Passive Ca2 Research Articles

Abstract 12087: KCa3.1 Channels Support Resting Potential and Proliferative Function During Store-Operated Ca 2+ -Entry in c-kit + Endogenous Cardiac Stem Cells

Introduction: Adult hearts contain c-kit + endogenous cardiac stem cells (eCSCs). Cultured eCSCs express a wide range of ion channels, but cell culture is known to affect phenotype. Here we defined ion currents in freshly isolated c-kit + eCSCs and their potential functional role. Methods: eCSCs isolated from healthy and heart failure (HF, ventricular tachypacing X 2 wks) dog hearts were magnetically purified with c-kit antibodies. Ion currents and membrane potential were recorded with patch clamp. Results: Contrary to cultured c-kit + eCSCs, ion currents were barely detectable with Ca 2+ i buffered (Fig A). Free Ca 2+ i activated prominent I KCa3.1 (TRAM34 sensitive, Fig B) in 82% of cells. I KCa3.1 was reduced in eCSCs isolated from HF dogs (Fig C), along with corresponding KCNN4 mRNA (Fig D). Under perforated patch to maintain spontaneous physiological [Ca 2+ ] i , eCSCs had a membrane potential (V mem ) of -73±10 mV. Ca 2+ i depletion with nominally 0 mM [Ca 2+ ] o strongly depolarized the membrane. After passive Ca 2+ i depletion, store-operated channel (SOC) mediated Ca 2+ entry (SOCE) caused a robust V mem hyperpolarization (from -21±4 to -79±7 mV, p<0.001). The SOCE effect on V mem was substantially reduced by both 2-APB (SOC inhibitor) and TRAM34 (Fig E). Inhibiting KCa3.1 (TRAM34, 0.1 μM) or SOCs (2-APB, 50 μM) in culture decreased c-kit + eCSCs proliferation by 21% (p<0.05) and 55% (p<0.001) respectively. Conclusions: In contrast to cultured eCSCs, voltage-dependent ion currents are virtually absent from freshly isolated c-kit + eCSCs. I KCa3.1 is prominent and maintains V mem in the face of substantial Ca 2+ entry from SOCs. This Ca 2+ entry would normally depolarize the cell, reduce the gradient for Ca 2+ entry and turn off the Ca 2+ signal. We hypothesize that the Ca 2+ -mediated KCa3.1 K + -conductance increase is necessary to maintain V mem in the face of the Ca 2+ entry that activates eCSC proliferation, and that KCa3.1 channels may therefore play an important role in eCSC function.

Supplementation with difructose anhydride III promotes passive calcium absorption in the small intestine immediately after calving in dairy cows

The incidence of hypocalcemia increases in high-parity dairy cows because resorption of bone Ca is delayed in these animals, and they appear to have a reduced ability to absorb Ca from the intestine during the early postpartum period. Difructose anhydride (DFA) III has been shown to promote the absorption of intestinal Ca via a paracellular pathway. However, past studies have not reported this effect in peripartum dairy cows. Therefore, we investigated the effect of DFA III supplementation on Ca metabolism during the peripartum period to determine whether DFA III promotes intestinal Ca absorption via this route. Seventy-four multiparous Holstein cows were separated into DFA and control groups based on their parity and body weight. The feed of the DFA group was supplemented with 40g/d of DFA III from −14 to 6d relative to calving. The control group did not receive DFA III. At calving (0h relative to calving), serum Ca declined below 9mg/dL in both groups. However, serum Ca concentrations were greater in the DFA group than in the control group at 6, 12, 24, and 48h relative to calving, and the time required for serum Ca to recover to 9mg/dL during the postpartum period was shorter in the high-parity cows in the DFA group than in those in the control group. Parathyroid hormone concentrations increased immediately after calving in both groups and were greater in the control group than in the DFA group at 12 and 24h relative to calving. Serum 1,25-dihydroxyvitamin D concentrations increased at 0 and 12h relative to calving in both groups and were higher in the control group than in the DFA group at 72h relative to calving. Serum concentrations of the bone-resorption marker cross-linked N-telopeptide of type I collagen (NTX) were not different between the groups during peripartum period, and serum NTX in all cows was lower at 0, 6, 12, 24, 48, and 72h relative to calving than at −21, 4, and 5d relative to calving. Thus, DFA treatment induced faster recovery of serum Ca, although bone resorption was restrained. In conclusion, DFA III promotes intestinal passive Ca absorption via the paracellular pathway during the early postpartum period; this absorption is unaffected by aging.

Asbestos-induced Disruption of Calcium Homeostasis Induces Endoplasmic Reticulum Stress in Macrophages

Although the mechanisms for fibrosis development remain largely unknown, recent evidence indicates that endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) may act as an important fibrotic stimulus in diseased lungs. ER stress is observed in lungs of patients with idiopathic pulmonary fibrosis. In this study we evaluated if ER stress and the UPR was present in macrophages exposed to chrysotile asbestos and if ER stress in macrophages was associated with asbestos-induced pulmonary fibrosis. Macrophages exposed to chrysotile had elevated transcript levels of several ER stress genes. Macrophages loaded with the Ca(2+)-sensitive dye Fura2-AM showed that cytosolic Ca(2+) increased significantly within minutes after chrysotile exposure and remained elevated for a prolonged time. Chrysotile-induced increases in cytosolic Ca(2+) were partially inhibited by either anisomycin, an inhibitor of passive Ca(2+) leak from the ER, or 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator known to deplete ER Ca(2+) stores. Anisomycin inhibited X-box-binding protein 1 (XBP1) mRNA splicing and reduced immunoglobulin-binding protein (BiP) levels, whereas BAPTA-AM increased XBP1 splicing and BiP expression, suggesting that ER calcium depletion may be one factor contributing to ER stress in cells exposed to chrysotile. To evaluate ER stress in vivo, asbestos-exposed mice showed fibrosis development, and alveolar macrophages from fibrotic mice showed increased expression of BiP. Bronchoalveolar macrophages from asbestosis patients showed increased expression of several ER stress genes compared with normal subjects. These findings suggest that alveolar macrophages undergo ER stress, which is associated with fibrosis development.

STIM1 regulates TRPC6 heteromultimerization and subcellular location.

STIM1 (stromal interaction molecule 1) regulates store-operated channels in the plasma membrane, but the regulation of TRPC (transient receptor potential canonical) heteromultimerization and location by STIM1 is poorly understood. STIM1 is a single transmembrane protein that communicates the filling state of the endoplasmic reticulum to store-operated channels. STIM1 has been reported to regulate the activity of all of the TRPC family members, except TRPC7. TRPC6 has been predominantly associated to second messenger-activated Ca2+ entry pathways. In the present paper we report that STIM1 regulates the expression of TRPC6in the plasma membrane and evokes translocation of this channel to the endoplasmic reticulum. Attenuation of TRPC6 expression in the plasma membrane resulted in a reduction in the association of this channel with TRPC1 and TRPC3. We have found that expression of TRPC6in the endoplasmic reticulum results in an increase in the passive Ca2+ efflux and basal cytosolic Ca2+ concentration, but not in the ability of cells to accumulate Ca2+ into the endoplasmic reticulum. We propose a novel mechanism for the regulation of TRPC6 channel location and function by STIM1, probably as a mechanism to modulate second messenger-operated Ca2+ entry while potentiating store-operated Ca2+ influx.

Reversal of Murine Epidermal Atrophy by Topical Modulation of Calcium Signaling

Cytosolic Ca(2+) signals are performed by Ca(2+) releases from the endoplasmic reticulum and Ca(2+) influx from the extracellular medium. Releases rely on the refilling of the intracellular Ca(2+) stores by the Ca(2+) influx "Store-Operated Calcium Entry" (SOCE) via the channel Orai1. Here we show that Orai1 expression, SOCE amplitude, and epidermal proliferation are decreased in the epidermis of patients with skin fragility when compared with aged nonatrophic skin. Epidermal atrophy was induced in mice by the inhibition of Orai1 with small interfering RNA and the topical application of a SOCE blocker BTP2. The inhibition of Orai1 impaired the heparin-binding epidermal growth factor (HB-EGF)-induced Ca(2+) influxes and fully prevented the mitogen effect of HB-EGF in primary human keratinocytes. Importantly, epidermal proliferation correlated with Orai1 expression in mice. Conversely, the topical application of an Orai1 activator, the benzohydroquinone (BHQ), increased the epidermal thickness and proliferation, whereas the pro-proliferative effect of BHQ was prevented by the inhibition of Orai1. Finally, the topical application of BHQ reversed the epidermal atrophy induced by corticosteroids in mice. The topical modulation of Ca(2+) signals may thus be a promising therapeutic strategy in dermatology.

Membrane topology of human presenilin-1 in SK-N-SH cells determined by fluorescence correlation spectroscopy and fluorescent energy transfer.

Presenilin-1 (PS1) protein acts as passive ER Ca(2+) leak channels that facilitate passive Ca(2+) leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer's disease (FAD). FADPS1 mutations abrogate the function of ER Ca(2+) leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487-500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca(2+)) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH2-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP-PS1, and PS1-CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca(2+) leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1-CFP was either expressed alone or together with YFP-PS1 into SK-N-SH cell line and the interaction between YFP-PS1 and PS1-CFP was determined by Förster resonance energy transfer analysis. Our results suggest interaction between YFP-PS1 and PS1-CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1-CFP and YFP-PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH2-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH2-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.

Myoplasmic resting Ca2+ regulation by ryanodine receptors is under the control of a novel Ca2+-binding region of the receptor

Passive SR (sarcoplasmic reticulum) Ca2+ leak through the RyR (ryanodine receptor) plays a critical role in the mechanisms that regulate [Ca2+]rest (intracellular resting myoplasmic free Ca2+ concentration) in muscle. This process appears to be isoform-specific as expression of either RyR1 or RyR3 confers on myotubes different [Ca2+]rest. Using chimaeric RyR3–RyR1 receptors expressed in dyspedic myotubes, we show that isoform-dependent regulation of [Ca2+]rest is primarily defined by a small region of the receptor encompassing amino acids 3770–4007 of RyR1 (amino acids 3620–3859 of RyR3) named as the CLR (Ca2+ leak regulatory) region. [Ca2+]rest regulation by the CLR region was associated with alteration of RyRs’ Ca2+-activation profile and changes in SR Ca2+-leak rates. Biochemical analysis using Tb3+-binding assays and intrinsic tryptophan fluorescence spectroscopy of purified CLR domains revealed that this determinant of RyRs holds a novel Ca2+-binding domain with conformational properties that are distinctive to each isoform. Our data suggest that the CLR region provides channels with unique functional properties that modulate the rate of passive SR Ca2+ leak and confer on RyR1 and RyR3 distinctive [Ca2+]rest regulatory properties. The identification of a new Ca2+-binding domain of RyRs with a key modulatory role in [Ca2+]rest regulation provides new insights into Ca2+-mediated regulation of RyRs.

TRPC6 participates in the regulation of cytosolic basal calcium concentration in murine resting platelets

Cytosolic-free Ca2+ plays a crucial role in blood platelet function and is essential for thrombosis and hemostasis. Therefore, cytosolic-free Ca2+ concentration is tightly regulated in this cell. TRPC6 is expressed in platelets, and an important role for this Ca2+ channel in Ca2+ homeostasis has been reported in other cell types. The aim of this work is to study the function of TRPC6 in platelet Ca2+ homeostasis. The absence of TRPC6 resulted in an 18.73% decreased basal [Ca2+]c in resting platelets as compared to control cells. Further analysis confirmed a similar Ca2+ accumulation in wild-type and TRPC6-deficient mice; however, passive Ca2+ leak rates from agonist-sensitive intracellular stores were significantly decreased in TRPC6-deficient platelets. Biotinylation studies indicated the presence of an intracellular TRPC6 population, and subcellular fractionation indicated their presence on endoplasmic reticulum membranes. Moreover, the presence of intracellular calcium release in platelets stimulated with 1-oleoyl-2-acetyl-sn-glycerol further suggested a functional TRPC6 population located on the intracellular membranes surrounding calcium stores. However, coimmunoprecipitation assay confirmed the absence of STIM1–TRPC6 interactions in resting conditions. This findings together with the absence of extracellular Mn2+ entry in resting wild-type platelets indicate that the plasma membrane TRPC6 fraction does not play a significant role in the maintenance of basal [Ca2+]c in mouse platelets. Our results suggest an active participation of the intracellular TRPC6 fraction as a regulator of basal [Ca2+]c, controlling the passive Ca2+ leak rate from agonist-sensitive intracellular Ca2+ stores in resting platelets.

Effects of endogenous cannabinoid anandamide on excitation–contraction coupling in rat ventricular myocytes

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca2+ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically evoked Ca2+ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca2+-uptake and Ca2+-ATPase activity in a biphasic manner. [3H]-ryanodine binding and passive Ca2+ release from SR vesicles were not altered by 10μM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1μM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2μg/ml for 4h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3μM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3μM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.

Calcium-induced calcium release mediates all-or-nothing responses of mesenchymal stromal cells to noradrenaline

By using Ca2+ imaging and Fluo-4 dye, we examined the capability of certain agonists of G-protein coupled receptors to stimulate Ca2+ signaling in cultured mesenchymal stromal cells (MSC) derived from the human adipose tissue. In particular, a small subpopulation (∼5%) MSC was found to respond to noradrenaline with Ca2+ transients. The all-or-nothing fashion was characteristic of adrenergic Ca2+ signaling in MSC, that is, while at low concentrations noradrenaline stimulated undetectable Ca2+ transients, virtually maximal responses were elicited by this agonist at any concentration above the threshold of 100–200 nM. In some experiments, MSC were loaded with the photosensitive Ca2+ chelator NP-EGTA to produce local or global jumps in cytosolic Ca2+ concentration by virtue of Ca2+ uncaging. Global uncaging eliciting a high enough Ca2+ jump triggered a Ca2+ transient in the MSC cytoplasm, which was similar to a noradrenaline response kinetically and by magnitude. When Ca2+ uncaging was produced locally, it initiated a Ca2+ signal that traveled along a cell with a speed that exceeded an expected one by two orders of magnitude, should Ca2+ signal transfer be mediated merely by passive Ca2+ diffusion in the presence of Ca2+ buffer. These findings implicated Ca2+-induced Ca2+ release (CICR) as a mechanism amplifying local Ca2+ signals in MSC. Of Ca2+ targets involved in CICR, the ryanodine receptor and IP3 receptor are only known. The inhibitory analysis revealed IP3 receptors to be principally responsible for CICR in MSC, whereas a contribution of ryanodine receptors was negligible. Altogether, our results suggest that an initial noradrenaline-dependent rise in cytosolic Ca2+ stimulates, should it reach the threshold level, IP3 receptors, thereby triggering an avalanche-like Ca2+ release from Ca2+ stores and underlying the all-or-nothing dependence of cellular responses on the agonist concentration.

The influence of gap junction network complexity on pulmonary artery smooth muscle reactivity in normoxic and chronically hypoxic conditions

Experiments on intrapulmonary arteries (IPAs) isolated from rats maintained in normoxia and chronic hypobaric hypoxia showed that in normoxia, the IPA contractile sensitivity to KCl was not modified by gap junction inhibition. In contrast, chronic hypoxia induced an endothelium-independent hypersensitivity, which was suppressed by gap junction inhibition. For the theoretical analysis of these results, we developed a model of interconnected myocytes. Given that smooth muscle cells in IPAs are known to communicate via gap junctions, we regard the cytoarchitecture of the IPA as a spatial network, in which nodes represent individual smooth muscle cells and the links signify intercellular communication. A single-cell model that drives the dynamics of individual nodes includes the major elements of voltage-dependent Ca(2+) signalling. In addition, interindividual variability of SMCs is introduced by distributing the reversal potentials for K(+). Cell-to-cell connection consists of passive Ca(2+) diffusion and electrical coupling, and connection between cells is determined by the topology of the intercellular network. Model predictions indicate that the experimental results can be explained by topological modifications and not by changes in the number of gap junctions. According to the model, in normoxia the myocytes are connected in a complex network, whereas chronic hypoxia is related to loss of complexity, leading to hypersensitivity. Our results thus indicate that chronic hypoxia entails gap junction network rearrangements, leading to disturbances in the intercellular communication pathways.

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca2+-calmodulin. In single channel measurements, the open Sec61 complex is Ca2+ permeable. It can be closed not only by interaction with BiP or Ca2+-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca2+ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.

Undernutrition Affects Cell Survival, Oxidative Stress, Ca2+ Handling and Signaling Pathways in Vas Deferens, Crippling Reproductive Capacity

BackgroundThe aim of this work was to investigate the mechanisms by which chronic malnutrition (CM) affects vas deferens function, leading to compromised reproductive capacity. Previous studies have shown that maternal malnutrition affects the reproductive tracts of adult male offspring. However, little is known about the effects of CM, a widespread life-long condition that persists from conception throughout growth to adult life.Methodology/Principal FindingsYoung adult male rats, which were chronically malnourished from weaning, presented decreased total and haploid cells in the vas deferens, hypertrophy of the muscle layer in the epididymal portion of the vas deferens and intense atrophy of the muscular coat in its prostatic portion. At a molecular level, the vas deferens tissue of CM rats exhibited a huge rise in lipid peroxidation and protein carbonylation, evidence of an accentuated increase in local reactive oxygen species levels. The kinetics of plasma membrane Ca2+-ATPase activity and its kinase-mediated phosphorylation by PKA and PKC in the vas deferens revealed malnutrition-induced modifications in velocity, Ca2+ affinity and regulation of Ca2+ handling proteins. The severely crippled content of the 12-kDa FK506 binding protein, which controls passive Ca2+ release from the sarco(endo) plasmic reticulum, revealed another target of malnutrition related to intracellular Ca2+ handling, with a potential effect on forward propulsion of sperm cells. As a possible compensatory response, malnutrition led to enhanced sarco(endo) plasmic reticulum Ca2+-ATPase activity, possibly caused by stimulatory PKA-mediated phosphorylation.Conclusions/SignificanceThe functional correlates of these cellular and molecular hallmarks of chronic malnutrition on the vas deferens were an accentuated reduction in fertility and fecundity.

Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia

Malignant hyperthermia (MH) susceptibility has been attributed to a leaky sarcoplasmic reticulum (SR) caused by missense mutations in RYR1 or CACNA1S, and the MH crisis has been attributed solely to massive self-sustaining release of Ca(2+) from SR stores elicited by triggering agents. Here, we show in muscle cells from MH-RyR1(R163C) knock-in mice that increased passive SR Ca(2+) leak causes an enlarged basal influx of sarcolemmal Ca(2+) that results in chronically elevated myoplasmic free Ca(2+) concentration ([Ca(2+)]i) at rest. We discovered that Gd(+3) and GsMTx-4 were more effective than BTP2 or expression of the dominant-negative Orai1(E190Q) in reducing both Ca(2+) entry and [Ca(2+)]i, implicating a non-STIM1/Orai1 SOCE pathway in resetting resting [Ca(2+)]i. Indeed, two nonselective cationic channels, TRPC3 and TRPC6, are overexpressed, and [Na]i is chronically elevated in MH-RyR1(R163C) muscle cells. [Ca(2+)]i and [Na(+)]i are persistently elevated in vivo and further increased by halothane in MH-RyR1(R163C/WT) muscle. These increases are markedly attenuated by local perfusion of Gd(+3) or GsMTx-4 and completely suppressed by dantrolene. These results contribute a new paradigm for understanding MH pathophysiology by demonstrating that nonselective sarcolemmal cation channel activity plays a critical role in causing myoplasmic Ca(2+) and Na(+) overload both at rest and during the MH crisis.-Eltit, J. M., Ding, X., Pessah, I. N., Allen, P. D., Lopez, J. R. Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia.

The Research of Automobile Automatic Stabilizer Bar Control System and Test Platform

In connection with the problems that the automobile passive stabilizer bar can’t reflect the road conditions in real time and it is difficult to simultaneously meet the requirements of ride comfort and manipulation stability, this paper describes a kind of control system and test platform of the automobile automatic stabilizer bar. The control system uses speed sensor, steering angle sensor, roll angle sensor, lateral acceleration sensor and displacement sensor to simulate a variety of vehicle’s real driving conditions in the laboratory environment. According to the formulated control strategy, the microcontroller will send different instructions to make the executive mechanism act reasonably and in time. Then the test platform is established in the laboratory environment by the method of semi-physical simulation. The system can be designed according to the different needs of the vehicle and ensures the vehicle’s safety and comfort to the maximum.

Acute effect of ethanol on hepatic reticular G6Pase and Ca2+ pool.

Hydrolysis of glucose 6-phosphate (G6P) via glucose 6-phosphatase (G6Pase) enlarges the reticular Ca(2+) pool of the hepatocyte. Exposure of liver cells to ethanol (EtOH) impairs reticular Ca(2+) homeostasis. The present study investigated the effect of acute EtOH administration on G6P-supported Ca(2+) accumulation in liver cells. Total microsomes were isolated from rat livers acutely perfused with varying doses of EtOH (0.01, 0.1, or 1% v/v) for 8 minutes. Calcium uptake was assessed by (45) Ca redistribution. Inorganic phosphate (Pi) formation was measured as an indicator of G6Pase hydrolytic activity. G6P-supported Ca(2+) uptake decreased in a manner directly proportional to the dose of EtOH infused in the liver, whereas Ca(2+) uptake via SERCA pumps was decreased by ~25% only at the highest dose of alcohol administered. The reduced accumulation of Ca(2+) within the microsomes resulted in a smaller inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. Kinetic assessment of IP(3) and passive Ca(2+) release indicated a faster mobilization in microsomes from EtOH-treated livers, suggesting alcohol-induced alteration of Ca(2+) releasing mechanisms. Pretreatment of livers with chloromethiazole (CMZ) or dithiothreitol (DTT), but not 4-methyl-pyrazole prevented the inhibitory effect of EtOH on G6Pase activity and Ca(2+) homeostasis. Liver G6Pase activity and IP(3) -mediated Ca(2+) release are rapidly inhibited following acute (8 minutes) exposure to EtOH, thus compromising the ability of the endoplasmic reticulum to dynamically modulate Ca(2+) homeostasis in the hepatocyte. The protective effect of CMZ and DTT suggests that the inhibitory effect of EtOH is mediated through its metabolism via reticular cyP4502E1 and consequent free radicals formation.

Functional TRPV6 channels are crucial for transepithelial Ca2+absorption

TRPV6 is considered the primary protein responsible for transcellular Ca2+ absorption. In vitro studies demonstrate that a negatively charged amino acid (D) within the putative pore region of mouse TRPV6 (position 541) is critical for Ca2+ permeation of the channel. To elucidate the role of TRPV6 in transepithelial Ca2+ transport in vivo, we functionally analyzed a TRPV6D541A/D541A knockin mouse model. After weaning, mice were fed a regular (1% wt/wt) or Ca2+-deficient (0.02% wt/wt) diet and housed in metabolic cages. Blood was sampled for Ca2+ measurements, and the expression of Ca2+ transport proteins was analyzed in kidney and duodenum. Intestinal 45Ca2+ uptake was measured in vivo by an absorption assay. Challenging the mice with the Ca2+-deficient diet resulted in hypocalcemia in wild-type and TRPV6D541A/D541A mice. On a low-Ca2+ diet both mouse strains displayed increased expression of intestinal TRPV6, calbindin-D(9K), and renal TRPV5. TRPV6D541A/D541A mice showed significantly impaired intestinal Ca2+ uptake compared with wild-type mice, and duodenal TRPV5 expression was increased in TRPV6D541A/D541A mice. On a normal diet, serum Ca2+ concentrations normalized in both mouse strains. Under these conditions, intestinal Ca2+ uptake was similar, and the expression levels of renal and intestinal Ca2+ transport proteins were not affected. We demonstrate that TRPV6D541A/D541A mice exhibit impaired transcellular Ca2+ absorption. Duodenal TRPV5 expression was increased in TRPV6D541A/D541A mice, albeit insufficient to correct for the diminished Ca2+ absorption. Under normal conditions, when passive Ca2+ transport is predominant, no differences between wild-type and TRPV6D541A/D541A mice were observed. Our results demonstrate a specific role for TRPV6 in transepithelial Ca2+ absorption.

Phospholamban phosphorylation increases the passive calcium leak from cardiac sarcoplasmic reticulum

Phospholamban (PLN) is a 52 amino acid integral membrane protein of the sarcoplasmic reticulum (SR) that exists in both monomeric and pentameric forms. In its unphosphorylated state, PLN inhibits the SR Ca(2+) ATPase (SERCA). This inhibition is relieved when PLN is phosphorylated as a result of β-adrenergic stimulation of the heart. Consistent with some predictions from molecular models and from functional studies of PLN incorporated into planar lipid bilayers, it has also been postulated that pentameric PLN can also form ion-selective channels. Other molecular models contradict this hypothesis, however. In the work reported here, we used the Ca(2+)-sensitive fluorescent dye Fura-2, to examine the passive Ca(2+) permeability of the SR membrane in vesicles derived from cardiac ventricle. We have found that phosphorylation of PLN by protein kinase A (PKA) leads to an increase in the rate of Ca(2+) leak from Ca(2+)-loaded SR vesicles. This enhanced rate of Ca(2+) leak from the SR is also observed when SR vesicles are incubated with a PLN specific antibody (A1) that mimics phosphorylation of PLN. The ryanodine receptor blocker ruthenium red does not affect the increased rate of Ca(2+) leak from the SR after PLN phosphorylation with PKA or after exposure to A1 antibody, arguing against a possible role of ryanodine receptors in mediating the enhanced leak. Our results are consistent with the hypothesis that phosphorylated PLN forms or regulates a Ca(2+) leak pathway in cardiac SR membranes in situ.

Repression of transcription of presenilin‐1 inhibits γ‐secretase independent ER Ca2+ leak that is impaired by FAD mutations

Genetic deletion or mutations of presenilin genes (PS1/PS2) cause familial Alzheimer's disease and calcium (Ca²⁺) signaling abnormalities. PS1/PS2 act as endoplasmic reticulum (ER) Ca²⁺ leak channels that facilitate passive Ca²⁺ leak across ER membrane. Studies with PS1/PS2 double knockout (PS1/PS2-DKO) mouse embryonic fibroblasts showed that PS1/PS2 were responsible for 80% of passive Ca²⁺ leak from the lumen of endoplasmic reticulum to cytosol. Transient transfection of the wild type PS1 expression construct increased cytoplasmic Ca²⁺ as a result of Ca²⁺ leak across ER membrane whereas the FADPS1 (PS1-M146V) mutation construct alone or in combination with the wild type PS1 expression construct abrogated Ca²⁺ leak in SK-N-SH cells. Inhibition of basal c-jun-NH2-terminal kinase (JNK) activity by JNK inhibitor SP600125 repressed PS1 transcription and PS1 protein expression by augmenting p53 protein level in SK-N-SH cells (Lee and Das 2008). In this report we also showed that repression of PS1 transcription by JNK inhibitor SP600125 inhibited passive Ca²⁺ leak across ER membrane which could be rescued by expressing PS1 wild type and not by expressing FADPS1 (PS1-M146V) under a SP600125 non-responsive promoter. Treatment of SK-N-SH cells with SP600125 also triggered InsP3R-mediated Ca²⁺ release from the ER by addition of 500 nM bradykinin, an agonist of InsP3 receptor (InsP3R1) without changing the expression of InsP3R1. This data confirms that SP600125 increases the Ca²⁺ store in the ER by inhibiting PS1-mediated Ca²⁺ leak across ER membrane. p53, ZNF237 and Chromodomain helicase DNA-binding protein 3 which are repressors of PS1 transcription, also reduced Ca²⁺ leak across ER membrane in SK-N-SH cells but γ-secretase inhibitor or dominant negative γ-secretase-specific PS1 mutant (PS1-D257A) had no significant effect. Therefore, p53, ZNF237, and Chromodomain helicase DNA-binding protein 3 inhibit the function ER Ca²⁺ leak channels to regulate both ER and cytoplasmic Ca²⁺ levels and may potentially control Ca²⁺-signaling function of PS1.

The Role of a Voltage-Dependent Ca2+Channel Intracellular Linker: A Structure-Function Analysis

Voltage-dependent calcium channels (VDCCs) allow the passage of Ca(2+) ions through cellular membranes in response to membrane depolarization. The channel pore-forming subunit, α1, and a regulatory subunit (Ca(V)β) form a high affinity complex where Ca(V)β binds to a α1 interacting domain in the intracellular linker between α1 membrane domains I and II (I-II linker). We determined crystal structures of Ca(V)β2 functional core in complex with the Ca(V)1.2 and Ca(V)2.2 I-II linkers to a resolution of 1.95 and 2.0 Å, respectively. Structural differences between the highly conserved linkers, important for coupling Ca(V)β to the channel pore, guided mechanistic functional studies. Electrophysiological measurements point to the importance of differing linker structure in both Ca(V)1 and 2 subtypes with mutations affecting both voltage- and calcium-dependent inactivation and voltage dependence of activation. These linker effects persist in the absence of Ca(V)β, pointing to the intrinsic role of the linker in VDCC function and suggesting that I-II linker structure can serve as a brake during inactivation.