Depletion Of Stores Research Articles

STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells.

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). The mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal interaction molecule 1), a single-transmembrane-domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER-PM junctions in response to depletion of ER Ca2+ stores and initiates store-operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR-PM interacting sites were decreased, and SR-dependent Ca2+-signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen-inducible, SMC-specific STIM1-knockout (Stim1-smKO) mice. VSMCs from Stim1-smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+-release sites on the SR and Ca2+-activated ion channels on the PM, accompanied by diminished channel activity. Stim1-smKO mice were hypotensive, and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 - independent of SR Ca2+ store depletion - is critically important for stable peripheral coupling in contractile VSMCs.

Functional communication between IP3R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER-plasma membrane (ER-PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER-PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER-PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER-PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry.

Iron Deficiency in Chronic Heart Failure Patients at the University Teaching Hospital in Lusaka, Zambia

Background: Iron plays a key role in oxygen uptake, transport, and storage, as well as oxidative metabolism in the muscle including myocardium and is essential for the formation of haemoglobin, myoglobin, cytochromes, cytochrome oxidase, peroxidase, and catalase. Iron deficiency (ID) often develop in chronic heart failure (CHF) patients due to depletion of iron stores, iron malabsorption and/or reduced availability of iron recycled in the reticuloendothelial system. Indeed iron content and transferrin receptor levels have been reported to be decreased in the myocardium of patients with ID. A cardinal manifestation of heart failure (both heart failure with reduced ejection fraction. HFrEF and heart failure with preserved ejection fraction, HFpEF) is impaired exercise capacity that is exaggerated in iron deficient patients. This easy fatigability is said to be a result of the reduced oxygen transport that is associated with the ID. ID leads to mitochondria and sarcomere dysfunction which reduces left ventricular functioning and hence reducing cardiac output. Reduced peak oxygen uptake has also been reported in patients withHFrEF.
 Objective: To determine iron status of CHF patients at University Teaching Hospital (UTH) in Lusaka.
 Methods: The study was conducted at UTH from May to August 2019. Using purposive sampling, data was collected from patients with CHF of New York Heart Association (NYHA) functional class II to IV. Information obtained included sex, age, height, weight and ejection fraction (reduced ifless than 45%). From the blood samples collected, serum iron levels, transferrin levels and ferritin concentration tests were done. The chi square test and logistic regression were used to analyze iron status in CHF patients.
 Results: Forty (40) patients were enrolled, 18 male and 22 female. The average age was 45.5 (19 to 60). 24 patients (60%) had (HFrEF) and 16 patients (40%) had (HFpEF). 22 patients (55%) had transferrin saturation of less than 20%, while 8 (20%) had ferritin concentration of less than l00ng/ml. Based on both factors, a total of22(55%) patients were iron deficient. Iron deficiency was negatively associated withHFrEF (p = 0.015).
 Conclusion: More than 55% of the participants in this study were iron deficient. However, ID may not be the primary cause of myocardial dysfunction as patients with HFrEF were less likely to be iron deficient. However, correction of ID in CHF patients in other studies has been reported to have clinical benefits. The observations of this study require further triangulation especially that the sample size was rather small.

Iron Deficiency in Chronic Heart Failure Patients at the University Teaching Hospital in Lusaka, Zambia

Background: Iron plays a key role in oxygen uptake, transport, and storage, as well as oxidative metabolism in the muscle including myocardium and is essential for the formation of haemoglobin, myoglobin, cytochromes, cytochrome oxidase, peroxidase, and catalase. Iron deficiency (ID) often develop in chronic heart failure (CHF) patients due to depletion of iron stores, iron malabsorption and/or reduced availability of iron recycled in the reticuloendothelial system. Indeed iron content and transferrin receptor levels have been reported to be decreased in the myocardium of patients with ID. A cardinal manifestation of heart failure (both heart failure with reduced ejection fraction. HFrEF and heart failure with preserved ejection fraction, HFpEF) is impaired exercise capacity that is exaggerated in iron deficient patients. This easy fatigability is said to be a result of the reduced oxygen transport that is associated with the ID. ID leads to mitochondria and sarcomere dysfunction which reduces left ventricular functioning and hence reducing cardiac output. Reduced peak oxygen uptake has also been reported in patients withHFrEF.
 Objective: To determine iron status of CHF patients at University Teaching Hospital (UTH) in Lusaka.
 Methods: The study was conducted at UTH from May to August 2019. Using purposive sampling, data was collected from patients with CHF of New York Heart Association (NYHA) functional class II to IV. Information obtained included sex, age, height, weight and ejection fraction (reduced ifless than 45%). From the blood samples collected, serum iron levels, transferrin levels and ferritin concentration tests were done. The chi square test and logistic regression were used to analyze iron status in CHF patients.
 Results: Forty (40) patients were enrolled, 18 male and 22 female. The average age was 45.5 (19 to 60). 24 patients (60%) had (HFrEF) and 16 patients (40%) had (HFpEF). 22 patients (55%) had transferrin saturation of less than 20%, while 8 (20%) had ferritin concentration of less than l00ng/ml. Based on both factors, a total of22(55%) patients were iron deficient. Iron deficiency was negatively associated withHFrEF (p = 0.015).
 Conclusion: More than 55% of the participants in this study were iron deficient. However, ID may not be the primary cause of myocardial dysfunction as patients with HFrEF were less likely to be iron deficient. However, correction of ID in CHF patients in other studies has been reported to have clinical benefits. The observations of this study require further triangulation especially that the sample size was rather small.

TM9SF4 Is a Crucial Regulator of Inflammation and ER Stress in Inflammatory Bowel Disease

Background & AimsInflammatory bowel disease (IBD) is a major intestinal disease. Excessive inflammation and increased endoplasmic reticulum (ER) stress are the key events in the development of IBD. Search of a genome-wide association study database identified a remarkable correlation between a TM9SF4 single-nucleotide polymorphism and IBD. Here, we aimed to resolve its underlying mechanism.MethodsThe role of TM9SF4 was determined with experimental mouse models of IBD. ER stress cascades, barrier functions, and macrophage polarization in colonic tissues and cells were assessed in vivo and in vitro. The expression of TM9SF4 was compared between inflamed regions of ulcerative colitis patients and normal colon samples.ResultsIn mouse models of IBD, genetic knockout of the TM9SF4 gene aggravated the disease symptoms. In colonic epithelial cells, short hairpin RNA–mediated knockdown of TM9SF4 expression promoted inflammation and increased ER stress. In macrophages, TM9SF4 knockdown promoted M1 macrophage polarization but suppressed M2 macrophage polarization. Genetic knockout/knockdown of TM9SF4 also disrupted epithelial barrier function. Mechanistically, TM9SF4 deficiency may act through Ca2+ store depletion and cytosolic acidification to induce an ER stress increase. Furthermore, the expression level of TM9SF4 was found to be much lower in the inflamed colon regions of human ulcerative colitis patients than in normal colon samples.ConclusionsOur study identified a novel IBD-associated protein, TM9SF4, the reduced expression of which can aggravate intestinal inflammation. Deficiency of TM9SF4 increases ER stress, promotes inflammation, and impairs the intestinal epithelial barrier to aggravate IBD.

Abstract- Poster

INTRODUCTION:Wernicke’s encephalopathy presents as a triad of ataxia, global confusion and ophthalmoplegia. It can have multiple causes; Hyperemesis gravidarum is one of the causative factors. It results in severe vomiting in pregnancy resulting in dehydration. This can lead to acute deficiency of thiamine, which can manifest as Wernicke’s encephalopathy. Poor nutritional intake, depletion of stores and and increased demands of pregnancy can also result in Wernicke’s.MRI Brain is both specific and sensitive for diagnosis.Case summary –A 25 year old married, primi-gravida hailing from urban background belonging to middle socio-economic status, presented at 16 weeks of gestation with complaints of 6 weeks history of severe vomiting, talking irrelevantly, disturbed sleep and weakness of all four limbs. Symptoms progressed over a week with total inability to ambulate and confusion. Clinically she had signs of minimal power and loss of tone, ataxic gait and disorientation. However no ophthalmoplegia was noted. Routine blood investigations were within normal limits. MRI Brain revealed hyperintensities in thalamus and mammillary bodies. A diagnosis of Wernicke’s encephalopathy was made and treatment was instituted with high dose parental thiamine. She reported significant improvement in symptoms over the next 2 weeks.DISCUSSION AND CONCLUSION-Hyperemesis gravidarum is one of the treatable causes of Wernicke’s encephalopathy. It requires a high degree of clinical suspicion. Neurologic outcomes are usually good, however pregnancy outcomes vary.

CGAS-STING Pathway Performance in the Vulnerable Atherosclerotic Plaque.

The important role of Ca2+ in pathogenic store-operated calcium entry (SOCE) is well-established. Among the proteins involved in the calcium signaling pathway, Stromal interacting molecule 1 (STIM1) is a critical endoplasmic reticulum transmembrane protein. STIM1 is activated by the depletion of calcium stores and then binds to another calcium protein, Orai1, to form a channel through which the extracellular Ca2+ can enter the cytoplasm to replenish the calcium store. Multiple studies have shown that increased STIM1 facilitates the aberrant proliferation and apoptosis of vascular smooth cells (VSMC) and macrophages which can promote the formation of rupture-prone plaque. Together with regulating the cytosolic Ca2+ concentration, STIM1 also activates STING through altered intracellular Ca2+ concentration, a critical pro-inflammatory molecule. The cGAS-STING pathway is linked with cellular proliferation and phenotypic conversion of VSMC and enhances the progression of atherosclerosis plaque. In summary, we conclude that STIM1/cGAS-STING is involved in the progression of AS and plaque vulnerability.

Ferric carboxymaltose for treatment of iron deficiency and iron deficiency anemia caused by abnormal uterine bleeding.

Heavy menstrual bleeding leads to depletion of iron stores, with subsequent iron deficiency (ID) and iron deficiency anemia (IDA). To evaluate the efficacy and safety of ferric carboxymaltose (FCM) in treatment of ID/IDA caused by abnormal uterine bleeding (AUB). One hundred and twenty women ≥ 40 years old with chronic AUB and ID/IDA were included in this study for correction of ID/IDA. Participants received FCM infusion for correction of ID/IDA. The pre-treatment ferritin, hemoglobin (Hb), red blood cell (RBC) mean corpuscular volume (MCV), and RBC mean corpuscular hemoglobin (MCH) values were compared with the 6- and 12-week post-treatment values. The pre-treatment ferritin and Hb levels significantly increased from 13.2 ±7.4 µg/l and 8.8 ±0.8 g/dl, respectively, to 111.5 ±5.6 µg/l and 13.9 ±0.6 g/dl, respectively, 6 weeks after FCM (p = 0.001 and 0.0009; respectively), and to 98.7 ±6.1 µg/l and 12.9 ±0.65 g/dl, respectively, 12 weeks after FCM (p = 0.01 and 0.01; respectively). In addition, the pre-treatment RBC MCV and RBC MCH values significantly increased from 74.3 ±2.3 fl and 26.6 ±5.3 pg, respectively, to 88.7 ±1.9 fl and 29.6 ±4.5 pg, respectively, 6 weeks after FCM (p = 0.01 and 0.03, respectively), and to 93.3 ±1.75 fl and 30.3 ±3.8 pg, respectively, 12 weeks after FCM (p = 0.001 and 0.0001, respectively). FCM was safe and effective for correction of ID/IDA caused by chronic AUB within 6 weeks. The serum ferritin, Hb, and RBC indices remained significantly high compared to the pre-treatment values 12 weeks after FCM infusion.

Orai1\u03b1, but not Orai1\u03b2, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

Assessment of iron status in regular blood donors in a tertiary care hospital in Southern India.

Regular blood donation depletes iron stores. The assertion is that the vulnerable donor population requires a predictive standard operative procedure for early detection of iron store depletion, preventing them from developing iron-deficiency anemia. This study aims to study the potential effects of blood donation in the regular donor group using hematological and biochemical estimation of iron status parameters. This was a prospective cross-sectional study on regular blood donors, defined as those who have donated at least 3 times, the last donation being within the last 12 months and continues to donate at least once a year, at a tertiary care teaching hospital in Southern India. The complete blood count (CBC) was performed on the Sysmex coulter, and the red cell indices were calculated. The ferritin and the soluble transferrin receptor (sTfR) assays were performed using Enzyme Immunoassays. The comparison of CBC, serum ferritin, and sTfR assay with donation frequency and time since the last donation was carried out using an independent student's t-test for two groups. The statistical analysis was performed using SPSS for Windows version 20. A total of 323 regular blood donors (6 were females) were included in the study of which they were categorized into three, 211 donors with less than or equal to 10 donations, 84 those who had donated between 11 and 20 times and 28 who had donated more than 20 times. The red cell indices were reduced and different in the groups but not statistically significant except for mean corpuscular volume. About 15% of the study population had a transferrin level of <15 ng/ml. The Ferritin levels showed a statistically significant negative correlation with the number of donations, the correlation coefficient being -0.27. Logarithmic ratios of sTfR/ferritin also correlated with a coefficient of 0.156 with the number of donations and were statistically significant. Our study found that regular blood donors had low iron stores, as shown by ferritin levels and other iron indicators. Using the current guidelines (hemoglobin >12.5 g/dL) for donation, or the red cell indices alone do not reflect the donor's actual iron status.

Tosoh reports on its consolidated results for 1H FY 2022: Speciality Group

Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

Manipulations of Glutathione Metabolism Modulate IP3-Mediated Store-Operated Ca2+ Entry on Astroglioma Cell Line.

The regulation of the redox status involves the activation of intracellular pathways as Nrf2 which provides hormetic adaptations against oxidative stress in response to environmental stimuli. In the brain, Nrf2 activation upregulates the formation of glutathione (GSH) which is the primary antioxidant system mainly produced by astrocytes. Astrocytes have also been shown to be themselves the target of oxidative stress. However, how changes in the redox status itself could impact the intracellular Ca2+ homeostasis in astrocytes is not known, although this could be of great help to understand the neuronal damage caused by oxidative stress. Indeed, intracellular Ca2+ changes in astrocytes are crucial for their regulatory actions on neuronal networks. We have manipulated GSH concentration in astroglioma cells with selective inhibitors and activators of the enzymes involved in the GSH cycle and analyzed how this could modify Ca2+ homeostasis. IP3-mediated store-operated calcium entry (SOCE), obtained after store depletion elicited by Gq-linked purinergic P2Y receptors activation, are either sensitized or desensitized, following GSH depletion or increase, respectively. The desensitization may involve decreased expression of the proteins STIM2, Orai1, and Orai3 which support SOCE mechanism. The sensitization process revealed by exposing cells to oxidative stress likely involves the increase in the activity of Calcium Release-Activated Channels (CRAC) and/or in their membrane expression. In addition, we observe that GSH depletion drastically impacts P2Y receptor-mediated changes in membrane currents, as evidenced by large increases in Ca2+-dependent K+ currents. We conclude that changes in the redox status of astrocytes could dramatically modify Ca2+ responses to Gq-linked GPCR activation in both directions, by impacting store-dependent Ca2+-channels, and thus modify cellular excitability under purinergic stimulation.

Pharmacological inhibition of translocon is sufficient to alleviate endoplasmic reticulum stress and improve Ca2+ handling and contractile recovery of stunned myocardium

IntroductionThe function of endoplasmic reticulum (ER), a Ca2+ storage compartment and site of protein folding, is altered by disruption of intracellular homeostasis. Misfolded proteins accumulated in the ER lead to ER stress (ERS), unfolded protein response (UPR) activation and ER Ca2+ loss. Myocardial stunning is a temporary contractile dysfunction, which occurs after brief ischemic periods with minimal or no cell death, being oxidative stress and Ca2+ overload potential underlying mechanisms. Myocardial stunning induces ERS response with negatively impact on the post-ischemic mechanical performance through an unknown mechanism. AimsIn this study, we explored whether ER Ca2+ efflux through the translocon, a major Ca2+ leak channel, contributes to Ca2+ mishandling and the consequent contractile abnormalities of the stunned myocardium. MethodsMechanical performance, cytosolic Ca2+, UPR markers and oxidative state were evaluated in perfused rat/mouse hearts subjected to a brief ischemia followed by reperfusion (I/R) in absence or presence of the translocon inhibitor, emetine (1 μM), comparing its effects with those of the chaperones TUDCA (30 μM) and 4-PBA (3 mM). ResultsEmetine treatment precluded the I/R-induced increase in UPR signaling markers and improved the contractile recovery together with a remarkable attenuation in myocardial stiffness when compared to I/R hearts with no drug. This alleviation of I/R-induced mechanical abnormalities was more effective than that obtained with the chemical chaperones, TUDCA and 4-PBA. Moreover, emetine treatment produced a striking improvement in diastolic Ca2+ handling with a partial recovery of the I/R-induced oxidative stress. ConclusionBlocking ER Ca2+ store depletion via translocon suppressed ER stress and improved mechanical performance and diastolic Ca2+ handling of stunned myocardium. Modulation of translocon permeability emerges as a therapeutic approach to face dysfunctional consequences of the I/R injury.

Adipocyte STAT5 deficiency does not affect blood glucose homeostasis in obese mice.

The aim of this study was to investigate whether the lack of signal transducer and activator of transcription 5 (STAT5) in mature adipocytes of obese mice (Stat5Adipoq mice) improves glucose and lipid metabolism as previously observed in lean mice. Male Stat5Adipoq mice and their wild type (WT) littermates were fed high-fat diet (HFD). Effects of adipocyte STAT5 deficiency on adiposity as well as on glucose and lipid metabolism were determined under ad libitum feeding and after weight loss induced by calorie restriction. Compared to WT mice, obese Stat5Adipoq mice showed modestly accelerated weight gain and blunted depletion of fat stores under calorie restriction (reduction in % body fat after 3 weeks: WT, -9.3±1.1, vs Stat5Adipoq, -5.9±0.8, p = 0.04). No differences were observed between Stat5Adipoq and WT mice with regard to parameters of glucose and lipid metabolism including basal glycaemia, glucose tolerance, and plasma triglycerides. In conclusion, STAT5 deficiency in the adipocyte of HFD-fed obese mice was associated with increased fat accumulation. In contrast to previous findings in lean mice, however, lipid accumulation was not associated with any improvement in glucose and lipid metabolism. Our results do not support adipocyte STAT5 as a promising target for the treatment of obesity-associated metabolic derangements.

Extracellular cysteines C226 and C232 mediate hydrogen sulfide-dependent inhibition of Orai3-mediated store-operated calcium entry.

The gaseous signaling molecule hydrogen sulfide (H2S) physiologically regulates store-operated Ca2+ entry (SOCE). The SOCE machinery consists of the plasma membrane-localized Orai channels (Orai1-3) and endoplasmic reticulum-localized stromal interaction molecule (STIM)1 and STIM2 proteins. H2S inhibits Orai3- but not Orai1- or Orai2-mediated SOCE. The current objective was to define the mechanism by which H2S selectively modifies Orai3. We measured SOCE and STIM1/Orai3 dynamics and interactions in HEK293 cells exogenously expressing fluorescently tagged human STIM1 and Orai3 in the presence and absence of the H2S donor GYY4137. Two cysteines (C226 and C232) are present in Orai3 that are absent in the Orai1 and Orai2. When we mutated either of these cysteines to serine, alone or in combination, SOCE inhibition by H2S was abolished. We also established that inhibition was dependent on an interaction with STIM1. To further define the effects of H2S on STIM1/Orai3 interaction, we performed a series of fluorescence recovery after photobleaching (FRAP), colocalization, and fluorescence resonance energy transfer (FRET) experiments. Treatment with H2S did not affect the mobility of Orai3 in the membrane, nor did it influence STIM1/Orai3 puncta formation or STIM1-Orai3 protein-protein interactions. These data support a model in which H2S modification of Orai3 at cysteines 226 and 232 limits SOCE evoked upon store depletion and STIM1 engagement, by a mechanism independent of the interaction between Orai3 and STIM1.

Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1.

The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it toward the plasma membrane to activate Orai and SOCE after store depletion.

Seipin: harvesting fat and keeping adipocytes healthy.

Seipin is a key protein in the assembly of cytoplasmic lipid droplets (cLDs) and their maintenance at endoplasmic reticulum (ER)-LD junctions; the absence of seipin results in generalized lipodystrophy. How seipin mediates LD dynamics and prevents lipodystrophy are not well understood. New evidence suggests that seipin attracts triglyceride monomers from the ER to sites of droplet formation. By contrast, seipin may not be directly involved in the assembly of nuclear LDs and may actually suppress their formation at a distance. Seipin promotes adipogenesis, but lipodystrophy may also involve postadipogenic effects. We hypothesize that among these are a cycle of runaway lipolysis and lipotoxicity caused by aberrant LDs, resulting in a depletion of fat stores and a failure of adipose and other cells to thrive.

THE BIDIRECTIONAL EFFECT OF CREATINE SUPPORTS THE MAINTENANCE OF OXIDANT-ANTIOXIDANT HOMEOSTASIS DURING EXERCISE

The importance of supplements used with exercise is increasing day by day. In this study, it was aimed to evaluate the effects of creatine monohydrate (CrM) supplementation with different intensities of exercise on oxidative stress through dynamic thiol disulfide homeostasis. Fourty two BALB/c mice were used and randomly divided into 6 groups; control (C), low-intensity exercise (LIE), high-intensity exercise (HIE), C+CrM (4% of daily diet), LIE+CrM, and HIE+CrM groups. Exercise groups were performed low-intensity (8m/min/30min/day) and high-intensity (24m/min/30min/day) exercise on a mouse treadmill for 8 weeks. At the end of the experimental period, the thiol disulfide homeostasis levels analyzed by using a new automated measurement technique. When the native thiol and total thiol values were examined the difference between the groups was statistically significant (respectively, p=0.029, p=0,035). Creatine intake with exercise decreased native thiol and total thiol levels. However, serum disulfide levels were lower in LIE+CrM compared to other study groups, but there was no statistically significant difference. It is thought that creatine supplementation with exercise reduces the thiol-disulfide homeostasis burden of the organism, and that after the depletion of creatine stores, the sustainability of oxidant-antioxidant homeostasis can be extended, thus prolonging the duration of antioxidant resistance.

Differential activation of Ca2+ influx channels modulate stem cell potency, their proliferation/viability and tissue regeneration

Stem cells have indefinite self-renewable capability; however, factors that modulate their pluripotency/function are not fully identified. Here we show that store-dependent Ca2+ entry is essential for modulating the function of bone marrow-derived mesenchymal stem cells (MSCs). Increasing external Ca2+ modulated cell cycle progression that was critical for MSCs survival. Additionally, Ca2+ was critical for stem proliferation, its differentiation, and maintaining stem cell potential. Ca2+ channel characterization, including gene silencing, showed two distinct Ca2+ entry channels (through Orai1/TRPC1 or via Orai3) that differentially regulate the proliferation and viability of MSCs. Importantly, NFκB translocation, but not JNK/ERK into the nucleus, was observed upon store depletion, which was blocked by the addition of Ca2+ channel inhibitors. Radiation lead to a decrease in saliva secretion, decrease in acinar cell number, and enlarged ducts were observed, which were restored by the transplantation of stem cells that were propagated in higher Ca2+. Finally radiation showed a decrese in TRPC1 expression along with a decrese in AQP5, which was again restored upon MSC tranplantation. Together these results suggest that Ca2+ entry is essential for stem cell function that could be critical for regenerative medicine.

Physiological Functions of CRAC Channels.

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol 1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity.