Venetoclax, a small-molecule B-cell lymphoma 2 (BCL-2) inhibitor, selectively eradicates leukemic stem cells (LSCs). While venetoclax has revolutionized the treatment of acute myeloid leukemia (AML), treatment failure and disease relapse are common. Mechanisms underlying venetoclax resistance are surprisingly heterogeneous. Venetoclax resistance encompasses a spectrum of genetic and epigenetic changes, with numerous pathways contributing to the upregulation of additional anti-apoptotic proteins. In this review, we address the mechanisms of venetoclax resistance in the context of signal transduction. We emphasize how aberrant cell signaling impairs apoptosis and predisposes to venetoclax failure.Commonly activated pathways, such as FLT3, PI3K/AKT/mTOR, and RAS, contribute to upregulated anti-apoptotic mediators and are frequently responsible for refractory disease or disease relapse. We highlight novel combination strategies aimed at disabling constitutively active signal transduction to augment response and overcome venetoclax resistance. Acute myeloid leukemia is a heterogeneous hematologic malignancy characterized by a maturation arrest of hematopoietic precursors . BecauseIDH1 or IDH2) is associated with a survival benefit when treated with venetoclax and azacitidine compared to azacitidine alone  [FLT3), rat sarcoma (RAS), or tyrosine-protein phosphatase non-receptor type 11 (PTPN11) are associated with decreased survival and higher rates of relapse following treatment with lower-intensity venetoclax-based strategies [Specific molecular subsets respond favorably to venetoclax augmentation. For example, AML with mutated isocitrate dehydrogenase  or directly activate apoptosis  [Additional BCL-2 family proteins potentiate apoptosis; they contain homologous structures to BAX and BAK, known as BCL-2 homology (BH) motifs. There are four BCL-2 homology motifs: BH1, BH2, BH3, and BH4 . These aD, PUMA) -25. AfteL, BCL-w, BFL-1, and MCL-1. By binding to BAX and BAK, BCL-2 and BCL-XL inhibit apoptosis and block the subsequent formation of the mitochondrial pore-forming subunits [In contrast to the pro-apoptotic mediators, the anti-apoptotic BCL-2 proteins bind BAX and BAK, rendering them unable to drive the intrinsic pathway. The anti-apoptotic proteins include BCL-2, BCL-Xsubunits ,26,27. Isubunits . An overPromoting leukemic cell death by activating apoptosis is an attractive therapeutic strategy. Owing to the overexpression of BCL-2 in LSCs, selective inhibition of BCL-2 leads to the sequestration of BAX and BAK and induces the eradication of quiescent LSCs . This diL [L is a platelet pro-survival protein, and inhibition of BCL-XL by navitoclax resulted in dose-limiting thrombocytopenia [L. Soon afterward, the landmark VIALE-A trial demonstrated the superiority of venetoclax and 7-day azacitidine compared to azacitidine alone, and cemented the use of combination therapy in patients with AML ineligible for intensive induction [Navitoclax was one of several BH3 mimetics investigated shortly after the discovery of BCL-2 inhibition as a therapeutic strategy. Navitoclax selectively targets BCL-2, BCL-w, and BCL-XL . Howeverytopenia . Rates onduction .L. Indeed, the upregulation of these anti-apoptotic proteins contributes directly to venetoclax resistance, and the downregulation of MCL-1 or BCL-XL restores venetoclax sensitivity [There is, however, a critical caveat to the use of venetoclax. Like navitoclax, venetoclax does not inhibit the anti-apoptotic mediator MCL-1; it also does not inhibit BFL-1 or BCL-Xsitivity . The uprsitivity . The redVenetoclax resistance is remarkably heterogeneous. Elevation of anti-apoptotic mediators is a frequent accompaniment of venetoclax-resistant disease. Many paths lead to persistent cell survival following venetoclax exposure, including aberrations in epigenetics, gene amplifications, or BCL-2 family mutations ,32 — butFMS-like tyrosine kinase 3 (FLT3) is among the most commonly mutated and well-understood signaling pathways in AML. FLT3 is a ligand-activated transmembrane tyrosine kinase that feeds into downstream pathways, including PI3K, RAS, and STAT5, promoting cell proliferation and survival . The FLTFLT3 are an internal tandem duplication (FLT3-ITD) of the juxtamembrane domain and tyrosine kinase domain mutations (FLT3-TKD) in the activation loop, although many other FLT3 mutations exist [FLT3-ITD and FLT3-TKD lead to constitutive activation of FLT3 kinase, driving leukemic proliferation and survival. As FLT3mut AML has an increased risk of relapse and shorter overall survival — particularly for FLT3-ITD [The two most commonly observed activating mutations in ns exist . FLT3-ITFLT3-ITD ,37 — theFLT3-ITD but not FLT3-TKD [FLT3-ITD and FLT3-TKD, improved overall survival when combined with an anthracycline and cytarabine [FLT3-ITD, which led to the development of more specific and potent next-generation FLT3 inhibitors, including quizartinib, crenolanib, and gilteritinib [FLT3mut AML [Sorafenib, a multi-kinase inhibitor with activity against FLT3-TKD , signifiFLT3-TKD . Subsequtarabine . Midostaeritinib . The mos3mut AML .FLT3mut AML are being investigated in the context of venetoclax-based induction without a FLT3 inhibitor. A recent subgroup analysis of the VIALE-A trial analyzed 42 patients with FLT3 mutations treated with venetoclax and azacitidine. The median overall survival of FLT3mut AML was shorter at 12.5 months compared to 14.7 months for wild-type FLT3 [FLT3-mediated venetoclax resistance. Additionally, new FLT3 mutations emerge following venetoclax treatment in patients without a FLT3 mutation at diagnosis, suggesting that FLT3 may be responsible for resistance by an adaptive mechanism [FLT3mut AML treated with venetoclax-based regimens.Outcomes of ype FLT3 , suggestechanism . These oFLT3 mutants occurs through several mechanisms in addition to the emergence of new FLT3 mutations. As venetoclax selectively inhibits BCL-2, upregulation of anti-apoptotic proteins other than BCL-2 is a primary cause of resistance. Indeed, constitutive FLT3 signaling upregulates the anti-apoptotic mediator MCL-1 [FLT3-ITD AML to therapy [Venetoclax resistance in or MCL-1 ; siRNA-m therapy .FLT3-mediated venetoclax resistance are similar to MCL-1 upregulation. The activity of another anti-apoptotic mediator, BCL-XL, is maintained in FLT3-ITD, resulting in leukemic stem cell persistence and survival. Targeting FLT3-ITD results in BCL-XL reduction [L appears to be an indirect consequence of downstream activation of PI3K, RAS, and STAT5 [Additional avenues of eduction . Togethend STAT5 ,46,47. TFLT3 mutations upregulate alternative anti-apoptotic mediators led to the design of several studies investigating the efficacy of venetoclax and FLT3 inhibition. Venetoclax with sorafenib, midostaurin, or gilteritinib led to synergistic apoptosis in FLT3mut AML through the downregulation of MCL-1 [The observation that of MCL-1 ,49. ThesFLT3mut AML, with no relapses observed during the follow-up period [Early phase trials of gilteritinib with 7-day azacitidine and venetoclax demonstrated an overall response rate of 100% in newly diagnosed p period . In the p period . ProlongFLT3 inhibitor, hypomethylating agent backbone, and duration of drug exposure to balance efficacy and toxicity. Nevertheless, preliminary results in the front-line setting are impressive. In relapsed or refractory AML, the combination approach is more problematic. The emergence of new FLT3 mutations frequently hampers outcomes through clonal evolution or constitutive activation of downstream parallel signaling pathways, such as PI3K or RAS.A similar study of sorafenib, midostaurin, or gilteritinib with 10-day decitabine and venetoclax yielded a composite complete remission rate of 92% in newly diagnosed AML, and 56% were negative for measurable residual disease (MRD). The overall survival was not reached at a median follow-up time of 14.5 months . The comCell signaling through the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) is one of the most central intracellular pathways in cancer. Mutations in receptor tyrosine kinases — such as FLT3 — or GTPases frequently lead to upregulation of the PI3K pathway and are associated with inferior overall survival in AML . PI3K isL, allowing these anti-apoptotic proteins to block the progression of mitochondrial outer membrane permeability and subsequent cell death [AKT-mediated cell survival occurs directly through interactions with the apoptosis-regulating proteins. AKT, a protein kinase, phosphorylates numerous substrates, including the apoptotic sensitizer BAD. Phosphorylated BAD frees BCL-2 and BCL-Xll death ,53. TherPI3K inhibition is unimpressive as monotherapy, and trials involving this class of agents have been limited by excessive toxicity. Gedatolisib, a dual PI3K/mTOR inhibitor, was evaluated in relapsed or refractory AML, and no objective response or clinical benefit was observed . ConsequInhibitors of mTOR were evaluated in AML. A caveat of mTOR inhibition is the resultant increased phosphorylation of AKT . To circThere is a relative paucity of data on outcomes of patients with AML harboring a PI3K pathway mutation treated with venetoclax-based strategies. The clinical impact of aberrations of PI3K is inferred from upstream effectors, such as FLT3 or KIT. These upstream kinase-activating mutations are associated with reduced survival when treated with venetoclax-based strategies, suggesting additive mechanisms of primary or adaptive resistance ,50.L, a third anti-apoptotic protein, MCL-1, is also upregulated by the PI3K pathway [Venetoclax resistance in AML with constitutively activated PI3K directly results from the upregulation of multiple anti-apoptotic mediators. In addition to AKT-dependent increases in BCL-2 and BCL-X pathway . TherefoTo combat multiple resistance mechanisms mediated through PI3K, apitolisib, a dual PI3K/mTOR inhibitor, was studied in AML cells undergoing treatment with concurrent BCL-2 inhibition. Venetoclax and apitolisib induced rapid AML cell apoptosis with MCL-1 downregulation and spared normal hematopoietic cells . The comFLT3mut AML undergoing treatment with FLT3 inhibition. FLT3-ITD AML cell lines resistant to sorafenib are enriched in the PI3K/mTOR pathway [Activation of the PI3K pathway also plays a unique role in  pathway . GedatolThe RAS proteins exhibit substantial crosstalk with the PI3K pathway and stimulate cell proliferation and survival through the cascade of RAF, MEK, and ERK, which constitute the RAS pathway . RAS patSimilar to PI3K, constitutively activated RAS signaling frequently occurs in the context of upstream FLT3 activity, with downstream RAS activation contributing to FLT3 inhibitor resistance ,64. In aRASmut AML have a significantly longer overall survival than RAS wild-type, perhaps partly due to a higher likelihood of association with favorable-risk cytogenetics [RASmut AML demonstrated significantly longer overall survival when treated with intensive cytarabine-containing induction regimens. Intensive chemotherapy with venetoclax did not significantly improve response rates in RASmut AML compared to intensive chemotherapy without venetoclax. In contrast, response rates, but not survival, were significantly lower in patients harboring a RAS mutation treated with a hypomethylating agent and venetoclax [In AML, RAS mutations confer a robust proliferative advantage and skew differentiation toward the myelomonocytic line ,67. Patigenetics . In partnetoclax . These fFLT3-ITD responded [FLT3-ITD AML suggests that the activation of parallel signaling pathways contributes to disease refractoriness, warranting the investigation of combination strategies. Indeed, MEK inhibition with trametinib enhanced the response to FLT3 inhibitors, improving survival in mouse models while sparing normal CD34+ cells [Clinical trials evaluated multiple inhibitors of the RAS pathway. Selumetinib, a MEK inhibitor, was associated with modest single-agent activity in relapsed or refractory AML, but no patient with esponded . Resista4+ cells . TherefoAML with myelomonocytic differentiation is implicated in venetoclax resistance, raising the hypothesis that RAS activation may be a driving factor . SimilarFurther studies in AML cell lines evaluated the combination of venetoclax and cobimetinib. Using this strategy, MCL-1 was downregulated following MEK inhibition, which sensitized cells to venetoclax and culminated in reduced leukemia cell burden in mouse models . Next, MRAS mutations and MAPK activation were discovered to stabilize MCL-1, resulting in persistent cell survival. To confirm the role of MCL-1 in RAS-mediated resistance to venetoclax, co-treatment with venetoclax and an MCL-1 inhibitor, AZD-5991, significantly reduced AML blasts in mice [RASmut AML.Subsequently,  in mice . These fRASmut AML. Sixteen patients received a median of four lines of prior therapy, and 81% received a prior hypomethylating agent with venetoclax. While 67% of patients that did not receive venetoclax-based treatment responded, only 15% of patients with previous venetoclax exposure had a response [Based on this data, a clinical trial evaluated trametinib with venetoclax and 7-day azacitidine in relapsed or refractory Therefore, more specific and less toxic inhibitors of the RAS pathway are needed to improve survival in this cohort.L. Signaling pathways commonly activated in AML frequently result in the elevation of these alternative anti-apoptotic mediators, culminating in treatment failure and disease relapse. These biological contributors of resistance are directly implicated in the observation of decreased overall survival in patients treated with venetoclax harboring constitutively activated signaling mutations [Venetoclax revolutionized the treatment of AML and ushered in new waves of questions. The most glaring question is readily apparent: how can we overcome or delay venetoclax resistance? Resistance to venetoclax has been consistently observed across molecular cohorts, primarily through the upregulation of MCL-1 and BCL-Xutations .Among the three commonly activated signaling pathways in AML — FLT3, PI3K, and RAS — the most promising therapeutic approach appears to be targeting FLT3 with a triplet regimen. The pre-clinical observation that FLT3 inhibitor-mediated MCL-1 downregulation is synergistic with BCL-2 inhibition illuminated the path to impressive early phase clinical trial results. Incorporating gilteritinib into triplet therapy with venetoclax offers a promising therapeutic advancement, particularly in the first-line setting. In this context, FLT3 inhibition may delay venetoclax resistance. In contrast, the optimal strategy at the time of disease relapse following exposure to a FLT3 inhibitor and venetoclax is unclear. Two therapeutic avenues can be envisioned: targeting the newly emerged mutation contributing to resistance or adopting a novel strategy. Innovative FLT3-directed therapies offer alternative methods of attack: FLT3 bispecific T-cell engagers (BiTEs) and FLT3-directed chimeric antigen receptor (CAR) T-cell therapy ,75.PI3K or RAS. Sequencing targeted therapy after the emergence of activating PI3K or RAS mutations following venetoclax exposure may improve survival and reduce toxicity. Alternatively, novel approaches hold promise — such as RAS-directed CAR T-cell therapy [Specific, efficacious, and potent inhibition of PI3K and RAS has yet to be fully realized in AML, and the optimal combinations and targets remain to be explored. This leads to a second question: what are the optimal targets for these pathways, and how should they be built into existing treatment strategies for patients who are not candidates for intensive chemotherapy? While venetoclax-based triplet combinations are compelling, clinical trials have not yet supported this approach for AML with mutated  therapy .The development of new therapies in AML has accelerated rapidly in recent years. Despite this, novel agents are still sorely needed and eagerly anticipated. Alternatively, direct inhibition of MCL-1 or other anti-apoptotic mediators may provide additional avenues for overcoming signaling pathway-mediated resistance. Regardless, it is an exciting era in the treatment of AML — one with many new therapeutic combination strategies waiting to be explored.


Connexin36 (Cx36) gap junctions electrically couple β-cells to coordinate glucose-stimulated Ca2+ and insulin secretion. Cx36 gap junction coupling can also protect against cytokine-induced apoptosis. Our goal was to determine how islet gap junction coupling and Ca2+ dynamics are altered in mouse models of T1D prior to diabetes. Glucose tolerance was assessed in NOD and immunodeficient NOD-RAG1KO mice at 6–12 weeks age. Glucose-stimulated insulin secretion, Ca2+ dynamics, and gap junction coupling were measured in islets isolated at each age. Gap junction coupling was also measured in islets from mice that underwent transfer of diabetogenic splenocytes and from chromograninA knockout NOD mice. Cell death was measured in islets isolated from wild-type, Cx36 knockout or Cx36 over-expression mice, each treated with a cocktail of pro-inflammatory cytokines and KATP or SERCA activators/inhibitors. NOD mice over-expressing Cx36 were also monitored for diabetes development, and islets assessed for insulitis and apoptosis. NOD and NOD-RAG1KO controls showed similar glucose tolerance at all ages. Ca2+ dynamics and gap junction coupling were disrupted in islets of NOD mice at 9 weeks, compared to controls. Transfer of diabetogenic splenocytes also decreased gap junction coupling. Islets from chromograninA knockout mice displayed normal coupling. Overexpression of Cx36 protected islets from cytokine-induced apoptosis. A knockout of Cx36 amplified cytokine-induced apoptosis, which was reversed by KATP activation or SERCA activation. Cx36 overexpression in NOD mice delayed diabetes development compared to NOD controls. However, apoptosis and insulitis were not improved. Decreases in islet gap junction coupling occur prior to T1D onset. Such decreases alter islet susceptibility to apoptosis due to altered Ca2+. Future studies will determine if increasing Cx36 gap junction coupling in combination with restoring Ca2+ homeostasis protects against islet decline in T1D.In type 1 diabetes (T1D), islet dysfunction occurs prior to diabetes onset. Pro-inflammatory cytokines can disrupt insulin secretion and Ca In vitro, pro-inflammatory cytokines reduce insulin secretion (2+) and ER calcium dynamics  gap junction coupling and disrupted the coordination of Ca2+ dynamics  is characterized by the progressive immune destruction of insulin producing β-cells in the pancreatic islets of Langerhans. Activated immune cells in the pancreas initiate β-cell death in part through secretion of high levels of Th1 cytokines, including tumor necrosis factor-α (TNF-α) from activated macrophages and interleukin-1β (IL-1β) and interferon-γ (IFN-γ) from activated T-cells . Early iecretion  and altedynamics . Our predynamics . However2+ signaling, and insulin secretion across the islet  induced β-cell death 1D]. Mice were housed in a temperature-controlled facility with access to food and water Cx36 knockout (KO) mice were generated as previously described . The ratvia the tail vein and 200 mg/kg glucose was given by intraperitoneal (IP) injection. Blood glucose was monitored over 2 h post glucose bolus.Mice were fasted with access to water ad lib for 16 h prior to performing the GTT. Fasting glucose was measured via exsanguination prior to inflating the pancreas with a collagenase solution and removing the pancreas, per our approved animal protocol. Islets were isolated by enzymatic digest of the pancreas with 2.5 mg/ml collagenase  at 37°C. Islets were handpicked into 1640 RPMI Medium (Millipore-Sigma) with 10% FBS, 10,000 U/mL Penicillin and 10,000 μg/ml Streptomycin and incubated overnight at 37°C and 5% CO2. Islet from WT, Cx36 KO, and RIP Cx36 mice were also cultured with a cytokine cocktail containing 10 ng/ml tumor necrosis factor-α , 5 ng/ml interleukin-1 β , and 100 ng/ml interferon-γ  for 24 h in culture media.Pancreata were isolated from female 6, 9, or 12-week old NOD and NOD-RAG1KO mice; male and female 12-week old WT-NOD, RIPCx36-NOD; or female 12-week old chromogranin A knockout NOD (ChGAKO-NOD) mice. Female NOD mice were selected for experiments investigating diabetes onset, as they more reliably progress to diabetes than their male counterparts . Male an6 leukocytes in HBSS was injected into each 12–14-week old NOD-scid  adoptive transfer recipient. NOD-scid vehicle controls were injected with an equal volume of HBSS. Islets were isolated as previously described 2 weeks after adoptive transfer.Splenocytes were isolated from female NOD mice with ad lib blood glucose levels >250 mg/dl by manual dissociation in cold Hanks Balanced Salt Solution (HBSS). Leukocytes were counted and one dose of 20 × 102, 1.2 mM MgCl2, 10 mM HEPES, 0.1% bovine serum albumin (BSA), and 2 mM glucose at room temperature for 2 h in the dark. Islets were transferred to MatTek glass-bottom imaging dishes  with fresh BMHH with either 2 mM or 5 mM glucose prior to imaging. Islets were imaged on one of three microscopes: 1) a Nikon Eclipse-Ti wide field microscope  with a x20 0.75 NA Plan Apo objective, a 490/40 nm band-pass excitation filter and a 525/36 nm band-pass emission filter, 2) a Zeiss 780 laser scanning microscope with a x40 1.2 NA water c-apochromat objective using a 488 nm Ar+ laser line and a QUASAR spectral detector, 3) a Zeiss 800 laser scanning microscope with a x40 1.2 NA c-apochromat objective using a 488 nm Ar+ laser with a 525/20 nm emission filter. Islets were imaged at 37°C, where images were acquired every second for 2.5–5 min following incubation at 2 mM glucose for 2 h, 5 mM glucose for 2 h, 11 mM glucose for 10 min, or 20 mM glucose for 10 min. The fraction of islet area active at 2 mM or 5 mM glucose and the fraction of islet area synchronized at 11 mM or 20 mM glucose was determined as previously described  in BMHH buffer containing 125 mM NaCl, 5.7 mM KCl, 2.5 mM CaClescribed  using MA+ laser, a 700/488 nm short pass dichroic beam splitter, a 490 nm long pass secondary beam splitter, and a 505 nm long pass emission filter or a Zeiss 800 LSM as previously described for Ca2+ imaging. As described in FRAP measurement of Cx36 coupling was performed as described in ATP channel activator Diazoxide (Millipore-Sigma), 20 mM of the K channel inhibitor tetraethylammonium , 1 µM of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) activator Ochratoxin A (Millipore-Sigma), and 1 µM of the SERCA inhibitor thapsigargin (Millipore). After incubation, islets were stained with 0.5 mg/ml fluorescein diacetate (Sigma) and 0.1 mg/ml propidium iodide (Sigma) in PBS to identify live and dead cells respectively. Islets were imaged on a Zeiss 800 scanning laser confocal microscope where 3 optical sections per islets were obtained and quantified manually for live and dead cells using ImageJ for 5–10 islets per condition.Islets from WT or Cx36 KO mice were cultured for 24 h with and without the cytokine cocktail mentioned previously. Islets were also cultured for 24 h with and without: 250 µM of the Kcontrol) and RIPCx36-NOD islets are the experimental samples (Ctexp). Relative changes in gene expression were calculated as follows:control = CtWT-NOD—CtHPRT11) ΔCtexp = CtRIPCx36-NOD—CtHPRT12) ΔCt-(ΔCtexp-AVG(ΔCtcontrol))3) ΔΔCt = 2Islets were isolated from 8 to 12-week old male and female NOD or RIPCx36-NOD littermates as previously described. Islet mRNA was isolated using the RNeasy mini kit  per the manufacturer’s instructions and RNA was eluted in 30 µl DI water. RNA was converted to cDNA using the 1st Strand cDNA synthesis kit  per the manufacturer’s instructions with 1 µg of RNA per reaction. Primers for Cx36 were purchased from OriGene  and primers for the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1) are as follows: 5′-TGGATACAGGCCAGACTTTGTT-3′ and 3′-GGGAAAATACAGCCAACACTGC-5′. PCR reactions were created using Eppendorf Matercycler Gradient Thermocycer and run on a CFX96 Real-Time System Thermocycler real-time PCR machine with Pre-soak 95°C for 10 min/Denaturation 95°C for 15 s/Annealing 60°C for 30 s for 40 cycles cycle/temp/time. Cycle time (Ct) values were calculated using Bio-Rad CFX Manager Software software and the relative change in Cx36 gene expression was calculated using the ΔΔCt method, where wild type (WT) NOD islets are the control samples  with ANDOR color camera with a x40 objective. Insulitis was determined from pancreatic sections stained with hematoxylin and eosin. Islets with no insulitis were given a score of 0, islets with peri-insulitis were given a score of 1, islets with <50% infiltrated area were given a score of 2, and islets with >50% infiltrated area were given a score of 3. The insulitis scores were averaged for all islets on 6 pancreatic sections taken from different regions of the pancreas (∼100 µm apart).Subsequent pancreas sections were deparaffinized, permeabilized with 0.25% Triton-X-100 (Sigma) and stained for TUNEL positive cells with the ClickiT Plus TUNEL Assay (ThermoFisher) per the manufacturer’s instructions. TUNEL staining was verified with DNase treated pancreatic tissue slices. Slides were then stained with guinea pig anti-insulin primary antibody  diluted 1:100 in PBS with 5% normal donkey serum (NDS) at 4°C overnight. Slides were rinsed in PBS, then stained with AlexaFluor 647 Donkey anti-guinea pig secondary antibody  diluted 1:500 in PBS with 5% NDS at room temperature for 2 h. Slides were rinsed, then mounted in Fluoromount with DAPI (ThermoFisher) and sealed. Insulin antibody staining was verified on pancreatic tissue slices from NOD-RAG1KO mice. Slides were imaged on a Nikon Eclipse Ti widefield microscope with x20 0.75 NA Plan Apo objective. DAPI was imaged with a 402/50 nm band-pass excitation filter and a 455/50 nm band-pass emission filter, TUNEL positive cells labeled with AlexaFluor488 were imaged with a 490/20 nm band-pass excitation filter and a 525/36 nm band-pass emission filter, and AlexaFluor647 was imaged with a 555/25 nm band-pass excitation filter and a 605/52 band-pass emission filter. Images of the whole pancreas slice were obtained using a tile scan and image stitching with 15% image overlap. TUNEL positive and insulin positive cells were quantified using ImageJ and insulin positive pancreas area was determined using MATLAB .p-value <0.05 were reported in all figures. To determine clustering of NOD coupling and Ca2+ data, k-means clustering was used in combination with a t-test to determine significance between identified clusters of data. To determine statistical differences in survival of the WT NOD and RIPCx36-NOD mouse colonies, Kaplan-Meier survival analysis with a Breslow test was used where indicated.To determine statistical significance, analysis of variance (ANOVA) with Tukey’s post hoc analysis or a student’s t-test was used where indicated. In each figure panel, all genotypes and treatments are compared against one another; however, only differences that were found to be statistically significant with a via GTT. No significant changes in glucose tolerance test  and the fraction of the islet that showed coordinated Ca2+ oscillations. At 2, 11, and 20 mM glucose there were no significant differences in the fraction of the islet that was active between NOD and NOD-RAG1KO islets (2+ oscillations from 6 to 9 weeks in NOD mice (p = 0.025), compared to NOD-RAG1KO controls which were unchanged with age  in NOD mice compared to NOD-RAG1KO controls (p = 0.007). We next determined if changes in β-cell coupling were predictive of a future diabetic phenotype. We used K-means clustering analysis of β-cell coupling from 2+ oscillations at 11 mM glucose from 2+ coordination (solid red squares) clustered with the NOD-RAG1KO control mice (2+ coordination (open red squares) clustered separate from both the NOD-RAG1KO islets and the NOD islets with higher coupling and coordination (2+ coordination (p = 0.003) difference in Ca2+ coordination between cluster 1 (mean = 0.58 ± 0.05 SEM) and cluster 2 (mean = 0.32 ± 0.03 SEM) identified using K-means clustering . NOD-ChgA KO mice have a competent immune system, but do not develop T1D as they lack the primary antigen causing autoimmune diabetes in this strain of NOD mouse, ChromograninA (p = 0.032) Cx36 gap junction coupling than age-matched NOD mice and showed similar levels of coupling as NOD-RAG1KO controls  in β-cell gap junction coupling compared to vehicle only control mice ; however, increasing intracellular Ca2+ by closing K+ channels, (including KATP channels) with TEA abolished this protective effect (p = 0.015) compared to WT controls, and this effect was completely abolished by decreasing intracellular Ca2+ by activating KATP channels with diazoxide  and increased ER Ca2+ improved the survival of cytokine treated islets in vitro. While overexpression of Cx36 in the β-cells of NOD mice protected against early diabetes onset (at ∼12 weeks), increased Cx36 gap junction coupling did not significantly improve islet function or survival, resulting in only modest protection against T1D development. Our results suggest that while decreases in β-cell Cx36 gap junction coupling and altered Ca2+ signaling dynamics are associated with the development of T1D in the NOD mouse, increasing gap junction coupling alone is not sufficient to prevent the onset of disease.The goal of this study was to determine the role of altered Cx36 coupling and Ca2+ oscillation coordination is mediated by electrical coupling between β-cells that coordinates membrane depolarization and subsequent Ca2+ influx across the islet (2+ oscillations directly correlated with decreases in Cx36 coupling. While we observed dysfunction to Cx36 coupling and Ca2+ oscillations in islets from NOD mice as early as 9 weeks of age, we did not observe dysfunction to insulin secretion at any of the time points studied. This is in line with previous studies where total KO of Cx36 in the islet disrupts insulin release pulsatility, but has a limited effect on overall insulin secretion levels (2+ oscillations without causing dysfunction to levels of insulin secretion (2+ dynamics with no significant effects on insulin secretion, as shown in this study. This is also supported by findings in type 2 diabetes, where low-grade inflammation caused impaired Ca2+ handling and islet dysfunction (2+ dynamics in T1D progression. Altogether, our results indicate a role for decreased Cx36 coupling in mediating islet dysfunction in early type 1 diabetes.Consistent with studies in prediabetic mouse models of type 2 diabetes , we founhe islet . This isn levels . Our preecretion . While dfunction . While m2+ oscillations. This heterogeneity in islet function was not present in the immunodeficient control mice and therefore is likely associated with diabetes pathogenesis. However, we were unable to follow these mice to determine if this increased dysfunction correlated with earlier development of diabetes. We also noted that there was no correlation between gap junction coupling and Ca2+ oscillation coordination in islet from NOD mice, suggesting that changes in gap junction coupling alone are not representative of islet dysfunction. The identified sub-population of NOD mice with lower Ca2+ oscillation coordination did not have significantly different levels of coupling compared to the population of NOD mice with increased Ca2+ oscillation coordination. This suggests that other factors, such as KATP channel function or ER/mitochondrial Ca2+ buffering may also be impacted in diabetic animals; however, further study is required to determine how Ca2+ handling is altered. Previous studies in twins who were at risk for developing T1D have shown a positive correlation between decreases in first-phase insulin release and onset of T1D, supporting our hypothesis that greater islet dysfunction prior to changes in glucose homeostasis is associated with disease progression (2+ signaling may predict disease onset.We also identified sub-populations of NOD mice with higher levels of dysfunction as determined by the decreased coordination of glucose-stimulated Cagression . As alte2+ dynamics as a measure of islet function and changes in electrophysiology provides an alternative technique to capture early decreases in function of islet in pre-diabetic NOD mice. Our results are in line with previous studies that showed islet dysfunction as measured by a reduction in first-phase insulin secretion in at-risk children who went on to develop T1D (2+ dynamics in vitro via activation of protein kinase C δ (Our results indicate that decreases in Cx36 coupling occur as a result of T1D onset, as levels of Cx36 are similar in NOD mice and immunodeficient controls at early ages but decrease starting at 9 weeks in the NOD mice. Deletion of chromogranin A in NOD mice, which lack autoreactive T-cells, preserves Cx36 coupling and transfer of diabetogenic splenocytes to immunodeficient mice decreases Cx36 coupling in islets . Thus auelop T1D . Activatnase C δ .2+ regulation are tightly coupled, suppressing spontaneous elevations at low glucose levels and coordinating pulsatile Ca2+ at high glucose. Elevations in Ca2+ can also exacerbate apoptosis, thus suggesting that Cx36 gap junction coupling protects against cell death via Ca2+ regulation (ATP channels, and OTA-activation of SERCA which will suppresses cytosolic Ca2+ elevations, counteracted the impact of a loss of Cx36 on cell death. The protection against cell death afforded by Cx36 overexpression was eliminated upon TEA inhibition of K+ channels, which will elevate cytosolic Ca2+ elevations. Together these findings indicate that a loss of Cx36 gap junction coupling exacerbates cytokine-mediated cell death via elevated spontaneous Ca2+ activity. This is supported by previous studies, where loss of gap junction coupling in islet β-cells resulted in increased intracellular Ca2+ at low glucose levels, likely due to heterogeneity in β-cell excitability in the islet (2+ (2+ has been linked to ER stress induced apoptosis in β-cells, suggesting that increasing ER Ca2+ with the SERCA activator may protect against cell death (2+ can protect against cytokine-mediated cell death by reducing ER stress in islets with decreases gap junction coupling. Overall, our results support a role for elevated intracellular Ca2+ as a result of a loss of Cx36 coupling that exacerbates cytokine-induced islet cell death. However, the exact mechanisms by which Cx36-regulated Ca2+ impacts Cx36-mediated protection require further studies.Our results indicate that modulation of Cx36 gap junction coupling in the islet can mediate the effects of pro-inflammatory cytokines on islet survival. Gap junction coupling and Cagulation . Both dihe islet . This coslet (2+ . Additioll death . Further2+ dynamics or insulin secretion. These results are in contrast to our in vitro studies, as well as a previous study that used glibenclamide to increase Cx36 gap junction coupling and observed protection against T1D onset in the NOD mouse (via KATP channel closure (-cell gap junction coupling, dysfunction to intracellular Ca2+ dynamics remained. This suggests that glucose-stimulated Ca2+ signaling can be disrupted by changes in ER Ca2+ uptake in addition to decreased Cx36 coupling, as has been suggested by previous studies in vitro (2+ handling.Our results indicate that decreases in Cx36 coupling are a result of T1D pathogenesis, therefore we also sought to determine if increasing Cx36 coupling could protect against the onset of disease in the NOD mouse. Overexpression of Cx36 did increase coupling at all time points studied; however, protection against disease onset was only observed early on from 10 to 12 weeks with a small difference in overall survival. We did not observe a significant improvement in islet CaOD mouse . Glibenc closure , which min vitro , and tha-cell death in vitro (in vivo (2+ handling which was not recovered by increased Cx36 gap junction coupling. For example, recovery of ER Ca2+ homeostasis can protect against cytokine-mediated β-cell death (in vitro with KATP and SERCA modulators. The lack of protection against β-cell death with increased Cx36 gap junction coupling may also be due to β-cell death mediated by Ca2+-independent activation of the Fas ligand on the β-cell surface by activated immune cells (2+ homeostasis may show greater protection against β-cell death in T1D. Future studies that investigate changes in cytosolic and ER Ca2+ using genetically encoded and organelle targeted fluorescent Ca2+ probes may help to determine if modulating both intracellular Ca2+ and gap junction coupling can maintain ER Ca2+ homeostasis and normalize β-cell electrophysiology in islets from diabetic NOD mice.Several studies have shown that Cx36 gap junction coupling between β-cells protects against cytokine-induced βin vitro  as well (in vivo . In contll death , which fne cells , as we d2+ dynamics across the islet and is mediated by autoreactive immune cells in the NOD mouse. In vitro, loss of Cx36 gap junction coupling exacerbates cell death and Cx36 overexpression protects against cell death via regulating cytosolic Ca2+. While increasing Cx36 gap junction coupling in the NOD mouse protected against early onset of T1D; long term survival, islet function, and β-cell death were not improved. Thus while islet dysfunction occurs prior to changes in glucose homeostasis in T1D, increased β-cell gap junction coupling alone is insufficient to recover islet function or prevent diabetes onset. Future studies will determine if increasing Cx36 coupling in combination with therapies which restore Ca2+ homeostasis can protect against islet dysfunction and death in the NOD mouse.In summary, we have shown that Cx36 gap junction coupling is reduced in the islets of NOD mice prior to changes in glucose homeostasis. This decrease in Cx36 gap junction coupling is accompanied by altered coordination of Ca


In vascular endothelial cells, SOCE regulates a plethora of functions that maintain cardiovascular homeostasis, such as angiogenesis, vascular tone, vascular permeability, platelet aggregation, and monocyte adhesion. The molecular mechanisms responsible for SOCE activation in vascular endothelial cells have engendered a long-lasting controversy. Traditionally, it has been assumed that the endothelial SOCE is mediated by two distinct ion channel signalplexes, i.e., STIM1/Orai1 and STIM1/Transient Receptor Potential Canonical 1(TRPC1)/TRPC4. However, recent evidence has shown that Orai1 can assemble with TRPC1 and TRPC4 to form a non-selective cation channel with intermediate electrophysiological features. Herein, we aim at bringing order to the distinct mechanisms that mediate endothelial SOCE in the vascular tree from multiple species . We propose that three distinct currents can mediate SOCE in vascular endothelial cells: (1) the Ca2+-selective Ca2+-release activated Ca2+ current (ICRAC), which is mediated by STIM1 and Orai1; (2) the store-operated non-selective current (ISOC), which is mediated by STIM1, TRPC1, and TRPC4; and (3) the moderately Ca2+-selective, ICRAC-like current, which is mediated by STIM1, TRPC1, TRPC4, and Orai1.Store-operated Ca The endothelial monolayer forms the innermost lining of all blood vessels and of the cardiac chambers, where it is known as endocardium. Endothelial cells provide a widespread interface that continuously perceives and integrates signals delivered by blood-borne humoral cues and by changes in shear stress and/or pulsatile stretch. Additionally, endothelial cells may sense even subtle alterations in local tissue microenvironment and/or in the signalling activity of neighbouring cells ,2,3,4,5.2+ concentration ([Ca2+]i) is crucial to select the most appropriate endothelial response to the multiple inputs that are simultaneously delivered either from blood flow or from adjoining tissue i drives ECFCs’ angiogenic activity both in vitro ER), which results in the activation of a Ca2+-permeable pathway in the plasma membrane (PM), known as store-operated Ca2+ entry (SOCE) ER could result in the development of a non-selective cation current that displays a slightly linear current-to-voltage (IV) relationship and is known as store-operated current (ISOC) i arises in the form of spatially restricted InsP3-dependent ER Ca2+ release events, known as Ca2+ puffs, that arise in the cell periphery and subsequently propagate as repetitive Ca2+ waves to engulf the whole cytoplasm through CICR ER that induced a gradual increase in SOCE activity; furthermore, SOCE underwent periodic cycles of rapid activation and slow deactivation during the slow intracellular Ca2+ oscillations that can be elicited by prolonged stimulation of CPAE cells with a submaximal concentration of ATP ER is sufficient to activate SOCE on the PM in an InsP3-independent manner ER leads to SOCE activation in endothelial cells [2+ concentration ([Ca2+]ER) that is activated upon ER Ca2+ emptying ER ER . In accomulation ,104. Furmulation  and is imulation ,104. STImulation ; a long mulation ; and a stivation .SOC encompasses a variety of store-operated non-selective cation currents that present a different electrophysiological fingerprinting as compared to the highly Ca2+-selective CRAC channels  IV relationship; permeability to K+ (outgoing) and Na+, Cs+, and Ca2+ (ingoing); and single-channel conductance in the pS range (~5–23 pS) i that engages a different transcription factor, i.e., nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) ,108SOC e5–23 pS) . Six TRP5–23 pS) . The avapS) ER and the intracellular Ca2+-buffering conditions , as shown in The first attempts to measure the ICRAC was provided by Cristina Fasolato and Bernd Nilius in 1998 ER, thereby resulting in the activation of a sub-pA ionic current . I. I158].  release ,159. An al cells . A subseal cells . These fal cells . Recent al cells ,163,164.al cells ,166, GABal cells ,168, andal cells , and neual cells  and aracal cells . Interesal cells , which ial cells . The phaal cells ,171. Oran HUVECs , but the2+]ER ER . The muttivation . These f2+ oscillations, to stimulate the nuclear translocation of NFAT, and to increase vascular permeability in a transgenic mouse model selectively lacking the endothelial STIM1  was inhibited by the genetic blockade of STIM1, TRPC1, and TRPC4 [2+]ER ER peripheral cisternae that is required to activate endothelial TRPC1 and TRPC4 channels, as recently shown for Orai1 in HeLa cells  -derived fragment, CGA47–66 . T. TSOC inn HUVECs . Likewisn hPAECs  201]. I. ISOC inn hPAECs . The ISOeability . Similar protein . TNF-α iCGA47–66 . FinallyCGA47–66 . Therefoeability .SOC through NF-κB activation is cell survival during thrombin stimulation. In accord, HUVECs challenged with a prolonged exposure to thrombin activate the transcriptional pro-inflammatory program, but do not undergo apoptosis, due to concomitant NF-κB-dependent A20 expression [SOC could be effectively targeted to treat PAH.An additional process that is seemingly mediated by the endothelial Ipression . More repression . Therefo2+ entry in MAECs [2+ entry in response to InsP3-dependent reduction in [Ca2+]ER ER . In addi[Ca2+]ER . Finally[Ca2+]ER ,217.Conversely, endothelium-dependent NO release and vasodilation were not affected in a transgenic mouse model devoid of TRPC1 . This fiSOC described a membrane current that actually displayed intermediate electrophysiological features between ISOC and ICRAC  and the occurrence of a small outward current (which reflects cytosolic K+ efflux) indicate that this current is also contributed to by monovalent cations and is therefore non-selective. The Nilius group also showed that depleting the ER by dialyzing the cytosol with InsP3 through the patch-pipette and adding tBHQ to the bath elicited a store-operated current in MAECs (2+ permeability (PCa/PNa ≈ 160 vs. PCa/PNa ≈ 1000) as compared to the archetypal ICRAC . Va. VaSOC dadykinin  44,189],189SOC dn rPAECs  . Th. ThSOC dal ICRAC ,222. Finchannels : (1) inw, ≈−7 mV . In our RAC-like , as suggRAC-like ,118, humRAC-like , and bovRAC-like .CRAC-like current hint at a model according to which the channel pore is contributed to by both TRPC and Orai1 subunits, which assemble into a heteromeric complex that is informed about ER Ca2+ content by STIM1  an anCRAC-leraction  69]. In. InCRAC-ent mice . A landmn rPAECs  51]. Th. ThCRAC- current . Thus, an rPAECs . The eviCRAC-like current has been mainly investigated in rat pulmonary vasculature [CRAC-like current is not detectable [CRAC-like currents in pulmonary artery, and promoted their occurrence in lung capillaries [CRAC-like current controls endothelial cell permeability [CRAC-like current could sensitize alveolar endothelium to Ca2+-dependent openings that result in pulmonary edema [2+ signals supported by both the ISOC  eNOcurrents ,159; andcurrents . TherefoCRAC, which is a Ca2+-selective current; (2) ISOC, which is a non-selective cation current; and (3) ICRAC-like, which bears intermediate electrophysiological features between ICRAC and ISOC. Orai1 incorporation into the channel signalplex is likely to make the difference between a Ca2+-selective and a non-selective cation channel. It is, however, not surprising that many compelling issues regarding the molecular architecture and the role of endothelial SOCE in cardiovascular physiology and pathology remain to be elucidated.The evidence discussed so far, which derives from almost three decades of intense research, indicates that the endothelial SOCE can be mediated by at least three different ionic currents: (1) I2+ pool, ICRAC and ISOC coexist in HSG cells, and that the ICRAC is activated first to stimulate the Ca2+-dependent insertion of TRPC1 on the PM [CRAC is masked by the larger TRPC1-mediated ISOC and can be easily detected only after genetic silencing of the latter conductance [CRAC-like current and then a larger ISOC in human gingival keratinocytes [CRAC-like current, also suppressed the subsequent ISOC [CRAC [SOC [CRAC [+-based intracellular solution, which is routinely employed to measure the ICRAC, while TRPC1 may display a poor permeability to Cs+ [SOC was based upon K+ [+ [CRAC [2+ entry through Orai1 from inducing TRPC1 exocytosis on the PM. Therefore, these authors might have found the ideal conditions to record the ICRAC long before the role of Orai1 in stimulating the ISOC was identified [SOC in HUVECs was inhibited by 1 µM La3+ [CRAC and ISOC coexist in HUVECs. Another popular model to investigate the role of SOCE in endothelial physiopathology is represented by pulmonary endothelium. Two independent studies showed that both the ICRAC [SOC [SOC recorded in hPAECs requires previous Orai1-mediated Ca2+ entry. Intriguingly, this ISOC is again sensitive to 1 µM La3+ [CRAC-like current that requires the assembly of a STIM1/TRPC1/TRPC4/Orai1 supermolecular complex (see CRAC-like currents (this feature would explain why SOCE is unaffected in mPMECs transfected with a siOrai1) [+ influx through CRAC-like channels can also regulate endothelial function  [First, may these three currents coexist in the same endothelial cell type? Pioneering work from the Ambudkar group showed that, following depletion of the ER Can the PM . Howeverductance . Intriguinocytes ,227. Thient ISOC . These fOC [CRAC  and ISOCRAC [SOC ,188,190 OC [CRAC . This iny to Cs+ . Convers upon K+  or Na+ [on K+ [+ ,193, whi[+ [CRAC , which mentified . In line µM La3+ ,152,229.he ICRAC  and the RAC [SOC  are presRAC [SOC , studiesRAC [SOC  and hPMERAC [SOC  suggest  µM La3+ . Intriguplex see . TherefosiOrai1) ; and (2)in mice) ,230. EleSOC or the ICRAC-like current? Is the endothelial ISOC strictly dependent on TRPC4 or can it be contributed to only by TRPC1? Ahmed et al. reported that the application of a specific antibody selectively targeting an extracellular epitope of TRPC1 significantly reduced, but not fully suppressed, InsP3-evoked ISOC in HUVECs [2+ entry only in sub-confluent, proliferating vascular endothelial cells. Conversely, TRPC4 did not support Ca2+ influx in either isolated or fully confluent endothelial cells [Second, why have only a few studies identified a clear role of TRPC4 in either the In HUVECs . The remn HUVECs . The Groal cells . Under s2+ microdomains that engage distinct Ca2+-dependent effectors, such as NFAT and NF-κB  mediate SOCE in ECFCs? SOCE supports ECFC proliferation, migration, and tube formation both in vitro ,241,242 nd TRPC1 ,39,239. tivation ,238 stro2+ response to extracellular stimulation and thereby regulates a plethora of endothelial functions, including angiogenesis, permeability, platelet aggregation, and NO release. Three distinct store-operated currents can mediate SOCE in vascular endothelial cells: (1) the Ca2+-selective ICRAC, which is mediated by STIM1 and Orai1; (2) the non-selective ISOC, which is mediated by STIM1, TRPC1, and TRPC4; and (3) the moderately Ca2+-selective, ICRAC-like current, which is mediated by STIM1, TRPC1, TRPC4, and Orai1. The incorporation of Orai1 into the STIM1/TRPC1/TRPC4 complex, therefore, turns the non-selective ISOC into a Ca2+ (semi)-selective current. As highlighted in 2+-permeable currents coexist in the same endothelial cell type or does their endothelial distribution change along the vascular network and/or from one species to another? (2) are the ICRAC, ISOC, and ICRAC-like currents coupled to distinct Ca2+-dependent decoders, and do they modulate different endothelial functions? (3) does Orai1-mediated extracellular Ca2+ entry result in TRPC1 translocation to the plasma membrane also in endothelial cells? Exploiting the novel technologies offered by both molecular biology  and imaging techniques  will help in solving these issues. Understanding the molecular architecture of this mechanism is indispensable in efficiently targeting endothelial SOCE in patients affected by life-threatening cardiovascular disorders, such as atherosclerosis, cerebrovascular dysfunction, sepsis-induced systemic inflammation, and PAH.SOCE maintains the endothelial Ca


Following activation of skeletal muscle contraction by the E-C coupling mechanism, replenishment of intracellular stores requires reuptake of cytosolic Ca2+ into the SR by the activity of SR Ca2+-ATPases, but also Ca2+ entry from the extracellular space, through a mechanism called store-operated calcium entry (SOCE). The fine orchestration of these processes requires several proteins, including Ca2+ channels, Ca2+ sensors, and Ca2+ buffers, as well as the active involvement of mitochondria. Mutations in genes coding for proteins participating in E-C coupling and SOCE are causative of several myopathies characterized by a wide spectrum of clinical phenotypes, a variety of histological features, and alterations in intracellular Ca2+ balance. This review summarizes current knowledge on these myopathies and discusses available knowledge on the pathogenic mechanisms of disease.In skeletal muscle, Ca The SR is composed of two distinct domains: the longitudinal SR (l-SR) and the junctional SR (j-SR). The l-SR is the main site of Ca2+ uptake from the sarcoplasm, thanks to the presence of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps, while the j-SR associates with sarcolemma transverse tubules (T-tubules) to form the triads, where the ryanodine receptor type 1 (RYR1) Ca2+ release channels are localized  mechanism, independently of the interaction with DHPR , triadin, junctin, and calsequestrin CASQ; . JPHs me complex . In mamm complex . Triadin complex . They ac complex , CASQ  mechanism, mediated by stromal interaction molecule 1 (STIM1), a Ca2+ sensor in the SR, and ORAI1, a selective Ca2+ channel on the plasma membrane, is also fundamental to refill intracellular Ca2+ stores and sustain prolonged activity of skeletal muscle fibers  is shown in red. Coincidentally, nearly all the mutations validated as causative by the EMHG are within the three hotspot regions. Recent cryo-EM studies revealed that several mutations are positioned near the fourfold symmetry axis, at sites of interaction between the RYR1 subunits or at sites of interaction between domains in the same subunit, resulting in destabilization of the closed state of the channel  mutations causing a gain of function, (2) mutations causing a loss of function, and (3) mutations causing a reduction in RYR1 protein content  and sex  linked to delayed inactivation of L-type currents through DHPR  that result in posttranslational modifications of RYR1, which in turn further enhance channel activity. In this way, a destructive feed-forward cycle is activated, leading (in acute conditions) to sudden death or inducing (over time) mitochondrial damage  production, and induction of apoptosis. According to the authors, since residue I4898 is localized in proximity of the RYR1 sequence involved in triadin binding, mutation in this residue causes a reduction in triadin binding and content. Given the interaction between triadin and CASQ, this also leads to mislocalization of CASQ, resulting in induction of ER stress/UPR. In support of this hypothesis, the use of the chemical chaperone 4-phenylbutyric acid resulted in a significant improvement of muscle function, suggesting that the ER stress/UPR pathway contributes significantly to development of CCD caused by the I4898T mutation , centronuclear myopathy (CNM), congenital fiber type disproportion (CFTD), dusty core disease (DuCD), and core rod myopathy  shows the main features of MmD-affected patients, with a reduction in muscle force and Ca2+ transients, associated with a decrease in RYR1 protein level of ∼65% (Ryr1A4329D) also shows a reduction in RYR1 protein content and muscle performance, but this was limited to slow-twitch muscle fibers. The reason for the selective effect of the homozygous A4329D mutation in slow-twitch fibers is not known; the authors suggested that a different epigenetic regulation of fast- versus slow-twitch muscle fibers may account, at least in part, for these differences  together with the missense mutation A4329D  expressed in type 1 muscle fibers and in the heart, are causative of ∼10% of CCD cases . Recessive and dominant mutations in CACNA1S were identified in patients showing MmD/CNM with structural alterations in T-tubules and SR. Although the pathogenetic mechanism has not been clearly defined, both recessive and dominant mutations correlate with a strong decrease in protein levels, suggesting instability or degradation of DHPR complexes. RYR1-mediated Ca2+ release was also impaired, although no change in cytosolic or SR Ca2+ levels was observed  are the most common cause of dominant CNM. DNM2 is a GTPase protein that can bind and organize the microtubular and actin cytoskeleton and associates with nascent vesicles to induce their release  and TPM3 encode two isoforms of tropomyosin expressed in skeletal muscles. TPM2 codes for β-tropomyosin expressed in type 1 muscle fibers and to a lesser extent in fast-twitch fibers, while TPM3 codes for slow α-tropomyosin expressed exclusively in slow-twitch fiber types  that are typical of nemaline myopathy. Rods are mainly composed of actin and α-actinin, probably deriving from Z disks; they can be assembled in clusters or widely distributed along the fibers . Longitue of CRM . Compounficiency .CFL2, ACTA1, and TPM3), although some of them are typically associated with nemaline myopathy, where cores are not present (CFL2), coding for the actin-binding protein cofilin-2, were identified in patients with nemaline myopathy with minicores and concentric laminated bodies . Mutations in ACTA1 are distributed along the entire gene, affecting stability, function, and conformation of the protein, so a clear correlation with genetic defect and disease onset and development is difficult to discern , exertional rhabdomyolysis, emotional stress, fatigue, and viral infection  is characterized by susceptibility to MH, delayed motor development, short stature, cryptorchidism, skeletal abnormalities, and variable dysmorphic features. Resting creatine kinase levels are elevated in some patients, and muscle biopsies show fiber size variation with atrophic type I muscle fibers and absence of cores. KDS can present with either a dominant or a recessive inheritance of mutations in netrance .RYR1 have been rarely identified in some other myopathies. For example, three different RYR1 mutations were identified in calf-predominant distal myopathy, a mild dominant distal myopathy characterized by fatty degeneration of medial gastrocnemius, elevated creatine kinase levels, and the presence of cores in muscle biopsies  that, by terminating Ca2+ entry, prevents the potentially harmful consequences of Ca2+ overload for cells  or STIM-ORAI–activating region (SOAR), interacts with ORAI1. The site in ORAI1 that mediates the interaction with STIM may require multiple separate domains, one of which has been recently proposed to correspond to a peptide in the cytoplasmic C-terminal extension that follows the fourth transmembrane helix in each of the six ORAI1 monomers  and two additional nd ORAI3 . STIM2 snd ORAI3 . Studiesr stores .STIM1 and STIM2 are expressed in different cells. Three spliced isoforms of STIM1 have been described that show tissue-specific patterns of expression and different regulatory properties: STIM1L, expressed in skeletal and cardiac muscle and in the brain  channels. Not much is yet known about the STIM2.3 splice variant, which differs from both STIM2.1 and STIM2.2 in the C-terminal region  of divalent and monovalent cations  family . The con cations .STIM and three ORAI genes and alternative spliced isoforms, some of which have dominant-negative effects on the ICRAC current, is strongly suggestive of the importance of fine-tuning SOCE-mediated Ca2+ signaling to regulate the variety of different functions that operate simultaneously in eukaryotic cells. That SOCE plays signaling functions in addition to refilling the empty Ca2+ stores is now supported by several studies that link modification of SOCE function with activation of specific transcription factors and signaling pathways, resulting in the regulation several specific cell functions, including regulation of metabolism. Indeed, SOCE has been shown to contribute to activation of T cell proliferation by triggering calcineurin-mediated activation of the NFAT transcription factor and of stimulation of the PI3K-AKT-mTOR pathways, resulting in upregulation of glucose transporters and glycolytic and mitochondrial enzymes that provide the metabolic support necessary for T cell expansion and activation of adaptive immune functions  binding to STIM1 , are formed by elongations of T tubules that run parallel to the l-SR. CEUs support increased Ca2+ entry via ORAI1 and contribute to improve fatigue resistance under continued muscle activity  during exercise, resulting in a decrease in SOCE, which may avoid cytosolic Ca2+ overload, help preserve energy for muscle contraction, and reduce fatigue  and NADH-tetrazolium reductase, but are negative for succinate dehydrogenase or cytochrome c oxidase staining . When vigregates .STIM1 have been identified, the majority of which affect different amino acids in the canonical and noncanonical EF-hand motifs . One of the two mutations has been also found in patients with CCD. The identification of mutations in the RYR1 gene in TAM patients is of interest, since RYR1 mutations may represent the genetic cause of myopathy in at least a fraction of the patients affected by TAM that do not carry mutations in STIM1, ORAI1, or CASQ1 and therefore represent a new diagnostic target for these patients. Moreover, since the phenotype of both patients is rather mild, it is possible that RYR1-related TAM may be currently underdiagnosed in the population.A recent study reported the identification of two different missense e in TAM . The twoRYR1-related disorders, result in several different myopathies characterized by a spectrum of clinical and histopathological phenotypes. At the same time, as in a mirror image, we have seen an increase in the number of genes that can be associated with the same disease (RYR1 mutations represents only the latest evidence of how complex it is to associate genetic data and histopathological patterns (The development of fast and cost-effective DNA sequencing technologies has tremendously increased our knowledge of the genetic basis of skeletal muscle pathologies. This has provided further evidence that mutations in one gene, as in  disease . The recpatterns .RYR1 or CACNA1S can be found in patients with MH or CCD and that mutations in STIM1 or ORAI1 are associated with TAM/Stormorken syndrome, less obvious are the cases where mutations in genes not involved in Ca2+-handling pathways, such as MYH7, TTN, or MEGF10, are detected in patients presenting with clinical symptoms and histopathological alterations like those present in RYR1-related myopathies. This apparent incongruency can be rationalized by evidence that, at least in some cases, mutations in genes apparently not directly connected with Ca2+ signaling may indirectly affect regulation of Ca2+ homeostasis, as proposed for SEPN1 (BIN1, MTM1, or DNM2 (2+ signaling mat induce a myopathy. Future advancements in studying functions, regulatory properties, and even more the network of interactions participated by proteins encoded by causative genes of interest will certainly help answer these questions. In this perspective, a significant contribution can be also provided by the identification of genetic or drug modifiers (Although it is easy to understand that mutations in or SEPN1  or BIN1, or DNM2 . Howeverodifiers . It can


Such variations affect the function of ion channels of the nervous system and consequently also neuronal signalling. We investigated here the mechanisms by which Ca2+ controls the activity of acid-sensing ion channel (ASIC) 3. ASICs are neuronal, H+-gated Na+ channels involved in several physiological and pathological processes, including the expression of fear, learning, pain sensation and neurodegeneration after ischaemic stroke. It was previously shown that Ca2+ negatively modulates the ASIC pH dependence. While protons are default activators of ASIC3, this channel can also be activated at pH7.4 by the removal of the extracellular Ca2+. Two previous studies concluded that low pH opens ASIC3 by displacing Ca2+ ions that block the channel pore at physiological pH. We show here that an acidic residue, distant from the pore, together with pore residues, controls the modulation of ASIC3 by Ca2+. Our study identifies a new regulatory site in ASIC3 and demonstrates that ASIC3 activation involves an allosteric mechanism together with Ca2+ unbinding from the channel pore. We provide a molecular analysis of a regulatory mechanism found in many ion channels.The extracellular Ca The regulatory role of extracellular Ca2+ is less widely known. Extracellular free Ca2+ concentrations are locally decreased during high neuronal activity or seizures and in ischaemia [2+ concentration affects the properties of many neuronal ion channels [2+-sensitive channel, the acid-sensing ion channel (ASIC) 3, is controlled by extracellular Ca2+. ASICs are H+-gated, Na+-conducting ion channels of the nervous system [Intracellular Caschaemia –3. Theseschaemia , since tchannels –8. In ths system –11. Extrs system ,10,12. Fs system ,10. Of ts system ,14, whils system .a). The ectodomain is composed of a central scaffold made up by the palm and the knuckle. The thumb and finger contain several α helices and are oriented towards the outside of the protein. The ASIC trimer contains two vestibules located along the central vertical axis between the three subunits, the ‘extracellular vestibule’, situated directly above the pore entry, and the ‘central vestibule’, enclosed by the β sheets of the lower palm domains (a). The ‘acidic pocket’, present three times in the trimer, is located at about 40 Å above the pore, enclosed by the finger, thumb and β-ball domains of one subunit, and the palm of a neighbouring subunit [The shape of an ASIC subunit is comparable to that of a hand holding a small ball, with the forearm being the transmembrane segment; the extracellular domains have accordingly been named as finger, knuckle, palm and thumb  figure a. The ec domains a. The ‘a subunit –19.Figu2+ concentration, suggesting that there is a competition between H+ and Ca2+ ions for common binding sites [2+-free solution at the same pH induced an inward current in ASIC3 [2+ and H+ concentrations and the observation that unitary current amplitudes were decreased by high Ca2+ concentrations, it was concluded that acidification activates ASIC3 by a displacement of Ca2+ ions bound to the pore entry at neutral and alkaline pH, without conformational changes [2+ removal at pH7.4 was strongly decreased, further supporting the hypothesis that the competition between H+ and Ca2+ occurs in the ASIC3 pore. The E435A mutation did, however, only partially suppress the low Ca2+-induced current. In the present work, we used several complementary approaches to determine the contribution of sites in and outside the ASIC3 pore to the gating control by Ca2+. We show that in addition to the previously identified Glu435 in the pore entry, a residue in the acidic pocket and an acidic residue deeper down in the channel pore contribute to the control of ASIC activation by Ca2+. Protons activate ASIC3 therefore by binding to H+-sensing residues that lead, via conformational changes, to the opening of the pore and by displacing Ca2+ bound into the pore entry.The ASIC pH dependence is inversely correlated with the extracellular Cang sites ,20. Chanin ASIC3 ,21. Base changes . A recen. 2. 2.1Xenopus laevis oocytes and its function was measured by two-electrode voltage clamp, extracellular acidification to pH5 resulted in large, transient inward currents (bi). If the pH was kept at 7.4, and the free Ca2+ concentration was lowered from 2 mM to 80 nM, a transient current of similar amplitude as that induced by the acidification was measured , as shown previously [2+ and pH7.4/80 nM Ca2+ were roughly equal (figure 1c). Xenopus oocytes express endogenous connexin hemichannels that are activated by lowering of the extracellular Ca2+ concentration [2+-activated currents had a much smaller amplitude  and slower kinetics than the ASIC currents and did therefore not interfere with the ASIC current measurements . To examine whether the low Ca2+-induced current can be explained by a changed pH sensitivity of ASIC3, the pH dependence of activation was determined at the two Ca2+ concentrations 2 mM and 80 nM. The pH dependence of ASIC activation was measured by stimulating the channels with a series of solutions of increasingly acidic pH. Fitting the pH dependence curves obtained at 80 nM free Ca2+ and at 2 mM Ca2+ yielded pH values of half-maximal activation (pH50) of 7.48 ± 0.14 (n = 62) and 6.14 ± 0.11 (n = 42), respectively, for the two Ca2+ concentrations, confirming therefore a strong alkaline shift of the ASIC3 pH dependence upon lowering of the Ca2+ concentration (figure 1d). According to the activation curve, pH7.4 induces in the 80 nM Ca2+ condition 64 ± 0.8% of the maximal current achieved at this Ca2+ concentration . By contrast, in the 2 mM Ca2+ condition, no current was measured at pH7.4 (figure 1d). In addition to its effects on pH dependence, Ca2+ can, at millimolar concentrations, inhibit ASICs by a pore block mechanism [2+ concentrations, the current ratio IpH5(2 mM Ca2+)/IpH5 (80 nM Ca2+) was 0.65 ± 0.15 , indicating that there is an approximately 35% inhibition of the maximal peak current amplitude at pH5 by 2 mM Ca2+. The similar amplitudes of the low Ca2+(pH7.4/80 nM Ca2+)- and low pH (pH5/2 mM Ca2+)-induced currents  are therefore due (i) to the shift in pH dependence of activation leading to an increase of the current amplitude in the low Ca2+ condition of approximately 65% of the maximal amplitude (figure 1d) and (ii) to the 35% pore block by 2 mM Ca2+ at pH5. Lowering of the extracellular Ca2+ concentration also shifts the pH dependence of the transition from the closed to the desensitized state, termed steady-state desensitization (SSD), to more alkaline values (SSD in the inset of figure 1d). To determine the pH dependence of SSD, ASIC3-expressing oocytes were perfused with conditioning pH solution for 55 s, before the fraction of not-desensitized channels was measured by a step to pH5; this protocol was repeated with increasingly acidic conditioning solutions. In these experiments, the Ca2+ concentration in the conditioning solution was 2 mM in one series, and 0.1 mM in the second series, while the pH of the stimulation solution was 5, and it contained in both series 2 mM Ca2+. The lowering of the Ca2+ concentration in the conditioning solution induced a substantial alkaline shift of the pH dependence of SSD. In the 2 mM Ca2+ condition, the SSD and activation curves did not overlap, while there was a large overlap in the low Ca2+ condition (figure 1d). To avoid channel activation by the conditioning solution, a concentration of 0.1 mM had been chosen for the low Ca2+ condition of the SSD, thus much higher than the 80 nM of the activation experiments. At a Ca2+ concentration of 80 nM this curve would be shifted to more alkaline values, and the overlap would be smaller. To estimate the affinity of the Ca2+ binding site, activation curves at different free Ca2+ concentrations were recorded (figure 1f). The plot of the pH50 values as a function of the Ca2+ concentration (figure 1g) indicates an IC50 of 41 µM. As another measure of Ca2+ binding affinity, the ASIC3 current was determined at pH6.6 at different free Ca2+ concentrations, showing smaller current amplitudes with increasing Ca2+ concentration. The IC50 measured under these conditions was 69 ± 52 µM . This IC50 depends on the pH, since there is a competition between Ca2+ and H+. At pH 7.0 the Ca2+ IC50 was 6.9 ± 6.8 µM .When ASIC3 was expressed in currents bi. If theviously . In thisntration . Controlechanism . When pH. 2.22+ concentration is likely due to a competition for binding sites between H+ and Ca2+ ions. The identification of the amino acid residues of ASIC3 to which Ca2+ binds would indicate where the competition occurs. As a first approach towards this aim, subdomain-based chimeras between ASIC3 and ASIC1a, which is much less Ca2+-sensitive, were constructed , and the subdomains indicated in figure 2a). The inverse chimeras (ASIC3 in ASIC1a) are named ‘CH#’. Most of the constructed chimeras were functional and expressed transient currents . The pH50 values of ASIC1a and ASIC3 WT are quite similar at 2 mM Ca2+ but different at 80 nM Ca2+ . Lowering the Ca2+ concentration from 2 mM to 80 nM Ca2+ induced an alkaline shift of the pH50 values (ΔpH50) of 1.19 ± 0.30 and 0.23 ± 0.22 , respectively, in ASIC3 and ASIC1a (figure 2ci). Of the ASIC1a-in-ASIC3 chimeras, CHB1, CHB2, CHB4 and CHB8 decreased the Ca2+-induced ΔpH50 , suggesting a possible role of the ASIC1a intracellular and transmembrane segments and lower palm , the palm and knuckle (CHB4), and the upper transmembrane and lower palm parts together with the palm-thumb loops and the thumb (CHB8) in reducing this Ca2+ sensitivity. Of the ASIC3-in-ASIC1a chimeras, CH8 showed an increased, and CH3 showed a tendency of increased ΔpH50 (p = 0.089) relative to ASIC1a WT . CH3 contains the upper transmembrane and lower palm parts of ASIC3, whereas CH8 contains the ASIC3 upper transmembrane and lower palm parts together with the palm-thumb loops and the thumb. The currents at 80 nM Ca2+ of both CH3 and CH8 were sustained. These currents activated rapidly upon the pH change; their kinetics and amplitude were thus different from the endogenous currents . The disruption of the desensitization in these chimeras may be due to the sequence changes in the palm, which controls desensitization. For selected chimeras, it was tested whether lowering of the Ca2+ concentration from 2 mM to 80 nM free Ca2+ at pH7.4 induced a current. Figure 2f compares representative current traces of these chimeras to those of non-injected or ASIC3 WT-expressing oocytes. Of the ASIC3-based chimeras, CHB2 generated a slowly increasing current, while the current of CHB4 and CHB8 was not different from that of non-injected oocytes, indicating that in these two chimeras, the activation by low Ca2+ was disrupted. The low Ca2+-induced current, normalized to the low pH-induced current in the same oocyte (the IpH7.4(80 nM Ca2+) / IpH5(2 mM Ca2+) ratio) was high for CH3 and CH8, as expected, even higher than in ASIC3 (figure 2g). Taken together, the chimera analysis highlights the importance of the pore and pore entry for the gating by Ca2+, as well as that of the palm, thumb and knuckle.The observed shift of pH activation curves by changes of the Castructed , table 1. 2.32+ binding sites, MD simulations with a structural model of ASIC3 were carried out in the presence of a high concentration of Ca2+ (100 mM). Analyses were performed to identify the Glu, Asp, Asn, His and Thr amino acid residues of ASIC3, in whose proximity (i.e. at a distance of less than 2.5 Å), a Ca2+ ion was found during a part of the simulation. Figure 3a plots the fraction of time a Ca2+ was close to the indicated residues of the transmembrane and extracellular parts of ASIC3 in two conditions, either without protonation of acidic side chains (Blue squares), or with a chosen protonation corresponding approximately to pH5.5 . We expect that acidification displaces Ca2+ from sites where Ca2+ and H+ compete, thus that at such sites, Ca2+ would not be found in the pH5.5 condition. In MD simulations, Ca2+ ions tend to stick to a site once they have bound. Residues to which Ca2+ bound in most of the experiments are therefore more likely than others to bind Ca2+. The computational analysis led together with inspection of the ASIC3 structural model to the identification of several clusters of residues in whose proximity Ca2+ ions were found. In the lower palm domain, at a subunit interaction site, Glu81, which had during most of the simulation time a Ca2+ ion bound, is close to Asn421 of the same subunit, and to ′Asn370 of a neighbouring subunit . ′Asp282, located further down just above the β-turn, had also a Ca2+ ion bound during most of the simulation time. Asp78 and Glu426 that also scored high are farther away. Glu79 and Glu423 of the lower palm are oriented towards the inside of the central vestibule where these residues of the three subunits may form a Ca2+ binding site (figure 3c). Glu423 did not score in our analysis but may be of interest because it has been suggested to bind 2-guanidine-4-methylquinazoline, which is known to compete with Ca2+ for binding sites [2+ binding site in ASIC1a [figure 3d). Of these, ′Glu212, Asp228, Glu230 and Asp358 all had frequently a Ca2+ in their proximity (figure 3a). The structural image shows that Glu230 and Asp358 are oriented towards each other, while ′Glu212 of a neighbouring subunit is oriented towards Glu235. ′Asp414 has a similar orientation as ′Glu212 and is located on an adjacent, antiparallel strand of the same palm β-sheet (figure 3d). A potentially interesting cluster was identified at the extracellular pore entry (figure 3e). Glu432 and Asp439 are homologous to the two Ca2+ pore-blocking binding residues identified in ASIC1a [To identify candidate Cang sites ,25. Furt. 2.42+ concentrations. Representative current traces of mutants that showed a difference to WT ASIC3 are shown at the two Ca2+ concentrations, at a pH close to the pH50 (b). The pH50 values measured at 2 mM Ca2+ were remarkably constant across mutants (black open circles in the figure 4aii). The largest shifts were observed with the mutations E423A and E426A. E79A produced only a very small current in these conditions, precluding the analysis of the pH dependence. The pH50 obtained at 80 nM free Ca2+ was more affected by the mutations. Mutation of Glu212, located in the palm and facing the acidic pocket (d), induced an acidic shift of the pH50  and decreased the ΔpH50 to 0.84 ± 0.29 , compared to 1.34 ± 0.25 in ASIC3 WT (n = 42–62). Mutation of the close residue Asp414 resulted in non-functional channels. If the ′E212A mutation was combined with the mutation of Glu235, which is oriented towards ′Glu212, the ΔpH50 was not further decreased (figure 4a). The E435A mutation (e), which was previously shown to decrease the low Ca2+-induced current [50 value to 0.77 ± 0.35 , thus similarly to E212A. In ASIC1a, the residues corresponding to Glu432 and Asp439 were identified as binding sites for pore block by Ca2+ [ΔpH50 was similar to the WT value (figure 4a). The D439A mutant did not produce any transient currents. Combination of the mutations of Glu435 of the pore and of Glu212 of the palm, with or without mutation of Glu235, resulted in a strong reduction of the pH50 shift upon lowering of the Ca2+ concentration, with ΔpH50 values of 0.24 ± 0.20  and 0.27 ± 0.27 . Comparison with the WT pH dependence (dashed lines in figure 4c) shows that these mutations affected mostly the channel properties in the 80 nM Ca2+ condition, and much less in the 2 mM Ca2+ condition. Other mutants with a significantly lower ΔpH50 than ASIC3 WT, but with smaller effects, were E230A/D358A, E322A and E426A . E230 and D358 are located in the acidic pocket (d), E322 just below, in the α4 helix of the thumb, while E426 is in the wrist (b). Measurement of the low Ca2+-induced current at pH7.4 demonstrated a decrease of the IpH7.4(80 nM Ca2+) / IpH5(2 mM Ca2+) ratio by the single mutations (figure 4d), confirming further the importance of these two sites for modulation by Ca2+. The low Ca2+-induced currents of the mutant E212A/E435A showed a slowly developing sustained current without a clear peak that could not be reliably quantified. The mutational analysis indicates therefore that two distinct Ca2+ binding sites, one in the pore entry and one in the palm/acidic pocket, contribute to the Ca2+ modulation of ASIC3 activation, consistent with the analysis of the chimeras.Based on the predictions of the MD simulations and of visual inspection of the ASIC3 structural model, approximately 30 residues—most of them acidic residues—were mutated to Ala, and the pH dependence of activation was determined at the two Cathe pH50 b. The pHc pocket d, inducemutation e, which c pocket d, E322 jhe wrist b. Measur. 2.5e) or D414A of the acidic pocket  in one or two of the subunits was not different from that of WT (a), thus it is unclear why the conventional mutants D282A, N421A and D414A, carrying these mutations in the three subunits, were not functional. The analysis of the concatamers provided the most interesting results in the pore region, where the presence of the mutation D439A or the double mutation E435A/D439A in one subunit of the trimer led to a ΔpH50 of 0.88 ± 0.39  and 0.69 ± 0.40 , respectively, compared to 1.20 ± 0.22 of the WT concatamer ABC . In the construct containing the D439A mutation in two of the three subunits, the ΔpH50 was 0.26 ± 0.38 , highlighting the importance of Asp439 for the Ca2+ modulation of the ASIC3 pH dependence. Representative current traces of concatamers with different activation pH dependence from WT are shown in figure 5b. Consistent with the above observations, the IpH7.4(80 nM Ca2+) / IpH5(2 mM Ca2+) ratio was low in the concatamers E435A/D439A-A and D439A-AB (figure 5d). This indicates that residues Glu435 and Asp439 contribute together to the Ca2+ binding site in the pore entry.Several mutations resulted in channels that did not express transient currents, among them mutations of Asp439, the residue homologous to a pore-blocking site in ASIC1a. To allow the study of such sites, concatamers were constructed in which residues of interest were mutated only in one or two subunits of the channel trimer. All tested concatamers carrying mutations in one or two subunits, except the one containing the double mutation E435A/D439A in two subunits of the trimer, produced measurable, transient currents. The pH dependence of concatamers carrying the mutations D282A or N421A of the wrist e or D414at of WT a, thus i2+ depended on the membrane potential, complete pH dependence curves were carried out with ASIC3 WT at either −100 or 0 mV. The pH50 values obtained were not different between these two conditions (e). To have a more direct comparison than activation curves obtained in different cells, the ratio of the current induced by a pH of the steep range of the pH-current curve, divided by the current induced by a pH generating maximal activation, was measured at −100 and 0 mV at different Ca2+ concentrations in paired experiments in the same cell (f). This IpH6.x/IpH4.5 ratio is sensitive to changes in the pH dependence. If Ca2+ binding to sites in the electrical field of the pore competes with pH-dependent activation, the IpH6.x/IpH4.5 ratio would be lower at the membrane potential of −100 mV. In contrast with these expectations, the IpH6.x/IpH4.5 ratio was significantly lower at 0 mV in the condition with 5 mM Ca2+ and showed no voltage dependence with 2 and 10 mM Ca2+ (f). This suggests that the Ca2+ binding site at residues Glu435 and Asp439 is located outside the electrical field of the pore, unlike the binding site of diminazene, which comprises residues corresponding to Gly442, Gly445 and Leu446 in ASIC3, and is closer to the selectivity filter [To test whether the modulation of the pH dependence by Canditions e. To havame cell f. This I mM Ca2+ f. This sy filter ,28.. 2.62+ concentrations in the conditioning solutions, 2 mM and 0.1 mM. The pHD50 shift induced by the lowering of the Ca2+ concentration from 2 to 0.1 mM was 0.27 ± 0.07 with ASIC1a, and 0.40 ± 0.07 with ASIC3 (n = 10–24 (a–c). The SSD curves showed some shifts in the pH dependence of SSD (b); however, the ΔpHD50 was in all cases except for CH6 close to the parent channel type value (c). Due to the high variability in the CH6 ΔpHD50 values, the CH6 ΔpHD50 was not significantly different from that of ASIC1a WT. In the chimera CH6, the thumb and part of the thumb-palm linkers is replaced by the corresponding sequence of ASIC3.The SSD of the chimeras that generated a transient current was measured at two different Ca = 10–24 a–c. The e of SSD b; howevepe value c. Due to50 values in the mutants E212A, E212A/E235A and E212A/E235A/E435A (d–f). Mutation of the pore residue Glu435 did not affect Ca2+ modulation of the SSD. This analysis identifies therefore E212 and E235 as important for SSD. These residues are located in the acidic pocket and are part of the palm and finger, respectively (d). Measurement of concatameric channels showed low ΔpHD50 values with N421A-AB and N421A/E423A-AB (g–i). Because the mutation E423A induced an alkaline shift of the pHD50 at both Ca2+ concentrations but did not decrease the ΔpHD50 , the observed decrease of the ΔpHD50 with N421A/E423A-AB identifies Asn421 as important for the Ca2+ modulation of the SSD. Asn421 is located in the lower palm (c). To combine the SSD-affecting mutations in the acidic pocket and wrist, we constructed a concatamer containing the mutations E212A and E235A in all three, and N421A in two subunits. This construct expressed only very small currents that were mostly sustained and did therefore not allow us to determine the Ca2+ dependence of SSD. Taken together, the analysis with chimeras and two mutagenic approaches identified Glu residues 212 and 235 of the acidic pocket, and Asn421 of the palm as contributing residues to Ca2+ modulation of SSD.Functional analysis of ASIC3 mutants showed low ΔpHD5A/E435A d–f. Mutaectively d. MeasurE423A-AB g–i. Becae ΔpHD50 e,f, the wer palm c. To com. 32+ tunes the pH dependence of ASIC3. We show here that at high Ca2+ concentrations, the pH dependence of activation and of SSD is shifted to more acidic values. We identify residues in the pore entry and in the acidic pocket that are critically involved in the regulation of ASIC3 pH dependence of activation by Ca2+, and residues of the acidic pocket and the palm that are involved in the modulation by Ca2+ of the pH dependence of SSD. Together, our data show that Ca2+ binding sites outside and within the pore entry are involved in the control of ASIC3 activity by Ca2+.Extracellular Ca2+ concentrations in the brain of mammals are between 1 and 2 mM. During high neuronal activity, this concentration decreases to approximately 0.8 mM or lower [2+ concentration (to approx. 0.1 mM) occurs [2+ chelator, this results in a lower concentration of free Ca2+ [2+ concentrations change under some conditions, and since Ca2+ and other ligands compete for ion channel binding, this can thereby affect the activity of ASICs and other ion channels.Basal interstitial Caor lower ,30. Foll2+ concentration shifts the voltage dependence of voltage-gated Na+ channels to more hyperpolarized potentials [2+ concentrations [2+ concentration. It is inhibited by extracellular Ca2+ with an IC50 of 320 µM at −80 mV; this inhibition is likely due to a pore block, since it is strongly suppressed by mutations of selectivity filter residues [2+ concentration. Reducing the Ca2+ concentration increases the channel open probability [50 for CALHM1 inhibition by Ca2+ is approximately 200 µM at −60 mV. Neutralization of an extracellular Asp residue that is not in the permeation pathway was shown to strongly alter the regulation by Ca2+ [Lowering of the extracellular Catentials ,31. It wtrations . The sodresidues . The calbability . The IC5 by Ca2+ .2+ modulation of ASIC3 activation are located in the acidic pocket and the pore entry (a). This is consistent with the analysis of chimeras indicating a contribution of the transmembrane segments, the thumb and the palm. Mutation of Glu435 in ASIC3 suppressed only partially the low Ca2+-induced current at pH7.4 in the study by Zuo et al. [2+-induced shift in the activation curve to a similar degree. In concatamers, the presence of the D439A mutation in two of three subunits inhibited the Ca2+ modulation to a higher degree than the single E212A and E435A mutation in the three subunits , underlining the importance of Asp439 for Ca2+ modulation of ASIC3. Of these three residues, both Glu212 and Asp439 are conserved between ASIC3 and ASIC1a; only Glu435 is unique to ASIC3. The different roles of these identical residues in ASIC1a and ASIC3 is therefore likely due to (i) differences in the amino acid residues or conformation in the proximity of these residues that may affect the Ca2+ affinity and/or (ii) a different coupling of these Ca2+ binding sites to ASIC activity.Our mutagenesis approach showed that the most important residues for Care entry a. This io et al.  and in o50 did this mostly by lowering the pH50 at the 80 nM Ca2+ condition, and with no, or only minor changes of the pH50 at 2 mM Ca2+. Since these mutations disrupt the binding of Ca2+, it would seem more logical that they have a stronger effect at the higher Ca2+ concentration. As a possible explanation for this apparent discrepancy, we hypothesize that the charge of the acidic side chains at the Ca2+ binding sites may allow the high pH sensitivity in the absence of Ca2+. If such acidic side chains are removed by mutagenesis, the channels may lose the possibility of gating with high pH sensitivity.The mutations that decreased the ΔpH2+ and H+ (b). The SSD analysis further highlighted the contribution of Asn421 located in the lower palm, which may form a Ca2+ binding site together with Glu79 and Glu423. These residues are also conserved between ASIC3 and ASIC1a.The analysis of SSD showed that the acidic pocket residues Glu212 and Glu235 are important for the competition between Ca+ and H+ b. The SS2+ block, corresponding to ASIC3-Glu432 and -Asp439, suppressed the pore block but not the Ca2+ modulation of the pH dependence, indicating that the Ca2+ binding site for gating is different from that for pore block in ASIC1a [+ and Ca2+ and concluded, based on a detailed functional analysis, that this channel is activated by an allosteric mechanism and not by a release of Ca2+ block [For ASIC1a, it was shown that mutation of the two residues in the ASIC1a pore that contribute to Can ASIC1a . A study2+ block ,34.2+ binding—the shift of the activation curve induced by lowering of the Ca2+ concentration was increased relative to that seen in WT cASIC1a [2+ activated the channel at pH7.4 [2+ concentration shifts the pH dependence of ASIC3.At the homologous position to ASIC3-Glu435, ASIC1a contains a Gly residue (Gly430). When the homologous Gly residue in chicken ASIC1a (cASIC1a) was mutated to Glu—in order to create a site favourable for Ca cASIC1a . In thisat pH7.4 , indicat+ and Ca2+ for the activation of ASIC3 concluded that this competition occurs in the pore entry, based on the good fit by a kinetic model of the H+ and Ca2+ dependence of the currents, and the dependence of the unitary current amplitude on the Ca2+ concentration [2+ bound to the pore entry, without any conformational changes involved. This conclusion was later supported by the demonstration of the involvement of Glu435 in the Ca2+ modulation [2+ binding site [2+ can bind with reasonable affinity even if it is hydrated. In such cases, the ion is placed 4–5 Å from the interacting side chains [2+ binds into the open ASIC3 pore. The ASIC3 structural model is based on the chicken ASIC1a structure [+/K+ permeability ratios, suggesting that at least at the selectivity filter they have similar pore radii. Assuming that the pore dimensions are correct in the ASIC3 open channel model, the three Asp439 side chains would not be able to coordinate binding of a Ca2+ ion between them, because of side chain O–O distances of greater than 12 Å. At the level of Glu435, the intersubunit carboxylic O–O distances are also close to 12 Å. The closest distance between side chain oxygens of Glu435 and Asp439 of the same subunit is 6.9 Å; therefore, it seems also unlikely that Glu435 and Asp439 would together coordinate a Ca2+ ion in the open ASIC3 pore. This indicates that in a pore conformation compatible with ion flow through the ASIC3 pore, the Glu435 or Asp439 residues of the three subunits cannot coordinate a Ca2+ ion, strongly suggesting that conformational changes in the pore accompany ASIC3 activation.The original study describing the competition between Hntration . It was dulation . A strucdulation . In the dulation , thus coing site ,37. In be chains . Accordie chains , Ca2+ bitructure . ASIC1a 2+ bound to the pore entry. At binding sites distant from the pore, Ca2+ interferes with allosteric gating by competing with H+. The competition for binding sites in the pore leads to release of Ca2+ from the pore when the pH is lowered.Based on our functional analysis and the comparison with the ASIC1a open pore dimensions, we conclude that ASIC3 activity is controlled by a hybrid mechanism involving on one hand conformational changes induced by protonation of an allosteric site, and on the other hand a release of Ca. 4. 4.1β globin to improve the stability in Xenopus laevis oocytes. The ASIC1a clone used in this study contains a G212D substitution [The rat ASIC3 clone  and the titution . Mutatiotitution  and weretitution . The lin. 4.2Xenopus laevis frogs were anaesthetized with 1.3 g L−1 MS-222 (Sigma-Aldrich). For the extraction of oocytes, a small incision (approx. 1 cm) was made on the abdominal wall. The procedures with the Xenopus laevis frogs were approved by the veterinary office of the canton de Vaud. Healthy stage V and VI oocytes were isolated and incubated with collagenase to isolate and defolliculate the oocytes. Oocytes were injected with 50 nL of cRNA at a concentration of 20–500 ng µL−1. During the protein expression phase, the oocytes were stored in modified Barth's saline containing (in mM) 85 NaCl, 1 KCl, 2.4 NaHCO3, 0.33 Ca (NO3)2, 0.82 MgSO4, 0.41 CaCl2, 10 HEPES and 4.08 NaOH at 19°C.Female Xenopus oocytes express endogenous connexin hemichannels, and that lowering of the extracellular Ca2+ concentration can induce inward currents [2+ to a solution containing 100 nM free Ca2+ induced a slowly developing inward current that still increased at the end of the 10 s solution change . Ca2+ was previously shown to inhibit these endogenous currents with an IC50 of approximately 100 µM at −60 mV [It has been reported that currents . Controlt −60 mV ; therefo50 of 5.41 ± 0.41 . The peak of ASIC currents appears normally within the first second after solution change (see e.g. b). The amplitude of the current induced in non-injected oocytes by lowering of the Ca2+ concentration to 100 nM at pH7.4, measured at 3.5 s after the solution change (which would be after the ASIC peak in ASIC-expressing oocytes) was −93 ± 50 nA . To estimate the consequences of the presence of endogenous low Ca2+-activated currents for the analysis of the ASIC3 pH dependence, pH dependence curves were generated in silico with Hill equations describing the pH dependence of the ASIC3 WT and of endogenous low Ca2+-activated currents (I = Imax/(1+(10−pH50/10−pH)nH), where Imax is the maximal current amplitude, pH50 is the pH inducing 50% of the maximal current amplitude and nH is the Hill coefficient). For ASIC3 WT, the pH50 and nH were 7.48 and 2.08, respectively, as determined in figure 1d. For the endogenous currents, the fit parameters determined in the electronic supplementary material, figure S1B were used. Activation curves were generated for pure ASIC3 and for conditions in which the endogenous current contributed 5, 10, 15 or 20% to the total maximal peak current amplitude . As expected from the opposite pH dependence of the two current types, contamination by endogenous currents affected the resulting pH dependence mostly at pH > 7.3). An endogenous current amplitude corresponding to 10% of the maximal total peak current induced for example an alkaline shift of the pH50 value of 0.07 pH units and a decrease of nH from 2.08 to 1.57. With the measured amplitude of the endogenous current of 93 nA, such an error would be expected for ASIC currents with a maximal peak amplitude of 0.93 µA. Of the ASIC constructs used in this study, the maximal peak current in activation curves with 80 nM Ca2+ was 0.92 µA for the chimera CHB6, 1.84 µA in the concatamer N421A/E423A-AB, and greater than 2 µA for all other constructs , indicating therefore that the endogenous currents did not affect the measurement of the ASIC pH dependence. The current traces of experiments with low ASIC current amplitudes at 80 nM Ca2+ were visually inspected and were only used if at pH conditions of greater than or equal to 7.3, the amplitude of the slowly activating endogenous current was less than 5% of the maximal low pH-induced current amplitude. For SSD experiments, the conditioning solution contained either 2 mM or 0.1 mM Ca2+. If at pH7.4, the Ca2+ concentration was lowered during 10 s to 0.1 mM, the current amplitude relative to the baseline at pH7.4/2 mM Ca2+ was 15 ± 9 nA at pH7.4 and 12 ± 8 nA at pH8.5, thus negligeable .Interestingly, the amplitude of the endogenous currents was smaller at pH6 and was inhibited with a pHsee e.g. b. The am2+ concentration. The pH was adjusted using NaOH or HCl. For Ca2+ concentrations greater than or equal to 0.1 mM, the Ca2+ was provided as CaCl2, and no Ca2+ chelator was added. For Ca2+ concentrations less than 0.1 mM, Ca2+ chelators at a concentration of generally 10 mM were used and total Ca2+ concentrations were chosen based on the MaxChelator program  together with an eightfold perfusion head was used to change solutions. Currents were filtered at 2 kHz and the sampling interval was 20 ms. The oocytes were exposed once per minute for 5–15 s (according to the specific protocol used) to the stimulation solution. The pH activation curves were fit to the Hill equation that is described above. An analogous equation was used to fit the SSD curves.Electrophysiological recordings were performed 1–3 days after cRNA injection as described . The curaxcS.htm ) to obta. 4.32+, to which Na+ and Cl− ions are added to neutralize the whole system and mimic a salt concentration of about 150 mM. Following an approach developed by Woolf & Roux [The structural model of ASIC3 used here was created with SWISS-MODEL  based onf & Roux , simulatf & Roux .2+ binding sites which are in competition with protonation of side chains, we ran two simulations with all acidic side chains deprotonated  for a total of 520 ns of simulation time. One of these two simulations additionally had a transmembrane potential of 200 mV to favour the entry of Ca2+ ions into the pore to identify potential residues involved in Ca-dependent pore block. A third simulation was run for 200 ns with protonation states of Glu and Asp residues adjusted to mimic pH5.5, thus with side chains with pKa ≥ 5.5 deprotonated; the pKa values were estimated by propKa [As we were interested in potential Cay propKa ,50. In t. 4.4t-test and differences between greater than two groups were analysed with ANOVA  followed by a Dunnett's post hoc test. Data are presented as mean ± s.d. or as individual data points. The significance of the difference of ΔpH50 or ΔpHD50 values between mutants and WT channels was assessed using a permutation test, with n = 10 000 permutations, using the R statistical language (version 4.2.1).The fits and the statistical analyses were carried out with Graphpad Prism, Version 9. Statistical differences between two groups were analysed with student's