Here, we show that TRPC3 null (−/−) mice presented with a reduction in Ca2+ entry and current in ductal cells with higher saliva [Ca2+] suggesting diminished transepithelial Ca2+ flux across the salivary ductal cells, leaving more Ca2+ in ductal fluid. Significantly, we found that TRPC3 was expressed in mice and human salivary ductal cells, while intraductal stones were detected in both mice (TRPC3−/−) and patient  salivary glands. To identify the mechanism, we found that TRPC3 was crucial in preventing the expression of calcification genes  in ductal cells which may be due to higher extracellular Ca2+ in SMG tissues. Similarly, inflammatory , fibrotic  and apoptotic (Bax1/Bcl2) markers were also elevated, suggesting that the loss of TRPC3 induces genetic changes that leads to salivary gland cell death and induction of inflammatory response. Overall, ablation of TRPC3−/− leads to higher saliva [Ca2+], along with elevated detrimental gene expressions, altogether contributing to salivary gland stone formation.Clinical studies and structural analyses of salivary stones strongly suggest a linkage between higher saliva calcium (Ca A large percentage of these sialoliths are found most frequently in the submandibular gland (SMG) ducts2. However, the exact etiology and pathogenesis of calculi formation are mostly unknown. Importantly, shock wave lithotripsy is a common procedure performed without damaging the adjacent tissue3. Although, failure of this procedure and the large size of an intraglandular stone often limit treatment and lead to the removal of the gland. Both of these therapeutic options carry risks such as use of anesthetic, facial nerve damage, surgical scar, Frey's syndrome, and dry mouth4. Therefore, understanding the molecular and cellular basis of sialolithiasis will help us to pinpoint the etiology and pathogenesis of this disease. It has been proposed that the genesis of Ca2+ rich saliva causes relative stagnation of salivary flow. This result in a deposition of Ca2+ salts around an initial organic nidus consisting of altered salivary mucins, bacteria, and desquamated epithelial cells, with subsequent progressive accretion forming a stone5.Sialolithiasis is a condition that involves the formation of calcified stone masses  in the salivary gland that eventually obstructs saliva flow and is considered to be one of the most commonly diagnosed salivary gland diseases2+ stones and vascular calcification in human salivary glands6. Furthermore, our studies on kidney stones revealed that the increased [Ca2+] in kidney tubular fluid can develop the risk of CaP crystal formation7, while elevated tubular epithelial cells [Ca2+]i invokes changes in Ca2+ signaling pathways and develop detrimental process by activating excessive reactive oxygen species (ROS) production, and ultimately apoptosis8. Moreover, both high Ca2+ diet or high luminal pH, exhibited a greater increase in Ca2+ entry, along with inflammation, oxidative stress, apoptosis, and fibrosis10. Since calcium overload is followed by an abnormal calcium homeostasis, leading to cell injury and apoptosis, the cell disintegration frequently serves as the nidus of calcification11. Importantly, apoptosis could also lead to inflammation, that can also induce calcification along with fibrosis12. Apoptosis was evident before the onset of calcification and was followed by an abnormal Ca2+ and Pi homeostasis to induce cell injury and apoptosis13. Calcification can also provoke immune response to injury site where tissue damage triggers an inflammatory response followed by collagen breakdown and calcification14. Moreover, calcium-containing crystals can also provoke inflammation by triggering the secretion of inflammatory cytokines15.We investigated the calcium phosphate (CaP) stone formation in a clinical setting and found the coexistence of ductal Ca2+]) in saliva and sialolithiasis. Sialolithiasis starts with the formation of calculi; and patients with calculi and sialolithiasis are shown to have higher [Ca2+] in their saliva16. We found that SMG ductal cells expressed calcium-sensing receptor (CaSR) which was co-localized with transient receptor potential canonical 3 (TRPC3) ion channel and mediated Ca2+ entry at the apical membrane17. Such process may constitute the transcellular pathway to represent a potential Ca2+ reabsorption mechanism that can account for the decrease in [Ca2+] in primary saliva as it flows down the ductal system. Additionally, SMG tissues from sialolithiasis patients revealed a greater area of fibrosis, inflammation, calcification, and apoptosis compared to the normal salivary gland12. Studies of intracellular microcalcification in ion-transporting epithelia revealed pathophysiological Ca2+ signaling pathways, in which disruption of Ca2+ regulation resulted endoplasmic reticulum (ER) and oxidative stress19. However, the link between disruption of Ca2+ signaling and the ROS mediated downstream mechanism (ER stress or oxidative stress) in SMG ductal cells is unclear. In this study, we utilized our TRPC3 knock out  mouse model to examine the physiological implications of the TRPC3 knockdown, and how its compromised Ca2+ signaling could potentially lead to fibrosis, inflammation, calcification, and apoptosis within the SMG tissue, while demonstrating an important role that TRPC3 has in potentially mitigating these effects, and eventually preventing stone formation in the salivary gland.Experimental and clinical studies strongly suggest a link between high calcium concentration , Pyr6, and Pyr10 were purchased from Sigma-Aldrich . Fura-2-acetoxymethyl ester (Fura-2-AM) was purchased from Invitrogen . All the chemicals used were analytical grade.-/- mice were purchased from the Jackson Laboratory  and were maintained and crossed previously described7. Mice were anesthetized using i.m. injection of ketamine [60–80 mg/kg] and xylazine [8–10 mg/kg] before subcutaneous injection of pilocarpine. All mice were euthanized by CO2 asphyxiation from a compressed gas tank followed by cervical dislocation (secondary measure). Animals were euthanized by gradual displacement in home cage to ensure comfort of the animal and no sudden change in environment. The displacement rate for CO2 was at a rate of 30% to 70% displacement per minute. This study has been performed under ARRIVE guidelines.Experimental procedures for mice are part of an approved protocol (MIRB-01422) designed according to the Guiding Principles in the Care and Use of Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) and the Research and Development Committee of DC Veterans Affair Medical Center. Both Wild Type (WT) and TRPC37. Extracellular solution was comprised of NaCl (140 mM), KCl (4 mM), MgCl2 (1 mM), CaCl2 (2 mM), D-glucose (5 mM), and HEPES . Intracellular solution was comprised of (in mM): CsCl (50 mM), NaCl (10 mM), CsF (60 mM), EGTA (20 mM), and HEPES . Data was obtained at 5.00 kHz and filtered at 2.873 kHz. Current–voltage (I–V) characteristic curve was obtained every 3 s, by application of voltage ramps (300 ms) from −100 mV to + 100 mV, with a holding potential of −80 mV. Patch clamp has membrane resistance > 500 MΩ.Whole-cell patch clamp recordings were performed using an EPC-10 digitally controlled amplifier and Patchmaster software  at room temperature as described2+]i measurements were performed as described17. After loading with Fura-2-AM, isolated SMG ductal cells were placed on an IX81 motorized inverted microscope equipped with an IX2-UCB control box . Time-lapse images were captured using IX81 microscope images that were fed into a C9100-02 electron multiplier CCD camera with an AC adaptor A3472-07 . Lambda-LS xenon arc lamp and 10–2 optical filter changer  were used as an illuminator capable of light output from 340 and 380 nm to a cutoff of 700 nm. Experiments were performed in a microincubator set at 37 °C with a gaseous mixture of 5% CO2/95% air. Ratiometric (340/380) measurements of [Ca2+]i were recorded using digital microscopy imaging software . Fura-2 ratiometric fluorescence measurements were recorded at an emission peak absorbance at 500 nm wavelength with an excitation peak absorbance alternating between wavelengths of 340 and 380 nm. Time-lapsed measurements were set to 120–500 time points at a 1 s interval. 50–150 cells were individually selected as regions of interest . Capture analyses and normalization were performed offline using Slidebook™ software and further quantitated with statistical analysis using Origin 6.1.Fura-2  for 30 min. Nuclear counterstaining of human SMG sections was performed according to manufacturer’s instructions by mixing a 4',6-diamidino-2-phenylindole  working solution of 100 ng/mL diluted in 1 × PBS. After washing the sections once with 1 × PBS, the working solutions was applied to the sections in the dark for 30 min at room temperature. Sections were visualized with Zeiss 710 confocal microscopes and were further analyzed using Zeiss software (Zen 2010). In our labeling controls (for background subtraction), isotypes were used instead of the primary anti-bodies and then the secondary antibodies  were added.Patient SMG sections were prepared as described12. Alizarin Red (AR) pH 4.3  stainings were performed as previously described22. Von Kossa stainings were performed to detected calcifications as described previously12. In situ apoptosis in mice tissue sections were performed using TACS•XL DAB in situ Apoptosis Detection Kit , as per manufacturer’s directions and our previously published method on mice kidney tissue section9.Human and mice tissue sections (∼5–7 µm) were prepared from SMG tissues collected from mice or biopsied from patients, which were fixed in a 10% formalin solution for 24 h and then dehydrated in ethanol and embedded in paraffin. Histochemistry was performed on paraffin sections of mice SMG tissues, as described previously−/− mice were anesthetized and whole saliva was collected after stimulation with pilocarpine (0.5 mg/kg) body weight as described23. The osmolality and ion concentrations were measured as described24.WT and TRPC3t-test, followed by a one-way ANOVA test as required, in Origin 6.1 software. Significant difference levels were set at p < 0.05 (*) orp < 0.01 (**). Details of all statistical analysis are mentioned in figures and figure legends.Data were quantitated in mean ± SEM and were analyzed using two-tailed Specimens are formalin fixed paraffin embedded (FFPE) de-identified tissue section  from Georgetown University tissue bank through an exempt Institutional Review Board (IRB) protocol . This IRB exemption was also approved by the IRB and the Research and Development committee of the Washington DC VA Medical Center.All methods were performed in accordance with the relevant guidelines and regulations.2+ homeostasis has been suggested to serve as the nidus of calcification in salivary gland5. Our previous studies have identified that TRPC3 could assist in renal stone formation; however, the molecular identity of the calcium channel in salivary gland and the mechanism leading to stone formation is not well known. Thus, we evaluated the role of TRPC3 in salivary gland function. Expression of TRPC3 was established at both gene (mRNA levels) and protein levels, where salivary glands isolated from control mice (WT) showed expression of TRPC3, which was absent in TRPC3−/−  compared to the WT mice  [Ca2+]i measurements, by utilizing L-Phe as a positive allosteric CaSR modulator, and two known pyrazole compounds Pyr6 and Pyr10, to inhibit the store-operated Ca2+ entry (SOCE) and receptor operated Ca2+ entry pathway (ROCE), respectively25. In WT cells, CaSR activation by L-Phe has yielded a small Ca2+ release (1.0 to 1.2), followed by a transient Ca2+ entry peak (1.0 to 2.23) upon Ca2+ replenishment  than by Pyr6 , suggesting that the majority of the Ca2+ entry pathway and possible means of extracellular Ca2+ regulation, upon CaSR activation, is through the ROCE pathway , but also a lower Ca2+ entry  cells, confirming the absence of TRPC3  control and stone patient SMG tissue sections , PMCA1, PMCA2, PMCA3, and PMCA4, exist in SMG ducts with no significant difference (p > 0.05) in expression levels between WT and KO mice  and calbindin (CaBP)-D9k and -D28k as well as anion solute carrier family 26  in SMG ductal cells from TRPC3 KO mice compared to WT mice -2, -6, and -7, osteocalcin 2 (OCL2), osteopontin 4 (OPN4), and runt-related transcription factor 2 (RUNX2) have been known to be osteogenic factors that can lead to stone formation29. RT-PCR analysis showed that the following genes were upregulated in SMG from KO mice when compared to WT mice: BMP2 (0.43 vs. 0.62), BMP6 (0.37 vs. 0.7), and RUNX (0.4 vs. 0.51)  upon absence of TRPC3  and nearly no effect on BMP-2, OCL2, and OPN4 expression in protein expression  compared to WT sections  and interleukin 6 (IL-6) were strongly increased by 1.8 and 1.5-fold in TRPC3 KO mice  and fibronectin 1 (FN-1) in SMG ductal cells in TRPC3 KO mice was increase by 1.2-fold while no significant changes were detected for other markers such as α-smooth muscle actin  which was once believed to be a passive process, is now accepted as an active and highly regulated process similar to bone formation as it shares many of the transcriptional and regulatory signaling pathways32. Salivary gland acinar cells create primary saliva, an isotonic fluid with a relatively high [Ca2+] which flows throughout the salivary duct system33. Although earlier studies have shown that salivary [Ca2+] decreases as it flows down the ductal tree into the oral cavity, ductal reabsorption of Ca2+ remains enigmatic34. Subsequently, the duct modifies the electrolyte composition to produce hypotonic final saliva, called secondary saliva, with no apparent loss in volume31. Like in other epithelia, ductal epithelia mediate Ca2+ reabsorption via two major pathways, paracellular and transcellular routes35. The data presented here clearly shows that loss of TRPC3 not only decreases saliva secretion, but also affect calcium reabsorption, which could be the main factor leading towards stone formation in salivary glands17. This observation indicates that the overall levels of Ca2+ and PO43− in saliva could play in the calcification/stone forming mechanism7. Other TRPCs, such as TRPC6 is structurally similar to that of TRPC3 and have been shown to present in apical and basolateral membrane of salivary ductal cells36. Unlike TRPC3 , TRPC6 although present apically, it is predominantly basolateral, but TRPC7 did not express in SMG ductal cells20. Moreover, we did not see any compensation (increase) in the expression of TRPC6 in TRPC3 KO mice . Moreover, our functional data in Fig. 2+ reabsorption due to CaSR activation since this activity was severely disrupted in TRPC3 KO mice. Furthermore, Pyr10, the TRPC3 inhibitor, we used, has been found to be more selective to TRPC3 than TRPC637.While the biomineralization of CaP is essential to the maintenance of healthy bones and teeth, this process also plays a considerable role in stone formation as the build-up of CaP crystals is the nidus for the development of salivary gland stones2+ level in saliva observed in TRPC3-/- mice could also potentially favor the formation of calcium deposits while salivary fluid moving along the duct17. Salivary secretion is regulated by the hypothalamus, mainly the lateral hypothalamus (LH) in the central nervous system, which controls SMG secretion through the superior and inferior salivatory nuclei in the brain stem38. TRPC channels are highly expressed in hypothalamus to maintain the excitability of hypothalamic neurons39. However, it is still unclear whether the absence of TRPC3 in the central nervous system is involved in this process since we collected the saliva after parasympathetic stimulation in global TRPC3−/− mice. It is possible that the systemic application of cholinergic (pilocarpine) agonist that we used to stimulate the parasympathetic secretion in mice may have lost some influence exerted by the LH in global TRPC3 KO mice. Although studies show minimal reduction (~ 20%) in SMG secretion due to lesions in those hypothalamic area, however, it is not clear how much of such reduced in salivation is due to the loss TRPC3 in LH40. Moreover, hypothalamic area is highly involved in the mastication-salivary reflex and the involvement of cholinergic pathway or TRPC channel in this process is unknown. Another point is that loss of TRPC3 in acinar cells may be involved in reduced salivation41, although drastic reduction in saliva secretion was shown in TRPC1-/-mice using similar method23. Thus, gradual autonomic denervation in TRPC3-/- mice may be the way to show the influence of salivary center of the midbrain, which is beyond the scope of the present study38.Reduction in the amount of saliva secretion as well as elevated Ca2+ flux and providing evidence that TRPC3 was critical for this process. Interestingly, concomitant increases in Ca2+, calcium-binding proteins, Pi, and phosphatases in blebs form matrix vesicles with membranous cellular degradation products has been shown to result from cell disintegration frequently serves as the nidus of calcification42. These previous studies are consistent with our data where increase in apoptosis was observed in TRPC3 KO mice. Moreover, the ER is sensitive to alterations in homeostasis from a variety of different stimuli, including perturbation of calcium homeostasis and CCAAT-enhancer-binding protein homologous protein (CHOP) has been a classical marker for apoptosis43. Interesting, increase in CHOP expression, along with increased Bax/Bcl2 ratio was observed in TRPC3 KO mice salivary glands. CHOP, a transcription factor induced under ER stress, has been reported to be involved in ER stress-induced apoptosis by reducing the expression of Bcl-244. Indeed, CHOP-deficiency causes resistance to ER stress-induced cell death both in vitro and in vivo45. Caspase-12 is also known to be essential for this ER stress-induced apoptosis, which was also increased in TRPC3 mice46. Together, these results suggest that loss of TRPC3 induces salivary gland cell death, which can contribute to stone formation.Immunofluorescent staining of mouse SMG sections further revealed distribution and localization of essential components involved in transcellular Ca47. Additionally, evidence shows that calcium-containing crystals can provoke inflammation by triggering the secretion of inflammatory cytokines48. Published data on physiological calcification are compared with findings in various dystrophic calcinoses. These findings led to the conclusion that apoptosis most likely underlies the mechanism of both physiological and pathological calcifications42. Indeed, human SMG stones are shown to be associated with inflammation, fibrosis, and microcalcifications in the surrounding tissues12. However, the mechanism underlying tissue degeneration leading to calcification is not fully known. Our data suggest that IL-1β-driven inflammation, that leads to the NLRP3 could be the possible mechanism that is activated upon the loss of TRPC3. Interestingly, crystal induced IL-6 activation was also observed in TRPC3 KO mice kidney cells9, suggesting a possible NF-κB–NLRP3–IL-1β pathway. Similarly, elevated IL-6 expression could be supported by our recent findings of ROS-induced activation IL-6 in human kidney cells48. Since CaP crystals can trigger the secretion of inflammatory cytokines which themselves are able to trigger the formation of calcifications, it is likely that there is a positive feedback loop between calcification and inflammation49.We further evaluated as to why cell death was observed. Importantly, previous studies have shown that pathological calcification and chronic inflammation induces an immune response to injury in which tissue damage triggers an inflammatory response followed by collagen breakdown and further calcification driving disease progression6. Fibrosis is primarily driven by inflammatory cytokines including the IL-1β and TGF-b150, which can influence apoptosis12. Moreover, fibrosis is a normal consequence of tissue injury and chronic inflammation and TGF-β1 is shown to stimulates the NLRP3 inflammasome51. Such process is thought to sense the disturbance of cellular homeostasis rather than directly recognizing a common motif present in its activators, and multiple cellular signals have been proposed to trigger its activation, including K+ efflux, Ca2+ signaling, mitochondrial dysfunction, and lysosomal rupture11. Fibrosis and inflammation in lung were also shown to be elicited via a Bax-dependent, Bid-activated Pathway52. These results are consistent with our data and taken together, these findings suggest that inflammation was more involved in SMG stone formation. Importantly, IL-6 is a known downstream target of IL-1β and is consistently increased in serum from patients with NLRP3 inflammasome-mediated conditions, elucidating the possible mechanism as how TRPC3 modulates the immune response that may be implied in salivary stone formation.While SMG calcification and stones co-exists, there is little information about the accompanying cell injury-repair process, apoptosis, and cell proliferationSupplementary Information.


However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca2+ leak.O-GlcNAcylation is the enzymatic addition of a sugar, O-linked β-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2.O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca2+ leak.We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in CaThese data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.The online version contains supplementary material available at 10.1186/s12933-023-02010-3. This mechanism is commonly known as Ca2+ -induced Ca2+ release (CICR) as Ca2+ is released from RyR2 in response to Ca2+ influx through L-type Ca2+ channels [2+ within the SR in a process termed store overload induced Ca2+ release (SOICR) [The cardiac ryanodine receptor RyR2) is responsible for the release of Cachannels . In addi is respo (SOICR) .2+/calmodulin dependent kinase II (CaMKII) and protein kinase G (PKG). Phosphorylation of these sites enhances Ca2+ release during CICR, and in excess, promotes SOICR [RyR2 can be regulated by post-translational modifications such as phosphorylation and oxidation –7. Of thes SOICR , 5.2+ cycling is O-GlcNAcylation (O-GlcNAc) [An emergent post-translational modification associated with regulation of cardiac function and Ca-GlcNAc) . O-GlcNA-GlcNAc) . Hence i-GlcNAc) , 10. O-G-GlcNAc) . The pre-GlcNAc) . Therefo-GlcNAc) , 12.We have previously shown that O-GlcNAcylation can increase SOICR indirectly through the activation of CaMKII and subsequent phosphorylation of RyR2 . Indeed,In this study we used a human embryonic kidney (HEK) 293 cell model, stably expressing RyR2, to determine the CaMKII independent effect of O-GlcNAcylation on SOICR. Next, we used site directed mutagenesis to establish the sites within RyR2 to mediate this effect. Our data show that RyR2 can be dynamically modified by O-GlcNAcylation and that this is associated with an increase in SOICR, independent of CaMKII activity. These findings suggest that O-GlcNAcylation of RyR2 promotes SOICR and is likely to contribute to the occurrence of arrhythmias seen in the diabetic heart. As O-GlcNAcylation of RyR2 has also been observed in the brain it is also likely to have a role there too.Human RAA were obtained from consenting patients (ethics: LRS/12/01/001/AM17), with or without Type 2 Diabetic Mellitus (T2DM), undergoing on-pump coronary artery bypass graft surgery. Patient characteristics are shown in Table Left ventricular tissue samples were collected from 5-month-old male ZDF rats or their lean littermate controls. Rats were housed at 20 ± 1 °C under a 12 h light–dark cycle and provided with food and water ad libitum. Animals were maintained on a Purina 5008 diet . Diabetic status was confirmed using a glucometer  when the hearts were removed. All procedures and animal handling were conducted with the approval of the University of Otago Animal Ethics Committee (AUP 19–250) in accordance with the New Zealand Animal Welfare Act (1999).Stable inducible human embryonic kidney (HEK 293) cells expressing wild-type RyR2 and S2808A were generated as previously described . Cells wProtein lysates were prepared from HEK293 cells as described previously , 15. In To prepare protein lysates from human RAA and ZDF rats, approximately 100 mg of tissue was added to 100 µL of stainless-steel beads (Lab supply) plus 300 µL of radioimmuno precipitation assay (RIPA) buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1 mM EDTA and protease inhibitors (as described above). The tissues were homogenized using a Bullet blender® (Next Advance) at speed 8 for 4 min followed by centrifugation at 15,000 g for 30 min at 4 °C. The supernatant was stored at -80 °C until required , 17.Briefly, 40 µg of protein lysates, prepared as described above, were incubated with protein G-Sepharose beads (15 µl) that were pre-bound with 1 µl total-RyR2 antibody (34 C) (Abcam) at 4 °C for 18–20 h. The supernatant was removed and retained, and the beads were washed 3x in PBS before storage at -80 °C until required.Briefly, 40 µg of protein lysates, or immunoprecipitated samples (beads and supernatant), were solubilized in Laemmli sample buffer before separation via 6% (RyR2) or 12% (GAPDH) SDS-PAGE. Following separation, proteins were transferred onto a 0.45 μm nitrocellulose membrane. The membranes were then blocked with PBS containing 0.5% Tween 20 (PBS-T) and 5% (w/v) dried non-fat skimmed milk powder for 1 h before protein levels were then detected with specific antibodies for total-RyR2 (34 C) (1:1000) (Abcam), total O-GlcNAc, clone 9D1.E4(10) (1:1000) (Abacus) which recognizes all O-GlcNAc modified proteins, and GAPDH (1:3000) (GeneTex). This was followed by detection with the appropriate peroxidase-coupled secondary antibody (1:10000) (Abcam). All bands were visualised using SuperSignal West Pico Plus Chemiluminescent Substrate (ThermoFisher Scientific) via a chemiDoc MP Imaging system (Bio-rad). The band intensities were analysed using ImageJ. O-GlcNAc modified RyR2 was normalised to total RyR2 levels.2+ measurements, the cells were loaded with 2 µM Fluo-4 before imaging. Cells were then continuously superfused with Krebs-Ringers HEPES (KRH) solution containing: 125 mM NaCl, 5 KCl, 25 mM HEPES, 5.5 mM glucose, 1.2 mM MgCl2 and variable CaCl2 (0–1 mM) (pH 7.4) at room temperature. When required, the drugs of interest were applied throughout the superfusion of the cells (100 nM Thm-G (O-GlcNAc promotor), 50 µM DON (O-GlcNAc inhibitor) and/or 2 µM of KN93 or KN92 . At the end of the experiments, 20 mM caffeine was applied to deplete intracellular Ca2 + store (the endoplasmic reticulum in HEK293 cells). Fluo-4 AM dye was excited at 470 nm (40 nm bandwidth) every 2 s with an exposure time of 100 ms using a pE-4000 CoolLED system . Fluorescence of Fluo-4 was detected using a Zyla 4.2 PLUS sCMOS camera (Andor) through a long pass dichroic mirror (495 nm) and emission filter (> 515 nm). Data capture and analysis were performed using NIS-Elements AR 4.00.03 64-bit software . The changes of Fluo-4 fluorescence expressed as F/F0, where F denotes the fluorescence intensity at a given time, while F0 is the average fluorescence intensity recorded in the first 30 s.Imaging was performed as described previously . To perm2+ was measured in cells grown as above with the additional transfection of 2 µg of the calcium-measuring organelle-entrapped protein indicator (CEPIA) cDNA [2+ phosphate precipitation, 24 h before RyR2 induction. During imaging the cells were perfused continuously at room temperature with KRH containing various concentrations of Ca2+ (0 and 2 mM), tetracaine (2 mM to block RyR2 from releasing Ca2+) and caffeine (20 mM to deplete the SR store) [max – Fmin, Release threshold = (FSOICR – Fmin) / Store size x 100%, Termination threshold = (Ftermi – Fmin) / Store size x 100% and Fractional release = Release – Termination threshold.Luminal CaIA) cDNA . CEPIA tR store) . O-GlcNAResults are presented as mean ± SEM. Statistical analysis is described within the corresponding figure legends. Differences were considered statistically significant if p < 0.05. All plotting, data analysis and curve fittings were performed using GraphPad Prism 10.0.2 .We have previously reported that O-GlcNAcylation can modify the activity of RyR2 indirectly via an increase in the activity of CaMKII and subsequent phosphorylation of RyR2 . HoweverUpon confirmation that RyR2 is directly O-GlcNAcylated, we next determined the impact of O-GlcNAcylation on the function of RyR2 using our well-established HEK 293 cell model. These cells have been extensively characterised, show spontaneous RyR2 activity comparable to cardiac cells and have served to investigate the impact of other post-translational modifications of RyR2 , 7. To m2+ imaging was performed  compared to cells exposed to Thm-G + KN-92 (control) . However, at 1mM Ca2+, the occurrence of SOICR was not affected by the presence of KN-93 (p = 0.8262) indicating that O-GlcNAcylation mediated changes in the occurrence of SOICR are largely CaMKII independent at this Ca2+ level. In the absence of O-GlcNAcylation (DON), the inhibition of CaMKII did not alter the occurrence of SOICR  [2+ store level  at which SOICR events terminate (FTermi). Measuring FSOICR and FTermi also allows us to determine whether O-GlcNAcylation alters the magnitude of SOICR  [To determine how O-GlcNAcylation increased SOICR independently of CaMKII at higher Ca(FSOICR) , 7. We arelease) .max represents the maximum Ca2+ store in the presence of 2 mM tetracaine and Fmin represents the minimum Ca2+ store in the presence of 20 mM caffeine. The free Ca2+ store size of each cell was obtained by Fmax-Fmin. FSOICR was determined by the mean fluorescence immediately prior to each SOICR event (downward deflection), FTermi was determined by the mean fluorescence of the nadir of SOICR. The release threshold  and fractional release compared to RyR2 in the absence of Thm-G (both p < 0.0001). Importantly, when treated with the CaMKII inhibitor KN-93, Thm-G retains a significant effect on SOICR , indicating a CaMKII independent effect of O-GlcNAcylation on SOICR. Although the effect of Thm-G on SOICR was retained after CaMKII inhibition, +KN-93 abrogated the effect, increasing both FSOICR and fractional release compared to cells treated with Thm-G without CaMKII inhibition . This indicates an additive effect of CaMKII and non-CaMKII O-GlcNAc mediated effects on SOICR. Interestingly, although it does not appear to translate to a change in the occurrence of SOICR , suggesting CaMKII might still have a partial role under these conditions.Figure old Fig. E and fraold Fig. F are repescribed . As showSOICR (p = 0.807) or fractional release (p = 0.790) in cells expressing S2808A, indicating that the CaMKII independent effect of O-GlcNAcylation requires this site.Phosphorylation of RyR2 by PKA at S2808 has been shown to modify the function of RyR2. As phosphorylation and O-GlcNAcylation can modify the same amino acids and mediate similar effects in other proteins , we next2+ handling in cardiomyocytes which can lead to arrhythmias [2+ release channel frequently linked to arrhythmias is other settings. The significant findings of our study are: (1) RyR2 is post-translationally modified by O-GlcNAcylation; (2) hyperglycemia induced O-GlcNAcylation can increase SOICR through a CaMKII independent pathway via a reduction in the threshold for SOICR; and (3) S2808 is required for O-GlcNAcylation mediated changes in SOICR.O-GlcNAcylation induced by hyperglycemia in diabetes mellitus is well established to have detrimental effects on Cahythmias , 12, 19.O-GlcNAcylation is a ubiquitous post-translational modification, with an enormous number of proteins being identified as O-GlcNAcylated . O-GlcNAAs other proteins commonly show differential changes at different sites occurring simultaneously, the overall integrated change does not reflect changes of individual sites , 23. Add2+ concentrations. This effect on SOICR at higher Ca2+ levels is consistent with other promotors of SOICR such as phosphorylation and pro-SOICR drugs [To determine the functional effect of O-GlcNAcylation of RyR2, we examined the occurrence of SOICR under the conditions we showed to modifiy the level of RyR2 O-GlcNAcylation. Under normoglycemic conditions promotion of O-GlcNAcylation by Thm-G had little effect on the occurrence of SOICR, presumably due to a limited amount of UDP-GlcNAc. However, a clear reduction in SOICR was observed when O-GlcNAcylation was inhibited. Under hyperglycemic conditions Thm-G caused a robust increase in the occurrence of SOICR at high extracellular CaCR drugs , 24. At 2+ concentrations KN-93 markedly reduced the occurrence of SOICR illustrating the previously observed influence of CaMKII on SOICR. However, at higher extracellular Ca2+ concentrations, CaMKII inhibition had no effect on the occurrence of SOICR indicating a CaMKII independent effect of O-GlcNAcylation. These data are interesting as they suggest that under conditions where SOICR is less common, O-GlcNAcylation is likely to induce SOICR via activation of CaMKII. However, under conditions where SOICR is already common, its occurrence can be further increased by O-GlcNAcylation independently of CaMKII. In many disease settings, such as diabetes, SOICR is already common due to excessive post-translational modifications such as phosphorylation and oxidation [The data above are consistent with our work in cardiac myocytes where we have previously shown O-GlcNAcylation promotes SOICR via CaMKII . To dissxidation –7. There2+ release due to a sensitization of RyR2 to activation by luminal Ca2+. Similarly, the sensitivity of RyR2 to luminal Ca2+ also impacts when SOICR terminates and consequently, the fractional release [2+ indicator CEPIA we found that CaMKII independent O-GlcNAc mediated regulation of SOICR (cells treated with KN-93 and Thm-G) also occurred due to a reduction in the release threshold. Interestingly, there was no change in the termination threshold, resulting in more but smaller SOICR events. This differential effect on release and termination thresholds is also shared by regulation of SOICR by FK506 binding proteins (FKBP12 and 12.6) which modify the termination threshold without effecting the release threshold and reduce the magnitude of SOICR [2+ (1 mM) and high O-GlcNAc conditions, CaMKII was still able to modify FSOICR and fractional release under similar conditions. The reason for the disparity is unclear but could be related to the already high incidence of SOICR and an insufficiently large increase in FSOICR to impact when SOICR occurs. Nevertheless, our cytosolic and luminal Ca2+ measurements clearly demonstrate a CaMKII independent, O-GlcNAc mediated, increase in SOICR and corresponding decrease in FSOICR and fractional release.Our previous work has shown that a common mechanism leading to SOICR is a reduction in the threshold for Ca release , 15. UsiIt is not unusual for O-GlcNAcylation to modify proteins at sites also phosphorylated . Arguabl2+ levels suggests CaMKII inhibition might be the best strategy early in the disease (when SOICR is less likely), but in the later stages of diabetes when SOICR is more likely due to other triggers, such as oxidation, inhibition of O-GlcNAcylation might be more beneficial.In conclusion, our data are the first to demonstrate that O-GlcNAcylation can regulate RyR2 and SOICR independently of CaMKII. We also show this CaMKII independent regulation requires the S2808 residue, thus providing new knowledge to develop combinatorial strategies, via inhibition of both CaMKII and O-GlcNAcylation, to reduce SOICR and arrhythmias in diabetes. Specifically, the more prevalent role of CaMKII in inducing SOICR at lower CaBelow is the link to the electronic supplementary material.Supplementary Material 1


Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca2+ oscillations are thought to originate from IP3 receptor-mediated Ca2+ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP3 receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca2+ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca2+ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca2+ oscillations in T-cells.T-cell receptor stimulation triggers cytosolic Ca IP3 subsequently activates IP3 receptors (IP3R) in the endoplasmic reticulum (ER) membrane, leading to the release of Ca2+ from the ER stores, which can then be pumped back into the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), leading to repeated cytosolic Ca2+ oscillations. However, to be sustained for long durations, these Ca2+ oscillations require the transport of extracellular Ca2+ into the cytoplasm by the store-operated calcium entry (SOCE) pathway, mediated by the Ca2+ release-activated Ca2+ (CRAC) channel  that only partially block SERCA activity elicit Ca2+ oscillations in T-cells with similar shape and amplitude to those activated by TCR stimulation , and the PM located and highly Ca2+-selective Orai channels, of which there are three isoforms, Orai1, Orai2, and Orai3 . However, when those cells were maintained in culture, their ER Ca2+ stores eventually refilled to near normal levels  isoforms resulting in the activation of equally varied transcriptional and metabolic programs and cellular responses , 41, 42.ignaling , 15, 17 ignaling . Notably nucleus , 44, 45. nucleus , 52, 53.2+ signaling in different ways. STIM2 responds to subtle depletion of ER Ca2+ stores while STIM1 responds to substantial store depletion  cells, we demonstrate the existence of a multiple-oscillator mechanism in Jurkat T-cells where both the IP3 receptors and CRAC channel activities oscillate, and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor to Ca2+ oscillations as proposed by Dolmetsch and Lewis . Genomic sequencing data of all our KO clones are included as a supplemental file. We also determined whether STIM KO resulted in any compensatory change in the Ca2+ handling proteins that are critical for store depletion, store refilling, and Ca2+ oscillations. We found no obvious compensatory changes in protein expression of the SERCA2 all three IP3R isoforms in all our STIM KO cell clones (A and B). Similarly, we observed no changes in mRNA expression of the three Orai isoforms, the three SERCA isoforms, and the three IP3R isoforms across all STIM KO cells . Addition of thapsigargin to cells loaded with the Ca2+ dye Fura-2 in the absence of extracellular Ca2+ led to passive depletion of the ER stores. This ER Ca2+ store depletion triggers the activation of SOCE, which can be visualized upon the reintroduction of Ca2+ (2 mM) to the extracellular milieu. We then used the lanthanide gadolinium at a relatively low concentration  to selectively block Orai channels (2+ entry is mediated by STIM-activated CRAC channels. The ionophore ionomycin (10 μM) was added at the end of the recordings to ensure maximal Fura-2 signal across different cell lines. This protocol revealed a significant reduction in SOCE in the single STIM KO clones than the parental WT Jurkat line.As T-cells are primarily dependent upon SOCE to propagate Cachannels , confirmC and D), suggesting that STIM2 has a more prominent role in mediating SOCE in Jurkat T-cells compared with other nonexcitable cells. This is similar to our previous studies in colorectal cancer cell lines showing that STIM2 has a large contribution to SOCE (F and G) and elicited by voltage ramps from −100 mV to +60 mV (E). As expected, KO of both STIM1 and STIM2 resulted in the ablation of SOCE (C and D), confirming that STIM1/2 are indeed the sole drivers of this pathway in Jurkat T-cells. Our repeated attempts to record CRAC currents from STIM1/2 DKO cells failed as these cells shrunk and died 1 to 2 min after contact with the recording pipette. These cells have low ER Ca2+ stores, exhibit increased cell death, and low proliferation rates and may likely have defects in membrane integrity and repair.It is notable that KO of STIM1 nearly eliminates SOCE in a number of primary cell types and cell lines, including primary CD4+ T-cells . While S to SOCE . Further to SOCE , F and Go +60 mV E. As exp of SOCE , C and D2+ recordings with Fura-2 above, it was readily apparent that the Ca2+ release phase, in the absence of extracellular Ca2+, was reduced in all STIM KO cells by comparison to the WT Jurkat cells (C), suggesting that the ER Ca2+ content is reduced in STIM KO cells. Therefore, we decided to perform direct measurements of ER Ca2+ using the genetically encoded ER-targeted dye CEPIAer  and subsequent reintroduction of 2 mM Ca2+ to the external milieu to determine the level of acute ER Ca2+ refilling . These experiments showed that the resting ER Ca2+ level and the level of acute ER refilling reached after store depletion with CPA were significantly lower in STIM KO cells compared to WT control cells with the following order of cell clones in basal ER Ca2+ content: STIM2KO>STIM1KO>STIM1/2 DKO (H and I).From the cytosolic Caat cells C, sugges CEPIAer . For thein vitro with anti-CD3 antibody, is a core mechanism by which T-cells are activated, and is known to produce cytosolic Ca2+ oscillations that drive NFAT translocation to the nucleus . Anti-CD3 stimulation produced a robust oscillatory response in the WT Jurkat cell line and in both STIM2 KO clones (A and B). However, each Ca2+ oscillation in WT cells had bigger amplitude and period than oscillations in the STIM2 KO clones (C and D), suggesting significant contribution to those oscillations in WT cells from STIM1-driven Ca2+ entry across the plasma membrane. By contrast, STIM1 KO clones showed mostly one initial oscillation (rarely two oscillations) while the STIM1/2 DKO clones showed almost no oscillations (at most one single low amplitude oscillation in some cells). These data suggest that both STIM proteins synergize for high amplitude optimal Ca2+ oscillations with STIM1 being the primary driver of TCR-induced Ca2+ oscillations in Jurkat T-cells (A–D). These data also suggest that, unlike the case of other nonexcitable cells, in Jurkat cells SOCE has a major contribution to Ca2+ oscillations both directly and indirectly through refilling of ER Ca2+ stores.TCR stimulation, typically simulated  nucleus , 11, 60.mulation . In one 2+ oscillations in an extracellular buffer lacking Ca2+ . The cells were kept in HBSS supplemented with 2 mM Ca2+ for the first minute of imaging. This external buffer was then replaced with Ca2+-free HBSS buffer prior to stimulation with α-CD3 where indicated by the arrow . In WT cells, we observed a reduction in Ca2+ oscillation frequency, amplitude, and period (E–G), which reflected the absence of the contribution from Ca2+ entry across the plasma membrane through SOCE. Similar trends were observed in the STIM2 KO clones but with smaller oscillation amplitudes. Of note, when WT cells were stimulated in Ca2+ free HBSS, the oscillations were faster to reach peak and showed a bursting behavior  compared to cells stimulated in 2 mM Ca2+ containing HBSS . The pattern of oscillations of WT cells in Ca2+ free HBSS mimicked that of the STIM2 KO cells whether they were stimulated in Ca2+ free or in 2 mM Ca2+ (A and H) solutions but with slightly smaller amplitude . These data suggest that the fast bursting oscillations in WT cells challenged with agonist in Ca2+ free HBSS are driven by IP3R-mediated Ca2+ release. The slightly reduced amplitude of these oscillations in STIM2 KO cells and their near absence in STIM1 KO and STIM1/2DKO cells (in either Ca2+ containing or Ca2+ free HBSS) reflect the relative contributions of STIM2 versus STIM1 to ER refilling.In the second set of experiments, we recorded Ca2+ responses (B–G). The differences between WT cells and either STIM1 KO cells or STIM1/2 DKO cells observed in nominally Ca2+-free bath solutions are likely due to the defects in ER store refilling in STIM KO cells, as documented in Figure 1H and quantified in Figure 1I. The fact that we observed decreased maximum amplitude and period in the oscillations of all Jurkat lines in the absence of extracellular Ca2+ compared to the presence of extracellular Ca2+ strongly supports the idea that SOCE has a major and direct contribution to Ca2+ oscillations in Jurkat T-cells. If antigen-activated oscillations were driven entirely by SOCE, Ca2+ oscillations recorded in nominally Ca2+ free solutions would have been eliminated. Our data suggest that there is a contribution of intracellular ER Ca2+ stores in maintaining Ca2+ oscillations through IP3 receptor-mediated Ca2+ release, and that some of the Ca2+ entry through STIM1/2-dependent SOCE serves to acutely refill the ER Ca2+ stores during agonist stimulation.However, the STIM1 KO clones and the STIM1/2 DKO cells had robust inhibition of Caesponses , B–G. Th2+ oscillations in response to α-CD3 stimulation , or GSK-7975a (10 μM). In a third condition, we added 80 mM KCl to the external solution to induce plasma membrane depolarization and limit the driving force for Ca2+ entry through Orai channels (A–C). These three Ca2+ entry inhibitors  were either added simultaneously with α-CD3 antibody , or 10 min after addition of α-CD3 antibody where indicated by arrows . When we stimulated WT Jurkat cells with α-CD3 (125 ng/ml) and allowed the oscillations to proceed for 10 min, the subsequent addition of either Gd3+, GSK-7975a, or KCl, caused the oscillations to cease immediately . In all conditions of Jurkat cells stimulated with α-CD3 antibody and treated simultaneously with either Gd3+, GSK-7975a, or KCl (at 1 min), we observed a significant reduction in frequency of Ca2+ oscillations that was similar between the three inhibitors, although KCl and Gd3+ were slightly more effective than GSK-7975a . The remaining oscillations when Ca2+ entry was inhibited likely reflect contributions from ER Ca2+ stores, which eventually ran out in the absence of Orai-mediated Ca2+ entry . When SOCE inhibitors were added 10 min after stimulation with α-CD3 , Ca2+ oscillations ceases immediately in this case because ER Ca2+ stores were likely mostly depleted during the first 10 min of α-CD3 addition.First, WT Jurkat cells were stimulated with α-CD3 (125 ng/ml) in the presence of 2 mM extracellular Cachannels , A–C. Th2+ recordings were conducted in nominally Ca2+ free external solutions (D–F). When Orai inhibitors or KCl were added at 10 min, the low amplitude and scarce Ca2+ oscillations proceeded largely unaffected by Orai inhibitors or membrane depolarization (E), although there was a small reduction in Ca2+ oscillation frequency in the presence of inhibitors that was not statistically significant. These results suggest that these oscillations in nominally Ca2+ free external solutions are driven strictly by ER Ca2+ release and are unaffected by membrane depolarization or Orai inhibitors. Surprisingly, when WT Jurkat cells stimulated with α-CD3 antibody in nominally Ca2+ free external solutions and treated simultaneously with either Gd3+, GSK-7975a, or KCl (at 1 min), we observed a slight but significant reduction in frequency of Ca2+ oscillations and this reduction was more pronounced with KCl than with Gd3+ or GSK-7975a (F). The simplest interpretation of these results is that prolonged exposure to nominally Ca2+ free solutions in combination with KCl or Orai blockers leads to more ER Ca2+ depletion (through Ca2+ leak) than exposure to nominally Ca2+ free alone.Second, we performed a similar set of experiments with the same protocols, stimuli and inhibitors, except that Caolutions , D–F. Whrization E, althouSK-7975a F. The si2+ signaling: IP3R-mediated and CRAC channel-mediated Ca2+ oscillations, and this conclusion is supported by a mathematical model that exhibits the same qualitative behaviors. When CRAC function is significant, large-period oscillations result from the interaction between ER depletion and CRAC channel activation ; these oscillations depend critically on a time delay between these two events, as originally proposed by Dolmetsch and Lewis (2+), the large-period oscillations are replaced by ones of qualitatively different shape, often taking the appearance of oscillations with bursts of faster oscillations on a raised plateau separated by quiescent periods . These bursts of faster oscillations on a raised plateau are clearly apparent in our experimental data when SOCE is inhibited through STIM KO or removal of extracellular Ca2+ (e.g. see A). In WT cells bathed in 2 mM extracellular Ca2+, the low-period oscillations are masked by the CRAC-based large-period oscillations, but are revealed when STIM1 or STIM2 are knocked out or when Ca2+ influx is reduced. However, passive depletion of the ER with low concentrations of thapsigargin (e.g. 15 nM) that partially block SERCA results in CRAC-based oscillations, in the absence of agonist stimulation  as experimentally documented by Dolmetsch and Lewis (2+ concentration. (2) There is a time delay between ER depletion and SOCE. (3) IP3R can mediate faster oscillations in the same manner as seen in multiple other cell types  the large-period CRAC-based oscillations; (2) the intermediate-period IP3R-based oscillations mediated by oscillating IP3 concentrations; (3) within these IP3R-based oscillations reside bursts of faster oscillations on a raised plateau that are driven by cycles of cytosolic Ca2+-mediated activation and inhibition of IP3Rs.Our experimental results suggest there are two concomitant and fundamentally different kinds of oscillations during TCR-mediated activation of Cand Lewis . When CRe.g. see A. In WT nd Lewis . Our matll types . (4) A rn of IP3 . Therefo2+ depend on ER depletion, the model predicts that the average ER concentration is slightly higher during these oscillations  than it is during the oscillations in the absence of Ca2+ influx. This is because of the lag between changes in ER Ca2+ and changes in Ca2+ entry. Upon application of agonist in extracellular Ca2+ containing solution , an initial Ca2+ depletion from the ER (through the IP3R) activates a large CRAC current, but with a time delay. This causes a large increase in cytosolic Ca2+, which is then taken up into the ER by SERCA pumps. However, because of the time delay, this does not immediately shut down the CRAC current. Since the SERCA pumps are still working, the ER overfills. Eventually, CRAC switches off and the ER Ca2+ slowly decreases, leading to another delayed activation of CRAC, and the cycle repeats. A hint of the underlying process can be seen at the very beginning of the red trace in of Figure 5A. As the ER is depleting initially (immediately after application of the agonist), but before CRAC activity has had enough time to start, the model exhibits a short period of much faster oscillations , reminiscent of those seen in Fig. 5B that are dependent on Ca2+ modulation of the IP3R. However, in the red trace of Fig. 5A, those initial IP3R-based oscillations are quickly masked by the CRAC-based oscillations.It is somewhat counterintuitive that, although the agonist-induced oscillations in the presence of extracellular Ca3R, the CRAC-based oscillations are independent of any modulation of IP3R-mediated Ca2+ release; indeed, the model shows (computations not shown) that if the IP3R flux is replaced by a constant leak, SOCE-based oscillations still occur. In other words, although an oscillatory cytosolic Ca2+ will necessarily cause oscillatory Ca2+ release through the IP3R, such modulation of the IP3R is not required for CRAC-based oscillations to occur. It occurs simply as a byproduct of the properties of the IP3R.Even though they are initiated by activation of the IP2+ influx from outside through CRAC channels is minimized or prevented, a qualitatively different kind of oscillations can occur (B), taking the form of spikes with bursts of rapid oscillations on a raised plateau separated by quiescent periods. In contrast to CRAC-based oscillations, these bursting oscillations are absolutely dependent on the biphasic modulation of the IP3R by Ca2+. Furthermore, the IP3R-based oscillations and their bursting pattern rely also on an additional negative feedback mechanism, whereby an increase in cytosolic Ca2+ causes an increase in the degradation of IP3. In the model this occurs through the action of Ca2+ to enhance IP3-3 kinase activity that produces IP4 from IP3.When Caan occur B, takingC. During thapsigargin-induced oscillations, the SERCA pumps continue to work (but more slowly), thus allowing for refilling of the ER, assisted by the absence of any leak through the IP3R. Similarly, during thapsigargin-induced oscillations, the average ER concentration is slightly higher than at rest, as a result of the time delay between ER depletion and SOCE activation, as explained above.CRAC-based oscillations can also occur in the absence of agonist, but some mechanism is needed to provide an initial store depletion. As shown by , applicaFigure 52+-dependent activation of NFAT isoforms to promote proliferation, T-cell activation, and cytokine production . After staining the cells, the ImageStream multispectral imaging flow cytometer was used to capture NFAT1 translocation in tens of thousands of cells for each population (A). We discovered significantly reduced nuclear translocation of NFAT1 in STIM1 KO and STIM2 KO cells in response to both thapsigargin and α-CD3 stimulation, with STIM1 KO cells showing slightly more inhibition of NFAT1 nuclear translocation (B and C). However, there was essentially no NFAT1 nuclear translocation in the STIM1/2 DKO cells in response to both thapsigargin and α-CD3 (B and C).A key role of the SOCE channel in T-cells is inducing the Caoduction , 64, 65.tivation , 66. Acctivation . Jurkat pulation A. We dislocation , B and Cnd α-CD3 , B and CA and B) than either the WT line or the single STIM KO clones. Indeed, we did not observe any difference between any single STIM KO clone and the WT line, suggesting that the cumulative effects of knocking out both STIM proteins are required to induce apoptosis. Similarly, the CyQUANT proliferation assay (C) revealed that the STIM DKOs display a heavily reduced proliferative rate in comparison to the WT cells and single STIM KOs. Nevertheless, the single STIM KO clones exhibited reduced proliferative rates in comparison to the parental WT line, with STIM1 KO cells displaying significantly more inhibition in proliferation than the STIM2 KO cells.As mentioned above, multiple attempts to generate and culture single clones of CRISPR/Cas9 STIM1/2 DKO cells in Jurkat cells were unsuccessful. While clones were selected in each attempt, we were unable to continuously culture them past two to three weeks as they failed to proliferate and eventually died out. During the fourth attempt to generate a stable population of STIM1/2 DKO cells, we observed a similar drop-off in colony survival after approximately 2 weeks. In an effort to prevent total loss of these new colonies, we reasoned that perhaps the low cell density in each population contributed to their failure to proliferate, and so we pooled the five surviving colonies into a single population to improve the odds of producing a stable population. This attempt proved successful, and we were able to maintain and use throughout this study the polyclonal STIM1/2 DKO population in cell culture, although this polyclonal population of STIM1/2 DKO cells grow significantly slower than the single STIM KO cells or their WT counterparts. To determine whether the consistent loss of STIM1/2 DKO clones was due to heightened apoptotic signaling or quiescence, we employed flow cytometry with Annexin V/7-amino-actinomycin D (7-AAD) co-staining and conducted a CyQUANT proliferation assay . These aon assay C reveale2+ oscillations in most cells is initiated by release of Ca2+ from the ER stores by IP3R and sustained by influx of extracellular Ca2+ into the cytosol by SOCE and the reuptake of this cytosolic Ca2+ by SERCA (e.g. 15 nM) that partially block SERCA can cause repetitive Ca2+ oscillations, which were noted to rely strictly on SOCE independently of IP3R activity  CRAC mediates the large period oscillations; (2) while the intermediate period IP3R-based oscillations are triggered by oscillating IP3 concentrations; (3) finally, within these IP3R-based oscillations reside bursts of faster oscillations on a raised plateau that are presumably driven by cycles of cytosolic Ca2+-mediated activation and inhibition of IP3Rs. We note that the data show that there must be a third oscillatory mechanism, not mediated by CRAC or by IP3R channels, operating on an intermediate time scale. Without this third oscillatory mechanism, it would be impossible to get fast oscillations occurring in bursts, with a quiescent period between the bursts. It is well established that feedback from rise of Ca2+ concentrations on the degradation of IP3 can cause such bursting behavior, and this is what our model assumes. This assumption can be considered a model prediction; there is, as yet, no direct evidence for this assumption, and yet it is necessary for the model to behave correctly.Our experimental results and mathematical modeling support the existence of two concomitant and fundamentally different kinds of oscillations during TCR-mediated activation of CaKO of either STIM1 or STIM2 significantly inhibits NFAT1 translocation in response to store depletion by either thapsigargin or anti-CD3 antibody, in agreement with our previous findings in other cells . Here, i2+ oscillations, low ER Ca2+ content, and no ability to induce the translocation of NFAT1 in response to thapsigargin or anti-CD3. Given that SOCE is the primary means through which T-cells sustain Ca2+ signals, and that Ca2+ signaling is central to numerous T-cell functions including proliferation, these results correspond well with our observation of increased cell death and inhibited proliferation in the STIM1/2 DKO cells. Curiously, however, this phenotype is not observed in primary CD4+ T-cells derived from KO mouse models. Instead, double STIM1/2 KO drastically affected the development and maturation of agonist-selected T-cells such as regulatory T-cells . Cells were cultured in RPMI-1640 with L-Glutamine (Corning) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and 1X penicillin-streptomycin (Corning). Cell lines were stored in a heated and humidified incubator under standard cell culture conditions . Cell lines were confirmed negative for mycoplasma contamination using a PCR detection kit (ABM: G238).The parental Jurkat E6-1 cell line was purchased from the 2+ imaging experiments.To generate STIM KO in Jurkat cells, we employed CRISPR/Cas9 using lentiCRISPR v2 vectors we described previously . The Jurvia centrifugation, washed with ice-cold phosphate-buffered saline, and re-pelleted via centrifugation prior to lysis with ice-cold RIPA buffer  supplemented with 1X Halt protease and phosphatase inhibitor (Thermo Fisher Scientific) for 20 min on ice. The crude protein lysates were clarified by centrifugation  and the supernatant was collected and total protein concentration was quantified by Pierce BCA assay (Thermo Fisher Scientific) according to the manufacturer’s protocols. Samples were then loaded onto precast NuPage 4% to 12% Bis–Tris gels, and the gels were run at 120V for 90 min. Proteins from completed gel runs were transferred to polyvinylidene difluoride (Millipore Sigma) membranes by wet transfer in Tris–glycine buffer  overnight in a cold room (4 °C). Polyvinylidene difluoride membranes were then blocked with Intercept Blocking Buffer (LI-COR) for 1 h at room temperature (RT) before incubating with primary antibodies diluted in Intercept Blocking Buffer  for 1 h at RT. The membranes were washed three times with Tris-buffered saline with 0.1% Tween 20 (5 min per wash) and incubated with secondary antibodies (800CW-conjugated anti-rabbit (LI-COR) and 680RD-conjugated anti-mouse (LI-COR)) diluted  in Intercept Blocking Buffer for 1 h at RT. The membranes were then washed an additional three times as before, and blots were imaged used an Odyssey CLx Infrared Imaging System (LI-COR). GAPDH or α-tubulin was used as loading controls for all Western blots.Cells were collected from culture flasks, pelleted 6 cells) using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocols. Recovered RNA was measured using a Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific) and treated with DNAse I (Thermo Fisher Scientific) for 15 min at RT followed by treatment with 25 mM EDTA to terminate the digestion.1 μg of treated RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following manufacturer’s protocols. Total complementary DNA was diluted 1:10 in nuclease-free water (Bio-Rad), mixed with target-specific primers, and amplified with SYBR Green qPCR Master Mix (Applied Biosystems) at a final volume of 10 μl. All samples were amplified with the same PCR protocol beginning with a hot-start incubation for 2 min at 95 °C followed by 40 PCR cycles consisting of a 15 s denaturation step at 95 °C, a 15 s annealing step at 55 °C, and an extension step of 30 s at 72 °C. Subsequently, a standard melt curve was generated to ensure primer specificity and the comparative cycle threshold method was used to analyze target and control samples. Samples were normalized to two reference genes, GAPDH and α-tubulin. All samples were run with three technical replicates per qPCR plate, and three biological replicates were run to ensure reproducibility.Total mRNA was collected from each cell line (5 × 10g for 5 min) before being resuspended in HBSS containing 140 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl2, 2.0 mM CaCl2, 10 mM glucose, and 10 mM Hepes with pH adjusted to 7.4 by NaOH. HBSS-suspended Jurkat cells were seeded at a density of 100,000 cells per coverslip. The Jurkat cells were allowed to adhere to the coverslips for a minimum of 15 min at RT to allow sufficient adhesion prior to mounting into an Attofluor cell chamber (Thermo Fisher Scientific). The mounted coverslips were incubated with 2 μM Fura-2-AM (Thermo Fisher Scientific) for 30 min at RT, and subsequently washed four times with HBSS to eliminate excess Fura-2. The chambers were then mounted on a Leica DMi8 Fluorescence Microscope. Cells were imaged using a 20X Fluor objective, and Fura-2 was excited at 340 and 380 nm using a fast shutter wheel (Sutter Instruments) with emissions captured at a wavelength of 510 nm with a Hamamatsu Flash 4 camera. The ratio of fluorescence at 340 and 380 nm (F340/F380) was analyzed using regions of interest drawn around the perimeters of 15 to 30 cells per coverslip using the Leica Application Suite X (LAS X) (https://www.leica-microsystems.com) software. The traces resulting from this analysis are represented as the mean ± SEM from at least three independent experiments.Glass coverslips with a diameter of 25 mm were coated with poly-L-Lysine  30 min prior to seeding. Jurkat cells were collected from cell culture flasks and pelleted by centrifugation  using 490 nm excitation and 528 emission wavelengths. Background fluorescence from unstained wells was subtracted from each reading. The plate was returned to the cell culture incubator for overnight incubations between recordings. Recordings were captured immediately after seeding (0 h) and then at 24, 48, and 72 h. All recordings were normalized to their respective 0 h recording, and this ratio was represented as RFU/RFU0. Displayed results represent the means of three technical replicates per experiment collected from three independent experiments.Proliferation of the Jurkat cells was measured using the CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific). A total of 2000 Jurkat cells from the WT line and each STIM KO population were seeded in complete media into separate wells of a 96-well tissue culture plate. For each recording, the 96-well plate was centrifuged for 5 min at 8006 Jurkat cells were collected per sample and pelleted by centrifugation prior to washing with ice cold PBS. The cells were then repelleted by centrifugation, and incubated with 100 μl of Annexin V binding buffer  containing 5 μl of Annexin V and 5 μl of 7-AAD. The samples were incubated for 30 min at RT, and then diluted with 400 μl of Annexin V binding buffer prior to collecting data on a LSRFortessa (BD Biosciences). Data were generated from three independent experiments, and results were analyzed by FlowJo (https://www.flowjo.com/) software.To assess Jurkat apoptosis, cells were costained with FITC-Annexin V (Tonbo) and 7-AAD (Tonbo) and fluorescence was measured using flow cytometry. A total of 2 × 106 Jurkat cells per condition were collected and transferred to 1.5 ml Eppendorf tubes. The cells were pelleted by centrifugation, and the media was replaced with 1X HBSS supplemented with 2 mM Ca2+. Cells were then treated for 30 min at RT with either thapsigargin (2 μM) to deplete the ER stores and induce SOCE or with 125 ng/ml α-CD3 to induce Ca2+ oscillations. After stimulation, the cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Kit following manufacturer protocols . After fixation and permeabilization, the cells were centrifuged, the supernatant was removed, and the cells were incubated with an NFAT1 primary antibody at a 1:100 dilution  for 1 h at RT. Unbound NFAT1 primary antibody was washed out, and the cells were stained using an Alexa-647-conjugated anti-rabbit secondary antibody  for 1 h at RT protected from light. Unbound secondary antibody was then washed out and the cells were resuspended in 40 μl of PBS. DAPI was used to stain the nucleus immediately before imaging by adding 10 μl of 1.25 μg/ml DAPI to the PBS .A total of 3 × 10https://www.emdmillipore.com/US/en/20150212_144049) software version 6.2 (Amnis Corporation) using the software’s transcription factor translocation guided analysis, identifying in-focus cells, excluding doublets, and ensuring cells were double-positive for DAPI and NFAT1. The raw nuclear similarity scores were exported to CSV files for statistical analysis. Displayed results represent similarity scores reported from three independent experiments.An Amnis ImageStream X Mk II Imaging Cytometer was used to capture NFAT1 translocation images. Bright field images were obtained with Channels 1 and 9 (430–480 nm), side scatter was monitored with Channel 6 , DAPI images were captured with Channel 7 , and Alexa-647 images were captured with Channel 11 . Cells were gated to exclude doublets and debris and excitation laser power were adjusted to prevent pixel saturation before beginning image acquisition. Data were analyzed using IDEAS (https://www.moleculardevices.com/) software. Before recordings, cells suspended in HBSS were seeded on round coverslips pretreated with poly-L-Lysine for 10 min to allow cell adhesion. HBSS solution was washed with the bath solution. We selected cells with tight seals (>3 GΩ) and with <15 MΩ series resistance for recording. Cells were maintained at +30 mV during the experiments and were stimulated by a voltage ramp from −100 mV to +60 mV, lasting 250 ms every 3 s. Bath solution (mM): CaCl2, 20; NaCl, 130; KCl, 4.5; MgCl2, 1; D-glucose, 10; Hepes, 5 (pH 7.4 with NaOH). Pipette solution (mM): Cs methanosulfonate, 150; MgCl2, 8; EGTA, 10; Hepes, 10 (pH 7.2 with CsOH).CRAC currents were measured using the whole cell configuration of the patch clamp technique on Jurkat WT, STIM1 KO, and STIM2 KO. Patch pipettes (2–4 MΩ) were pulled from borosilicate glass capillaries (World Precision Instruments) with P-1000 Flamming/Brown micropipette puller (Sutter Instrument Company). Data acquisition was performed with the Axopatch 200B, the Digidata 1440A (Molecular Devices) and was monitored with pCLAMP10  influx are the result of oscillatory Ca2+ fluxes through the IP3R. On the other hand, oscillations in WT cells have a higher amplitude and a larger period; these oscillations are caused primarily by transport of Ca2+ across the plasma membrane of the cell, and thus called SOCE oscillations.Ca3R model used here is taken from . Parameters of the model are listed in The first term represents production of IPllations B. Paramehttps://www.graphpad.com/features). Data are presented as the mean ± SEM unless otherwise specified, and each point presented in scatter plots represents a single independent cell. All comparisons were analyzed using either one-way or two-way ANOVA, with parametric statistical tests used for normally distributed data, and nonparametric tests used when data were not normally distributed. GraphPad Prism 9 was employed to assess data normality . Statistical test results are displayed on each graph where ns, ∗, ∗∗, ∗∗∗, ∗∗∗∗ indicate p-values that are not significant, < 0.05, <0.01, <0.001, and <0.0001 respectively.All statistical analyses were conducted using GraphPad Prism 9 (All raw data related to this manuscript are included as an online supplement.This article contains J. Biol. Chem. The remaining authors declare no competing interests.Mohamed Trebak is a consultant for Seeker Biologics Inc, and is a member of the editorial board of the


Ca2+, a universal signaling ion, passively diffuses into the mitochondrial intermembrane space (IMS) through voltage‐dependent anion channels (VDAC), where uptake into the matrix is tightly regulated across the inner mitochondrial membrane (IMM) by the mitochondrial Ca2+ uniporter complex (mtCU). In recent years, immense progress has been made in identifying and characterizing distinct structural and physiological mechanisms of mtCU component function. One of the main regulatory components of the Ca2+ selective mtCU channel is the mitochondrial Ca2+ uniporter dominant‐negative beta subunit (MCUb). The structural mechanisms underlying the inhibitory effect(s) exerted by MCUb are poorly understood, despite high homology to the main mitochondrial Ca2+ uniporter (MCU) channel‐forming subunits. In this review, we provide an overview of the structural differences between MCUb and MCU, believed to contribute to the inhibition of mitochondrial Ca2+ uptake. We highlight the possible structural rationale for the absent interaction between MCUb and the mitochondrial Ca2+ uptake 1 (MICU1) gatekeeping subunit and a potential widening of the pore upon integration of MCUb into the channel. We discuss physiological and pathophysiological information known about MCUb, underscoring implications in cardiac function and arrhythmia as a basis for future therapeutic discovery. Finally, we discuss potential post‐translational modifications on MCUb as another layer of important regulation.Mitochondrial calcium (Ca ADAlzheimer's diseaseALLacute lymphoblastic leukemiaAMPKAMP‐activated protein kinaseATPadenosine 5′‐triphosphate−Ccarboxyl2+CacalciumCAMKIIcalmodulin‐dependent protein kinase IICCDC109Bcoiled‐coil domain containing 109B proteincryoEMcryoelectron microscopyDIME motifAsp‐Ile‐Met‐Glu motifDMDDuchenne muscular dystrophyEMREessential MCU regulatorERendoplasmic reticulumGBMglioblastoma multiformeHAhemagglutininHFheart failureHIF1αhypoxia‐inducible factor‐1‐alphaI/Rischemia/reperfusionIMMinner mitochondrial membraneIMSintermembrane spaceIP3Rinositol 1,4,5‐trisphosphate receptorJMLjuxtamembrane loopKDequilibrium dissociation constantKOknockout+LilithiumMAMmitochondria‐associated membraneMCUmitochondrial calcium uniporterMCUbmitochondrial calcium uniporter dominant‐negative beta subunitMCUBMCUb geneMCUR1MCU regulator‐1MICU1/2/3mitochondrial calcium uptake 1, ‐2, and ‐3mPTPmitochondrial permeability transition poremROSmitochondrial reactive oxygen speciesmtCUmitochondrial calcium uniporter complex−Namino+NasodiumNCLXsodium/calcium/lithium exchangerOMMouter mitochondrial membranePISAprotein, interfaces, structures, and assemblies serverPXP motifPro‐X‐Pro motifPyk2proline‐rich tyrosine kinase 2Ru360/265Ruthenium red derivative 360 and ‐265RyR2ryanodine receptor 2shRNAshort hairpin ribonucleic acidsiRNAsmall interfering ribonucleic acidSRsarcoplasmic reticulumSTZstreptozotocinTM1/2transmembrane domain 1 and ‐2VDACvoltage dependent anion channelsWTwild‐type12+) transients, with mitochondrial Ca2+ uptake into the matrix fundamentally regulating all of these processes.2+ buffering capacity, taking up Ca2+ upon elevated cytosolic levels and protecting cells from Ca2+ overload.2+ plays a role in regulating key enzymes of the citric acid cycle and oxidative phosphorylation, such as pyruvate dehydrogenase, isocitrate dehydrogenase, α‐ketoglutarate dehydrogenase, and perhaps even the ATP synthase complex, to regulate cellular bioenergetics.2+ can be pathological due to mitochondrial permeability transition pore (mPTP) opening, the release of pro‐apoptotic factors, and a progressive signaling cascade leading to cell death.Mitochondria play essential roles in ATP production, cell death, and shaping cytosolic calcium  Ca2+ release channels, generating microdomains of elevated Ca2+  at the outer mitochondrial membrane (OMM).2+ freely diffuses into the intermembrane space (IMS) through voltage‐dependent anion channels (VDAC).2+ uptake is tightly regulated across the inner mitochondrial membrane (IMM) by the mitochondrial Ca2+ uniporter complex (mtCU).2+ uptake across the IMM through mtCU is the highly negative membrane potential .The endoplasmic reticulum (ER) and mitochondria are in close contact through mitochondria‐associated membranes (MAMs).2+ uptake phenomenon by DeLuca, Engstrom, Vasington, and Murphy,2+ uptake‐1, 2 and 3 (MICU1/2/3) gatekeeping regulators by the Mootha group,2+ uniporter (MCU) essential pore‐forming subunit by the Mootha and Rizzuto groups,After ~6 decades since the discovery of the mitochondrial CaThe most complete structural views of the human mtCU have been revealed by cryoelectron microscopy (cryoEM) and include MICU1, MICU2, MCU, and EMRE components.22+ permeation into the matrix.2+ selectivity of the channel is located at the end of the loop, forming the start of TM2  and a preference for binding Ca2+ over other cations.2+ into the mitochondrial matrix, Glu288 and Val290 from the JML of each pore‐forming MCU subunit circle the pore exit to form the opening, with EMRE believed to promote the open conformation of this luminal gate.MCU is the pore‐forming subunit that is essential for Ca2+ uptake.2+ and undergo conformational changes that may be involved in regulating the accessibility of Ca2+ to the mtCU channel pore or otherwise regulate channel open probability  mouse studies highlight a complex gatekeeping function as fibroblast KO mitochondria at low and high cytosolic Ca2+ show higher and lower rates of Ca2+ uptake, respectively,2+ uptake rates after stimulus2+ uptake at subthreshold cytosolic Ca2+ but reduced Ca2+ uptake at high cytosolic Ca2+.The MCU tetramer interacts with MICU1 directly or indirectly.ow; Ref. , 52, 53)2+, cryoEM revealed mtCU arrangements with MICU1 tightly occluding the pore entrance through electrostatic interactions between Lys/Arg residues of MICU1 and three of the DIME Asp261 residues of the MCU tetramer.2+ mtCU arrangements have also been identified with MICU1 completely displaced from the pore, remaining part of the complex only via EMRE interactions. In this context, non‐occluded dimers of tetrameric channels, bridged through N‐terminal domain interactions of MCU in the matrix and MICU2 interactions in the IMS, have been resolved.2+.2+ occluded/high Ca2+ non‐occluded states provide a convenient explanation for MICU gatekeeping. Yet, this simple model is not only incompatible with the observed non‐blocked states in low Ca2+, but also compelling electrophysiology data collected in the absence of Ca2+ showing sodium (Na+) currents are similar in wild‐type (WT) and MICU1‐KO mitoplasts.2+ currents in WT mitoplasts are ~2× that of MICU1‐KO mitoplasts due to MICU1‐dependent increases in the pore open probability.2+ structures, we speculate that increased open probability occurs due to Ca2+‐dependent structural changes in the MICUs, which allosterically couple to the JML luminal gate of MCU via EMRE.In low Ca2+ permeable channel in metazoans.Although not resolved in the mtCU cryoEM structures, the polyaspartate, IMS‐facing C‐terminus of EMRE interacts with a polybasic domain of MICU1, and the matrix‐oriented N‐terminus forms a beta‐hairpin motif, interacting with MCU.2+ permeation through the mtCU channel.Finally, MCUb, the dominant‐negative beta subunit, is a paralog of the MCU pore‐forming subunit.33.12+ permeation through the mtCU channel.2+ permeation through the mtCU channel.MCUb consists of two coiled‐coil domains and two TM domains that are separated by a short IMS loop.MCU−/− KO HeLa cells revealed that EMRE fused with a C‐terminal FLAG tag interacts with both MCU and MCUb fused to C‐terminal HA tags.2+/non‐occluded  and lowdb; Ref. ) human M2+  at Val48 of EMRE. Note that residues 1–47 of EMRE comprise the mitochondrial transit sequence of the resolved model, while UniProtKB accession NX_Q9H4I9 reports the mitochondrial transit sequence of human EMRE as residues 1–52. In contrast, while a salt bridge was predicted between Glu314 on MCUb and Arg52 on EMRE  but sinAspergillus fischeri  MCU structure, suggested that there are fewer hydrophobic interactions between TM1 and TM2 from the adjacent MCU subunit of the complex, resulting in a widening of the IMS side of the pore.2+ permeation pathway through mtCU are the Asp and Glu rings of DIME at the IMS pore entrance and the luminal gate at the matrix pore exit. In our models based on resolved human mtCU structures, the Glu ring of DIME did not exhibit any notable diameter changes after MCUb integration in either the occluded or non‐occluded states, in contrast to the DIME Asp ring distances, which changed. Specifically, The high Ca2+/non‐occluded human mtCU template structure showed an average shortest pore diameter of 6.2 Å, measured between Asp261 of opposite MCU subunits. This distance increased to 6.6 Å upon integration of MCUb, modeled with 2 × MCU:2 × MCUb, and further increased to 6.8 Å in the MCUb homotetramer , aberrant mitochondrial Ca2+ signaling impairment.+/Ca2+/lithium (Li+) exchanger (NCLX) expression, the primary mitochondrial Ca2+ efflux mediator in excitable cells, along with a decrease in MICU1 and MCUb.2+ overload reported in neurons during disease progression.2+ levels preceded neuronal death and thus, may be a target for the development of neuroprotective therapies for AD.Alzheimer's disease (AD) has also been associated with neuronal mitochondrial Ca2+ homeostasis within cancer cells has been correlated with continuous cell proliferation and inhibition of cell death.MCUB gene.MCUB using shRNAs in U87MG and U251 human glioma cell lines inhibited proliferation, migration, and invasion and led to decreased tumor volume and prolonged overall survival in orthotopic tumor models of nude mice.MCUB mRNA levels, and further treatment with siRNAs targeting HIF1α reduced mRNA levels of MCUB.MtCU function has been highly implicated in cancer. The disturbance of CaMCU expression and HIF1α was shown in triple‐negative breast cancer samples.2+ uptake due to increased MCU and decreased MCUb expression may promote HIF1α signaling, leading to increased tumor size and lymph node infiltration in breast cancer.MCUb overexpression has been reported in drug‐resistant acute lymphoblastic leukemia (ALL).2+ dysfunction.2+ uptake increased ~1.4‐fold in hepatic cells from type 1 diabetic rats, 2 weeks after diabetes induction using streptozotocin (STZ).2+ uptake within liver mitochondria of diabetic rats may be related to the decrease in MCUb expression.Diabetes mellitus is also associated with mitochondrial Ca2+ overload‐induced injury.2+ uptake and suppressed mPTP opening, thus reducing cardiac tissue damage.Finally, MCUb expression may be an endogenous cardioprotective mechanism, reducing mitochondrial Ca3.4.12+ dysregulation in cardiomyocytes.2+ released from the sarcoplasmic reticulum (SR).2+ uptake can interfere with Ca2+ diffusion between RyR2 clusters, reducing the probability of arrhythmogenic spontaneous Ca2+ wave initiation and propagation.2+ transporters, such as mtCU, may be candidate pathways for novel anti‐arrhythmic therapeutics with fewer adverse side effects.Arrhythmia is a common cause of cardiovascular disease‐related deaths, and there are limited treatment options due to adverse anti‐arrhythmic drug side effects.2+ uptake and cardiac function.MCUB with a dominant‐negative MCUB transgene (MCUBW245R/V251E) in vivo rescued cardiomyocytes from relying almost solely on mitochondrial fatty acids for energy production and increased cardiac contractile function.W245R/V251E mutant was found to interact with MICU1, but similar to the proteomic data  triggered by Ca42+ uptake and mitochondrial reactive oxygen species (mROS) production, triggering mPTP opening and cell death  phosphorylation site.Post‐translational modifications regulate mtCU channel oligomerization and function,S‐glutathionylation of Cys97 within the MCU N‐terminal domain has been shown to regulate mtCU channel activity  that may be susceptible to oxidative modifications.The high sequence homology with MCU implies that post‐translational modifications of MCUb could also contribute to the regulation of MCUb and mtCUs in which the dominant‐negative beta subunit has been incorporated. Thus, understanding the mechanisms and structural consequences of post‐translational modifications within MCUb will provide a deeper understanding of the subunit regulation and function, both within physiological and pathophysiological states.52+ overload.2+ regulation and the MCUb subunit will lead to a greater understanding of arrhythmogenesis and help in the development of safer therapeutics.Since the discovery of mtCU components, MCUb has emerged as a critical regulator, despite the lack of understanding surrounding the precise molecular mechanisms that govern the complex assembly and negative regulation by this inhibitor.Danielle M. Colussi: writing original draft, investigation, editing/revising, conceptualization, and model analyses. Peter B. Stathopulos: editing, review, supervision, modeling methodology, and funding. Both authors read and approved the submitted versionThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Supplementary Material S1Click here for additional data file.Supplementary Material S2Click here for additional data file.


The objective of this study was to report real-life clinical outcomes in adult patients with chronic urticaria who had an inadequate response to omalizumab and were switched from omalizumab to cyclosporine. Medical records of adult patients with chronic urticaria who had an inadequate response with omalizumab and were later treated with cyclosporine were reviewed retrospectively. Data pertaining to treatment method, clinical response, and adverse effects were recorded.While both the AAAAI/ACAAI and the EAACI/GAFive patients with omalizumab-refractory chronic urticaria, three of whom also had angioedema and one with an inducible urticaria, were treated with low doses of oral cyclosporine (1–3 mg/kg/d). Four of five patients in this case series had complete resolution of symptoms with oral cyclosporine, while continuing other standard therapies. Systemic side effects occurred in three patients which prompted drug discontinuation in two patients.Cyclosporine alone was effective in inducing urticaria control in adult patients with chronic urticaria who had an inadequate response to omalizumab, though the impact of cyclosporine was limited by reversible adverse effects. Adverse effects were associated with pre-existing medical conditions. As novel chronic urticaria therapies are being investigated, this experience highlights the importance of uncovering chronic urticaria subtypes which tend to respond to cyclosporine, while providing alternative treatments with better tolerability. For paty agents .Omalizumab, an IgE-targeting monoclonal antibody, was FDA approved for antihistamine refractory CSU in 2014. Unfortunately approximately 30% of patients do not achieve adequate control of their symptoms with omalizumab . The pat2LEN/EuroGuiDerm/APAAACI guidelines recommend cyclosporine as an option for patients who have had an inadequate response to omalizumab, many clinicians are hesitant to initiate cyclosporine treatment due to concerns for side effects and safety. Furthermore, there is little data regarding outcomes with cyclosporine after omalizumab is failed. This case series aims to provide clinicians with real-life clinical outcomes of cyclosporine in patients with omalizumab-refractory chronic urticaria.Cyclosporine, a calcineurin inhibitor, lacks FDA approval for CSU in the United States but can be used off-label. Clinical response rates of 40–100% have been reported with cyclosporine for CSU , 7. WhilBaseline investigations included complete blood count, urea, creatinine, serum electrolytes, blood sugar, and urine examination. Repeat tests were performed every 4 weeks. Blood pressure was monitored at least every 2 weeks during the treatment period. Clinical response was based upon patient observation.This study was approved by the Institutional Review Board. Table The objective of this study was to report real-life clinical outcomes in adult patients with chronic urticaria who underwent treatment with omalizumab, had an inadequate response after a period of at least three months, and were switched from omalizumab to cyclosporine.Medical records of adult patients with chronic urticaria who had an inadequate response with omalizumab and were later treated with cyclosporine were reviewed retrospectively. Data pertaining to treatment method, clinical response, and adverse effects were recorded.Patient #1 A 24 year-old female with no significant past medical history presented with 7 months of hives and angioedema. She was diagnosed with chronic spontaneous urticaria. Her symptoms were refractory to maximum-dose H1- and H2-antihistamines, and omalizumab 300 mg every 4 weeks. She had been intermittently reliant upon oral corticosteroids for control of her hives. After discontinuation of omalizumab, she was started on cyclosporine 3 mg/kg/d, which resulted in complete remission of symptoms within 2 weeks. Cyclosporine was used for a total of 4 months without adverse effects. She remains in remission 6 years later and she is off all medications, including antihistamines.Patient #2 A 35 year-old male with a past medical history of vitiligo and chronic rhinitis presented with 1 year of hives. He was diagnosed with chronic spontaneous urticaria. His symptoms were refractory to maximum-dose H1- and H2-antihistamines, sulfasalazine, hydroxychloroquine, and omalizumab 300 mg every 4 weeks. After discontinuation of omalizumab, he was started on cyclosporine 1 mg/kg/d, which resulted in complete remission of symptoms within 1 week. Cyclosporine was used for 4 months without adverse effects. Patient was lost to follow up after hives went into remission.Patient #3 A 41 year-old female with a past medical history of hypertension, headaches, and chronic rhinitis presented with 9 months of hives and angioedema. She was diagnosed with chronic spontaneous urticaria. Her symptoms were refractory to maximum-dose H1- and H2-antihistamines, montelukast, and omalizumab 300 mg every 4 weeks. She had been dependent upon prednisone 20 mg daily to control her hives. After discontinuation of omalizumab, she was started on cyclosporine 1 mg/kg/d, which resulted in complete remission of her symptoms within 2 weeks. Cyclosporine was used for 7.5 months without adverse effects. She was in clinical remission and off all medications for 2 years, after which she experienced a recurrence. H1 and H2-antihistamines were restarted and after no improvement cyclosporine 1 mg/kg/d was restarted and she again achieved remission. She developed elevated blood pressure 6 weeks after re-starting cyclosporine and was started on amlodipine. Cyclosporine was used for a total of 8 months. She remains in remission over 2 years later and she is off all medications.Patient #4 A 39 year-old male with a past medical history of asthma, chronic rhinitis, and hyperthyroidism presented with one and a half years of inducible hives. He was diagnosed with pressure and cholinergic urticaria based on history (provocation testing was not performed). His symptoms were refractory to maximum-dose H1- and H2-antihistamines, montelukast, sulfasalazine, hydroxychloroquine, dapsone, and omalizumab 300 mg every 4 weeks. Omalizumab was used off-label for chronic inducible urticaria. After discontinuation of omalizumab, he was initially started on cyclosporine 1 mg/kg/d. After no improvement in 6 months the cyclosporine dose was increased to 3 mg/kg/d, which resulted in near-complete improvement of cholinergic urticaria and moderate improvement of pressure-induced urticaria (improvement based upon patient observation). Cyclosporine was used for 4 additional months until he developed hypertension, hyperglycemia, hematuria, and hyperuricemia which prompted cessation. These adverse effects had developed after 44 weeks of treatment with cyclosporine. Blood pressure, blood glucose, serum uric acid, and hematuria normalized after cessation of cyclosporine.Patient #5 A 58 year-old female with a past medical history of asthma, chronic rhinitis, thyroid cancer (status post thyroidectomy), and hypertension presented with one year of hives and angioedema. She was diagnosed with chronic spontaneous urticaria. Her symptoms were refractory to maximum-dose H1- and H2-antihistamines, montelukast, hydroxychloroquine, dapsone, and omalizumab 300 mg every 4 weeks. She had been intermittently reliant upon oral corticosteroids for control of her hives. After discontinuation of omalizumab, she was started on cyclosporine 1.25 mg/kg/d, which resulted in complete remission of her symptoms within 1 week. Cyclosporine was used for 6 months without adverse effects. She was in clinical remission and off all medications for two years, after which she experienced a recurrence. H1- and H2-antihistamines were restarted and after no improvement cyclosporine 1.25 mg/kg/d was restarted. After minimal improvement the dose was increased to cyclosporine 3 mg/kg/d, which caused elevated creatinine approximately 4 months after cyclosporine was re-started. The creatinine elevation had prompted the drug to be discontinued. Serum creatinine returned to baseline after cyclosporine was discontinued.Severe chronic urticaria refractory to omalizumab is exceedingly challenging for both the clinician and patient. Few studies have examined the clinical outcomes using cyclosporine alone in omalizumab-refractory chronic urticaria. Both Rosenblum et al  and SancFour of five patients in this case series had complete resolution of symptoms with oral cyclosporine, while continuing other standard therapies  according to the AAAAI/ACAAI guideline . SystemiThere are inherent limitations of this case series including the small sample size and lack of control group. The clinical course of chronic urticaria is variable and spontaneous remission may occur which was not accounted for given the nature of this study. Use of patient observation and comments to assess treatment response, rather than using validated measures of urticaria control, is also a limitation . BiomarkIn summary, cyclosporine was effective for five patients with omalizumab refractory chronic urticaria. In this cohort, the benefit of cyclosporine was limited by reversible adverse effects which tended to occur in patients with pre-existing medical conditions. This highlights the importance of laboratory and blood pressure monitoring during cyclosporine therapy. Further research is needed to better understand the pathophysiology of refractory chronic urticaria, to better predict the tolerability of cyclosporine, and to reliably identify predictors of response to omalizumab, cyclosporine and other therapies in the clinical setting. As novel chronic urticaria therapies are being investigated , 15 this