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Huntington's Disease (HD) is caused by an abnormal expansion of the polyglutamine (polyQ) domain within the first exon of the huntingtin protein (htt). This expansion promotes disease-related htt aggregation into amyloid fibrils and the formation of proteinaceous inclusion bodies within neurons. Fibril formation is a complex heterogenous process involving an array of aggregate species such as oligomers, protofibrils, and fibrils. In HD, structural abnormalities of membranes of several organelles develop. In particular, the accumulation of htt fibrils near the endoplasmic reticulum (ER) impinges upon the membrane, resulting in ER damage, altered dynamics, and leakage of Ca 2+ . Here, the aggregation of htt at a bilayer interface assembled from ER-derived liposomes was investigated, and fibril formation directly on these membranes was enhanced. Based on these observations, simplified model systems were used to investigate mechanisms associated with htt aggregation on ER membranes. As the ER-derived liposome fractions contained residual Ca 2+ , the role of divalent cations was also investigated. In the absence of lipids, divalent cations had minimal impact on htt structure and aggregation. However, the presence of Ca 2+ played a key role in promoting fibril formation on lipid membranes despite reduced htt insertion into and association with lipid interfaces, suggesting that the ability of divalent cations to promote fibril formation on membranes is mediated by induced changes to the lipid membrane physicochemical properties. With enhanced concentrations of intracellular calcium being a hallmark of HD, the ability of divalent cations to influence htt aggregation at lipid membranes may play a role in aggregation events that lead to organelle abnormalities associated with disease.2+ played a key role in promoting fibril formation on lipid membranes despite reduced htt insertion into and association with lipid interfaces, suggesting that the ability of divalent cations to promote fibril formation on membranes is mediated by induced changes to the lipid membrane physicochemical properties. With enhanced concentrations of intracellular calcium being a hallmark of HD, the ability of divalent cations to influence htt aggregation at lipid membranes may play a role in aggregation events that lead to organelle abnormalities associated with disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn2+ > Mn2+ > Mg2+ > Ca2+ >> Cu2+. Remarkably, Cu2+ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations.

Calcium and magnesium are the most abundant divalent cations in the body. The plasma level is controlled by coordinated interaction between intestinal absorption, reabsorption in the kidney, and, for calcium at least, bone storage and exchange. The kidney adjusts urinary excretion of these ions in response to alterations in their systemic concentration. Free ionized and anion-complexed calcium and magnesium are filtered at the glomerulus. The majority (i.e., >85%) of filtered divalent cations are reabsorbed via paracellular pathways from the proximal tubule and thick ascending limb (TAL) of the loop of Henle. Interestingly, the largest fraction of filtered calcium is reabsorbed from the proximal tubule (65%), while the largest fraction of filtered magnesium is reclaimed from the TAL (60%). The paracellular pathways mediating these fluxes are composed of tight junctional pores formed by claudins. In the proximal tubule, claudin-2 and claudin-12 confer calcium permeability, while the exact identity of the magnesium pore remains to be determined. Claudin-16 and claudin-19 contribute to the calcium and magnesium permeable pathway in the TAL. In this review, we discuss the data supporting these conclusions and speculate as to why there is greater fractional calcium reabsorption from the proximal tubule and greater fractional magnesium reabsorption from the TAL., (© 2022 New York Academy of Sciences.)

Abiogenic metals Pb and Hg are highly toxic since chronic and/or acute exposure often leads to severe neuropathologies. Mn2+ is an essential metal ion but in excess can impair neuronal function. In this study, we address in vitro the interactions between neuronal calcium sensor 1 (NCS1) and divalent cations. Results showed that non-physiological ions (Pb2+ and Mn2+) bind to EF-hands in NCS1 with nanomolar affinity and lower equilibrium dissociation constant than the physiological Ca2+ ion. (Kd, Pb2+ = 7.0 ± 1.0 nM; Kd, Mn2+ = 34.0 ± 6.0 nM; K). Native ultra-high resolution mass spectrometry (FT-ICR MS) and trapped ion mobility spectrometry-mass spectrometry (nESI-TIMS-MS) studies provided the NCS1-metal complex compositions-up to four Ca2+ or Mn2+ ions and three Pb2+ ions (M⋅Pb1-3Ca1-3, M⋅Mn1-4Ca1-2, and M⋅Ca1-4) were observed in complex-and similarity across the mobility profiles suggests that the overall native structure is preserved regardless of the number and type of cations. However, the non-physiological metal ions (Pb2+, Mn2+, and Hg2+) binding to NCS1 leads to more efficient quenching of Trp emission and a decrease in W30 and W103 solvent exposure compared to the apo and Ca2+ bound form, although the secondary structural rearrangement and exposure of hydrophobic sites are analogous to those for Ca2+ bound protein. Only Pb2+ and Hg2+ binding to EF-hands leads to the NCS1 dimerization whereas Mn2+ bound NCS1 remains in the monomeric form, suggesting that other factors in addition to metal ion coordination, are required for protein dimerization., (© The Author(s) 2022. Published by Oxford University Press.)

Objective: The mechanisms by which glucose stimulates insulin secretion from β-cells are well established and involve inhibition of ATP-sensitive K + (K ATP ) channels, followed by a rise in [Ca 2+ ] c that triggers exocytosis. However, the mechanisms by which glucose controls glucagon release from α-cells are much less known. In particular, it is debated whether the sugar controls glucagon secretion by changing α-cell [Ca 2+ ] c , and whether K ATP channels or paracrine factors are involved. The present study addresses these issues., Methods: We tested the effect of a decrease or an increase of glucose concentration (Gx, with x = concentration in mM) on α-cell [Ca 2+ ] c and glucagon secretion. α-cell [Ca 2+ ] c was monitored using GluCreGCaMP6f mice expressing the Ca 2+ -sensitive fluorescent protein, GCaMP6f, specifically in α-cells. [Ca 2+ ] c was compared between dispersed α-cells and α-cells within islets to evaluate the potential contribution of an indirect effect of glucose. The same protocols were used for experiments of glucagon secretion from whole islets and [Ca 2+ ] c measurements to test if changes in glucagon release mirror those in α-cell [Ca 2+ ] c ., Results: Blockade of K ATP channels by sulfonylureas (tolbutamide 100 μM or gliclazide 25 μM) strongly increased [Ca 2+ ] c in both dispersed α-cells and α-cells within islets. By contrast, glucose had no effect on [Ca 2+ ] c in dispersed α-cells, whereas it affected it in α-cells within islets. The effect of glucose was however different in islets expressing (Sst +/+ ) or not somatostatin (SST) (Sst -/- ). Decreasing glucose concentration from G7 to G1 modestly increased α-cell [Ca 2+ ] c , but to a slightly larger extent in Sst +/+ islets than in Sst -/- islets. This G1-induced [Ca 2+ ] c rise was also observed in the continuous presence of sulfonylureas in both Sst +/+ and Sst -/- islets. Increasing glucose concentration from G7 to G20 did not affect α-cell [Ca 2+ ] c in Sst +/+ islets which remained low, whereas it strongly increased it in Sst -/- islets. The observations that this increase was seen only in α-cells within islets but never in dispersed α-cells and that it was abrogated by the gap junction inhibitor, carbenoxolone, point to an indirect effect of G20 and suggest that, in Sst -/- islets, G20-stimulated β-cells entrain α-cells whereas, in Sst +/+ islets, the concomitant release of SST keeps α-cell [Ca 2+ ] c at low levels. The [Ca 2+ ] c lowering effect of endogenous SST is also supported by the observation that SST receptor antagonists (SSTR2/3) increased [Ca 2+ ] c in α-cells from Sst +/+ islets. All these [Ca 2+ ] c changes induced parallel changes in glucagon release. To test if glucose also controls glucagon release independently of [Ca 2+ ] c changes, additional experiments were performed in the continuous presence of 30 mM K + and the K ATP channel opener diazoxide (250 μM). In these conditions, α-cell [Ca 2+ ] c within islets was elevated and its steady-state level was unaffected by glucose. However, decreasing the glucose concentration from G7 to G1 stimulated glucagon release whereas increasing it from G1 to G15 inhibited it. These effects were also evident in Sst -/- islets, and opposite to those on insulin secretion., Conclusions: We propose a model according to which glucose controls α-cell [Ca 2+ ] c and glucagon secretion through multiple mechanisms. Increasing the glucose concentration modestly decreases [Ca 2+ ] c in α-cells independently of their K ATP channels and partly via SST. The involvement of SST increases with the glucose concentration, and one major effect of SST is to keep α-cell [Ca 2+ ] c at low levels by counteracting the effect of an entrainment of α-cells by β-cells when β-cells become stimulated by glucose. All these [Ca 2+ ] c changes induce parallel changes in glucagon release. Glucose also decreases the efficacy of Ca 2+ on exocytosis by an attenuating pathway that is opposite to the well-established amplifying pathway controlling insulin release in β-cells., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Calcium (Ca 2+ ) is an important second messenger molecule in the body, regulating cell cycle and fate. There is growing evidence that intracellular Ca 2+ levels play functional roles in the total physiological process of erythroid differentiation, including the proliferation and differentiation of erythroid progenitor cells, terminal enucleation, and mature red blood cell aging and clearance. Moreover, recent research on the pathology of erythroid disorders has made great progress in the past decades, indicating that calcium ion hemostasis is closely related to ineffective erythropoiesis and increased sensitivity to stress factors. In this review, we summarized what is known about the functional roles of intracellular Ca 2+ in erythropoiesis and erythrocyte-related diseases, with an emphasis on the regulation of the intracellular Ca 2+ homeostasis during erythroid differentiation. An understanding of the regulation roles of Ca 2+ homeostasis in erythroid differentiation will facilitate further studies and eventually molecular identification of the pathways involved in the pathological process of erythroid disorders, providing new therapeutic opportunities in erythrocyte-related disease., Competing Interests: Conflict of interest disclosure The authors declare no competing interests., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Giant unilamellar vesicles (GUVs) have been used as a material for bottom-up synthetic biology. However, due to the semi-permeability of the membrane, the need for methods to fuse GUVs has increased. To this aim, methods that are simple and show low leakage during fusion are important. In this study, we report a method of GUV fusion by a divalent cation (Ca 2+ ) enhanced with a long chain polyethylene glycol (PEG20k). The methods showed significant GUV fusion without leakage of internal components of GUVs and maintained cell-free transcription-translation ability inside the GUVs without external supplementation of macromolecules. We demonstrate that the Ca-PEG method can be applied for switching ON of transcription-translation in GUVs in a fusion-dependent manner. The method developed here can be applied to extend bottom-up synthetic biology and molecular robotics that use GUVs as a chassis., (© 2021 Wiley-VCH GmbH.)

The volitional movement of skeletal is controlled by the motor neuron at the site of neuromuscular junction (NMJ) where the retrograde signals are also passed back from muscle to the motor neuron. As the normal function of muscle largely depends on mitochondria that determine the fate of a skeletal muscle myofiber, there must exist a fine-controlled functional coupling between NMJ and mitochondria in myofibers. This mini-review discusses recent publications that reveal how spatiotemporal profiles of intracellular free Ca 2+ could couple mitochondrial function with the activity of NMJ in skeletal muscle myofibers., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Microdomains formed by proteins of endoplasmic reticulum and plasma membrane play a key role in store-operated Ca 2+ entry (SOCE). Ca 2+ release through inositol 1,4,5-trisphosphate receptor (IP 3 R) and subsequent Ca 2+ store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca 2+ , which, in turn, open the Orai channels in plasma membrane. Downstream to this process could be activated TRPC (transient receptor potential-canonical) calcium permeable channels. Using single channel patch-clamp technique we found that a local Ca 2+ entry through TRPC1 channels activated endogenous Ca 2+ -activated chloride channels (CaCCs) with properties similar to Anoctamin6 (TMEM16F). Our data suggest that their outward rectification is based on the dependence from membrane potential of both the channel conductance and the channel activity: (1) The conductance of active CaCCs highly depends on the transmembrane potential (from 3 pS at negative potentials till 60 pS at positive potentials); (2) their activity (NPo) is enhanced with increasing Ca 2+ concentration and/or transmembrane potential, conversely lowering of intracellular Ca 2+ concentration reduced the open state dwell time; (3) CaCC amplitude is only slightly increased by intracellular Ca 2+ concentration. Experiments with Ca 2+ buffering by EGTA or BAPTA suggest close local arrangement of functional CaCCs and TRPC1 channels. It is supposed that Ca 2+ -activated chloride channels are involved in Ca 2+ entry microdomains.

The binding of cytoplasmic Ca 2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca 2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca 2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca 2+ .

The anion channel TMEM16A is activated by intracellular Ca 2+ in a highly cooperative process. By combining electrophysiology and autocorrelation analysis, we investigated the mechanism of channel activation and the concurrent rearrangement of the gate in the narrow part of the pore. Features in the fluctuation characteristics of steady-state current indicate the sampling of intermediate conformations that are successively occupied during gating. The initial step is related to conformational changes induced by Ca 2+ binding, which is ensued by rearrangements that open the pore. Mutations in the gate shift the equilibrium of transitions in a manner consistent with a progressive destabilization of this region during pore opening. We come up with a mechanism of channel activation where the binding of Ca 2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation.

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca 2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca 2+ -sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca 2+ and Mg 2+ and a regulatory site (II) that has been thought to bind only Ca 2+ . Binding of Ca 2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca 2+ /Mg 2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca 2+ /Mg 2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg 2+ caused a 1.4-fold lower affinity for Ca 2+ . These experiments strongly suggest that cytosolic-free Mg 2+ occupies a significant population of the available site II. Interaction of Mg 2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg 2+ , such as secondary hyperparathyroidism or ischemia, respectively., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca 2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca 2+ channel that conducts L-type Ca 2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca 2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα 1S subunit abolishing Ca 2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn 2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca 2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca 2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca 2+ , Mn 2+ entry and subsequent quenching did occur because Mn 2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba 2+ and Ba 2+ currents were strongly blocked by external Ca 2+ . Ba 2+ permeation was smaller, current kinetics slower and Ca 2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca 2+ impeding in this way permeation. Because Ba 2+ binds with lower affinity to D, Ba 2+ currents occur, but with reduced amplitudes as compared to Ba 2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Large-conductance Ca 2+ and voltage-activated K + (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca 2+ sensors. While it is known that BK channels are activated by voltage and Ca 2+ , and that voltage and Ca 2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca 2+ activation for both Ca 2+ bowl and RCK1 Ca 2+ sites, suggesting that both high-affinity Ca 2+ sites transduce their effect, at least in part, through the αB helix. Mg 2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca 2+ binding and voltage depolarization to pore opening and that shared Ca 2+ and voltage transduction pathways involving the αB helix may be involved., Competing Interests: The authors declare no competing interest.

Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca 2+ homeostasis. This work examines the kinetic parameters of Ca 2+ transport and the opening of the Ca 2+ -dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca 2+ uniport, with the kinetics of Na + -dependent Ca 2+ efflux not changing. The data obtained indicate that the decreased rate of Ca 2+ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca 2+ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca 2+ homeostasis of skeletal-muscle mitochondria., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca 2+ ) influx and spontaneous Ca 2+ oscillations. The oscillations cease during maturation but Ca 2+ influx continues, as the oocytes' internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca 2+ influx has not been completely determined. GV and matured oocytes are known to express three Ca 2+ channels, Ca V 3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca 2+ homeostasis, suggesting a complex regulation of Ca 2+ influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. Ca V 3.2 and TRPM7 channels contributed the majority of Ca 2+ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca 2+ entry. Sr 2+ influx was promoted by Ca V 3.2 channels, as Sr 2+ oscillations were negligible in Ca V 3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn 2+ entry relied on expression of Ca V 3.2 and TRPM7 channels, but Ni 2+ entry depended on the latter. Ca V 3.2 and TRPV3 channels combined to fill the Ca 2+ stores, although Ca V 3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca 2+ and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress-inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca 2+ oscillations. As ER stress is known to reduce ER Ca 2+ levels, we tested the hypothesis that the observed increase in Ca 2+ oscillations occurred because of reduced ER Ca 2+ levels and, in turn, increased store-operated Ca 2+ entry. TM-induced cytosolic Ca 2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca 2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca 2+ depletion-activated Ca 2+ current in mediating Ca 2+ -induced insulin secretion in response to ER stress., (© 2020 Zhang et al.)

Mechanical forces play a key role in essential biological processes including cell growth, division, deformation, adhesion, migration and intra-cell interactions. The effect of mechanical forces in modulating the structure and properties of metal-occupied protein binding sites has not been fully understood. Here, by employing a combination of density functional theory (DFT) calculations and polarizable continuum model (PCM) computations applied on model metal-loaded EF-hand binding sites, we shed light on the intimate mechanism of the Mg 2+ /Ca 2+ competition impacted by the application of mechanical stimuli. Applying mechanical force with a specific directionality and magnitude may shift the balance between the competing metal cations in favor of a given contestant depending on the composition and strength of the coordinative bonds and robustness of the metal binding site. Furthermore, the calculations help to determine the range of mechanical rupture forces typical for these structures: these range from 0.4 to 1.5 nN depending on the nature of the metal and amino acid residue. This positions the strength of the Mg 2+ -O and Ca 2+ -O coordinative bonds between that of typical covalent and hydrogen bonds. The bonds between the metal cation and the charged amino acid residue rupture at higher forces (∼1.2-1.5 nN) relative to those of their metal-noncharged counterparts which dissociate at ∼0.2-0.4 nN.

We previously demonstrated that loss of Cdk5 in breast cancer cells promotes ROS-mediated cell death by inducing mitochondrial permeability transition pore (mPTP) opening (Oncogene 37, 1788-1804). However, the molecular mechanism by which Cdk5 loss causes mPTP opening remains to be investigated. Using primary mouse embryonic fibroblasts (MEFs) isolated from Cdk5 -/- mouse embryos, we show that absence of Cdk5 causes a significant increase in both mPTP opening and mitochondrial Ca 2+ level. Analysis of subcellular fractions of MEFs demonstrates that Cdk5 localizes in the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and Cdk5 loss in MAMs causes increased ER-mitochondria tethering, a process required for Ca 2+ transfer from the ER to the mitochondria. Loss of Cdk5 also causes increased ATP-mediated mitochondrial Ca 2+ uptake from the ER. Inhibition of ER Ca 2+ release or mitochondrial Ca 2+ uptake in Cdk5 -/- MEFs prevents mPTP opening, indicating that mPTP opening in Cdk5 -/- MEFs is due to increased Ca 2+ transfer from the ER to the mitochondria. Altogether, our findings suggest that Cdk5 in MAMs regulates mitochondrial Ca 2+ homeostasis that is disturbed upon Cdk5 loss, which leads to mPTP opening.

Ryanodine receptors (RyR) are ion channels responsible for the release of Ca 2+ from the sarco/endoplasmic reticulum and play a crucial role in the precise control of Ca 2+ concentration in the cytosol. The detailed permeation mechanism of Ca 2+ through RyR is still elusive. By using molecular dynamics simulations with a specially designed Ca 2+ model, we show that multiple Ca 2+ ions accumulate in the upper selectivity filter of RyR1, but only one Ca 2+ can occupy and translocate in the narrow pore at a time, assisted by electrostatic repulsion from the Ca 2+ within the upper selectivity filter. The Ca 2+ is nearly fully hydrated with the first solvation shell intact during the whole permeation process. These results suggest a remote knock-on permeation mechanism and one-at-a-time occupation pattern for the hydrated Ca 2+ within the narrow pore, uncovering the basis underlying the high permeability and low selectivity of the RyR channels.

N-Acyl-phosphatidylethanolamines (NAPEs) are known to be precursors of bioactive N-acylethanolamines (NAEs), including the endocannabinoid arachidonoylethanolamide (anandamide) and anti-inflammatory palmitoylethanolamide. In mammals, NAPEs are produced by N-acyltransferases, which transfer an acyl chain from the sn-1 position of glycerophospholipid to the amino group of phosphatidylethanolamine (PE). Recently, the ɛ isoform of cytosolic phospholipase A 2 (cPLA 2 ɛ) was found to be Ca 2+ -dependent N-acyltransferase. However, it was poorly understood which types of phospholipids serve as substrates in living cells. In the present study, we established a human embryonic kidney 293 cell line, in which doxycycline potently induces human cPLA 2 ɛ, and used these cells to analyze endogenous substrates and products of cPLA 2 ɛ with liquid chromatography-tandem mass spectrometry. When treated with doxycycline and Ca 2+ ionophore, the cells produced various species of diacyl- and alkenylacyl-types of NAPEs as well as NAEs in large quantities. Moreover, the levels of diacyl- and alkenylacyl-types of PEs and diacyl-phosphatidylcholines (PCs) decreased, while those of lysophosphatidylethanolamines and lysophosphatidylcholines increased. These results suggested that cPLA 2 ɛ Ca 2+ -dependently produces NAPEs by utilizing endogenous diacyl- and alkenylacyl-types of PEs as acyl acceptors and diacyl-type PCs and diacyl-type PEs as acyl donors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca 2+ -uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca 2+ -uptake function. Thus, it is essential for the Ca 2+ uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca 2+ -uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel carbohydrate binding module (CBM) was identified in a β-1,3-xylanase from Flammeovirga pacifica, which showed only 25.0% sequence identity with the reported CBMs with the coverage of 36.4%. To verify its function, a truncated β-1,3-xylanase (Xy13088-T) and a carbohydrate binding module (CBM3088) were expressed and purified. The thermostability and catalytic efficiency of the Xy13088-T declined significantly when compared with the full-length one, with the decreasing of the half-life and catalytic efficiency (Kcat/K m ) by 90%. Interestingly, the CBM3088 showed the binding ability to β-1,3-xylan only when Ca 2+ existed, which was different from the reported CBMs of β-1,3-xylanases. The maximum amount of CBM3088 binding to β-1,3-xylan was 9.65 μmol/g of β-1,3-xylan. The residues probably involved in the binding to the β-1,3-xylan and Ca 2+ were addressed by bioinformatics analysis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We develop an iterated map model to describe the bifurcations and complex dynamics caused by the feedback between voltage and intracellular Ca^{2+} and Na^{+} concentrations in paced ventricular myocytes. Voltage and Ca^{2+} can form either a positive or a negative feedback loop, while voltage and Na^{+} form a negative feedback loop. Under certain diseased conditions, when the feedback between voltage and Ca^{2+} is positive, Hopf bifurcations occur, leading to periodic oscillatory behaviors. When this feedback is negative, period-doubling bifurcation routes to alternans and chaos occur.

The βγ -crystallin superfamily contains both β- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate βγ -crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial βγ -crystallins bind calcium ions. The βγ -crystallin from the tunicate Ciona intestinalis ( Ci - βγ ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci - βγ is valuable for investigating the evolution of the βγ -crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca 2+ and other divalent cations on the stability and aggregation propensity of Ci - βγ and human γS-crystallin (HγS). Beyond Ca 2+ , Ci - βγ is capable of coordinating Mg 2+ , Sr 2+ , Co 2+ , Mn 2+ , Ni 2+ , and Zn 2+ , although only Sr 2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci - βγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn 2+ , Ni 2+ , and Co 2+ induce aggregation by interacting with cysteine residues, whereas Cu 2+ -mediated aggregation proceeds via a different binding site.

The α7 subtype of neuronal nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that is vital to various neurological functions, including modulation of neurotransmitter release. A relatively high concentration of extracellular Ca 2+ in the neuronal environment is likely to exert substantial structural and functional influence on nAChRs, which may affect their interactions with agonists and antagonists. In this work, we employed atomistic molecular dynamics (MD) simulations to examine the effects of elevated Ca 2+ on the structure and dynamics of α7 nAChR embedded in a model phospholipid bilayer. Our results suggest that the presence of Ca 2+ in the α7 nAChR environment results in closure of loop C-in the extracellular ligand-binding domain, a motion normally associated with agonist binding and receptor activation. Elevated Ca 2+ also alters the conformation of key regions of the receptor, including the inter-helical loops, pore-lining helices and the "gate" residues, and causes partial channel opening in the absence of an agonist, leading to an attendant reduction in the free energy of Ca 2+ permeation through the pore as elucidated by umbrella sampling simulations. Overall, the structural and permeability changes in α7 nAChR suggest that elevated Ca 2+ induces a partially activated receptor state that is distinct from both the resting and the agonist-activated states. These results are consistent with the notion that divalent ions can serve as a potentiator of nAChRs, resulting in a higher rate of receptor activation (and subsequent desensitization) in the presence of agonists, with possible implications for diseases involving calcium dysregulation., (© 2019 Wiley Periodicals, Inc.)

Ganoderic acids produced by Ganoderma exhibit anticancer and antimetastatic activities. A novel approach by combining Vitreoscilla haemoglobin (VHb) expression and calcium ion induction was developed to enhance ganoderic acid (GA) production in liquid static cultures of G. lingzhi. The maximum contents of GA-O, GA-S and GA-Me were 1451.33 ± 67.50, 1431.23 ± 79.74 and 1283.81 ± 85.13 μg per 100 mg cell weight, respectively under the integrated approach, which are the highest contents as ever reported in Ganoderma. The contents of squalene and lanosterol were increased by 2.0- and 3.0-fold in this case compared with those in the control. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl-diphosphate synthase, squalene synthase and cytochrome P450 CYP5150L8 were upregulated by 2.56-, 3.31-, 2.59- and 6.12-fold respectively. Additionally, the expression of VHb improved the ratio of type I to type II GA in liquid static cultivation of G. lingzhi. The transcription levels of cyp512a2, cyp512v2 and cyp512a13, candidate cytochrome P450 genes involved in oxidative modification of the lanostane skeleton in GA biosynthesis, were also increased by 2.28-, 2.65- and 3.54-fold in the VHb-expressing strain respectively. Our results illustrated that the approach described here efficiently improved GA production in G. lingzhi fermentation., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca 2+ entry (SOCE). Here, we report that SOCE, peak Ca 2+ transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca 2+ entry coincident with a modest decrease in total releasable Ca 2+ store content. Importantly, this decrease in releasable Ca 2+ store content observed after exercise was reversed by repetitive high-frequency stimulation, consistent with enhanced SOCE. The functional benefits of exercise on SOCE, constitutive Ca 2+ entry and muscle force production were lost in mice with muscle-specific loss of Orai1 function. These results indicate that TT association with SR-stacks enhances Orai1-dependent SOCE to optimize Ca 2+ dynamics and muscle contractile function during acute exercise., Competing Interests: AM, SB, LP, MG, TT, SM, RD, FP No competing interests declared, (© 2019, Michelucci et al.)

TRPM2 is critically involved in diverse physiological processes including core temperature sensing, apoptosis, and immune response. TRPM2's activation by Ca 2+ and ADP ribose (ADPR), an NAD + -metabolite produced under oxidative stress and neurodegenerative conditions, suggests a role in neurological disorders. We provide a central concept between triple-site ligand binding and the channel gating of human TRPM2. We show consecutive structural rearrangements and channel activation of TRPM2 induced by binding of ADPR in two indispensable locations, and the binding of Ca 2+ in the transmembrane domain. The 8-Br-cADPR-an antagonist of cADPR-binds only to the MHR1/2 domain and inhibits TRPM2 by stabilizing the channel in an apo-like conformation. We conclude that MHR1/2 acts as a orthostatic ligand-binding site for TRPM2. The NUDT9-H domain binds to a second ADPR to assist channel activation in vertebrates, but not necessary in invertebrates. Our work provides insights into the gating mechanism of human TRPM2 and its pharmacology., Competing Interests: YH, BR, WL, JD No competing interests declared, (© 2019, Huang et al.)

Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca 2+ ] i ). The extremely complex calcium machinery includes plasma membrane bound channels as well as intracellular receptors distributed in distinct cytosolic compartments that act jointly to maintain a physiological [Ca 2+ ] i , which is crucial for triggering many cellular mechanisms. Here, we first survey the overall notions related to intracellular Ca 2+ mobilization and later highlight the involvement of this second messenger in crucial ECs functions with the aim at stimulating further investigation that link Ca 2+ mobilization to ECs in health and disease.

Allosteric interactions between the voltage-sensing domain (VSD), the Ca 2+ -binding sites, and the pore domain govern the mammalian Ca 2+ - and voltage-activated K + (BK) channel opening. However, the functional relevance of the crosstalk between the Ca 2+ - and voltage-sensing mechanisms on BK channel gating is still debated. We examined the energetic interaction between Ca 2+ binding and VSD activation by investigating the effects of internal Ca 2+ on BK channel gating currents. Our results indicate that Ca 2+ sensor occupancy has a strong impact on VSD activation through a coordinated interaction mechanism in which Ca 2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca 2+ -binding sites contained in the C-terminus domains, RCK1 and RCK2, contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca 2+ sensors and VSDs., Competing Interests: YL, WC, KC, OA, RL No competing interests declared, (© 2019, Lorenzo-Ceballos et al.)

The endoplasmic reticulum ( ER ) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about how this vital ATP transport occurs across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca 2+ -antagonized mechanism, or CaATiER ( Ca 2+ - A ntagonized T ransport i nto ER ). Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca 2+ gradient across the ER membrane is necessary for ATP transport into the ER, through SLC35B1/AXER. We propose that under physiological conditions, increases in cytosolic Ca 2+ inhibit ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation ( OxPhos ) inhibitors and that ER protein misfolding increases ATP uptake from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e. an ' anti-Warburg ' effect., Competing Interests: JY, HB, SB, MS, AM, RM, RK No competing interests declared, (© 2019, Yong et al.)

The Glutamate Receptor Ionotropic NMDA-Associated Protein 1 (GRINA) belongs to the Lifeguard family and is involved in calcium homeostasis, which governs key processes, such as cell survival or the release of neurotransmitters. GRINA is mainly associated with membranes of the endoplasmic reticulum, Golgi, endosome, and the cell surface, but its presence in the nucleus has not been explained yet. Here we dissect, with the help of different software tools, the potential roles of GRINA in the cell and how they may be altered in diseases, such as schizophrenia or celiac disease. We describe for the first time that the cytoplasmic N-terminal half of GRINA (which spans a Proline-rich domain) contains a potential DNA-binding sequence, in addition to cleavage target sites and probable PY-nuclear localization sequences, that may enable it to be released from the rest of the protein and enter the nucleus under suitable conditions, where it could participate in the transcription, alternative splicing, and mRNA export of a subset of genes likely involved in lipid and sterol synthesis, ribosome biogenesis, or cell cycle progression. To support these findings, we include additional evidence based on an exhaustive review of the literature and our preliminary data of the protein-protein interaction network of GRINA., Competing Interests: The authors declare no conflict of interest.

Many biological processes are based on molecular recognition between highly charged molecules such as nucleic acids, inorganic ions, charged amino acids, etc. For such cases, it has been demonstrated that molecular simulations with fixed partial charges often fail to achieve experimental accuracy. Although incorporation of more advanced electrostatic models (such as multipoles, mutual polarization, etc.) can significantly improve simulation accuracy, it increases computational expense by a factor of 5-20×. Indirect free energy (IFE) methods can mitigate this cost by modeling intermediate states at fixed-charge resolution. For example, an efficient "reference" model such as a pairwise Amber, CHARMM, or OPLS-AA force field can be used to derive an initial estimate, followed by thermodynamic corrections to a more advanced "target" potential such as the polarizable AMOEBA model. Unfortunately, all currently described IFE methods encounter difficulties reweighting more than ∼50 atoms between resolutions due to extensive scaling of both the magnitude of the thermodynamic corrections and their statistical uncertainty. We present an approach called "simultaneous bookending" (SB) that is fundamentally different from existing IFE methods based on a tunable sampling approximation, which permits scaling to thousands of atoms. SB is demonstrated on the relative binding affinity of Mg 2+ /Ca 2+ to a set of metalloproteins with up to 2972 atoms, finding no statistically significant difference between direct AMOEBA results and those from correcting Amber to AMOEBA. The ability to change the resolution of thousands of atoms during reweighting suggests the approach may be applicable in the future to protein-protein binding affinities or nucleic acid thermodynamics.

P2X3 receptor channels expressed in sensory neurons are activated by extracellular ATP and serve important roles in nociception and sensory hypersensitization, making them attractive therapeutic targets. Although several P2X3 structures are known, it is unclear how physiologically abundant Ca 2+ -ATP and Mg 2+ -ATP activate the receptor, or how divalent cations regulate channel function. We used structural, computational and functional approaches to show that a crucial acidic chamber near the nucleotide-binding pocket in human P2X3 receptors accommodates divalent ions in two distinct modes in the absence and presence of nucleotide. The unusual engagement between the receptor, divalent ion and the γ-phosphate of ATP enables channel activation by ATP-divalent complex, cooperatively stabilizes the nucleotide on the receptor to slow ATP unbinding and recovery from desensitization, a key mechanism for limiting channel activity. These findings reveal how P2X3 receptors recognize and are activated by divalent-bound ATP, aiding future physiological investigations and drug development., Competing Interests: ML, YW, RB, FM, SS, MH No competing interests declared, JF, KS Reviewing editor, eLife

Ca 2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca 2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of Ca 2+ signal transduction in cyanobacteria are poorly understood, and very few protein components are known. The current study describes a previously unreported Ca 2+ -binding protein which was called the Ca 2+ Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind Ca 2+ , which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction centre protein complexes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ca 2+ -transcription coupling controls gene expression patterns that define vascular smooth muscle cell (VSMC) phenotype. Although not well understood this allows normally contractile VSMCs to become proliferative following vessel injury, a process essential for repair but which also contributes to vascular remodelling, atherogenesis and restenosis. Here we show that the Ca 2+ /HCO 3 - -sensitive enzyme, soluble adenylyl cyclase (sAC), links Ca 2+ influx in human coronary artery smooth muscle cells (hCASMCs) to 3',5'-cyclic adenosine monophosphate (cAMP) generation and phosphorylation of the transcription factor Ca 2+ /cAMP response element binding protein (CREB). Store-operated Ca 2+ entry (SOCE) into hCASMCs expressing the FRET-based cAMP biosensor H187 induced a rise in cAMP that mirrored cytosolic [Ca 2+ ]. SOCE also activated the cAMP effector, protein kinase A (PKA), as determined by the PKA reporter, AKAR4-NES, and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and CREB. Transmembrane adenylyl cyclase inhibition had no effect on the SOCE-induced rise in cAMP, while sAC inhibition abolished SOCE-generated cAMP and significantly reduced SOCE-induced VASP and CREB phosphorylation. This suggests that SOCE in hCASMCs activates sAC which in turn activates the cAMP/PKA/CREB axis. sAC, which is insensitive to G-protein modulation but responsive to Ca 2+ , pH and ATP, may therefore act as an overlooked regulatory node in vascular Ca 2+ -transcription coupling.

Ca 2+ -stimulated translocation of cytosolic phospholipase A 2 α (cPLA 2 α) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLA 2 α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLA 2 α C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl- sn -glycero-3-phosphocholine (DHPC) and Ca 2+ ions. Two Ca 2+ are complexed at previously reported locations in the lipid-free C2-domain. One of these Ca 2+ ions, along with a third Ca 2+ , bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid headgroup recognition via cation-π interaction with the PC trimethylammonium group. Mutagenesis analyses confirm that Tyr96 and Asn65 function in PC binding selectivity by the C2-domain and in the regulation of cPLA 2 α activity. The DHPC-binding mode of the cPLA 2 α C2-domain, which differs from phosphatidylserine or phosphatidylinositol 4,5-bisphosphate binding by other C2-domains, expands and deepens knowledge of the lipid-binding mechanisms mediated by C2-domains., Competing Interests: YH, YG, DS, NV, LM, DS, CC, DP, RB No competing interests declared, (© 2019, Hirano et al.)

Manganese (Mn 2+ ) is extruded from the cell by the zinc transporter 10 (ZnT10). Loss of ZnT10 expression caused by autosomal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs. Here, combining fluorescent monitoring of cation influx in HEK293-T cells expressing human ZnT10 with molecular modeling of ZnT10 cation selectivity, we show that ZnT10 is exploiting the transmembrane Ca 2+ inward gradient for active cellular exchange of Mn 2+ In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn 2+ and Ca 2+ fluxes. We found that ( a ) application of Mn 2+ -containing Ca 2+ -free solution to ZnT10-expressing cells triggers an influx of Mn 2+ , ( b ) reintroduction of Ca 2+ leads to cellular Mn 2+ extrusion against an inward Mn 2+ gradient, and ( c ) the cellular transport of Mn 2+ by ZnT10 is coupled to a reciprocal movement of Ca 2+ Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn 2+ /Ca 2+ exchange to an uncoupled channel mode, permeable to both Ca 2+ and Mn 2+ The findings in our study identify the first ion transporter that uses the Ca 2+ gradient for active counter-ion exchange. They highlight a remarkable versatility in metal selectivity and mode of transport controlled by the tetrahedral metal transport site of ZnT proteins., (© 2019 Levy et al.)

Bacteria sense environmental chemicals using chemosensor proteins, most of which are present in the cytoplasmic membrane. Canonical chemoreceptors bind their specific ligands in their periplasmic domain, and the ligand binding creates a molecular stimulus that is transmitted into the cytoplasm, leading to various cellular responses, such as chemotaxis and specific gene expression. Vibrio cholerae , the causative agent of cholera, contains about 44 putative sensor proteins, which are homologous to methyl-accepting chemotaxis proteins involved in chemotaxis. Two of them, Mlp24 and Mlp37, have been identified as chemoreceptors that mediate chemotactic responses to various amino acids. Although most of the residues of Mlp37 involved in ligand binding are conserved in Mlp24, these chemoreceptors bind the same ligands with different affinities. Moreover, they have distinct cellular roles. Here we determined a series of ligand complex structures of the periplasmic domains of Mlp24 (Mlp24p). The structures revealed that Ca 2+ binds to the loop that forms the upper wall of the ligand-binding pocket. Ca 2+ does not bind to the corresponding loop of Mlp37, implying that the structural difference of the loop may cause the ligand affinity difference. Isothermal titration calorimetry (ITC) measurements indicated that Ca 2+ changes the ligand binding affinity of Mlp24p. Furthermore, Ca 2+ affected chemotactic behaviors to various amino acids mediated by Mlp24. Thus, Ca 2+ is suggested to serve as a cosignal for the primary signal mediated by Mlp24p, and V. cholerae fine-tunes its chemotactic behavior depending on the Ca 2+ concentration by modulating the ligand sensitivity of Mlp24. IMPORTANCE Mlp24 and Mlp37 are homologous chemoreceptors of Vibrio cholerae that bind various amino acids. Although most of the residues involved in ligand interaction are conserved, these chemoreceptors show different affinities for the same ligand and play different cellular roles. A series of ligand complex structures of the periplasmic region of Mlp24 (Mlp24p) and following ITC analysis revealed that Ca 2+ binds to the loop of Mlp24p and modulates the ligand binding affinity of Mlp24p. Moreover, Ca 2+ changes the chemotactic behaviors mediated by Mlp24. We propose that Ca 2+ acts as a cosignal that modulates the affinity of Mlp24 for the primary signal, thereby changing the chemotactic behavior of V. cholerae ., (Copyright © 2019 American Society for Microbiology.)

Novel Ca 2+ -independent C-type lectins, SPL-1 and SPL-2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL-2 composed of two B-chains) or distinct (SPL-1 composed of A- and B-chains) polypeptide chains, and show affinity for N-acetylglucosamine (GlcNAc)- and N-acetylgalactosamine (GalNAc)-containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C-type lectin family. X-ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate-recognition domain (CRD) of the C-type lectin family. Nevertheless, these lectins (especially SPL-2) showed Ca 2+ -independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL-2/GalNAc complex revealed that bound GalNAc was mainly recognized via its acetamido group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate-recognition motifs among the C-type CRD (the QPD [Gln-Pro-Asp] and EPN [Glu-Pro-Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate-binding specificities of individual A- and B-chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α-glycosidic linkages with slightly different specificities., (© 2019 The Protein Society.)

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific Tbx5 -mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca 2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5 -deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca 2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca 2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca 2+ -dependent pathway for AF risk., Competing Interests: WD, BL, LT, KS, RN, FA, SM, SL, MG, YW, YL, HV, LS, MB, IM, CW No competing interests declared, (© 2019, Dai et al.)

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca 2+ influx via Ca 2+ release-activated Ca 2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca 2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca 2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

Development of the nervous system relies on a balance between axon and dendrite growth and subsequent pruning and degeneration. The developmental degeneration of dorsal root ganglion (DRG) sensory axons has been well studied in part because it can be readily modeled by removing the trophic support by nerve growth factor (NGF) in vitro . We have recently reported that axonal fragmentation induced by NGF withdrawal is dependent on Ca 2+ , and here, we address the mechanism of Ca 2+ entry required for developmental axon degeneration of mouse embryonic DRG neurons. Our results show that the transient receptor potential vanilloid family member 1 (TRPV1) cation channel plays a critical role mediating Ca 2+ influx in DRG axons withdrawn from NGF. We further demonstrate that TRPV1 activation is dependent on reactive oxygen species (ROS) generation that is driven through protein kinase C (PKC) and NADPH oxidase (NOX)-dependent pathways that become active upon NGF withdrawal. These findings demonstrate novel mechanistic links between NGF deprivation, PKC activation, ROS generation, and TRPV1-dependent Ca 2+ influx in sensory axon degeneration.

Ca 2+ homeostasis is crucial for the entire life of eukaryotic cells from the beginning to the end. Mishandling in Ca 2+ homeostasis is indeed linked with a large number of pathological conditions. Thus, the possibility to specifically monitor cellular calcium fluxes in different subcellular compartments represents a key tool to deeply understand the mechanisms involved in cellular dysfunctions. To cope with this need, several Ca 2+ indicators have been developed allowing to accurately measure both basal Ca 2+ concentration and agonist-induced Ca 2+ signals in a wide spectrum of organelles. Among these, the genetically encoded GFP-based indicators are routinely used to measure Ca 2+ transients thanks to their ability to change their spectral properties in response to Ca 2+ binding. In this chapter, we will describe a protocol that utilizes the GCaMP6f probe targeted to mitochondria (4mtGCaMP) to measure mitochondrial calcium levels in resting conditions in HeLa cells. This method allows to easily and quickly register alterations of mitochondrial Ca 2+ homeostasis in different cell populations and experimental settings, representing a precious tool to unravel the pathological pathways leading to pathogenic conditions.

Basal-like breast cancer (BLBC) is the most aggressive subtype with a poor clinical outcome; however, the molecular mechanisms underlying aggressiveness in BLBC remain poorly understood. Methods: The effects of gamma-aminobutyrate aminotransferase (ABAT) on GABA receptors, Ca 2+ -NFAT1 axis, and cancer cell behavior were assessed by Ca 2+ imaging, Western blotting, immunostaining, colony formation, and migration and invasion assays. We elucidated the relationship between ABAT and Snail by luciferase reporter and ChIP assays. The effect of ABAT expression on BLBC cells was determined by in vitro and in vivo tumorigenesis and a lung metastasis mouse model. Results: We showed that, compared to other subtypes, ABAT was considerably decreased in BLBC. Mechanistically, ABAT expression was downregulated due to Snail-mediated repression leading to increased GABA production. GABA then elevated intracellular Ca 2+ concentration by activating GABA-A receptor (GABA A ), which contributed to the efficient activation of NFAT1 in BLBC cells. ABAT expression resulted in inhibition of tumorigenicity, both in vitro and in vivo , and metastasis of BLBC cells. Thus, loss of ABAT contributed to BLBC aggressiveness by activating the Ca 2+ -NFAT1 axis. In breast cancer patients, loss of ABAT expression was strongly correlated with large tumor size, high grade and metastatic tendency, poor survival, and chemotherapy resistance. Conclusions: Our findings have provided underlying molecular details for the aggressive behavior of BLBC. The Snail-mediated downregulation of ABAT expression in BLBC provides tumorigenic and metastatic advantages by activating GABA-mediated Ca 2+ -NFAT1 axis. Thus, our results have identified potential prognostic indicators and therapeutic targets for this challenging disease., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Calcium ion (Ca 2+ ) is a ubiquitous intracellular messenger able to generate versatile intracellular signals that modulate a large variety of functions in virtually every cell type. Chemical and genetic biosensors, targeted to different subcellular compartments, have been developed and continuously improved to monitor Ca 2+ dynamics in living cells. Here we describe the usage of Förster resonance energy transfer (FRET)-based Cameleon probes to investigate Ca 2+ influx across the plasma membrane (PM) or Ca 2+ release from the main intracellular Ca 2+ store, the endoplasmic reticulum (ER).

The store-operated calcium (Ca 2+ ) entry (SOCE) pathway is an essential Ca 2+ signaling pathway in non-excitable cells that serve many physiological functions. SOCE is mediated through the plasma membrane (PM) protein, Orai1, and the endoplasmic reticulum protein, stromal interaction molecule 1 (STIM1). One of the most well-established methods to study SOCE is using the Ca 2+ -sensing dye, fura-2. Here we describe a detailed protocol on how to use fura-2 to study Ca 2+ signaling from SOCE in human embryonic kidney (HEK) cells.

In brain networks, neurons are constantly involved in a dynamic interaction with the other cell populations and, particularly, with astrocytes, the most abundant glial cells in the brain. Astrocytes respond to neurotransmitters with Ca 2+ elevations which represent a key event in the modulation of local brain circuits played by these glial cells. Due to technical limitations, the study of Ca 2+ signal dynamics in astrocytes has focused for decades almost exclusively on somatic and perisomatic regions. Accordingly, Ca 2+ signal in astrocytic fine protrusions, which are in close contact with the synapse, has been poorly investigated. Over the last years, the diffusion of novel tools such as the viral vector gene delivery of genetically encoded Ca 2+ indicators (GECI), the optogenetics, and multiphoton laser scanning microscopy has boosted significantly our capability to study astrocytic Ca 2+ signals in the different subcellular compartments. Here we report a protocol that combines these techniques to study astrocyte Ca 2+ signaling in response to somatostatin (SST)-expressing interneurons, one of the main classes of GABAergic inhibitory interneurons.

Lysosomes are emerging as calcium store organelles that can modulate various intracellular processes such as the regulation of nutrient signaling through the activation of TFEB, a master gene for lysosomal function, or very specialized functions like lysosomal exocytosis. Here, we describe two different techniques that can be used to study these processes. In the case report, we described two studies where these methodologies allowed us to unravel the role of calcineurin in the dephosphorylation of TFEB as well as the involvement of TFEB in lysosomal exocytosis, respectively.