Protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Arabidopsis NADPH oxidases and may function as a trigger for the positive feedback regulation of Ca2+ and reactive oxygen species

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca2+. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca2+-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca2+ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca2+. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca2+-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Rboh. Positive feedback regulation of Ca2+ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca2+-ROS signalling network.     

Apoptosis of tiger shrimp (Penaeus monodon) haemocytes induced by Escherichia coli lipopolysaccharide

This study was aimed at investigating the toxicity mechanism of lipopolysaccharide (LPS) on Penaeus monodon haemocytes at a cellular level. Reactive oxygen species (ROS) production, nitric oxide (NO) production, non-specific esterase activity, cytoplasmic free-Ca^2 + (CF-Ca^2 +) concentration, DNA damaged cell ratio and apoptotic cell ratio of in vitro LPS-treated haemocytes were measured by flow cytometry. Two concentrations of Escherichia coli LPS (5 and 10 μg mL^? 1) were used. Results showed that ROS production, NO production and CF-Ca^2 + concentration were significantly induced in the LPS-treated haemocytes. Ratio of DNA damaged cell and apoptotic cell increased caused by LPS, while esterase activity increased at the initial 60 min and dropped later. The initial increase in esterase activity suggested that LPS activated the release of esterase, and the later decrease might result from apoptosis. These results indicated that LPS would induce oxidative stress on shrimp haemocytes, and cause Ca^2 + release, DNA damage and subsequently cell apoptosis. This process of ROS/RNS-induced Ca^2 +-mediated apoptosis might be one of the toxicity mechanisms of LPS on shrimp haemocytes.     

How do reactive oxygen species and calcium trigger mitochondrial membrane permeabilisation?

BackgroundMitochondrial membrane permeabilisation (MMP) is classically considered as a point of no return in several forms of cell death and is involved in numerous diseases such as cancer, neurodegenerative disorders or ischemia/reperfusion injuries. Many studies established that reactive oxygen species (ROS) and Ca^2 + were the prominent inducers of MMP. However, the mechanisms connecting ROS and Ca^2 + to the players of MMP are still a matter of debate.Scope of reviewThe aim of this review is to summarise the various studies related to the mechanisms of ROS- and Ca^2 +-induced MMP. Several lines of evidence suggest that ROS and Ca^2 + cooperate to induce MMP but the molecular details of the ROS–Ca^2 +-MMP network remain controversial. We then discuss recent data depicting this topic.Major conclusionsCytotoxic stimuli may be transduced within the cell by ROS and Ca^2 + increases. In most models, Ca^2 + and ROS can cooperate to induce MMP. Moreover, several data suggest that MMP increases mitochondrial Ca^2 + and ROS which therefore amplify the cytotoxic signal. Intriguingly, many reports have identified players of MMP as direct ROS targets. On the contrary, direct targets of Ca^2 + remain elusive. At the same time, the mechanisms by which mitochondrial Ca^2 + overload induces ROS generation are well documented. Upon these observations, we hypothesise that Ca^2 + cannot directly induce MMP and requires ROS production as a mandatory step.General significanceGiven the importance of Ca^2 +- and ROS-induced MMP in diseases, we expect that a better understanding of this process will lead to the development of novel therapies.     

Ca2 +-dependent permeabilization of mitochondria and liposomes by palmitic and oleic acids: A comparative study

In the present work, we examine and compare the effects of saturated (palmitic) and unsaturated (oleic) fatty acids in relation to their ability to cause the Ca^2 +-dependent membrane permeabilization. The results obtained can be summarized as follows. (1) Oleic acid (OA) permeabilizes liposomal membranes at much higher concentrations of Ca^2 + than palmitic acid (PA): 1 mM versus 100 μM respectively. (2) The OA/Ca^2 +-induced permeabilization of liposomes is not accompanied by changes in the phase state of lipid bilayer, in contrast to what is observed with PA and Ca^2 +. (3) The addition of Ca^2 + to the PA-containing vesicles does not change their size; in the case of OA, it leads to the appearance of larger and smaller vesicles, with larger vesicles dominating. This can be interpreted as a result of fusion and fission of liposomes. (4) Like PA, OA is able to induce a Ca^2 +-dependent high-amplitude swelling of mitochondria, yet it requires higher concentrations of Ca^2 + (30 and 100 μM for PA and OA respectively). (5) In contrast to PA, OA is unable to cause the Ca^2 +-dependent high-amplitude swelling of mitoplasts, suggesting that the cause of OA/Ca^2 +-induced permeability transition in mitochondria may be the fusion of the inner and outer mitochondrial membranes. (6) The presence of OA enhances PA/Ca^2 +-induced permeabilization of liposomes and mitochondria. The paper discusses possible mechanisms of PA/Ca^2 +- and OA/Ca^2 +-induced membrane permeabilization, the probability of these mechanisms to be realized in the cell, and their possible physiological role.     

Mechanism of dopamine D2 receptor-induced Ca2 + release in PC-12 cells

Intracellular Ca^2 + levels are tightly regulated in the neuronal system. The loss of Ca^2 + homeostasis is associated with many neurological diseases and neuropsychiatric disorders such as Parkinson's, Alzheimer's, and schizophrenia. We investigated the mechanisms involved in intracellular Ca^2 + signaling in PC-12 cells. The stimulation of NGF-differentiated PC-12 cells with 3 μM ATP caused an early Ca^2 + release followed by a delayed Ca^2 + release. The delayed Ca^2 + release was dependent on prior ATP priming and on dopamine secretion by PC-12 cells. Delayed Ca^2 + release was abolished in the presence of spiperone, suggesting that it is due to the activation of D2 dopamine receptors (D2R) by dopamine secreted by PC-12 cells. This was shown to be independent of PKA activation but dependent on PLC activity. An endocytosis step was required for inducing the delayed Ca^2 + release. Given the importance of calcyon in clathrin-mediated endocytosis, we verified the role of this protein in the delayed Ca^2 + release phenomenon. siRNA targeting of calcyon blocked the delayed Ca^2 + release, decreased ATP-evoked IP₃R-mediated Ca^2 + release, and impaired subsequent Ca^2 + oscillations. Our results suggested that calcyon is involved in an unknown mechanism that causes a delayed IP₃R-mediated Ca^2 + release in PC-12 cells. In schizophrenia, Ca^2 + dysregulation may depend on the upregulation of calcyon, which maintains elevated Ca^2 + levels as well as dopamine signaling.     

Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca^2 + properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca^2 + release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca^2 + properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca^2 + anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.     

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.     

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca^2 +-induced (Pal/Ca^2 +) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca^2 + with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca^2 + from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca^2 + influx, seems to be partly due to the opening of Pal/Ca^2 + pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca^2 +-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca^2 + (Sr^2 +) also induced an outflow of K⁺ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K⁺/H⁺ exchange blocker, and the reuptake was prevented by an ATP-dependent K⁺ channel inhibitor. It was also shown that the addition of Sr^2 + to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr^2 + and K⁺ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca^2 +-dependent phospholipase A₂, and a new Sr^2 + cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr^2 +. All of the Ca^2 +- and Sr^2 +-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca^2 + pores in the maintenance of cell ion homeostasis.     

The use of transgenic mouse models to reveal the functions of Ca2 + buffer proteins in excitable cells

BackgroundCytosolic Ca^2 + buffers are members of the large family of Ca^2 +-binding proteins and are essential components of the Ca^2 + signaling toolkit implicated in the precise regulation of intracellular Ca^2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca^2 + buffers or mice with ectopic expression.Scope of ReviewIn this review, results obtained with knockout mice for the three most prominent Ca^2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.Major ConclusionsThe absence of Ca^2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca^2 + buffer-specific changes in intracellular Ca^2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca^2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca^2 + buffers, but have revealed that the absence of Ca^2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.General SignificanceThe complex phenotype of Ca^2 + buffer knockout mice arises from the direct effect of these proteins on Ca^2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca^2 + signaling, a global view on Ca^2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

The role of plasma membrane STIM1 and Ca2 +entry in platelet aggregation. STIM1 binds to novel proteins in human platelets

Ca^2 + elevation is essential to platelet activation. STIM1 senses Ca^2 + in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca^2 + entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca^2 + entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca^2 + entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca^2 +-sensing role of STIM1 is served by the protein in the ER.     

Flow-induced activation of TRPV5 and TRPV6 channels stimulates Ca2 +-activated K+ channel causing membrane hyperpolarization

TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca^2 + reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinases A and C, membrane phospholipid PIP₂, protons, and divalent ions Ca^2 + and Mg^2 +. Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2 min. Fluid flow stimulated TRPV5 and 6-mediated Ca^2 + entry and increased intracellular Ca^2 + concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La^3 +. In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channel. These results reveal a new mechanism for the regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K⁺ secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria.     

Functional validation of Ca2 +-binding residues from the crystal structure of the BK ion channel

BK channels are dually regulated by voltage and Ca^2 +, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca^2 + concentration is sensed by a large cytoplasmic region in the channel known as “gating ring”, which is formed by four tandems of regulator of conductance for K⁺ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca^2 + coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca^2 +. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca^2 + in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca^2 + sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca^2 + coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca^2 + binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca^2 + binding sites regulating the Ca^2 + dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.     

Large conductance Ca2+-activated K+ channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects

Large conductance calcium activated potassium channels (BK_Ca) are fundamental in the control of cellular excitability. Thus, compounds that activate BK_Ca channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BK_Ca channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BK_Ca channel α-subunit alone or α + β1-subunit complex.Channel activity was determined using a non-radioactive Rb⁺ efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb⁺ efflux both in cells expressing α-subunit alone or α + β1-subunits. Co-expression of the β1-subunit modified the Rb⁺ efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC₄₀ values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BK_Ca channel α + β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC₄₀ values when tested on the BK_Ca α + β1-subunit expressing cells compared to BK_Ca α-subunit expressing cells. Further, the E_max values for BKPr4, BKVV and BKH1 were lower in the BK_Ca channel α + β1-subunit expressing cells.In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BK_Ca channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles.     

Alterations in ryanodine receptors and related proteins in heart failure

Sarcoplasmic reticulum (SR) Ca^2 + release plays an essential role in mediating cardiac myocyte contraction. Depolarization of the plasma membrane results in influx of Ca^2 + through l-type Ca^2 + channels (LTCCs) that in turn triggers efflux of Ca^2 + from the SR through ryanodine receptor type-2 channels (RyR2). This process known as Ca^2 +-induced Ca^2 +release (CICR) occurs within the dyadic region, where the adjacent transverse (T)-tubules and SR membranes allow RyR2 clusters to release SR Ca^2 + following Ca^2 + influx through adjacent LTCCs. SR Ca^2 + released during systole binds to troponin-C and initiates actin–myosin cross-bridging, leading to muscle contraction. During diastole, the cytosolic Ca^2 + concentration is restored by the resequestration of Ca^2 + into the SR by SR/ER Ca^2 +-ATPase (SERCA2a) and by the extrusion of Ca^2 + via the Na⁺/Ca^2 +-exchanger (NCX1). This whole process, entitled excitation–contraction (EC) coupling, is highly coordinated and determines the force of contraction, providing a link between the electrical and mechanical activities of cardiac muscle. In response to heart failure (HF), the heart undergoes maladaptive changes that result in depressed intracellular Ca^2 + cycling and decreased SR Ca^2 + concentrations. As a result, the amplitude of CICR is reduced resulting in less force production during EC coupling. In this review, we discuss the specific proteins that alter the regulation of Ca^2 + during HF. In particular, we will focus on defects in RyR2-mediated SR Ca^2 + release. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.     

Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut

We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of ～ 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50–60 °C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca^+ 2, Mn^+ 2 and Mg^+ 2 somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.     

Thapsigargin decreases the Na+- Ca2 + exchanger mediated Ca2 + entry in pig coronary artery smooth muscle

Na⁺- Ca^2 + exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca^2 + pool along with the SER Ca^2 + pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca^2 + depletion on NCX–SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na⁺-loaded and then placed in either a Na⁺-containing or in a Na⁺-substituted solution. Subsequently, the difference in Ca^2 + entry between the two groups was examined and defined as the NCX mediated Ca^2 + entry. The NCX mediated Ca^2 + entry in the smooth muscle cells was monitored using two methods: Ca^2 +sensitive fluorescence dye Fluo-4 and radioactive Ca^2 +. Ca^2 +-entry was greater in the Na⁺-substituted cells than in the Na⁺-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca^2 + entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na⁺-substituted solution with or without thapsigargin. SER Ca^2 + depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca^2 + entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca^2 + entry may protect the cells against Ca^2 +-overload during ischemia–reperfusion when SERCA2 is known to be damaged.     

Arabidopsis KEA2, a homolog of bacterial KefC,encodes a K+/H+ antiporter with a chloroplast transit peptide

KEA genes encode putative K⁺ efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K⁺ efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast; however, a truncated AtKEA2 protein (AtsKEA2) lacking the N-terminal domain complemented disruption of the Na⁺(K⁺)/H⁺ antiporter Nhx1p to confer hygromycin resistance and tolerance to Na⁺ or K⁺ stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H⁺ exchange with preference for K⁺ = Cs⁺ > Li⁺ > Na⁺. When a conserved Asp⁷²¹ in transmembrane helix 6 that aligns to the cation binding Asp¹⁶⁴ of Escherichia coli NhaA was replaced with Ala, AtsKEA2 was completely inactivated. Mutation of a Glu⁸³⁵ between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K⁺/H⁺ antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids.     

Transient receptor potential ankyrin-1 (TRPA1) modulates store-operated Ca2 + entry by regulation of STIM1-Orai1 association

TRPA1 is a non-selective Ca^2 + permeable channel located in the plasma membrane that functions as a cellular sensor detecting mechanical, chemical and thermal stimuli, being a component of neuronal, epithelial, blood and smooth muscle tissues. TRPA1 has been shown to influence a broad range of physiological processes that involve Ca^2 +-dependent signaling pathways. Here we report that TRPA1 is expressed in MEG01 but not in platelets at the protein level. MEG01 cells maturation induced by PMA results in attenuation of TRPA1 protein expression and enhances thapsigargin-evoked Ca^2 + entry without altering the release of Ca^2 + from intracellular stores. Inhibition of TRPA1 by HC-030031 results in enhancement of both thrombin- and thapsigargin-stimulated Ca^2 + entry. Co-immunoprecipitation experiments revealed that TRPA1 associates with STIM1, as well as Orai1, TRPC1 and TRPC6. Downregulation of TRPA1 expression by MEG01 maturation, as well as pharmacological inhibition of TRPA1 by HC-030031, results in enhancement of the association between STIM1 and Orai1. Altogether, these findings provide evidence for a new and interesting function of TRPA1 in cellular function associated to the regulation of agonist-induced Ca^2 + entry by the modulation of STIM1/Orai1 interaction.     

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP⁺ induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP⁺ injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca^2 + concentration ([Ca^2 +]_cyt) and Ca^2 + in endoplasmic reticulum (ER) ([Ca^2 +]_ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca^2 + release up to 120 min after MPP⁺ injury. Furthermore, decrease of [Ca^2 +]_cyt induced by Homer1 knockdown in MPP⁺ treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP⁺ insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.