric is finer (280 g/m2) and is made of thinner threads;A comparison of materials A1 and B1 demonstrated that, especially for lower cutting speeds, lower values of amplitude of the cutting-force component Fz has an impact on the cutting-force component Fz. For all tested GFRP materials, an increase in feed per tooth led to an increase in the amplitude of the cutting-force component Fz;The feed per tooth fz were obtained for the lowest tested feed per tooth value equal to 0.04 mm/tooth for all tested materials. It can be observed that the materials produced with the use of a special pressing device are characterized by lower values of the cutting-force component Fz;The lowest amplitude values of the cutting-force component Fz for all tested GFRP materials;Feed per tooth also has the greatest impact on delamination. The delamination factor increases with an increase in feed per tooth fThe lowest delamination factor increase, of about 12.5%, was obtained for the B1 material;Based on the images of the drilled holes, it can be concluded that the smallest amount of damage to the edges of the drilled holes was observed for materials A1 and B1. This proves that the manufacturing of composites with the use of special pressing device has a positive effect on delamination;c = 182 m/min and fz = 0.04 mm/tooth;The minimal delamination was obtained for B1 that was machined with the following machining parameters: vz, both the delamination factor and the value of Fz cutting-force component increase for all types of GFRP;With the increase in the feed per tooth fc and the feed per tooth fz.Since the error margin of the obtained numerical results does not exceed 15%, it can be concluded that artificial neural networks are a suitable tool for modeling cutting-force amplitudes depending on the cutting speed vThe results of the study investigating the cutting forces and delamination in drilling GFRPs leads to the following conclusions:z cutting-force component in the drilling process and higher quality of the machined holes.Apart from the technological parameters of drilling, other factors that affect the cutting forces and delamination in this process include the manufacturing method of GFRP composites and the type of material. The materials manufactured using a special pressing device were characterized by a lower F


Low-voltage electrical installations are increasingly saturated with power electronic converters. Due to very high popularity of photovoltaic (PV) installations and the spread of electric vehicles (EV) as well as their charging installations, DC–AC and AC–DC converters are often found in power systems. The transformerless coupling of AC and DC systems via power electronic converters means that an electrical installation containing both these systems should be recognized from the point of view of earth fault current waveform shapes. In such installations, various shapes of the earth fault current may occur—a DC component of a high value may especially flow. The DC component included in the earth fault current influences the tripping threshold of residual current devices (RCDs)—the devices which are mandatory in certain locations. This paper presents results of the AC-type, A-type, and F-type RCDs sensitivity testing under residual currents of various compositions of the DC component. This testing has shown that the DC component may both degrade and improve the sensitivity of RCDs. Moreover, unexpected positive behaviors of RCDs in some circumstances under DC residual current is discussed. Therefore, recognizing the real sensitivity and behavior of RCDs from the point of view of the DC component is important for effective protection against electric shock, in particular, in PV installations and EV charging systems. The research results provide a new insight into the real behavior of RCDs in modern power systems and, consequently, the safety of people. Air pollution coming from the classic way of power production as well as from utilization of combustion engines in transportation systems has nowadays resulted in a rapid development of photovoltaic (PV) power generation and an increased number of electric vehicles (EV). PV systems and EV charging systems include installations composed of alternating current (AC) circuits and direct current (DC) circuits. To utilize power generated in PV systems, the use of the DC–AC power electronic converters is necessary. These converters (inverters) may have various topologies ,2,3 and Taking properties of PV installations and EV charging installations into account, one can conclude that evaluation of the effectiveness of electrical safety, i.e., protection against fire and especially protection against electric shock in case of an insulation fault in such installations is not an easy task. Deep analysis of phenomena occurring during earth faults is very important especially when residual current devices (RCDs) are applied. Electrical installations composed of both DC and AC systems which are coupled via transformerless converters (inverters), as well as installations having other types of converters, e.g., variable-speed drive systems, may generate the earth fault currents of various waveform shapes. Problems with protection against electric shock, including residual current protection in circuits with power electronic converters refer mainly, in the literature, to high-frequency components of the earth fault current and their negative effect on the tripping of RCDs. With reference to high frequency currents, this negative effect is studied in ,26,27, wA review of the literature has shown that in the case of installations containing direct current sources, very little attention is paid to the sensitivity of RCDs and their tripping testing under direct currents, although there are more and more PV and charging EV installations having DC circuits. Moreover, there is a fairly common opinion that the earth fault current with a large share of the DC component can be detected only by the most technically advanced and the most expensive B-type RCDs. Some results for B-type RCDs are discussed in . The simHowever, the authors of this paper conducted a wide testing of A-type and F-type RCDs under earth fault currents of high DC component and results of tests have shown that in the condition of a real earth fault these types of RCDs may react unexpectedly positive. This gives a new perspective and a new evaluation of the possible sensitivity and behavior of RCDs under the earth fault/residual current having a high DC component, in particular with regard to the state of the art contained in international standards. The research results and conclusions contained in this paper are very important for the safety of people. The results of the analyses and tests obtained by the authors are also a guideline for manufacturers/constructors of RCDs dedicated to circuits in which a high DC component may appear in the earth fault current.The rest of the paper is organized as follows. is of enough high value has to flow through relay RL  and properties of the iron core of the current transformer CT. Therefore, it is evident that for RCDs containing an iron core transformer , the main problem is the transformation of the unidirectional currents. To increase the ability of RCDs to detect such currents or ensure the delay in their tripping, secondary circuit of the RCD may include additional matching electronic elements (MEE).An RCD is a protective device, the use of which is recommended for protection against electric shock, and in some installations its use is even mandatory. The mandatory use of RCDs, especially those of a rated residual operating current equal to 30 mA, is specified in standards, e.g., in ,35. Howerelay RL . The valFrom the point of view of the ability to detect various shapes of the earth fault/residual currents, RCDs are divided into four groups , as As shown in (1)TEST 1: verification of the tripping level of RCDs for slowly rising AC sinusoidal waveform in presence of a smooth DC component;(2)TEST 2: verification of the tripping level of RCDs for slowly rising pulsating  component in presence of a smooth DC component;(3)TEST 3: verification of the tripping level of RCDs in the case of the suddenly applied pure direct current of the predetermined value. This type of test reflects a real accident of the electric shock and the most probable behavior of RCDs. If there is an earth fault (damage to insulation of the current-using equipment), the current suddenly starts flowing towards the earth. It is similar when a person touches an enclosure of the faulty current-using equipment—the body current appears suddenly.Taking the aforementioned into account, AC-type, A-type, and F-type RCDs are interesting to verify their real behavior under DC waveforms (wider than normative). Therefore, the following tests of the RCDs have been performed:The aim of the above-described tests is to find out the real ability of A-type, F-type, and AC-type RCDs for detection of earth fault currents containing a high-value DC component as well as the RCD’s response to the pure direct current which is suddenly applied. Determination and evaluation of the real behavior of such types of RCDs would enable their broader use instead of the most expensive B-type RCDs.DC, the generator GDC and rheostat RDC are used for setting the proper value of the DC component . When predetermined DC component IDC is forced, then the switch S1 is closed and the generator G1 forces the sinusoidal waveform I1, whose value is increased until the RCD trips. This way of forcing the waveforms enables mutual superimposition of the pure DC component and AC sinusoidal component. In this test, the rms value of the AC sinusoidal component, which initiates tripping of the RCD, is measured.The testing of RCDs has been performed with the use of the laboratory setup of the structure presented in 1, generator G2 is used, and a pulsating direct current  is forced. In this test, the rms value of the pulsating current is measured.In the case of TEST 2, a similar principle applies. The difference is that instead of generator GDC is used. After the circuit parameters are set to force a prospective smooth DC current of one of the following values: 15, 20, 30, 60, 90, 150, 300, or 600 mA, the switch SDC is closed, which means there is a sudden DC current flow (it reflects a sudden contact of person with enclosure under dangerous voltage). During this test, the tripping/no tripping of the RCD is recorded.TEST 3 is performed on a different principle. Here, only the generator GI∆n = 30 mA; these RCDs are marked RCD30_1, RCD30_2, and RCD30_3;three A-type RCDs of a rated residual operating current I∆n = 30 mA; these RCDs are marked RCD30_4, RCD30_5;two F-type RCDs of a rated residual operating current I∆n = 100 mA; these RCDs are marked RCD100_1, RCD100_2;two A-type RCDs of a rated residual operating current I∆n = 300 mA; these RCDs are marked RCD300_1, RCD300_2;two A-type RCDs of a rated residual operating current I∆n = 30 mA; these RCDs are marked RCD30_AC1, RCD30_AC2.two AC-type RCDs of a rated residual operating current For the testing under composite currents containing a smooth DC component, the following RCDs have been specified:I∆n, i.e., for a 30 mA RCD it is 15–30 mA . For considerations conducted in the paper this range is assumed as a proper one and a reference.Results of TEST 1 are presented in I∆n = 30 mA (upper assumed limit). The most favorable behavior refers to RCD30_2 for which the tripping threshold is within the normative AC range when the DC component is equal up to 60 mA. This RCD has the best behavior for the 150 mA DC component as well.The testing of 30 mA A-type RCDs under a composite current having a sinusoidal AC and a smooth DC has shown a that thI∆n = 30 mA even when the DC component is five times higher, i.e., 150 mA.F-type 30 mA RCDs b have beI∆n. This behavior of the RCDs is very favorable. The comparison of the results for 30 mA vs. 100 mA, and vs. 300 mA RCDs shows that the ratio of DC component/rated residual operating current I∆n is important. The RCDs with a greater value of I∆n will be relatively more resistant to a DC component of a given value.The results obtained for 100 mA RCDs  and 300 The analysis of the results obtained during TEST 2 (the A-type RCDs and AC-type RCDs have been tested) allows us to present conclusions that the composition of the pulsating  component and smooth DC component may give unexpected behavior of RCDs—(1)the half-wave component is positive (+) and the smooth DC component is positive (+) as well;(2)the half-wave component is positive (+) but the smooth DC component is negative (−).If the polarities of the half-wave component and the smooth DC component are opposite, the positive behavior of RCDs may be met. Paradoxically, the smooth DC component may improve sensitivity of the RCD then. It is clearly seen in In case (1), the tripping threshold rises when the smooth DC component is being increased. The opposite phenomenon is recorded for the aforementioned case (2)—the higher the DC component, the lower the tripping threshold is. This is because increasing the smooth DC component to some extent makes the resultant/composite waveform bidirectional (see example waveforms in I∆n = 30 mA (RCD30_AC2) had a highest tripping current of as much as 2.3 A. This is unacceptable from the point of view of protection against electric shock and fire.As aforementioned in To explain the behavior of RCDs in the presence of the DC component as well as to recommend the parameters of these protective devices to improve their sensitivity against this component, the RCD model, according to the diagram presented in is has a high enough value to trigger the relay RL. However, this secondary current strictly depends on the secondary voltage es induced in the secondary winding of the CT. Therefore, the production of the voltage es can be assumed as a crucial factor to ensure the operation of the RCD.The RCD operates if the secondary current es of the current transformer depends on the following parameters (for symbols see Induced secondary voltage Φ in the iron core of the current transformer is a function of the magnetic induction B and cross-section sFe of the core of the current transformer:Magnetic flux H is the magnetic field strength in the iron core.The flow law has the form:B) and magnetic field strength (H) strictly depends on the type of iron core of the CT. Properties of the commonly used magnetic cores are presented in The relation between magnetic induction ;only sinusoidal of value I∆n + smooth DC component of value I∆n—(step 2);sinusoidal of value I∆n + smooth DC component of value 2I∆n—(step 3).sinusoidal of value es decreases if the DC component increases. This makes deterioration of the sensitivity of RCDs, as shown in For both types of iron cores of the CT ;only pulsating DC  of value I∆n-half + smooth DC component of value I∆n—(step 2);pulsating DC  of value I∆n-half + smooth DC component of value 2I∆n—(step 3).pulsating DC  of value The situation changes when the induced voltage rrent ip :only pulWhile in the case of the A-type core (flat hysteresis loop), the induced voltage appears even when the DC component is relatively high , step 2,Brem, so that, with a high share of the DC component, the highest change in the magnetic induction can be achieved—compare ∆BDC in Thus, a key element in detecting waveforms with a DC component is the magnetization characteristics of the iron core of the CT. One should strive for the production and use of cores with the lowest possible value of remanence induction es is induced in the secondary winding of the CT , only a pure direct current is forced. After adjusting parameters of the testing circuit (generator GSelected results (the most interesting because they are varied) for four A-type RCDs are depicted in The results obtained for RCD30_6 a inform If one assesses the results obtained for F-type RCDs b. The reAccording to the standard , F-type es voltage in is initiates tripping of the relay RL—hence, also the RCD. Analysis of the oscillograms for various values of predetermined residual direct current enables to conclude that this current has a form of a rectangular half-wave and its flow time is very short (up to 14 ms). Moreover, this time depends to a small extent on the value of the current. This is the effect of a very high rise rate of the wave just after the circuit is switched on.For A-type RCDs with tripping results presented in es, described by (1), than the sinusoidal waveform. This voltage es is the function of the residual (primary) current rise rate di∆/dt (dip/dt). Such a property may give an unexpectedly positive reaction to a DC component that is suddenly applied.If comparing a sinusoidal waveform vs. a rectangular waveform , it is eI∆n is probable, although they are not normatively designed to detect such a waveform.Thus, as the investigation has shown, the detection of suddenly applied direct current by no-delayed A-type RCDs is relatively easy and their tripping for direct current of value equal to even The respective oscillograms recorded for F-type RCD30_5  confirmeRCDs are widely used in low-voltage electrical installations. Undoubtedly, they contribute to the improvement of electrical safety, but their selection must be considered carefully. The main issue determining the correctness of the earth fault/residual current detection in circuits with power electronic converters is the selection of RCDs due to their ability to react to specific waveform shapes of the current. In PV installations and in EV charging installations, one should take into account the possibility of the appearance of a DC component in the residual current.I∆n = 100 or 300 mA) may operate, as it has been experimentally verified, within the range (0.5–1.0)I∆n, even if a DC component is many times higher than the normative value (6 mA or 10 mA) specified in current international standards. Another important factor is the type of iron core of the current transformer installed inside the RCD. It is highly recommended that RCD manufacturers select magnetic cores with a particularly low remanence magnetic induction. Considering that in modern installations one can expect a large share of the DC component in the earth fault current, such a core  will allow the detection of a current with a very high DC component.As shown by the results of above-presented laboratory tests, A-type and F-type RCDs are able to detect slowly rising residual current with a very high content of the DC component. Many of the tested RCDs met the requirements of the relevant standards to a large excess—e.g., for A-type RCDs the max normative value of the smooth DC component is equal to only 6 mA  but some of them tripped properly for the DC component up to a maximum forced value of 150 mA. Therefore, it seems reasonable for RCDs’ manufacturers to include, along with their products, the information about the real ability of RCDs to detect residual currents with a DC component. It is especially important for proper use of RCDs, because devices with higher rated residual operating currents , which have very good properties for a slowly rising residual current containing a DC component. Therefore, it is strongly postulated that two types of F-type RCDs should be introduced into the standardization: the 1st type—a no-delayed RCD; the 2nd type—a short-delayed RCD (as it is now). It is expected then that F-type RCDs operating without any delay could detect the suddenly applied direct current very well, which is very important for the effective protection against electric shock in installations containing direct current sources. This is also an economic advantage as in some cases, F-type RCDs may be installed instead of B-type.


Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP3R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP3, Ca2+ and ATP, revealing conformational changes that couple ligand-binding to channel opening. Using a deep-learning approach and 3D variability analysis we extracted molecular motions of the key protein domains from cryo-EM density data. We find that IP3 binding relies upon intrinsic flexibility of the ARM2 domain in the tetrameric channel. Our results highlight a key role of dynamic side chains in regulating gating behavior of IP3R channels. This work represents a stepping-stone to developing mechanistic understanding of conformational pathways underlying ligand-binding, activation and regulation of the channel.Inositol-1,4,5-trisphosphate receptors (IP 3R1 which provide atomic details of IP3, Ca2+ and ATP binding. Molecular motions of key domains and sidechains were found to regulate ligand binding and gating, which are validated by functional assays.Here authors report cryo-EM structures of IP IP3Rs play fundamental roles in the physiology of a diverse array of organisms as they mediate the release of Ca2+ from ER Ca2+ stores in response to diverse physiological stimuli. Decades of studies have revealed the central role that IP3Rs have in generating complex intracellular Ca2+ signals, which are responsible for many complex physiological processes ranging from gene transcription and secretion to the more enigmatic brain activities such as learning and memory. Dysfunction of IP3Rs leads to aberrant Ca2+ signaling that is associated with a multitude of human diseases such as Alzheimer’s disease, hereditary ataxias, cardiac hypertrophy, heart failure, Parkinson’s and Huntington’s diseases, atherosclerosis, hypertension and some migraines3.Inositol-1,4,5-trisphosphate receptors  is predominantly expressed in the central nervous system, especially in Purkinje cells of the cerebellum. All IP3R channels are tetrameric assemblies of monomers of either identical or different subtypes, resulting in channel complexes with a wide range of functional properties. Each IP3R subunit comprises the pore-forming transmembrane domains (TMDs) and cytoplasmic (CY) domains containing binding sites for ligands and multiple modulators of the channel. These nonselective Ca2+ channels are tightly linked to essential phospholipase C-signaling pathways that mediate generation of IP3 and control a wide range of Ca2+-dependent cellular processes in a highly coordinated manner. Complex cross talk occurring between these pathways and IP3R channels leads to precise regulation of intracellular Ca2+ levels. However, an in-depth understanding of the comprehensive mechanism governing control of intracellular Ca2+ dynamics is still lacking.In mammals, there are three subtypes of IP3Rs work as signaling hubs through which diverse cellular inputs like IP3, Ca2+, ATP, thiol modifications, phosphorylation and interacting proteins are processed and then integrated to result in cytosolic Ca2+ signals with precise temporal and spatial characteristics. IP3 and Ca2+ are the primary agonists of IP3R channels and both are required for channel opening. Moreover, to fulfill their many physiological roles in vivo, IP3Rs associate with an array of regulatory molecules ranging from ions and small chemical compounds to proteins4. Unique interactions with these intracellular messengers contribute to the specificity, duration and shape of Ca2+ signals generated by IP3Rs and the channel’s capacity to integrate signals from different pathways. A single IP3 binding site exists at the N-terminus on each IP3R subunit and functional studies suggest that binding of IP3 to each monomer is required for channel opening5. Important, IP3 has little effect in the absence of its co-agonist Ca2+. Multiple Ca2+ binding sites have also been predicted7. IP3R channel opening is modulated with a biphasic dependence on cytosolic Ca2+ concentrations, suggesting that the channel has distinct types of Ca2+ binding sites for activation and inhibition. Electrophysiological studies showed that the IP3-evoked activity of IP3R is enhanced by modest increases in cytosolic Ca2+ concentration in the sub-μM range (<1 μM), while higher micromolar concentrations of Ca2+ are inhibitory to IP3 responses9. ATP is known to enhance the IP3-induced Ca2+ release, by specific binding to IP3R15.IP3Rs, the detailed molecular and structural mechanisms underlying the complex interplay between ligands, allosteric modulators and channel gating remains unresolved. With the resolution revolution in single particle cryogenic electron microscopy (cryo-EM), there have been remarkable advances in structural characterization of IP3R21. These studies reported that the IP3R structure undergoes conformational changes upon ligand binding, suggesting structural flexibility that allows the channel to switch from a closed state, capable of interacting with its ligands such as IP3 and Ca2+, to an open state, capable of transferring Ca2+ ions across the ER membrane. However, all of these 3D cryo-EM structures represent defined static conformations of the channel, and the mechanistic insights are derived based on interpolations between discrete structures, each of them likely a mixture of states from a dynamic conformational ensemble. Therefore, mechanistically it remains poorly understood how ligands engage the ligand-binding domains and how multiple signals are coordinated and processed within the IP3R channel. This implies the necessity to consider the dynamic conformational landscape of the channel protein in determining the molecular mechanisms underlying its function.Despite more than three decades since the discovery of IP3R channel trapped in physiologically relevant states using key ligands that target channel gating. Here, we report cryo-EM structures of IP3R1 bound to IP3, ATP, Ca2+ and IP3R1 bound to Ca2+ alone at overall resolutions of 3.50 Å and 3.26 Å, respectively. In addition, we used a deep neural network approach22 and 3D variability analysis23 to extract functionally relevant conformational motions of the IP3R1 domains directly from 2D cryo-EM images. The ratcheting mechanism for IP3-binding was built via deep-learning approach and corroborated by experimental single-particle cryo-EM analysis. This combined approach allowed for a mechanistic understanding of how conformational motions of the ARM2 domain are coupled with structural changes in the IP3-binding pocket in the context of the tetrameric IP3R channel. From these studies, we correlate protein structural changes that connect the ligand-binding regulatory sites to the channel gate via the IP3R conformational landscape. Our structural findings and hypothesis were validated through mutagenesis and electrophysiology . Our study provides a structural framework for understanding the allosteric mechanisms underlying ligand-mediated IP3R activation and regulation.In this work, we determined the structures of the IP3R channel, we isolated native, Ca2+ permeable IP3R1 channels and used cryo-EM single particle analysis to solve the channel structures in distinct ligand-bound conformations. As the membrane environment is relevant for the functionality of channels, we studied IP3R1 reconstituted into lipid nanodisc20. IP3 and Ca2+ are imperative ligands to trigger the channel opening and ATP increases the open probability of IP3R1 by synergizing with the activating effect of these two primarily ligands15. Purified IP3R1 binds IP3 in a stoichiometric manner (one IP3 per IP3R1 monomer) with an affinity of 2–100 nM while functional IP3 affinities vary from the nanomolar to low micromolar range with mean values for maximum effect around 10 μM, dependent on the technique used26. Ca2+ regulates IP3R1 gating with positive and negative feedback acting in a bell-shaped manner with maximum channel open probability at low μM free Ca2+, while high μM Ca2+ inhibits the channel28.To elucidate allosteric coupling mechanisms underlying ligand-binding and gating kinetics of the IP3R1 channels were determined in the presence of saturating IP3 concentration (10 μM), ~2 μM Ca2+ and 1 mM ATP , and using 20 μM Ca2+, as the sole ligand , at estimated overall resolutions 3.50 and 3.26 Å, respectively  from the same subunit  had the highest resolution at 2.4-3 Å, while the cytoplasmic armadillo domain 2 ARM2; L1103-G1567) had the lowest resolution, suggesting that this domain possesses structural flexibility 22. When taken together, the set of raw 2D particles, each in a known orientation, does not just define a single structure, but a complete set of related structures embodying the motion and composition of the macromolecule in solution. Using a set of Gaussian spheres in space as a proxy, deep-learning makes it possible to relate these 2D particles most similar to each of these 3D states. We can then reconstruct 3D maps using particle subsets, representing actual points along motion pathways, directly from the particle data without any structural interpolations. That is, each structure along a motion pathway was reconstructed purely from experimental data and the computational model is used only to select which data to include.To gain insights into the conformational landscape of IP3R1 protein exhibits substantial dynamic motions involving both the cytoplasmic and pore domains  exhibits pronounced apical motions, and the linker domain  and intervening lateral domain  that form a nexus at the cytosolic-membrane interface, dilate. Noteworthy, these conformational changes are propagated to the HD-ARM3 interaction interface also undergoing structural rearrangements. In the TMD region the global movements are quite notable exhibiting a rotation of the TM assembly relative to the cytosolic LNK/ILD nexus (Supplementary Movie 3-binding pocket properties (discussed below). Overall, the observed conformational motions are in good agreement with our earlier proposed model for long-range allosteric conformational coupling between ligand-binding and activation of the channel gate17.As expected, the IP3-binding pocket is formed at a cleft between the β-trefoil domain, βTF2 (W226-V435) and the ARM1 domain  form a triangular architecture at the apex of the CY assembly17. In the CIA-IP3R1 structure, we observe a strong density bridging the βTF2 and ARM1 domains at the site where IP3 is expected to bind   becomes unraveled. Additionally, the two helices in the ARM1 domain , which residues directly contribute to the coordination of IP3, move ~20° when engaged with IP3.Superimposition of a single subunit from the apo-, CaSD) Fig. . In contket Fig. . In part3R1, CIA-IP3R1 and apo-IP3R1 structures  and in the presence of 20 μM Ca2+ that produces channel inhibition (Ca-IP3R1). A comparison of the cryo-EM density maps of Ca-IP3R1 and apo-IP3R1 revealed strong non-protein densities in Ca-IP3R1 structure at locations consistent with putative Ca2+-binding sites predicted in earlier mutagenesis and functional studies29. The ascribed Ca2+ densities were validated as described in Methods, establishing the locations of five occupied Ca2+- binding sites in each IP3R1 subunit , which we assigned to a bound Ca2+ ion, and this Ca2+-binding site is referred to here as Ca-IIIS  Ca2+-binding proteins. Moreover, alignment of 3D structures of the Ca-IIIS site in IP3R1, IP3R3 and RyR channel confirms a structural conservation of this Ca2+ binding site across both families of Ca2+ release channels as demonstrated in our previous studies39. In our companion study, mutations E1978 or E2042 in the Ca-IIIS site markedly affected single channel activity such that altering the negative charge on either aspartate residue, shifted the [Ca2+] dependence for activation to the left, consistent with an important role of this site in Ca2+ activation of IP3R1 activity24. A conserved E2101 (E4033 in RyR1 and E2005 in IP3R3) was previously proposed to be responsible for Ca2+ sensitivity for activation of IP3R1 and RyR1 channels41, however, this residue does not directly contribute to coordination of a Ca2+ ion in the binding pocket, and appears to exert an effect via allosteric interactions with surrounding residues.Another strong non-protein density is observed in the putative Ca2+ binding sites, Ca-IILBD and Ca-IIIS, were occupied with Ca2+ ions. The structures of the two cytosolic sites are nearly identical to those identified in Ca-IP3R1 except for subtle rearrangements in the loop regions  and inhibitory (20 μM) Ca2+ concentrations, it would thus appear that Ca2+ should bind with higher affinity to these two Ca2+-binding sites compared to the Ca-ILBD site, which is occupied only at an inhibitory Ca2+ concentration. We found that in the CIA-IP3R1 structure, the D426 side chain moved away from the Ca-ILBD binding pocket and the positively charged R170 shifted toward the location of bound Ca2+. This raises the possibility that the conformational differences in Ca-ILBD site between Ca-IP3R1 and CIA-IP3R1 structures might reflect changes in Ca2+-binding affinities. Consistent with our data, mutagenesis of D426 in the Ca-ILBD site abolished Ca2+ binding42. Hence, one could speculate that the Ca-ILBD site is a possible candidate for an inhibitory Ca2+-binding site, and two other cytosolic Ca2+-binding sites (Ca-IILBD and Ca-IIIS) are activating. As it might have been anticipated, binding of IP3 “tunes” Ca2+-inhibition of the IP3R channel37.By contrast, in the CIA structure, only two cytosolic Ca3R1 and CIA-IP3R1 structures, two clear densities, which were interpreted as the bound Ca2+ ion, are found in the luminal vestibule leading to the selectivity filter (SF) and the central cavity above the SF. We assigned these densities to bound Ca2+ ions (see Methods). One of these Ca2+-binding sites, named Ca-IVL, is shaped by the side chains of residues D2551, G2550, R2544, H2541, S2545 contributed across the interface between two neighboring subunits  is located in the central cavity, and Ca2+ ion bound at this site is ~2.9 Å away from the amide group in the side chain of N2583 suggesting that the bound Ca2+ ion is stabilized in a fully hydrated state. Both identified luminal sites, Ca-IVL and Ca-VL, hint at a high affinity binding site as they are occupied at relatively low Ca2+ concentration. Moreover, the N2583 residue in rodent IP3R1 corresponds to N2543 in humans, and it has been found that the N2543I mutation is closely associated with Gillespie syndrome43.In both the Ca-IP3R channels45. Previous sequence analysis identified three glycine-rich regions (GXGXXG) in IP3R, reminiscent of the Walker-A motif found in many ATP-binding proteins47, and were presumed to account for the ATP-sensitivity of IP3Rs based on functional studies15. Although binding of ATP to these motifs has been shown with expressed polypeptides, site-directed mutagenesis and single channel measurements revealed that the Walker-A motifs are not required for ATP modulation of IP3R111 or IP3R312. At present, there is a substantial lack of structural knowledge about how ATP regulates the channel gating.ATP is known to be a crucial extracellular signaling molecule that increases the open probability of the IP3R1 channel bound to ATP in the presence of its primary activating ligands (CIA-IP3R1). The CIA-IP3R1 structure reveals a previously unseen ATP-binding site located at the interface between the ILD and LNK domains, where clear density is seen   Fig. .Fig. 4St2+-bound structures with the CIA-structure reveal that binding ATP induces the most significant change in the side chain conformation of W2639. In the Ca-IP3R1 and apo-IP3R1 structures, we found two clear densities where alternative W2639 rotamers can be fitted  and widened to ~9.4 Å and 10.7 Å in apo- and CIA-structures, respectively  state , which SF is narrower (~8.5 Å) than that in apo-IP3R117. These structural differences might reflect the separate contexts in which these structures were determined, CIA-bound in a nanodisc and the other in the detergent. Noteworthy, the narrow diameter of the SF in apo-IP3R1 likely does not reflect any physiologically relevant conformation as the luminal vestibule of the channel pore is normally exposed to the ER lumen containing calcium in the low millimolar range54. While the Ca2+ binding to the luminal sites may have a regulatory function56, it remains to be established whether Ca2+ store depletion could lead to dissociation of Ca2+ from the luminal vestibule.Overall, the conformation of the ion conduction pore in the CIA-IPate Fig. . In addi3R1 (hR1). The F2586K mutation replaces a large, hydrophobic Phe with a positively charged Lys, while the I2590N and I2550T mutations are associated with human disease58. The mutations were stably overexpressed in cells engineered via CRISPR/Cas9 to lack all three endogenous IP3Rs (HEK-3KO)5 . The presence of neutral T or hydrophilic N at the I2590 position is clearly not favorable for inter-subunit hydrophobic side-chain packing that will perturb the pore stability thereby affecting the channel open probability. Thus, our results confirm the critical nature of the F2586 and I2590 residues in IP3R1 channel function.To further investigate functionality of the amino acids at the constriction site, mutants of residues F2586 (F2586K) and I2590 (I2590N and I2550T) were generated in human IPO)5 Fig. . All thrase Fig.  when comons Fig. . Replaci2+- and Ca2+/IP3/ATP-bound IP3R1 presented in this study highlight the correlation between the protein dynamics and its propensity for accepting multiple cellular signals that regulate the channel activities, such as ligand-binding, gating and regulation. Considering the remote distances between the cytoplasmic ligand-binding sites and ion conduction pathway in the channel, the intrinsically flexible 3D architecture of the IP3R provides the basic premise behind the allosteric regulation of the channel activity that involves the transfer of ligand-evoked signals from the distinct ligand-binding domains to the ion conducting channel gate  held in place by its contacts with βTF1 and ARM1 domains from the neighboring subunit  state, which transitions to an α-helix in the presence of IP3 and Ca2+. Strikingly, unlike IP3R3, the ligand-bound structures of IP3R1 reconstituted into lipid nanodisc solved in this study, appear to favor the π-helix upon the channel activation. Together, these data suggest that the lipid environment plays an essential role in formation and maintenance of π-helix structure.The presence of the π-gating hinge in IP3R1 protein. Studies suggest that the three IP3R isoforms exhibit different sensitivity to ATP with IP3R1 being enhanced by micromolar ATP and IP3R3 being augmented by millimolar ATP, whereas IP3R2 is more sensitive to ATP but only at sub-micromolar IP366. Our analysis revealed a side chain conformational preference for W2639 that contributes to ATP binding. This observation supports the hypothesis that ATP-binding specificity of IP3Rs might be mediated by the recognition of specific rotameric states of key residues in the ATP-binding pocket. Additionally, the presence of conserved lysine residues responsible for the coordination of ATP phosphates in IP3R1 likely contribute to isoform specific ATP binding affinity. Decoding the structural basis of exquisite isoform specificity with respect to a modulatory role of ATP in the IP3R family will certainly be critical in future studies.Furthermore, the results of the present study are supportive of the importance of side chain dynamics in ATP binding by IP3R1 to binding of IP3, based on the conformational selection of the ligand-binding pocket, which may adapt different conformations in its unbound state for which IP3 binds selectively to one of these conformations. We propose that IP3/Ca2+-evoked gating involves ‘a conformational wave’ that unequivocally propagates via the cytoplasmic domains towards the pore-forming TMDs  and 0.1% (w/v) l-α-phosphatidylcholine  for 2 h at 4 °C. Non-solubilized material were cleared by ultracentrifugation  and the supernatant was applied to an immunoaffinity matrix pre-coupled with monoclonal antibodies  in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.02 mM LMNG. Reconstitution of IP3Rs into lipid-nanodiscs occurred “on-column” as we described in ref. 3R1-bound immunoaffinity column matrix followed by the addition of 0.6 mg/ml MSP1E3D1 (Cube Biotech). Detergent removal was achieved by the addition of 0.25 mg/ml activated Bio-Beads-SM2 (BioRad) for 16 h at 4 °C. IP3R1 was eluted with 500 μM of T433 peptide and concentrated using a 100 kDa Amicon centricon (Millipore).Purification of neuronal IP3R1 (2 mg/ml) was vitrified after 30 min in incubation with 20 μM free Ca2+ to obtain the structure of IP3R1 bound with Ca2+ (Ca-IP3R1) or with 2 μM free Ca2+, 10uM of IP3 and 1 mM ATP to obtain Ca/IP3/ATP-bound IP3R1 (CIA-IP3R1). Vitrification of the IP3R1 samples was performed using a Vitrobot Mark IV  as described earlier20. Blotting was done with filter paper Whatman 542 containing 8–14 μg Ca/g paper. The final free Ca2+ concentrations were determined using MaxChelator (http:// maxchelator.stanford.edu/oprog.htm). CryoEM data acquisition was performed with a Titan Krios microscope  operated at 300 keV, equipped with a BioQuantum energy filter  with zero-loss energy selection slit set to 20 eV. All data sets were acquired using the EPU software  at a nominal TEM magnification of 130,000X and recorded on a K2 Summit direct electron detector  operated in super-resolution counting mode with a calibrated physical pixel size of 1.07 Å. The data acquisition parameters  are summarized in Table The nanodisc reconstituted purified IP68. The motion-corrected micrographs were evaluated by ‘e2evalimage.py’ in EMAN269 and were used for all the following image processing. Contrast transfer function (CTF) determination was performed using Gctf70. Particles were picked using the NeuralNet autopicking procedure implemented in EMAN2 and the .box files were imported to RELION371 and were subjected to iterative 2D and 3D classification. Our previously resolved cryo-EM map of apo-IP3R1 (Electron Microscopy Data Bank (EMDB: EMD-23337) was low-pass filtered to 60 Å as a starting reference map. After 3D auto-refinement, two overall 3D reconstructions at 3.50 Å and 3.26 Å resolution (using FSC at 0.14372) were generated for the CIA-IP3R1 and Ca-IP3R1 respectively 20. To improve the map quality of ARM2 domain for all three states , we performed symmetry expansion by running the command “relion_particle_symmetry_expand” with C4 symmetry, followed by multiple rounds of 3D classification with a mask including only ARM2 domain, and the particles from the best featured classes were subjected to local refinement  were combined and a single averaged structure with C4 symmetry was reconstructed from the particles, which was used as the neutral state structure for GMM based heterogeneity analysis. To analyze the movement of each subunit independently, each IP3R1 particle was duplicated four times, each time with orientation assigned to a different asymmetrical unit. During the heterogeneity analysis, the model was constrained such that Gaussian functions corresponding to the ARM2 domain could only translate/rotate together as a rigid body, and only a single subunit of IP3R1 was allowed to deviate from the neutral state position. After training the network model, the first eigen-vector on the conformational space exhibited a clear extended-retracted motion of the ARM2 domain. The three distinct particle subsets all exhibited motion on the same pathway in the ARM2 domain, but to differing degrees, with the CIA data set exhibiting more motion than the other two states.Overall analysis was performed as described in23. For each cryo-EM data set, five modes of variability were calculated. Ten maps for each mode were generated using a 4 Å low-pass filter. In each state, the ARM2 domain displayed the largest motions of similar trajectories, regardless of symmetric and symmetry-breaking variability. To visualize the conformational variability across the channel protein assembly, the maps from the modes representing C4 motion  were examined in UCSF Chimera75 as volume series and recorded as movies  module3R1 and Ca-IP3R1 cryo-EM density maps. Our previously published cryo-EM structure of apo-IP3R1 in nanodisc  was used for rigid body fitting in UCSF Chimera75 followed by an initial round of flexible fitting of the IP3R1 subunit in COOT. Density modification “phenix.resolve_cryo_em” was performed on the half-maps resulting in improved resolvability in the density maps allowing for further model optimization. AlphaFold76 was used to generate optimized models for each of the ten domains within an IP3R1 subunit using the initial IP3R1 model for each domain as a template. The results were then concatenated and tetramerized in Chimera, refined against the density map with “phenix.real_space_refine” with default options and manual optimization in Coot to maximize fit to density, minimize Ramachandran angle outliers and eliminate steric clashes. Ligands, including lipids, calcium, IP3 and ATP, were identified using “pw_ligands.py”, a stand-alone ligand identification tool based on Pathwalking77. Briefly, “pw_ligands.py” identifies all non-protein density, analyzes the geometry of the un-modeled density and masks out ligands and water/ion density from the cryoEM map. At the reported resolutions, identification of non-ion/non-water ligands is robust, though it is difficult to distinguish waters from ions based purely on density profile. All prospective water/ion densities were examined; only clearly distinguishable densities with obvious coordinating chemistry were considered as ions. The ligands were fit to the masked densities and refined along with the protein model using “phenix.real_space_refine”. Model validation was carried out using EMRinger and MolProbity in PHENIX. Map-model visualization was performed in Coot and UCSF Chimera. Interfaces described in the paper were identified with PDBePisa78, ChimeraX79 LigPlot+80 and HOLE81. Figures and movie were prepared using UCSF Chimera; ChimeraX and VMD 1.9.482.We utilized a consistent approach for protein model building in both CIA-IP3R1 ). Mutagenesis and all DNA modifications were carried out using Pfu Ultra II Hotstart 2X Master Mix (Agilent). Mutagenesis primers for hIP3R1 F2586K forward (TCTTCATGGTCATCATCATTGTTCTTAACCTGATTAAGGGGGTTATCATTGACACT), hIP3R1 F2586K reverse (AGTGTCAATGATAACCCCCTTAATCAGGTTAAGAACAATGATGATGACCATGAAGA), hIP3R1 I2590N forward (TTGGGGTTATCAATGACACTTTTGCTGACCTACGTAGTGAGAAGCAG), hIP3R1 I2590N reverse (CTGCTTCTCACTACGTAGGTCAGCAAAAGTGTCATTGATAACCCCAA), hIP3R1 I2590T forward (TTGGGGTTATCACTGACACTTTTGCTGACCTACGTAGTGAGAAGCAG), and hIP3R1 I2590T reverse (CTGCTTCTCACTACGTAGGTCAGCAAAAGTGTCAGTGATAACCCCAA), were synthesized by Integrated DNA Technologies (IDT). In addition to encoding the specified mutations, primers also silently introduced a restriction site for verification purposes. The coding regions for all constructs were confirmed by sequencing.A two-step QuikChange mutagenesis protocol was used to introduce amino acid substitutions into cDNA encoding the human IP3R isoforms83 and HEK293 cells modified by CRISPR/Cas9 to only express endogenous hIP3R1 (Endo. hR1) were grown at 37˚C with 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco/Life Technologies). Transfection of HEK-3KO cells to exogenously express desired hIP3R1 WT or mutant constructs stably was performed as previously described83. In brief, five million cells were pelleted, washed once with PBS, and resuspended in either Nucleofector Solution T (Lonza Laboratories) or a homemade transfection reagent . 4–6 µg of DNA was mixed with the resuspended cells and electroporated using the Amaxa cell nucleofector (Lonza Laboratories) program Q-001. Cells were allowed to recover for 48 h before passage into new 10 cm plates containing DMEM media supplemented with 2 mg/ml Geneticin sulfate . Following 7 days of selection, cell colonies were either picked and transferred to new 24-well plates or diluted into 96 well-plates, both of which contained DMEM media supplemented with 2 mg/ml G418. Diluted wells in the 96-well plate were screened after 7 days for the presence of a single colony of cells growing in each well. Those wells that exhibited multiple colonies growing were excluded from further consideration. 10–14 days after transfection, wells—both 96 and 24—that exhibited growth were expanded and those expressing the desired constructs were confirmed by western blotting.HEK-293s were originally obtained from ATCC (CRL-3022). HEK-3KO cells, HEK293 cells engineered through CRISPR/Cas9 for the deletion of the three endogenous IP3R constructs were harvested by centrifugation (200 x g for 5 min), washed once with PBS, and solubilized in membrane-bound extraction lysis buffer containing 10 mM Tris-HCl, 10 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100 (v/v), 0.5% sodium deoxycholate (w/v), and 10% glycerol supplemented with a cocktail of protease inhibitors . Lysates were incubated for a minimum 30 min on ice and cleared by centrifugation  at 4 °C. Protein concentrations in cleared lysates were determined using Dc protein assay kit (Bio-Rad) and 4x SDS gel loading buffer was subsequently added to 5 µg of lysate. Proteins were resolved using 8% SDS-PAGE and subsequently transferred to a nitrocellulose membrane . Membranes were probed with a rabbit polyclonal antibody against the C-terminal 19aa of IP3R1 (custom generated by Antibody Research Corporation) at a 1:1000 dilution, GAPDH  at a 1:75,000 dilution, and the appropriate Dylight™ 800CW secondary antibodies at a 1:10,000 dilution . Membranes were imaged with an Odyssey infrared imaging system and quantified using Image Studio Lite (LICOR Biosciences).HEK-3KO cells and HEK-3KO cells stably expressing IP2+ imaging was performed in intact cells as described previously83. Glass coverslips were plated with HEK-3KO, Endo. hR1, Exo. hR1, or HEK-3KO cells stably expressing IP3R mutant constructs at least 18 h prior to imaging experiments. Subsequently, the glass coverslips were mounted onto a Warner chamber and the cells were loaded with 2 µM Fura-2/AM (Molecular Probes) in Ca2+ Imaging Buffer  at room temperature for 25 min. Following loading, cells were then perfused with Ca2+ IB which provided a basal 340/380 ratio of the [Ca2+] and stimulated with 3 µM, 30 µM, and 100 µM carbachol (CCh) to obtain measurements of agonist-induced Ca2+ release into the cytoplasm.Single cell Ca2+ imaging was performed using an inverted epifluorescence Nikon microscope with a 40x oil immersion objective. Cells were alternately excited at 340 and 380 nm, and emission was monitored at 505 nm. Images were captured every second with an exposure of 15 ms and 4 × 4 binning using a digital camera driven by TILL Photonics software. Image acquisition was performed using TILLvisION software and data was exported to Microsoft Excel where the means were calculated. Statistical analysis of at least n = 3 experiments for each cell line was performed in GraphPad Prism (one-way ANOVA with Tukey’s test).CaFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting Summary