CACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient +/−Cacna1c rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of +/−Cacna1c rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular +/−Cacna1c myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in +/−Cacna1c rats. The β-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both +/−Cacna1c and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in +/−Cacna1c myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in +/−Cacna1c than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in +/−Cacna1c compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, +/−Cacna1c myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in +/−Cacna1c cardiomyocytes. CACNA1C gene encodes the pore-forming α1C subunit of the L-type calcium (Ca2+) channel, Cav1.2, which is widely expressed in neurons, smooth muscle cells, and cardiac myocytes [2+ channels (LTCCs) are macromolecular complexes consisting of the α1C subunit, which is the pore-forming subunit containing the voltage sensor, and auxiliary subunits , which modulate biophysical and trafficking properties of the cardiac Cav1.2 channel [The alcium Ca+ channel2+ entry and Ca2+-mediated regulation of transcription [2+ entry upon depolarization during an action potential. This initial Ca2+ entry triggers the opening of cardiac ryanodine receptors (RyR2) located on the sarcoplasmic reticulum (SR), with subsequent SR Ca2+ release. Cytosolic Ca2+ binds to myofilaments and initiates contraction [2+ levels are decreased by Ca2+ re-uptake into the SR mediated by the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2a), whose activity is regulated by the small inhibitory protein phospholamban (PLB), and by Ca2+ extrusion from the cell mediated by the sarcolemmal Na+/Ca2+ exchanger (NCX) [2+ handling proteins are modulated by β-adrenergic signaling, which results in the stimulation of protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). PKA and CaMKII phosphorylate Cav1.2, RyR2, and PLB (among other proteins) to mediate the positive-inotropic and -lusitropic effects of sympathetic stimulation [In neurons, Cav1.2 is primarily located on postsynaptic membranes, dendrites, and somata, where it controls activity-dependent Cacription . In cardtraction . Afterwaer (NCX) . During mulation .CACNA1C has emerged as a major target of interest in neuropsychiatric and cardiovascular disease. In genome-wide association studies, single-nucleotide polymorphisms of the gene were associated with psychiatric disorders, such as major depression, bipolar disorder, and schizophrenia [CACNA1C can lead to Timothy syndrome, a genetic disease comprising autism spectrum symptoms, arrhythmias and heart disease [2+ current in  cardiomyocytes [2+ channel blockers, such as verapamil, are inhibitors of Cav1.2/LTCCs, and they have been used for decades as anti-arrhythmic and anti-hypertensive drugs. A recent study involving patients with bipolar disorder, schizophrenia, and nonaffective psychosis has revealed that those patients taking LTCC blockers exhibited reduced rates (up to 70% reduction) of psychiatric hospitalizations and self-harm admissions [CACNA1C/Cav1.2 is involved in psychiatric and cardiac disease and that the two entities might be linked.Due to its pivotal role in neurons and cardiac myocytes, ophrenia . Therefoophrenia . Further disease , Brugada disease ,7. In ca disease ,9. On thmyocytes ,11,12. Cmissions . Moreovemissions . Thus, t+/−Cacna1c rats represent a recently developed animal model for studying the role of Cav1.2 in psychiatric and cardiovascular disease [+/−Cacna1c rats exhibit deficits in pro-social behavior and communication together with other behavioral alterations consistent with autism-like symptoms [+/−Cacna1c rats has not been studied to date. Hence, we set out to characterize the cardiac phenotype of these animals with a focus on cardiomyocyte Ca2+ signaling. Our results reveal that, despite apparently normal L-type Ca2+ currents and Ca2+ transients, there is profound remodeling of Ca2+ handling proteins under baseline conditions, which underlies an impaired response to sympathetic stress.Haploinsufficient  disease . Thus fasymptoms ,16,17, t+/−Cacna1c rats differed from WT  from WT and +/−Cacna1c myocytes obtained during the recording of a current-voltage (I-V) relationship were similar  were all comparable between WT and +/−Cacna1c rats for both linescan  were studied in isolated ventricular myocytes loaded with Fluo-4 either by means of linescan confocal imaging or by fast 2D confocal imaging. The results are shown in linescan A and 2D linescan B data. B imaging C. Neithe2+ indicator. +/−Cacna1c myocytes under baseline conditions (left panels), and average results are shown in +/−Cacna1c myocytes. Diastolic sarcomere length (SL), fractional shortening, time-to-peak 90%, and relaxation time 90% were all comparable between WT and +/−Cacna1c myocytes.CaTs underlie contraction. Hence, we also studied the contractile activity of ventricular myocytes by means of sarcomere shortening. The protocol was identical to the one used for imaging of CaTs, i.e., 1 Hz electrical stimulation, except the myocytes were not loaded with any Ca+/−Cacna1c myocytes exhibit Ca2+ handling  and contractions very similar to WT myocytes.Thus, under baseline conditions, 2+ handling proteins and their major phosphorylation sites in WT and +/−Cacna1c rats. +/−Cacna1c rats. However, +/−Cacna1c rats developed a moderate but significant up-regulation of both major Ca2+ extrusion proteins, i.e., NCX (by ≈20%) and SERCA2a (by ≈50%). Furthermore, the phosphorylation of PLB at both S16 and T17 was unaltered. By contrast, RyR2 exhibited altered phosphorylation. While phosphorylation at S2814 did not differ, there was a highly significant up-regulation of phosphorylation at S2808 by ≈120% in +/−Cacna1c rats.Next, we performed a Western Blot analysis of LV tissue to investigate expression levels of the most important Ca2+ current  and/or an increase in stimulation frequency.+/− rats ,16,17 an+/− rats . Therefo+/−Cacna1c myocytes before (left panels) and during (right panels) stimulation with ISO. In both groups, ISO greatly increased systolic Ca2+ and CaT amplitude and accelerated CaT decay. In the presence of ISO, diastolic Ca2+ did not differ between WT and +/−Cacna1c myocytes  compared to WT (≈92%) myocytes. Decay of the caffeine-induced CaT, again, was comparable between WT and +/−Cacna1c myocytes and similar to baseline conditions  causes a large, Ca+/−Cacna1c myocytes stimulated at 1 Hz. Ventricular myocytes from both genotypes were treated with progressively increasing ISO concentrations ranging from 1 to 100 nM. Following rundown correction, the ISO response on CaT amplitude was normalized to the response at 100 nM ISO (see +/−Cacna1c myocytes are illustrated in +/−Cacna1c myocytes (EC50 = 13.0 nM) as compared to WT (EC50 = 4.1 nM).Using linescan imaging, we investigated the concentration dependence of the ISO-induced increase in CaTs in WT and  ISO see . Figure +/−Cacna1c myocytes stimulated at 1 Hz. The results are shown in +/−Cacna1c myocytes (EC50 = 6.1 nM) as compared to WT (EC50 = 3.9 nM).In similar experiments, we determined the concentration dependence of the ISO-induced increase in fractional shortening in WT and +/−Cacna1c compared to WT myocytes.Thus, ISO exhibits a reduced potency to stimulate CaT amplitude and fractional shortening in 2+ handling proteins, Langendorff-perfused hearts were treated with ISO (100 nM), and LV tissue homogenates of these hearts were probed for phosphorylation of RyR2 and PLB at PKA and CaMKII sites by means of Western blotting. The results are shown in +/−Cacna1c rats, where phosphorylation at S2808 was not increased in a statistically significant manner  myocyte before and during ISO exposure. Baseline CaTs were very small in amplitude, and CaT decay was slow in both cells because SERCA function was blocked, the SR was depleted of Ca2+, and sarcolemmal Ca2+ influx contributes only a minor fraction (≈7%) to the cytosolic CaT (with intact SR function) in rat ventricular myocytes [2+ in both cells, but the increase appeared to be less pronounced in the +/−Cacna1c myocyte. 2+ influx did not differ between WT (+/+) and +/−Cacna1c (+/−) myocytes, in line with our observation of identical L-type Ca2+ currents  of ISO to increase CaTs and contractions in myocytes . Applicaents see C. FollowCacna1c haploinsufficient rats represent a unique animal model with a global heterozygous knockout of the gene encoding the Cav1.2 channel, found in neurons, cardiomyocytes, and other cell types. Previous studies have established a behavioral phenotype of the animals [2+ handling, which is unmasked during sympathetic stress-induced changes in RyR2-mediated SR Ca2+ release. The results imply that alterations in CACNA1C per se can predispose to alterations in both behavior and cardiac function, providing a potential molecular link between neuropsychiatric disorders and cardiac disease. animals ,16,17. H+/−Cacna1c rats appeared unaltered under basal conditions. L-type Ca2+ current, sarcolemmal Ca2+ influx, electrically stimulated CaTs, SR Ca2+ load and release, and sarcomere shortenings were all very similar in +/−Cacna1c and WT myocytes. Moreover, ventricular myocyte size (membrane capacitance) was also very similar, as was heart (and LV and RV) size, suggesting no gross structural and functional alterations of the heart and its cardiomyocytes. Therefore, it appears that +/−Cacna1c rats were able to compensate for reduced expression of Cav1.2 to maintain cardiomyocyte and heart function under basal conditions.Despite reduced expression of Cav1.2 (by ≈30%), cardiomyocyte function of 2+ current observed here, are in line with findings of the mouse model with an inducible knockout of one Cav1.2 allele [2+ current were found. However, Goonasekera et al. [2+ current in their haploinsufficient mouse model, along with reduced SR Ca2+ load, CaTs, and contractions.This is consistent with the findings of a mouse model with an inducible knockout of one Cav1.2 allele . Another2 allele , in whica et al. , observe+/−Cacna1c myocytes exhibit reduced expression of Cav1.2 but unaltered L-type Ca2+ current? Our present results do not provide an answer to this question, and we can only speculate on the underlying cellular mechanisms. Cav1.2 expression, location, and function are regulated in a complex fashion by auxiliary subunits, regulatory proteins (such as RAD), phosphorylation events , membrane trafficking, turnover or degradation of the channel, e.g., [+/−Cacna1c myocytes despite reduced global expression. Alternatively, Cav1.2 channels in +/−Cacna1c myocytes may exhibit increased single-channel conductance or open probability. This latter scenario seems unlikely, however, as it would result in a leftward shift of the ICa-V curve, which we did not observe.How is it possible that l, e.g., ,22,23. P2+ handling  under basal conditions, LV myocardium from +/−Cacna1c rats exhibited profound alterations in the expression and phosphorylation of major Ca2+ handling proteins. These alterations consisted of reduced expression (by ≈30%) of Cav1.2, increased expression of Ca2+ removal proteins NCX (by ≈20%) and SERCA2a (by ≈50%), and increased phosphorylation of RyR2 at S2808 (by ≈120%). This remodeling of Ca2+ handling proteins may be the reason why +/−Cacna1c myocytes were able to maintain normal CaTs under basal conditions. RyR2 exhibited larger phosphorylation at S2808, a site predominantly phosphorylated by PKA, in +/−Cacna1c myocytes. By contrast, phosphorylation of PLB at either S16 or T17 was unaltered. Thus, only proteins in the junctional cleft, i.e., RyR2, exhibited higher PKA-dependent phosphorylation suggesting subcellularly restricted regulation of protein activity in +/−Cacna1c myocytes possibly to promote Ca2+-induced SR Ca2+ release in order to compensate for the reduced Cav1.2 protein levels.Despite unaltered cardiomyocyte Ca+/−Cacna1c myocytes were able to maintain normal CaTs and contractions. Mimicking sympathetic stress by applying the β-adrenergic receptor agonist isoprenaline, however, unmasked a Ca2+ handling phenotype in +/−Cacna1c myocytes. Following β-adrenergic receptor activation by isoprenaline, rising cAMP levels and PKA activity lead to positive-inotropic and -lusitropic effects through phosphorylation of Ca2+ handling proteins. Isoprenaline exhibited both lower potency and efficacy in increasing CaTs and sarcomere shortenings in +/−Cacna1c myocytes. Thus, the isoprenaline-induced positive-inotropic effect was attenuated in +/−Cacna1c myocytes, and this could be explained by a smaller increase in sarcolemmal Ca2+ influx and fractional SR Ca2+ release resulting in a smaller increase in CaTs and hence contractions. Sarcolemmal Ca2+ influx is largely carried by L-type Ca2+ channels and acts as a trigger for SR Ca2+ release. Sarcolemmal Ca2+ influx was identical between WT and +/−Cacna1c myocytes under baseline conditions, but the isoprenaline-mediated increase in sarcolemmal Ca2+ influx was attenuated in +/−Cacna1c myocytes suggesting impaired β-adrenergic regulation of L-type Ca2+ channels in +/−Cacna1c myocytes. This also means reduced Ca2+-induced SR Ca2+ release in +/−Cacna1c myocytes. SR Ca2+ release is mediated by RyR2, and RyR2 activity, in turn, is regulated by SR Ca2+ load  and RyR2 phosphorylation (among other regulators) [2+ load in the presence of isoprenaline was similar in +/−Cacna1c and WT myocytes and is thus unlikely to account for the observed difference in a fractional release. As the PKA-dependent phosphorylation of RyR2 at S2808 was elevated already under basal conditions in +/−Cacna1c myocytes, we suggest that a smaller phosphorylation reserve was accountable for the attenuated response to isoprenaline. In line with this hypothesis, experiments with Langendorff-perfused hearts directly showed that, in +/−Cacna1c LV myocardium, isoprenaline caused no significant increase in RyR2 phosphorylation at S2808, but a highly significant increase in WT LV myocardium (by ≈310%), matching our cellular findings. Similarly, isoprenaline elicited increased RyR2 phosphorylation at S2814 in WT (by ≈160%) but not +/−Cacna1c LV myocardium.Under basal conditions, ulators) ,25. SR C+/−Cacna1c and WT myocytes. In both genotypes, isoprenaline caused a comparable acceleration of CaT decay and relaxation time. The main actor for CaT decay and relaxation is the SERCA pump, whose activity is regulated by PLB phosphorylation [+/−Cacna1c hearts suggesting a full-blown increase in cAMP levels and PKA and CaMKII activities with subsequent phosphorylation of certain target proteins following stimulation with isoprenaline. (2) It implies that there are compartmentalized alterations of PKA- and CaMKII-dependent phosphorylation in the junctional cleft and the microenvironment of the RyR2 in +/−Cacna1c myocytes following stimulation with isoprenaline. The latter notion is in line with previous studies demonstrating local regulation of RyR2 activity by enzymes  in its microenvironment, e.g., [In contrast to the positive-inotropic effect, the positive-lusitropic effect elicited by isoprenaline did not exhibit any differences between rylation . PLB phot, e.g., ,28.CACNA1C gene are associated with both psychiatric and cardiac disease [2+-dependent regulation of transcription and excitation-contraction coupling. Therefore, alterations in expression and function of Cav1.2 are likely to impair both neuronal and cardiac function, suggesting that Cav1.2 may be a mechanistic link between psychiatric disorders and cardiac disease.Clinically, there are mutual associations between neuropsychiatric disorders and cardiac diseases. For example, depression or anxiety are associated with several cardiac diseases, including coronary artery disease, myocardial infarction, heart failure, and atrial fibrillation ,31,32,33 disease ,6. In boCacna1c haploinsufficient rats. Previous studies have unraveled a behavioral phenotype of this animal model, including deficits in pro-social behavior and communication together with other behavioral alterations consistent with autism-like symptoms [+/−Cacna1c rats also exhibit a cardiac phenotype. Hence, reduced expression of Cav1.2 appears to be sufficient to induce both a behavioral and a cardiac phenotype. The cardiac phenotype occurred in the absence of gross structural alterations as both ventricular myocytes and whole hearts (LV and RV) of +/−Cacna1c rats were similar in size compared to WT.Here, we provide further evidence for this notion using a unique rat model, i.e., symptoms ,16,17. T2+ handling proteins was observed in +/−Cacna1c hearts, suggesting that alterations in Cav1.2 expression are sufficient to induce remodeling. Some features of this Ca2+ handling remodeling in +/−Cacna1c myocytes are reminiscent of the remodeling observed in other cardiac diseases. In particular, increased expression/activity of NCX and elevated PKA-dependent phosphorylation of RyR2 are hallmarks of heart failure [+/−Cacna1c myocytes, is also found in hypertrophy and heart failure [+/−Cacna1c model clearly differ from the Ca2+ handling remodeling in hypertrophy and heart failure, such as the increased expression of SERCA  or the  failure ,45). Inc failure ,47,48. T2+ influx and the CaT. In addition, CaMKII and PKA have various targets other than Ca2+-regulating proteins. Notably, these targets include sarcolemmal ion channels, such as voltage-dependent Na+ and K+ channels [2+ regulation [+/− myocytes should certainly aim at characterizing a potential electrophysiological phenotype in this model.Action potential duration and shape are important determinants of sarcolemmal Cachannels ,50,51. Agulation . We did +/−Cacna1c myocytes are able to maintain normal CaTs and contractions. There is remodeling of Ca2+ handling proteins though, consisting of increased expression of NCX and SERCA and elevated phosphorylation of RyR2 at S2808. +/−Cacna1c myocytes display an attenuated positive-inotropic—but not positive-lusitropic—response to isoprenaline stimulation mediated by an impaired ability to increase sarcolemmal Ca2+ influx and SR Ca2+ release. This attenuated β-adrenergic response is caused by a reduced phosphorylation reserve of RyR2 at S2808 and S2814. This Ca2+ handling remodeling in +/−Cacna1c rats under basal conditions may alter the heart’s response to stressors and thus cause an increased susceptibility to cardiac disease under conditions of mechanical or mental stress. Since CACNA1C is a prominent risk factor for many psychiatric disorders, which often go along with heightened stress or anxiety levels, it is interesting to see that a heterozygous knockout of the gene in rats also leads to an altered reaction to stress at the level of the heart. Thus, alterations in CACNA1C/Cav1.2 may constitute a molecular link between neuropsychiatric disorders and cardiac disease.Under baseline conditions, A detailed description of methods can be found in the +/−Cacna1c rats were generated by means of zinc finger technology by SAGE Labs  on a Sprague Dawley (SD) background, following a previously established protocol [+/−Cacna1c rats [-/-Cacna1c rats are embryonically lethal [Cacna1c Cel-1 F) and CCTCCTGGATAGCTGCTGAC (Cacna1c Cel-1 R). After weaning on postnatal day 21, rats were socially housed in groups of 4–6 with same-sex littermate partners under standard laboratory conditions (22 ± 2 °C and 40–70% humidity) with free access to standard rodent chow and water. In this study, female rats were investigated at the age of 9–15 months. All experiments, except for immunoblotting, were performed with the experimenter blinded to the genotype of the animals/cells. Rats were anesthetized by isoflurane inhalation , AbbVie, Wiesbaden, Germany). Deep anesthesia was confirmed by abolished pain reflexes. Rats were sacrificed by decapitation using a guillotine. Following the sacrifice of the animals, hearts were excised quickly and used for tissue or cell isolation.The study was approved by local animal welfare authorities and was performed in accordance with the European Union Council Directive 2010/63/EU and the German Animal Welfare Act. protocol . Cacna1c+/− rats  exhibit y lethal . Rats wey lethal . The dayy lethal . The folFollowing decapitation, hearts were quickly excised and placed in an ice-cold solution. The chambers (LV) were separated, weighed, frozen in liquid nitrogen, and stored at −80 °C. Tissue lysates were used for investigating protein expression using a standard Western Blot technique.Ventricular myocytes were isolated by means of a standard Langendorff protocol with enzymatic perfusion as previously described ,56. For 2+ current of isolated ventricular myocytes was recorded using whole-cell patch clamp. Ca2+ transients (CaTs) were recorded using the linescan mode of a confocal microscope  or at a 2D-array scanning microscope . Myocytes were loaded with 6.6 μM Fluo-4/AM , field stimulated (40 V) at 1 Hz, and superfused with recording solution containing (mM): 140 NaCl, 5.4 KCl, 1.5 CaCl2, 0.5 MgCl2, 10 HEPES, 10 glucose, pH 7.4, at RT. By means of a caffeine bolus application (20 mM), SR Ca2+ content and fractional SR Ca2+ release were estimated. Sarcomere shortening was measured in unloaded myocytes using an IonOptix setup .L-type CaStress was mimicked by increasing frequencies of stimulation  or by adding Isoprenaline  to the recording solution. A concentration-response curve was obtained by usage of ISO concentrations ranging from 1–100 nM.n”, and the number of animals as “N”. Normally distributed data sets were compared by means of an unpaired, two-tailed Student’s t-test, not-normally distributed data with a Mann–Whitney U test and considered significant when p < 0.05. For multiple group comparisons, ANOVA was applied. Concentration-response curves were analyzed with a non-linear four-parameter (4PL) regression model.Statistical analysis was performed with GraphPad Prism . Data are presented as scatter plots with bar graphs indicating mean ± SEM. The number of cells is provided as “


Aging-related impairment of the blood brain barrier (BBB) and neurovascular unit (NVU) increases the risk for neurodegeneration. Among various cells that participate in BBB and NVU function, calcium signals in astrocytic endfeet are crucial for maintaining BBB and NVU integrity. To assess if aging is associated with altered calcium signals within astrocytic endfeet of the dorsolateral striatum (DLS), we expressed GCaMP6f in DLS astrocytes of young (3–4 months), middle-aged (12–15 months) and aging (20–30 months) mice. Compared to endfeet in young mice, DLS endfeet in aging mice demonstrated decreased calreticulin expression, and alterations to both spontaneous membrane-associated and mitochondrial calcium signals. While young mice required both extracellular and endoplasmic reticulum calcium sources for endfoot signals, middle-aged and aging mice showed heavy dependence on endoplasmic reticulum calcium. Thus, astrocytic endfeet show significant changes in calcium buffering and sources throughout the lifespan, which is important for understanding mechanisms by which aging impairs the BBB and NVU. A large body of evidence strongly suggests that aging-related impairment of the blood brain barrier (BBB) and neurovascular unit (NVU) potentiates and possibly triggers neurodegenerative processes in the brain12. Among the multiple brain regions prone to neurodegeneration, the striatum is particularly vulnerable to neurovascular dysfunction and neurodegeneration because of its rich vascular supply13. This notion is supported by reports showing a higher percentage of lacunar infarcts in the striatum compared to other brain regions14, increased propensity for accumulating toxic striatal protein aggregates16, and an increase in striatal BBB permeability during PD, AD, and stroke19. Furthermore, aging-related changes in the striatal BBB and NVU can lead to parkinsonian symptoms20, enlarged perivascular spaces21, and PD due to dopaminergic neuron loss22. Although these studies indicate that striatal BBB and NVU dysfunction are important contributing factors for various forms of neurodegeneration, little is known about the changes to BBB and NVU function that occur across the lifespan.Advanced age is the single greatest risk factor for neurodegenerative conditions such as Parkinson’s disease (PD), Alzheimer’s disease (AD)26. Among these cell types, astrocytes are unique in that they simultaneously contact neurons and vasculature, thus enabling these cells to alter vascular structure and function in response to brain activity30. Astrocytes perform this vital function via spontaneous Ca2+ signals within endfeet that completely ensheathe capillaries32. Indeed, Ca2+ signals in astrocytic endfeet can alter vasoconstriction and dilation33, mediate vascular repair after injury34, regulate brain volume35, alter aging-related cognitive behavior via intracellular IP3R2-specific Ca2+ signals36, as well as maintain NVU coupling37. Thus, at a functional level, astrocytic endfoot Ca2+ signals govern critical aspects of BBB integrity and NVU function. It follows that aging-related changes in striatal astrocytic endfoot Ca2+ signals likely contribute to BBB and NVU dysfunction during neurodegeneration. Based on the strong body of evidence pointing to striatal vascular dysfunction in various aging-related neurodegenerative diseases as well as a central role for astrocytic endfoot Ca2+ signals in maintaining BBB integrity and NVU function, we asked if aging is associated with alterations in spontaneous astrocytic endfoot Ca2+ events within the dorsolateral striatum (DLS) of acutely extracted live brain slices from young, middle-aged, and aging mice.The BBB and NVU are comprised of distinct cellular elements, viz., neurons, endothelial cells, pericytes and astrocytes, each of which plays important and specific roles in neurovascular function2+ events, as well as changes in Ca2+ influx into astrocytic endfoot mitochondria. These aging-related alterations in endfoot Ca2+ events are associated with significant reductions in the expression of a major endoplasmic reticulum (ER) Ca2+ buffering protein, calreticulin (CALR) and astrocyte endfoot mitochondrial mass. Our findings have important implications for understanding how an aging-related reduction in CALR expression can alter astrocytic endfoot Ca2+ signals, eventually resulting in vascular dysfunction during aging and aging-related neurodegeneration.We show that when compared to young mice, DLS brain slices of aging mice display dramatic alterations in the kinetics, dynamics, and sources of spontaneous membrane-associated Ca2+ signals in the DLS were altered across the lifespan. To measure spontaneous membrane-associated astrocytic endfoot Ca2+ events in the DLS, young, middle-aged, and aging mice were stereotaxically injected with an AAV expressing membrane localized GCaMP6f (Lck-GCaMP6f), driven by an astrocyte-specific GfaABC1D promoter (AAV2/5-GfaABC1D-Lck-GCaMP6f). Intraventricular administration of red fluorescent tomato lectin (TL) into the mouse heart prior to live brain slicing enabled visualization of blood vessels (BV) in the DLS    Fig. . Conversce) Fig. . Next, wice Fig. . Aging mice Fig. . Togethe2+ events in all mice occurred as either static or expanding waves adjacent to TL BVs39  but no differences in the distance traveled or area covered 40 To assess this possibility, young and aging striatal brain sections were co-immunostained for CALR and the astrocyte endfoot marker, aquaporin 4 (AQP4). In both young and aging mice, astrocytic AQP4 labeling clearly outlined BVs, while CALR staining appeared as punctate structures throughout AQP4 stained endfeet and into the surrounding neuropil  and is associated with cognitive decline50, decreased ATP production51, changes to mitochondrial morphology51, and increased accumulation of misfolded proteins52. Moreover, a study by Biwer et al. demonstrated that conditional deletion of CALR from endothelial cells in aging mice increased the spatial spread of spontaneous endothelial Ca2+ signals and impaired Ca2+ mobilization47. Thus, we hypothesize that our observation of decreased CALR expression is upstream of the changes to spontaneous Ca2+ events in the cytosol and mitochondria of DLS astrocyte endfeet reported in Figs We found that aging astrocyte endfeet in the DLS demonstrate a significant reduction in CALR Fig. . ER-locaddle-age 2 months 2+ signals in aging mice rely heavily on ER Ca2+ stores 2+ events are downstream of reduced CALR expression. This study does not address whether changes in Ca2+ signals and CALR/AQP4 expression in the aging endfoot are directly associated with BBB dysfunction. In particular, studies characterizing the relationship between ER stress, CALR expression, and changes to vascular permeability are justified. In support of this idea, reports show that ER stress is associated with vascular endothelial cell dysfunction68 and angiogenesis in the context of disease69. Therefore, because reduced CALR can increase ER stress60, it is possible that CALR-induced alterations in ER stress within the aging astrocyte endfoot can affect BBB integrity. Future work will focus on dissecting the relationship between CALR in astrocytic endfeet, ER stress, and BBB permeability.In summary, we show that aging-related abnormalities in spontaneous astrocyte endfoot cytosolic and mitochondrial CaMale and female C57BL/6J mice  were used for all experiments. Young mice were 3–4 months of age, middle-aged mice were 12-15 months of age, and aging mice were 20–30 months of age. Mice were aged in-house in the animal vivarium. All experiments were conducted in compliance with all relevant ethical regulations for animal testing and research approved by the Texas A&M University Institutional Animal Care and Use Committee. Food and water were provided ad libitum. All mice were maintained on a 12 h light-dark cycle.70. AAVs were injected into the right DLS using a glass injection pipette, at a rate of 750 nl/min using a Harvard Apparatus Pump 11 Pico Plus Elite, 70-41506 . To image near membrane astrocyte endfoot Ca2+ events in young, middle-aged, and aging mice, 1 μl of AAV2/5-GfaABC1D-Lck-GCaMP6f (1013 genome copies/ml)  was injected into the DLS. In young, middle aged, and aging mice, astrocyte endfoot mitochondrial Ca2+ events were imaged by injecting 2 μl of AAV2/5-GfaABC1D-mito7-GCaMP6f (1013 genome copies/ml)41 into the DLS. Coordinates for stereotaxic injection were 0.8 mm rostral to bregma, 2 mm lateral to the midline and 2.4 mm ventral to the pial surface.Young, middle-aged, and aging mice were deeply anesthetized using isoflurane dispensed from a SomnoSuite Low Flow Anesthesia System , and a craniotomy was performed as previously describedLycopersicon esculentum lectin  was injected into the apex of the beating left ventricle. 1 min following TL injection into the left ventricle, the mouse was decapitated, the brain was extracted and then immediately blocked for obtaining live brain slices. 400 µm coronal striatal slices were cut using a Microslicer 01 N (Ted Pella) in a solution comprising 194 mM sucrose, 30 mM NaCl, 4.5 mM KCl, 1.2 mM NaH2PO4, 26 mM NaHCO3, 10 mM D-glucose, and 1 mM MgCl2 saturated with 95% O2 and 5% CO2. Live brain slices were incubated in artificial cerebrospinal fluid (aCSF) composed of 126 mM NaCl, 2.5 mM KCl, 1.24 mM NaH2PO4, 1.3 mM MgCl2, 2.4 mM CaCl2, 26 mM NaHCO3, 10 mM D-glucose at 33 °C for 30 min and then maintained at 23 °C in aCSF for the duration of the experiment.Young, middle-aged, and aging mice were deeply anesthetized with isoflurane and the left ventricle was rapidly accessed. 200 μl of DyLight 594 labeled 2+ events. A 25 mW HeNe 594 nm excitation laser line at 10% of maximum intensity was used to visualize blood vessels (BVs) and localize astrocytic endfoot Ca2+ events in the DLS. Membrane-associated endfoot and mitochondrial Ca2+ events near TL-labeled BVs were identified as astrocytic endfoot Ca2+ events and imaged for 10 min at 1 frame per second. Imaging parameters  were maintained constant across all imaging sessions.Live mouse striatal brain slices were imaged with an Olympus FV1200 upright laser-scanning confocal microscope using a 40× water immersion objective lens (NA 0.8), and a digital zoom of 3. We used a 488 nm excitation wavelength at 10% of maximum intensity of a 100 mW Argon laser to record membrane-associated astrocyte endfoot and mitochondrial Ca2+ events in astrocyte endfeet was performed by first imaging spontaneous Ca2+ events, followed by sequential depletion of extracellular Ca2+ and ER Ca2+ in the slice. To deplete extracellular Ca2+, spontaneous Ca2+ events were recorded for 5 min in bath perfused aCSF, followed by bath perfusion of zero Ca2+ aCSF, in which CaCl2 was omitted. To deplete ER Ca2+ stores, 20 µM cyclopiazonic acid  was perfused into the bath for 15 min, after which Ca2+ events were recorded for 5 min from the same field of view.Imaging of Ca13 genome copies/ml), three weeks later mice were perfused, and brains from young and aging mice were collected. 40 µm striatal sections were obtained and then stained with rabbit anti-AQP4 and goat anti-rabbit Alexa Fluor 595. Following all staining, sections were mounted on glass slides and imaged using an FV1200 inverted confocal microscope equipped with a 60× oil immersion objective. Settings for laser power, high voltage (HV), gain, offset, and aperture diameter were maintained across all sessions.Mice were deeply anesthetized with isoflurane and transcardially perfused with 1X PBS, followed by 10% formalin. Brains were extracted and stored in 10% formalin for 48 h at 4 °C then dehydrated in 30% sucrose in 1X PBS  for 48 h. A Microm HM 550 cryostat was used to cut 40 µm coronal sections of the striatum that were stored at 4 °C in 0.01% sodium azide  until the day of immunostaining. Sections were washed 3× in 1X PBS, then permeabilized and blocked in 0.5% Triton X-100 and 10% normal goat serum at room temperature for 45 min. To prevent cross reactivity, sections were sequentially stained with calreticulin (CALR) and aquaporin 4 (AQP4) primary and secondary antibodies. Sections were first incubated overnight at 4 °C in rabbit anti-CALR , washed twice in 1X PBS, then incubated in goat anti-rabbit Alexa Fluor 594  secondary antibody for 1 h at room temperature. After 3 washes in 1X PBS, sections were stained with rabbit anti-AQP4 1:5000  overnight at 4 °C, followed by goat anti-rabbit Alexa Fluor 488  for 1 h at room temperature. For astrocyte mitochondria images, the right DLS of young and aging mice was injected with 400 nl of AAV2/5-GfaABC1D-mito7-eGFP  for endfoot Ca2+ events were generated as previously described, using the ImageJ plugin, GECIquant71. dF/F traces of GCaMP6f fluorescent signals were generated from the acquired ROIs and analyzed with Minianlysis 6.0.07 (Synaptosoft) to generate amplitude (dF/F), and duration (s) values for each trace. Frequency (events/min) was determined by manually counting the number of peaks associated with Ca2+ events that initiated at a specific ROI. Expanding Ca2+ waves coming from adjacent ROIs were visually identified and omitted from the ROI being used for determining event frequency.Membrane-associated and mitochondrial astrocytic endfoot Ca2+ waves, the velocity (µm/s), distance traveled (µm), and area (µm2) of membrane-associated astrocytic endfoot Ca2+ events were analyzed using ImageJ. All confocal movies for calculating expansion velocity were acquired at a frame rate of 1 frame/s. Expansion velocities were determined by counting the number of frames taken for Ca2+ events to attain maximum area within the astrocytic endfoot. The distance covered by expanding endfoot Ca2+ signals was determined by using the line tool on ImageJ to trace the maximum length attained by Ca2+ events along the corresponding BV. The area of propagating Ca2+ events were quantified using the polygon tool in ImageJ. Using the polygon tool, the total area covered by a propagating Ca2+ event along the BV was manually outlined from the final frame of the Ca2+ event. In the case of ROIs with multiple areas, the areas were averaged.For expanding endfoot Ca72 was used to determine directionality of expanding Ca2+ events. Prior to analysis videos were cropped to specifically encompass BVs, and BVs were rotated to the horizontal orientation. Videos were then loaded into AQuA using the default GCaMP-ex vivo-lck preset with a nominal spatial resolution of 0.21 µm/pixel and a temporal resolution of 1 frame per second. Two ROIs were then manually drawn to demarcate the area to be analyzed and to identify the BV as a landmark. The top of the FOV was assigned as north and this direction was maintained for each video. All parameters during the detection pipeline were adjusted to minimize the detection of noise for each video. Specific values for each parameter did not differ more than 5% from the software presets. During post processing, detected events smaller than 10 µm2 or that occurred more than 5 µm away from the BV ROI were excluded. An overall propagation score, defined as an arithmetic sum of 4 directions for each endfoot calcium event was generated using AQuA. Only events with an overall propagation score greater than 1 were determined to be expanding, while any event with a propagation score less than 1 was considered a static event and was excluded. For expanding events, we utilized a ratio of posterior to anterior propagation score as a method to determine overall directionality. For example, an event with a posterior/anterior value greater than 1 was determined to be moving towards the BV.AQuAt test or paired sample t test was used to compare normally distributed datasets. p < 0.05 was considered statistically significant. Sample sizes, statistical tests used, and exact p values are reported in figures and figure legends.In all graphs each datapoint represents a single DLS BV whereby all ROIs pertaining to that BV have been averaged. Statistics were performed using Origin 2022 . For statistical testing, datasets were first tested for normality. Non-normal datasets were subjected to either a Mann-Whitney or paired sample Wilcoxon signed rank test. A two-sample Further information on research design is available in the Reporting SummarySupplemental Movie 1Supplemental Movie 2Supplemental Movie 3Supplemental Movie 4Supplemental Movie 5Supplemental Movie 6Supplemental Movie 7Supplemental Movie 8Supplemental Movie 9Supplemental Movie 10Supplemental Movie 11


Rhipicephalus linnaei transmits pathogens such as Ehrlichia canis, Babesia vogeli, and Hepatozoon canis. The veterinary market has synthetic acaricides to ticks control. Esters derived from castor oil are efficient. However, there is little information about their effects on non-target organisms. This work consisted of a clinical  and histological and histochemical analysis (liver and spleen) of female rabbits exposed to these esters and afoxolaner. The rabbits were divided into three groups: control group (CG) received Bandeirante® rabbit feed; the afoxolaner treatment (TG1) received rabbit feed and two doses of afoxolaner; castor oil esters treatment (TG2) received rabbit feed enriched with esters (1.75 g esters/kg). No alterations were observed in the AST, ALT, and ALP enzymes in exposure to esters TG2. Rabbits from TG1 showed changes in AST. The liver of rabbits exposed to afoxolaner underwent histological and histochemical changes, such as steatosis and vacuolation, as well as poor protein labeling. Polysaccharides were intensely observed in the group exposed to esters. The spleen showed no changes in any of the exposure. Esters from castor oil caused fewer liver changes when incorporated into the feed and fed to rabbits than exposure to afoxolaner.In dogs,  Rhipicephalus linnaei, formerly known as the tropical lineage of R. sanguineus, known as the brown dog tick, has canids as its primary hosts . In addition, protozoa such as Babesia vogeli and Hepatozoon canis can also cause serious illness . These bioactives act as a natural acaricide against ticks, changing the morphology and physiology of organs, such as salivary glands and ovaries. Pilot studies have indicated that they would not cause damage to the hosts or the environment  were used. They were divided into three groups: GC (control group), TG1 (treatment with afoxolaner) and TG2 (treatment with castor oil). They were placed in individual cages made of galvanized wire, for adaptation, under controlled temperature and photoperiods, receiving water and food ad libitum.To evaluate the effects of esters, the rabbits were fed 175g of ester-enriched feed daily at a concentration of 1.75 g ester/kg of Bandeirante® commercial feed, a concentration previously established in a pilot study conducted by Control Group (CG): the rabbits were provided daily with Bandeirante® commercial feed for 60 days.Afoxolaner treatment (TG1): rabbits were fed Bandeirante® commercial feed and exposed to two doses in 0.5 g chewable form containing 11.3 mg afoxolaner per Nexgard® tablet for 60 days with a 30 day interval between administrations.Castor oil treatment (TG2): the rabbits received daily the Bandeirante® commercial feed enriched with castor oil ricinoleic acid esters at a 1.75 g of esters/Kg of feed for 60 days.By jugular puncture, 2 mL of blood was collected from each rabbit using a 3 mL syringe and 25x0.08 mm needle (sterile) placed in a dry Vacuntainer® with separating gel, for biochemical tests of liver enzymes AST, ALT, and ALP.The CG, TG1, and TG2 rabbits were euthanized in a room of the Department of General and Applied Biology Viverium by veterinarian Letícia Maria Graballos Ferraz Hebling, CRMV nº 5.412, using ketamine and xylazine, applied intraperitoneally in dosages of 300 mg/kg and 30 mg/kg, respectively, to collect the fragments of the liver and spleen.2HPO4 2.38 g/L, and KH2PO4 2.72 g/L), where they remained for 24 hours. Subsequently, they were dehydrated in increasing series of 70%, 80%, 90%, and 95% ethyl alcohol (baths of 30 minutes each), soaked in Leica historesin for 24 hours, and placed in plastic molds containing historesin plus polymerizer (Leica Historesin Kit®). The blocks were sectioned (3 µm thick) in Leica RM2265 microtome (Leica®), the sections were rehydrated for 1 minute in distilled water and then stained by Harris hematoxylin for eight minutes. After washing for three minutes in running water, they were stained by aqueous eosin for five minutes and again washed in running water. After drying, the slides were dipped in xylol and then covered with Entellan and coverslip. The permanent slides were examined and photographed under a Leica DM750 bright field microscope (Leica®).The host rabbits’ liver and spleen fragments were fixed in 4% paraformaldehyde for 72 hours at 4 ºC. They were then transferred to a phosphate buffer solution . Later, the blocks were sectioned (3 µm thick) in Leica RM2265 microtome (Leica®), and the sections were rehydrated for 1 minute in distilled water and transferred to a 4% periodic acid solution for 10 minutes. Washing was performed in distilled water for 1 minute and immersion for 1 hour in Schiff's reagent. They were then washed for 15 minutes under running water, dried at room temperature, and mounted on Entellan. The permanent slides were examined and photographed under a Leica DM750 (Leica®) bright-field microscope.Fragments of the liver and spleen were fixed in 4% paraformaldehyde for 72 hours to detect total proteins. They were dehydrated in increasing series of ethyl alcohol 70%-95% (30 minutes each), soaked in Leica historesin for 24 hours, and placed in plastic molds containing historesin plus polymerizer (Leica Historesin Kit®). The blocks were sectioned (3 µm thick) in Leica RM2265 microtome (Leica®). The sections were bromophenol blue staining for one hour at room temperature. After being washed under running water for 15 minutes, they were dried, mounted on Entellan, and covered with a coverslip. The permanent slides were examined and photographed under a Leica DM750 (Leica®) bright-field microscope.The data obtained from the analysis of blood samples from rabbits from the control groups (CG), the group exposed to afoxolaner (TG1), and the group exposed to esters derived from castor oil (TG2) at times 0, 15, 30, 45, and 60 days are arranged in The data obtained from the enzymes AST, ALT, and ALP of the rabbits allocated in the (CG) served as a reference for comparison with the results obtained from the rabbits allocated in Treatment Groups 1 and 2 -3.The rabbits allocated to this group were fed with Bandeirante® commercial feed and exposed to two doses of afoxolaner. The first dose was administered at T0 (baseline time) and the second after 30 days at T30. The rabbits (TG2) were fed with the Bandeirante® commercial feed enriched with castor oil ricinoleic acid esters at a concentration of 1.75 g of esters/Kg on days 0, 15, 30, 45, and 60. The blood sample data did not differ from those found in the control group (CG) used as a reference for the enzymes AST, ALT, and ALP.The results showed that the liver of the rabbits of the CG, showed preserved liver tissue, with hepatocyte cords parallel to each other and evident cell boundaries. Hepatocytes showed the presence of one or two rounded nuclei and a nucleolus -1B.The cytoplasm of the hepatocytes was generally homogeneous, i.e., with no vacuoles or lipid droplets of significant size .A large amount of red blood cells was observed inside the hepatic blood vessels, which was also expected.Nuclei with elongated shapes belonging to Kupffer cells, known as liver macrophages, can be observed between the strands of hepatocytes  A-1B.CG) individuals confirmed that the liver tissue was preserved , the cytoplasm of these cells was more heterogeneous, that is, with lipid droplets distributed throughout the cell -2B. The Contrary to what was observed in the CG rabbits, the detection of proteins in the liver tissue of the rabbit exposed to afoxolaner was less intense . The cytCG. Many vacuolated areas could be observed in the cytoplasm of hepatocytes and are composed of strongly positive clusters of fine granulations (carbohydrate aggregates) distributed over almost the entire extent of the hepatocytes  was not observed at the time of exposure to esters . On the Also under the histochemical aspect, the protein detection in the liver tissue of rabbits exposed to esters showed more significant preservation of elements of a protein nature in the cytoplasm of hepatocytes than was observed in exposure to afoxolaner , 3D.Although cytoplasmic vacuolation was evidenced, strong (coarse) granulation of protein elements was observed. Additionally, contrary to what was observed in exposure to afoxolaner, the hepatocyte nuclei were strongly marked by bromophenol blue, as were the nucleoli .). The most evident change occurred in the group exposed to castor oil ricinoleic acid esters (TG2) , afoxolaner treatment (TG1), and castor oil treatment (TG2rs (TG2) -6, with rs (TG2) .The spleen is considered the highest concentration of lymphoid tissue in the body, representing an organ of defense against microorganisms and the destroyer of erythrocytes. This organ is subdivided into white pulp (lymphoid tissue) and red pulp (tissue rich in cells and reticular fibers) rich in blood.The data obtained showed that there were only minor morphological changes in the spleen when comparing the two exposure groups (TG1 and TG2) to the control group (CG) -7. Histologically, no significant changes were detected when applying the techniques for detecting proteins and carbohydrates -7B.Rabbits and dogs share anatomical, physiological, and immunological similarities, making them relevant models for studying the effectiveness of tick control products and strategies in dogs. This approach allows a better understanding of the effects and safety of treatments in a similar biological system. In addition, rabbits are widely used in research and readily available in many laboratories and institutions, facilitating access to these experimental models . By usinRicinus communis) have demonstrated their effectiveness in the main organs related to tick feeding  and reproduction (ovaries), as demonstrated by Tick control is necessary to eliminate this ectoparasite, especially in urban environments where hosts, such as dogs, are in constant contact with humans. Because these “pets” are often considered family members, concern about ectoparasite control increases due to the potential for humans to become targets for tick parasitism. Research focusing on the bioactive compounds found in castor oil esters  and synthetic (afoxolaner) acaricides by analyzing the enzymes AST, ALT, and ALP -3, demonOn the other hand, the data obtained from rabbits allocated in TG1, exposed to the synthetic acaricide afoxolaner, showed an increase in the parameters of the enzyme aspartate aminotransferase (AST), suggesting the occurrence of drug poisoning, probably due to the synthetic chemical. Aspartate aminotransferase (AST) is a liver enzyme that has its blood levels increased in the presence of liver cell injury. Its specificity is lower than that of ALT, and the increase in serum levels can occur in cases of intoxication, mainly due to drug intake (1 and 2). An increase in this enzyme in the blood of TG1 rabbits was found in the experiment conducted as a result of the drug administration, indicating liver poisoning. However, the blood levels of this enzyme in TG2 remain similar to those of CG, compatible with the reference values indicative of normality. This means that there is no impairment of the liver and that ricinoleic acid esters are more indicated for use as an acaricide.Mus musculus) due to the exposure to synthetic acaricides fipronil, thymol, and permethrin, which could suggest a strategy of hepatocyte preservation in response to the presence of the toxic element in the tissue. In other words, this vacuolation would be removing damaged organelles or even certain regions of the cytoplasm that were altered by the presence of the toxic element, sheltering them in vacuoles that would later be digested and/or eliminated, thus preserving the content that was still intact in the cytoplasm. The vacuolation around the nucleus could indicate that the liver cell, under the action of the toxic product, would be preserving its genetic material by removing any residue that could alter its DNA, since changes in nuclear physiology, lead to the deregulation of essential characteristics that contribute to the function of hepatocytes  or macrovesicular  . The resatocytes . The expThe histochemical evaluation of the liver of rabbits exposed to afoxolaner (TG1) to mark proteins and polysaccharides showed in CG strong positivity for proteins in the cytoplasm of hepatocytes, corroborating Histochemistry to detect polysaccharides in the liver of rabbits exposed to afoxolaner showed hepatocytes with cytoplasmic areas containing polysaccharide granulation aggregates distributed over almost the entire extent of the cells, data different from those found in the CG, which presented preserved liver tissue, with homogeneous cytoplasm. According to some authors , the incAgeratum conizoydes. The hepatocytes of rabbits of TG2 presented well-preserved nuclei and nucleoli indicating that esters did not cause toxicity to cells, since that changes are directly linked to the occurrence of cell death, a cellular strategy to preserve the tissue. Exposure to esters did not cause cytoplasmic or nuclear vacuolation in hepatocytes, also absent or minimal between the strands of hepatocytes, indicating that the esters at the concentration in question were not hepatotoxic (The morpho-histology and histochemistry of the liver of rabbits exposed to the castor oil ricinoleic acid esters (TG2) were also analyzed. The results showed that, although of natural origin, they still caused changes in liver tissue, but less aggressively than the rabbits exposed to the synthetic drug. The liver of rabbits exposed to esters showed hepatocytes organized in hepatic cords and remained intact since their cellular outlines could be evidenced. The cytoplasm of these cells remained as in the CG without the presence of lipid droplets. This same pattern was described in the study of atotoxic .The liver rabbits exposed to castor oil esters showed strong PAS positivity in the cytoplasm of hepatocytes due to an increase in the intensity of polysaccharide granulation, most likely an accumulation of glycogen. According to the literature, this suggests that the esters somehow interfered in the production and storage of this element when compared with the CG and TG1. Glycogen, abundant in hepatocytes shows the individual’s nutritional status , or the The observations regarding proteins indicated no change in the histochemical pattern when compared with the data obtained in the CG, demonstrating that the liver continued to synthesize and release/store proteins, even in the presence of esters, corroborating the morphological information described for the mammalian liver .The spleen here analyzed, in addition to other functions, is involved with immunology, acting against the presence of antigens that may be transported via blood circulation . MorpholThe spleen analysis revealed no changes in the tissue of rabbits exposed to afoxolaner (TG1) since the same morphological characteristics found in the control group were observed. The exposure to castor oil esters showed vacuolation, not cellular, but tissular of the red pulp, data not found in other studies with spleen exposure to other bioactive compounds. The histochemical techniques to detect proteins and polysaccharides in this organ showed results similar to those found in CG, i.e., the absence of changes due to exposure to synthetic and natural acaricides.In conclusion, the data obtained in this study revealed that the castor oil esters caused no detectable toxicity in the rabbits exposed to them when incorporated into the feed, data confirmed by clinical analysis of the hepatic parameters of the enzymes. The morphological data of the liver and spleen showed that the castor oil esters did not induce detectable alterations the cells or tissue of these organs indicating that esters may be an alternative for tick control that causes minimum damage to the hosts and is potentially cleaner due to its lower contamination compared to synthetic chemical acaricides that leave toxic residues in the environment. On the other hand, the administration of afoxolaner resulted in a significant increase in the AST enzyme, indicating an alleged occurrence of drug poisoning in rabbits exposed to it. In addition, morphohistological changes in the liver were observed, including the presence of steatosis (accumulation of fat) and cellular and tissue vacuolizations. These findings corroborate the hypothesis that afoxolaner can cause liver damage in cases of improper administration.


STIM1 is also activated by temperature independent of ER Ca2+ depletion. Here we provide evidence, from advanced molecular dynamics simulations, that EF-SAM may act as a true temperature sensor for STIM1, with the prompt and extended unfolding of the hidden EF-hand subdomain (hEF) even at slightly elevated temperatures, exposing a highly conserved hydrophobic Phe108. Our study also suggests an interplay between Ca2+ and temperature sensing, as both, the canonical EF-hand subdomain (cEF) and the hidden EF-hand subdomain (hEF), exhibit much higher thermal stability in the Ca2+-loaded form compared to the Ca2+-free form. The SAM domain, surprisingly, displays high thermal stability compared to the EF-hands and may act as a stabilizer for the latter. We propose a modular architecture for the EF-hand-SAM domain of STIM1 composed of a thermal sensor (hEF), a Ca2+ sensor (cEF), and a stabilizing domain (SAM). Our findings provide important insights into the mechanism of temperature-dependent regulation of STIM1, which has broad implications for understanding the role of temperature in cellular physiology.Intracellular calcium signaling is essential for many cellular processes, including store-operated Ca Stromal interaction molecule 1 (STIM1), a type 1 transmembrane protein in the ER membranes, plays a central role in SOCE, an intricate process involving the cooperation of multiple partners. The two main factors therein are STIM1 in the ER membrane and the Orai1 Ca2+ channel in the plasma membrane (PM)  and formation of "Ca2+ release activated Ca2+ channels" (CRAC) that subserve SOCE (2+ sensor is connected by the transmembrane (TM) region to the cytoplasmic domains. The latter presents (a) the coiled-coil domain, which includes the CAD/SOAR region that binds to and activates Orai1; (b) the serine-proline-rich (S/P) domain, important for the correct targeting of the STIM1 cluster to the ER-PM junctions; and (c) the lysine-rich polybasic (K) domain. The latter is involved in multiple interactions with regulatory factors   , 6, 7, 8rve SOCE , 11, 12.rve SOCE . The int factor) , 14, wit factor) , and wit factor) , 17.Figu2+ sensors responding to changes in cytosolic [Ca2+], in the range 10 to 1000 nM, stromal interaction molecules have evolved for sensing [Ca2+] changes in the ER, in the millimolar to micromolar range  and the non-Ca2+-binding hidden EF-hand (hEF)  , 20. Thend (hEF) , 23, 24 nd (hEF) , 26, 27.residues , 20. EF-residues  to bind residues  allows tresidues , 32, 33.residues .2+ depletion  clearly requiring its interaction with Orai1 and (b) an explanation for mammals’ ability to sense very small changes (<1 °C) from normal skin temperature  heat ability to activate STIM1. More puzzling and opposed to previous experimental evidence, recent far-UV circular dichroism spectroscopy and FRET measurements  the luminal EF-SAM loss of structure and exposure of hydrophobic core residues in low Caurements  indicate2+-binding proteins, such as calmodulin , the results did not allow for a more detailed description of EF-hands involvement therein, or for an explanation of how the hydrophobic interface between EF-hands and SAM is affected by Ca2+ un-binding. In the second study, Mukherjee et al. , residues in the cEF and hEF domains of STIM1 that can bind multiple Ca2+ ions. However, they also found that a single Ca2+ ion is sufficient to stabilize the luminal domain. Furthermore, their MD simulations revealed that the F108I mutation destabilizes the hEF hand on a timescale of hundreds of nanoseconds. While all these studies did not directly address the role of temperature in STIM1 activation, they provide valuable insights into the structural dynamics of EF-SAM and its response to mutations.Molecular dynamics (MD) simulations constitute a well-established method to examine atomistic aspects of protein dynamics. MD simulations have been successfully used in the past to understand the mechanisms underlying the functions of other Calmodulin , 43, 44,lmodulin , parvalblmodulin , and trolmodulin . Many ofulations , 51, 52.  simulations, offered a possible mechanistic explanation for STIM1 temperature sensing and its decoupling from Ca2+ sensing. The results obtained helped us to design a functional model of the EF-SAM sensor, with the following specific roles of the subdomains: hEF-SAM is the temperature sensor, cEF-hand is the [Ca2+] sensor, and SAM is a stabilizer for both sensors in the inactive state of STIM1.Here we used conventional MD and enhanced sampling MD to investigate molecular mechanisms of temperature sensing by STIM1 and the crosstalk between temperature- and Ca2+ and thermal sensing of STIM1 was also addressed. Probing for the inceptive structural rearrangements induced by Ca2+ dissociation at different temperatures gave us valuable insights on the molecular “hot-spots” critical for the activation process. Thus, we first used conventional MD simulations , followed by enhanced sampling replica exchange with solute tempering (REST2) simulations, the latter being necessary to describe the large-scale conformational changes of EF-SAM in response to the studied stimuli (i.e. Ca2+ depletion and/or heating).In view of a mechanistic explanation for the thermal sensitivity of STIM1, we examined the conformational changes in the EF-SAM domain at different temperatures. The interplay between Ca2+ removal, two types of MD simulations were initially considered. The first type examined the EF-SAM domain with Ca2+ removed (APO-37) and the second one considered the cEF site occupied by one Ca2+ cation (HOLO-37), the latter being used as reference. Both simulation types were performed at 37 °C to mimic physiological conditions. However, as preliminary simulations evolved, two facts became evident. First, at 37 °C with Ca2+, the RMSD of the protein backbone displayed large deviations from the NMR structure over the first several hundred nanoseconds, analogous to those recorded for the Ca2+ free form. Deviations were as high as 6 Å, reflecting large conformational drifts in both cases. This could be anticipated for the Ca2+ free form, but not for the Ca2+ bound form and it was attributed to the temperature difference between the simulations (37 °C) and the NMR experiment (20 °C) (2+ binding site loop developed early (50–150 ns) in the Ca2+-free form. While the Ca2+ binding site expansion is an expected result of Ca2+ removal in EF-hand proteins, the extensive unfolding of the EF-SAM binding loop is atypical although previously mentioned for some EF-hand proteins (2+ sensitive EF-hand (cEF) and a Ca2+ insensitive one (hEF), may impose uncommon structural responses upon either Ca2+ loss or temperature rise or both.The overall simulation approach for the conventional MD simulations is schematized in periment  °C (20).oved APO- and the proteins , 58. Thu2+ (HOLO-20), T = 20 °C without Ca2+ (APO-20), T = 37 °C with Ca2+ (HOLO-37), and T= 37 °C without Ca2+ (APO-37). Initially, for each combination, 12 short individual simulations (150 ns) were performed, starting with different initial conditions (atom velocity distributions). This was necessary to test the reproducibility of the observed large binding loop expansion in the Ca2+ free form and its stability in the Ca2+ loaded form and how this is affected by temperature. To investigate longer time scale behavior, four simulations (one per each set of 12) were extended to 1.8 μs.Simulations at the NMR experiment temperature were thus required to further clarify such aspects. Therefore, four simulation conditions were used: T = 20 °C with Ca2+ stability was carefully evaluated on the microsecond timescale. The results  are undersampled in conventional MD, even on the μs time scale. Therefore, we used REST2 to evaluate EF-SAM conformations at different reference temperatures and also the EF-SAM thermal unfolding curves in the presence and absence of Ca2+. The outcome of the latter could be directly compared with experimental data from the literature.The correlations in low-frequency atomic displacements -Ser(-4)-His(-3)-Met(-2)-Ala(-1)-Ser(0) added to its N-t Ser58. This sequence, together with Ser58-Lys62, was extremely flexible, lacking any defined secondary structure, in both the NMR and the MD ensembles, so they were not included in RMSF analysis. The least well-defined residue in the NMR ensemble is Asp112, belonging to the hEF α3-β2 loop, together with Glu111 and Lys113. The protein backbone displayed large fluctuations in this region in the MD ensemble too. Still, the Ca2+ binding site residues showed good stability in both the calculated and the experimental data. Hydrophobic cleft residues of the EF-hand subdomain  and α10 anchoring residues  also displayed comparable low fluctuations in both ensembles (MD and NMR). One exception was Leu96 in the C-t region of α2, with larger RMSF in the MD ensemble. Also, the α2-α3 loop and the N-t part of α6 were more flexible in MD trajectories versus their neighboring sequences, a feature that was not apparent in the NMR ensemble. Within the SAM domain, NMR and MD RMSF showed correlated increased flexibility of α6-α7 loop, α7 helix, and α9-α10 loop. Figure 3B shows a direct comparison between the MD and experimental NMR conformational ensembles. The secondary structure analysis, detailed in 2+ presence at room temperature.EF-SAM had a well-preserved structure along the entire 1.8 μs of HOLO-20 simulation, with an average RMSD relative to the NMR structure lower than 0.35 . For furFigure 32+ removal affects EF-SAM stability at different temperatures.These results show that the chosen NMR structure and the force field used to describe the molecular models constitute a reasonable basis for modeling. Next, we used these models to investigate how Ca2+ removal on the binding site of EF-SAM at 20 °C and 37 °C, respectively. The cEF coordinates Ca2+ in a pentagonal bipyramidal configuration. In the 2K60 NMR structure, the residues involved in Ca2+ binding include Asp76, Asp78, Asn80, Asp84, and Glu87 . Position “9” is probably occupied by a water molecule, H2O , while the Asp82 side chain has no contact with Ca2+ in the NMR structure.Here we discuss the structural impact of Ca2+ binding site loop (1): the α carbon RMSD (2); the radius of gyration Rg of the sequence Leu74 to Ser88 (3); the distance d1 between C-α of Asp76 and C-α of Glu87 (4); and the distance d2 between C-α of Asn80 and C-δ of Glu87 (A). All four parameters showed larger deviations for the APO vs HOLO simulations at both 20°C  and 37°C . The Rg showed Ca2+-dependent variations, larger for the Ca2+-free forms. These variations correlated well with the increased RMSD values in the same conditions. The larger values of C-α RMSD indicate that Ca2+ removal induces backbone destabilization, while elevated Rg suggests an expansion of the binding pocket and an increased side chain disorder, as confirmed by visual inspection of the MD trajectories. Distances d1 and d2 gave a more detailed picture of the local rearrangements following Ca2+ removal. The entering and exiting α helices within cEF (α1 and α2 respectively) come into contact through Asp76 and Glu87, residues of the Ca2+ binding pocket. The distance d1 between the C-α of these residues was considered a measure of the binding pocket expansion. There are significant differences between d1 measured in APO vs. HOLO, up to d1 APO– d1 HOLO ≈ 12 Å in some of the simulations (B and D). Thus, the removal of Ca2+ was shortly followed by marked expansion of the Ca2+ binding pocket at both 37 °C and 20 °C.The initial effects of calcium removal could be evaluated from the repeated short-length MD simulations (150 ns each) . A combiof Glu87 A. All foulations , B and Dd2, between the C-δ of Glu87 and the C-α of Asn80 was chosen as a metric of preservation or loss of this orientation. As seen in Figure 4B, the d2 values are higher for APO than for HOLO both at 37 °C and at 20 °C. This is due to Glu87 side-chain reorientation in the absence of Ca2+, in a position facing away from the binding site. Simulations displaying highly increased d2 also showed increased d1 values (i.e. BLE). In contrast, the Ca2+ binding loop showed good structural stability in the HOLO forms, at both 37 °C and 20 °C, with the conservation of all analyzed structural parameters.The bidentate side chain ligands of the 12th loop residue (Glu87) are known to be of critical importance to the EF-loop structure stability. Despite the Glu87 location at three residues away from the last chelating amino acid, the side chain of Glu87 strongly interacts with the cation, facing toward the rest of the chelating residues. The distance 2+-loaded form. Each EF-hand has a core of hydrophobic residues between the entering and exiting helices, including Met75, Val83, Val85, Leu92, and Leu96 for cEF and Phe108, Leu114, and Ile115 for hEF, respectively. The EF-pairing is also stabilized by these residues, as they come into close contact in the resting folded state (A). Three hydrophobic residues, strategically placed on the α10 helix of SAM , anchor the α10 helix inside the hydrophobic cleft provided by the EF-hand pair. These interfaces allow for “tripartite” cEF-hEF-SAM hydrophobic interactions hat stabilize the compact, well-folded conformation of EF-SAM. The stability of the inter-EF hand (B) and EF pair–SAM (C) hydrophobic interfaces was monitored by constructing inter-residue hydrophobic contact maps at different times during APO and HOLO simulations. Both the cEF–hEF interface and the EF pair–SAM interface were undermined by Ca2+ removal, with many direct contacts between the above-mentioned hydrophobic residues being lost . Several hydrophobic interactions stabilize the well-folded helical structure in the Caed state A. Three -EF hand B and EF pair–SAM C hydrophThe time evolution of RMSD for the Cα atoms of EF-SAM in HOLO-37 displayed large variations, reflecting important conformational drifts from the experimental structure . After tA and B). Our simulations revealed that certain regions of the EF-SAM domain displayed higher RMSF values, indicating that local structural changes occurred due to increased temperature. Given that STIM1 can be activated by temperature even in the presence of Ca2+, it was important to carefully evaluate the temperature-induced conformational changes of EF-SAM in more detail. The most flexible fragment was Thr107-Phe108-His109-Gly110. The high local flexibility of α3-β2 could be related to its observed solvent exposure. The solvent-accessible surface area (SASA) of two representative residues from this region, Phe108 and Gly110, was plotted against time (C). These two residues were postulated to play an important role in the Ca2+ sensor function, because replacing Phe108 and Gly110 with Asn in EF-SAM mutants rendered the protein constitutively (±Ca2+) aggregated (2) (2); surface adsorbed (0.25–1 nm2); buried (<0.25 nm2). Along the 1.8 μs of simulation, the side chain of Phe108 displayed little or no solvent exposure in HOLO-20. In contrast, in HOLO-37, Phe108 stays with its phenyl group lying at the protein surface for the initial ∼1.2 μs and then it fully exposes its side chain to the solvent in the last 0.6 μs. Gly110 also displays lower backbone SASA at 20 °C vs 37 °C.More insights were provided by the fluctuations of individual residues of the protein chain. As expected, RMSF analysis showed higher spatial fluctuations for HOLO-37 vs HOLO-20 , A and Bnst time C. These ated (2) . Based o2+ and temperature sensitivity (A), located in the cEF, corresponds to the Ca2+ binding site and is highly stable in both HOLO-20 and HOLO-37, showing no temperature-dependent instabilities. However, it becomes highly disordered in APO-37, as shown by much higher locally fitted RMSFs (B).The above-discussed RMSFs were calculated for the entire protein and thus give information about the overall fluctuations of the polypeptide chain. However, due to large structural drifts in the HOLO and APO forms at 37 °C, globally fitting the protein backbone may hinder functionally important local fluctuations. These include fluctuations of polypeptide chain segments between conserved secondary structure elements. Based on global RMSF data and on secondary structure plots for HOLO-20 , severalsitivity : (δ1) α1sitivity A, locateed RMSFs B.Figure B): constantly present in HOLO-20; partially compromised in HOLO-37; completely disrupted in APO-37, followed by Phe108 side chain solvent exposure. Not only the Phe108-Leu199 pair is destabilized, but consequently the Leu199-Leu195 contacts are also affected, as snapshots representing their side chains are more scattered in Figure 6A. Thus, the hEF of STIM1 is highly temperature sensitive. Moreover, hEF contains in the “X” position a highly conserved hydrophobic residue among STIM1 proteins (Phe108) , located mainly in the hEF, corresponds to the EF-hands intervening loop α2-α3, the α3 helix, and the α3-α4 loop. This region has increased flexibility and disorder, accompanied by loss of α3 secondary structure in both HOLO-37 and APO-37. Phe108 is located in the highly flexible α3-α4 loop, and its solvent exposure disrupts the interaction with Leu199, one of the main anchoring residues on α10. As suggested by the visual inspection of MD snapshots, the data confirmed that the close contact between Phe108 and Leu199 is B: consta(Phe108) , instead2+; without Ca2+ the disorder is significantly more extended (including N-t of α10), as proven by the locally fitted RMSFs. This is in line with previous findings (2+ is lost.The third region (δ3) is highly stable for HOLO-20, but at 37 °C, it becomes disordered even in the presence of Cafindings  that ide2+, obtained by enhanced sampling MD techniques.Based on the results obtained so far by equilibrium MD at two fixed temperatures, we further extended our analysis to the full thermal unfolding profiles of EF-SAM ± CaIt is well known that conventional MD simulations suffer from the “sampling problem”, that is, the system becomes trapped in local minima, where it can remain for long periods of time, hindering the efficient sampling of the phase space. Conventional MD is not well suited to study the folding and unfolding of large proteins, occurring along tenths of microseconds to seconds. Conventional MD is limited to the description of the initial steps of such long events. To explore the unfolding and the thermal behavior of EF-SAM on a wider time scale, we used replica exchange with solute tempering (REST2) simulations. REST2 uses a number of “N” copies of the simulated system, called “replicas”, which are all simulated in parallel. Each replica is simulated at the same reference temperature but has a modified Hamiltonian with the soft degrees of freedom  scaled down by a factor λ. The modified Hamiltonian is applied only to a part of the system, namely, the studied protein. This allows for an efficient passage over the conformational energy barriers using a small number of replicas compared with the original parallel tempering method . At spec2+-free form of EF-SAM is much less stable than the Ca2+-loaded one, as shown by the unfolding profiles expressed as a fraction of protein helical content (m) values between the Ca2+-free and Ca2+-loaded states was included as a single-parameter quantitative expression of the stability difference between the two unfolding curves, as the relative difference between the two states is likely to be less affected. The calculated value of 39 °C resembles well the experimentally measured value (26 oC). As expected, Ca2+ had an important stabilizing effect on EF-SAM structure, maintaining the protein in a compact folded conformation at much higher temperatures vs the Ca2+ free form.Based on the higher stability of the HOLO forms encounter in experiments and our conventional MD simulations, we have chosen the REST2 reference temperatures: 20 °C for HOLO and 15 °C for APO. The information contained in the “warmer” replicas (λ < 1) were used to construct the thermal unfolding profiles for the HOLO and APO forms. The Ca content . The sha content . Despite content , the difA). As predicted by the conventional MD simulations, Ca2+ removal destabilized cEF, moving down its stability profile curve . However, analyzing the plots in Figure 8B, it is obvious that Ca2+ un-binding had a drastic effect on the overall structure of the EF-hand pair, compromising the stability of both cEF and hEF, even at low temperatures. This reflects the cooperative interactions between the two pairs of the EF-hand motif. On the other hand, SAM displayed high stability , maintaining high helical content even up to 80 °C in simulations.The profiles in Fig. 82+ sensor of STIM1, which is known to be activated also by heat, and into the interplay between temperature sensing and Ca2+ sensing. The effect of heating on the EF-SAM Ca2+ sensor was evaluated at different temperatures, both in the resting state and after Ca2+ removal. The main results are summarized here. 1. We identified key residues and interaction interfaces that are important for the unfolding initiation. 2. There were large discrepancies in EF-SAM conformational preference at different temperatures in the resting state. These were not the overall effects of increased flexibility due to higher temperature. 3. Specifically, while the overall folding was preserved, the thermal-driven structural disorder appeared in the hEF-hand, which contains the hydrophobic Phe108, a highly conserved residue in this position among STIM1 proteins. Phe108 became solvent exposed in a temperature-dependent manner, an effect that promotes EF-SAM dimerization. 4. We identified Phe108 as a thermal “hot spot”. Using replica exchange simulations, we constructed in full atomistic detail the unfolding profiles of EF-SAM ±Ca2+, with results that strongly support the conventional MD findings. 5. We propose a modular architecture for EF-SAM, containing a Ca2+ sensor (cEF), a temperature sensor (hEF), and a stabilizing module (SAM).Here we used advanced molecular dynamics simulations to gain insight into the thermal behavior of the intraluminal Cai.e. not capable of the heat-response) show significantly lower basal Ca2+ levels and STIM1-dependent gene expression compared to the cells expressing the normal STIM1/Orai1. Wild-type cells instead showed heat-induced clustering of STIM1 at temperatures above 35 °C without depleting Ca2+ stores  using flooding simulations at the same temperature and high Ca2+ concentrations found hEF to be stable. This may be explained by the fact that the simulations of Schober et al. (2019) were performed in extremely high Ca2+ concentration (150 mM) to identify multiple Ca2+ binding sites, some of which were located in the hEF. Another reason may be related to the length of the simulations. An important unfolding event, consisting of Phe108 side chain exposure to solvent, occurred at around 1200 ns in our simulations (C), a time point that exceeds the length of the simulations presented in Schober et al. A direct comparison, however, must be regarded with caution as even on the microsecond time scale, the sampling of the conformational space is still probably insufficient. However, both studies found the SAM subdomain to be well-folded both in HOLO and APO forms, underlining the stability of this domain, which was also observed by us when assessing the thermal stability of EF-SAM. Our REST2 simulations further support our findings and provide a more comprehensive view on the hEF propensity to unfold at physiological and near-physiological temperatures. This may well explain the mentioned STIM1-involving behavior of temperature-responsive cells observed by others  show that the temperature responses of the two EF-hands are uncoupled. The cEF is stable in the physiological range of temperatures if Ca2+ is present, while the hEF presents partial disorder and prompt unfolding response to moderate temperature increase. Thus, the hEF may become unfolded due to moderate temperature rise, even in the presence of Ca2+, while the overall structure of EF-SAM is still preserved. The hEF unfolding is accompanied by the unmasking of the hydrophobic Phe108 side chain toward the solvent. So, this side chain is now unable to participate in the stabilizing “tripartite” hydrophobic interactions, while it becomes free to interact with other unfolded hEF-hands. This type of response indicates that hEF-hand is a possible temperature sensor within STIM1. Two findings strongly support this supposition (1): hEF is highly unstable thermally, compared to the rest of EF-SAM, in the physiological temperature range (2); hEF contains the highly conserved hydrophobic Phe108. Sequence alignments of hEF reveal that position 108 is persistently occupied by Phe residues in different known STIM1 proteins, while cEF motifs have a highly invariant Asp for Ca2+ binding in position 108 . These results are consistent with current perspectives on cooperative protein folding/unfolding, which acknowledge: (1) the presence of significant dispersion in folding/unfolding rates across different protein structure sites, illustrating asynchronous structural changes occurring in multiple steps  is used by the Ca2+ sensor (cEF), its close neighbor.The molecular mechanisms underlying temperature sensing in proteins are still a matter of debate. While the function of all macromolecules is altered by temperature, protein thermosensors provide information about the thermal environment due to their peculiar thermal properties, which allow them to trigger an appropriate physiological response upon heating or cooling. A particularly interesting example is the transient receptor potential vanilloid member 1 (TRPV1), a Can humans . For a ln humans , using cn humans , 72. Liket al. ; hEF-hand is a temperature sensor (3); SAM is a stabilizing domain. Such an architecture would explain the function of this complex and versatile sensor, which not only incorporates sensitivity for two distinct parameters but also allows reciprocal functional adjustments by allosteric influences between the two different detectors involved. Our data suggest a peculiar type of cooperation between these two detector elements of EF-SAM. Although not as a direct conclusion of our results, the functional impact of such roles of cEF and hEF, actually correlated, does open a new perspective upon the mechanism of SOCE: the temperature sensor seems to render the calcium sensor optimal at physiologically elevated temperatures, for the rapid activation of SOCE by a rather small decrease of reticular Ca2+ concentration.Therefore, we propose a modular functional architecture for the multiple responsivities of the EF-SAM sensor, with three components  (1): cEF2+ concentration, which has important implications for understanding the physiological role of STIM1 in temperature-dependent calcium signaling.The present study brings together two different aspects of STIM1 sensing capabilities, temperature sensing, and calcium signaling, providing input for more extended analyses of their interplay. Understanding how STIM1 senses temperature is critical for gaining insight into the underlying mechanisms of temperature-dependent cellular processes essential in immune responses, fever response, gene expression, temperature sense, and metabolism. Proteins exist in a dynamic equilibrium between multiple conformations, and their population depends on factors such as temperature, pH, metal binding, and interaction partners. Our simulations showed that at 37 °C, the hEF subdomain of EF-SAM tends to occupy a partially unfolded conformation, which may expose the Phe108 residue to solvent. At lower temperatures, this tendency disappears, while at higher temperatures, it becomes more favorable. This suggests that slightly increasing the temperature above 37 °C may shift the equilibrium toward the unfolded state of hEF, which itself is sufficient to promote dimerization, as shown in previous experimental studies. Moreover, our findings also suggest that the temperature sensor renders the calcium sensor optimal at physiologically elevated temperatures for the rapid activation of SOCE by a decrease of reticular Ca2+-loaded EF-SAM domain (Protein Data Base (PDB) entry: 2K60) , constant pressure (1 atm), water as a solvent, and 150 mM KCl ionic strength. [Ca2] was not explicitly considered, low or high Ca2+ ionic strengths being modeled by either the absence or the presence of Ca2+ in the binding site.EF-SAM is the intraluminal N-terminal (N-t) topological domain of STIM1 (663 residues), with a length of 138 amino acid residues . Here we used the NMR structure of a recombinant construct of human Ca protein , 20. Thu2+ (HOLO form) was solvated in a cubic simulation box with 7 nm edges, containing 10,165 water molecules. We added 41 K+ and 31 Cl- ions to the simulation box, by replacing the adequate number of water molecules, in order to achieve a physiological salt concentration of 150 mM and an electrically neutral system. Everything was similarly done for the simulation of EF-SAM behavior after Ca2+ loss (APO form) but omitting Ca2+ from the 2K60 structure and compensating the respective charge loss by adding only 29 Cl− ions to the solvent instead of 31 Cl− ions. The systems were energy minimized, followed by a suite of consecutive equilibration MD stages in which the restraints applied on subgroups of protein atoms were gradually relaxed . In these equilibration simulations, the water molecules together with K+ and Cl− ions were free to move and adapt to the local protein microenvironment. The total equilibration period was 14 ns, enough for the solvent to intimately equilibrate with the protein surface.Much attention was paid to system construction and equilibration, to ensure that these steps do not introduce any unnatural conformational artifacts prior to production runs. For protein and ions description we used the OPLS  all-atom force field . TIP4P w2+, 37 °C), APO-37 , HOLO-20 , and APO-20 . Each set contained 12 independent simulations thermalized with randomly generated velocities from a Maxwell distribution at the specified temperature. One simulation from each set was continued up to 1.8 μs. Production simulations were performed without any atomic position restraints. All bond lengths were constrained via the LINCS (linear constraint solver) algorithm . Pressure was constrained, by coupling the simulation box to an external Berendsen barostat, at 1 atm and a relaxation time constant of τ = 0.5 ps. For thermal coupling, we followed the common scheme with two different thermostats, one coupled to the solvent (including ions) and the other coupled to the simulated protein  and root-mean-square fluctuations (RMSF) of the protein backbone. These were computed using the g_rmsf and g_rms utilities from the GROMACS 4.6 suite, after the removal of protein overall rotation and translation by a prior fitting of the trajectory snapshots to the starting structure. The fitting involved only the C-α atoms of the residues Ser64 to Leu199, as the N-t (Gly(-5) to Leu63) is not well-defined in the NMR structure and highly fluctuates during simulations. RMSFs were averaged per amino acid residue. Conformational changes were also monitored by radius of gyration (Rg) analysis using the g_gyrate program in GROMACS suite. Residue contact maps were evaluated by computing the inter-residue distance matrices consisting of the smallest distances between each residue pair in selected subsets. Contact maps were evaluated using the g_mdmat program for seven frames equally distributed at 300 ns intervals along the μs range simulations. Secondary structure evolution during the simulations was monitored using the DSSP program (http://www.ks.uiuc.edu/Research/vmd/)  .et al.  SAM’s α10 is anchored in between α1 and α2. For each replica, a cluster analysis was performed using GROMACS software suite. The median conformation of each cluster was analyzed with respect to the above criteria. If all the requirements were fulfilled, the cluster was considered to represent a folded conformation, and the number of its members was added to the total number of folded conformations. The individual thermal stability curves for cEF and hEF were represented as the percentage of unfolded conformations, from the total number of conformations, versus the effective temperature. For SAM, which was much more stable, the α-helical content proved to be enough for constructing the thermal unfolding profiles (Solute tempering (REST2)  simulatiet al. . The α-het al. . To disset al. . Usual Rprofiles .The datasets/trajectories and computer scripts used in this study are available upon request to the corresponding author.This article contains The authors declare that they have no conflicts of interest with the contents of this article.