Involvement of envelope-bound calcium in the transient depolarization of the Escherichia coli cytoplasmic membrane induced by bacteriophage T4 and T5 adsorption

We previously showed that adsorption of bacteriophages T4 and T5 to their respective outer membrane receptors induced a partial depolarization of the cytoplasmic membrane. As these membrane potential changes were independent of phage properties, we proposed that phage adsorption triggered the emission of a signal which must be transmitted between the two membranes. We show here that these two phages use different mechanisms of transmission of this stimulation signal. In the case of T4, but not of T5, a specific requirement for envelope-bound calcium was found. Indeed, addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the membrane potential changes induced by T4. This envelope-bound calcium became accessible to the chelator only as a consequence of phage adsorption and remained in this state during the depolarization and repolarization. Membrane potential changes again occurred if calcium was added after the addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phage. The same concentration (300 microM) of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the T4-induced depolarization between multiplicities of infection of 6 and 30. This suggests that phage adsorption triggers both a conformational change of membrane components, the number of which reflects the number of stimuli (phages), and the liberation of a definite amount of calcium. This liberated calcium would, in turn, activate these modified membrane components to induce the depolarization. The fact that depolarization may be induced several times after a unique adsorption implies that these membrane components remain irreversibly modified.     

Role of phospholipid hydrolysis in the mechanism of toxic cell death by calcium and ionophore A23187

Manganese chloride inhibits the hydrolysis of arachidonate-containing phospholipids stimulated in 3T3 mouse fibroblasts by ionophore A23187 in the presence of extracellular calcium. The inhibition is reduced by increasing extracellular calcium concentrations. Stimulation by A23187 of this phospholipid hydrolysis and cell killing are inhibited at similiar concentrations by (i) manganese chloride or (ii) reduced extracellular calcium. These results indicate an important role for the phospholipid hydrolysis in the mechanism of cell killing by A23187 plus calcium. Analysis of the rates of the two processes indicates that phospholipid hydrolysis triggers cell killing, but it is not itself the membrane permeabilizing step.     

Intravesicular calcium transient during calcium release from sarcoplasmic reticulum

The time course of changes in the intravesicular Ca2+ concentration ([Ca2+]i) in terminal cisternal sarcoplasmic reticulum vesicles upon the induction of Ca2+ release was investigated by using tetramethylmurexide (TMX) as an intravesicular Ca2+ probe. Upon the addition of polylysine at the concentration that led to the maximum rate of Ca2+ release, [Ca2+]i decreased monotonically in parallel with Ca2+ release. Upon induction of Ca2+ release by lower concentrations of polylysine, [Ca2+]i first increased above the resting level, followed by a decrease well below it. The release triggers polylysine, and caffeine brought about dissociation of calcium that bound to a nonvesicular membrane segment consisting of the junctional face membrane and calsequestrin bound to it, as monitored with TMX. No Ca2+ dissociation from calsequestrin-free junctional face membranes or from the dissociated calsequestrin was produced by release triggers, but upon reassociation of the dissociated calsequestrin and the junctional face membrane, Ca2+ dissociation by triggers was restored. On the basis of these results, we propose that the release triggers elicit a signal in the junctional face membrane, presumably in the foot protein moiety, which is then transmitted to calsequestrin, leading to the dissociation of the bound calcium; and in SR vesicles, to the transient increase of [Ca2+]i, and subsequently release across the membrane.     

On the overshoot of calcium accumulation in fragmented sarcoplasmic reticulum induced by thymol.

The mechanism was studied of the overshoot of calcium accumulation in fragmented sarcoplasmic reticulum (FSR) which is observed when the active transport of calcium into FSR is performed in the presence of thymol; the amount of calcium in FSR increases steeply during the first minute of the reaction and then decreases markedly. In contrast to this behavior, the amount of calcium in FSR increases monotonically and then reaches saturation in the absence of thymol. It is shown that the amount of calcium accumulated in FSR is determined by the balance between the rapid influx and efflux of calcium, and that both processes are depressed by thymol. The overshoot of calcium accumulation can be explained as follows: thymol so markedly depresses the efflux of calcium uncoupled with Ca2+-ATPase activity that the amount of calcium is increased in FSR in spite of partial deactivation of Ca2+-ATPase. However, the state of the FSR membrane is rapidly changed when the concentration of accumulated calcium exceeds a certain critical value. Concomitant with this change, the calcium permeability of the membrane is increased, leading to a decrease in the amount of accumulated calcium. The effects of magnesium and temperature on the overshoot of calcium accumulation can be accounted for by this proposed mechanism.     

Voltage-dependent calcium channels at the plasma membrane, but not vesicular channels, couple exocytosis to endocytosis

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.     

Localization of intracellular calcium during the acrosome reaction in ram spermatozoa

Calcium was identified by a pyroantimonate-osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39°C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time. Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a “sawtooth” arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion. © 1995 Wiley-Liss, Inc.     

Absence of blocking effects on cardiac slow calcium channels by the intracellular calcium antagonist 2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene

Extensive pharmacological evidence supports the contention that 2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene hydrochloride (pr-MDI) is a calcium antagonist with a predominantly intracellular site of action. On the other hand, electro-physiological evidence points to a possible membrane slow inward calcium channel blocking property of this agent. To gain further insight as to the site of action of pr-MDI, the interactions between the negative inotropic action of this agent and the positive inotropic actions of excess extracellular calcium (which directly penetrates the myocardial cells through the slow calcium channels), isoproterenol (which indirectly augments calcium influx through the slow calcium channels), and ouabain (which enhances calcium influx through membrane calcium entry routes distinct from the slow calcium channels) were investigated in the isolated, electrically drive guinea pig left atrium. Although excess extracellular calcium, isoproterenol, and ouabain reversed the negative inotropic effect of pr-MDI, an analysis of the concentration-response relationships to all three positive inotropic agents in the presence and the absence of pr-MDI demonstrated that this agent did not significantly inhibit the contractile effects of calcium, isoproterenol, or ouabain, at pr-MDI concentrations which exhibit intrinsic negative inotropic effects. It is concluded that pr-MDI does not block the membrane slow inward calcium channel nor other presumptive membrane routes of calcium entry into myocardial cells at concentrations of 10(-4) M or less. At very high concentrations (3 X 10(-4) M) some inhibition of slow channel calcium influx may occur.     

Action of cyclosporine on mitochondrial calcium fluxes

Cyclosporine (Cys A) is a potent immunosuppressor used to reduce rejection in transplantation surgery. We studied its action upon mitochondrial functions: oxidative phosphorylation and Ca²⁺ movements through mitochondrial membrane. We show that Cys A exhibits an inhibitory effect upon mitochondrial respiration. This result is in good agreement with previous works and may be correlated with Cys A toxicity. The action of cyclosporine on calcium fluxes is more pronounced. Indeed it blocks mitochondrial calcium efflux and allows mitochondria to accumulate a large amount of calcium. If this effect occurs in the cell, it would induce a Ca²⁺ decrease in cytosol. This action might be correlated with the inhibitory effect of Cys A upon the mitogenic stimulation of T lymphocytes.     

Mechanism of store-operated calcium entry

Activation of receptors coupled to the phospholipase C/IP₃ signalling pathway results in a rapid release of calcium from its intracellular stores, eventually leading to depletion of these stores. Calcium store depletion triggers an influx of extracellular calcium across the plasma membrane, a mechanism known as the store-operated calcium entry or capacitative calcium entry. Capacitative calcium current plays a key role in replenishing calcium stores and activating various physiological processes. Despite considerable efforts, very little is known about the molecular nature of the capacitative channel and the signalling pathway that activates it. This review summarizes our current knowledge about store operated calcium entry and suggests possible hypotheses for its mode of activation.     

Effect of calcium ion on triiodothyronine binding to kidney outer mitochondrial membrane in vitro

The effect of calcium ion on 3,5,3'-triiodothyronine (T3) binding to rat kidney outer mitochondrial membranes was examined in vitro. The outer mitochondrial membranes were prepared by using a discontinuous sucrose density gradient centrifugation. The membrane fraction, which is enriched with monoamine oxidase activity, contained specific binding sites for T3. Scatchard analysis of T3 binding to outer mitochondrial membranes gave an association constant (Ka) of 0.53 X 10(10)M-1. The binding of [125I]-T3 to the membranes was inhibited by the addition of CaCl2(0.25 X 10(-4)--2.5 X 10(-3)M). 50% inhibition was obtained by 0.75 X 10(-4)M CaCl2 in the presence of 0.1 mM EGTA. When outer mitochondrial membranes were solubilized with Triton X-100, four main T3 binding activities were isolated by a gel filtration study. On the other hand, the binding of [125I]-T3 to the solubilized T3 receptors derived from outer mitochondrial membranes was not strongly inhibited by calcium. When outer mitochondrial membranes were preincubated in the presence of 1 mM calcium, the number of T3 binding sites in the membranes was decreased, and this was associated with an increase in the number of T3 binding sites in the supernatants of the incubation mixture. Scatchard analysis showed that the number of T3 binding sites in the membranes is decreased by calcium ion without any change in the association constant. In studies with gel filtration of receptors which are released by Ca2+ from outer mitochondrial membranes, three main T3 binding activities were isolated. Mg2+, Mn2+, Zn2+ and Cu2+ did not affect T3 binding to outer mitochondrial membranes. The results indicate that calcium ion regulates T3 binding to the outer mitochondrial membrane through the release of T3 receptors from the membranes.     

Light-activated calcium release from sonicated bovine retinal rod outer segment disks.

Calcium trapped within sonicated and resealed bovine rod outer segment disks is released upon light exposure with a stoichiometry of 0.75 +/- 0.05 calcium for each rhodopsin bleached. The amount of calcium liberated is proportional to the amount of bleaching in the range of 20 to 100% bleaching and is relatively insensitive to the internal trapped calcium concentration. The results are obtained using a flow system in which the disk membrane vesicles are adsorbed on glass particle supported by a filter. The external calcium is washed away and subsequent calcium release is monitored by collecting fractions of the effluent before, during, and after light exposure. Disks that are sonicated and allowed to reseal prior to incubation with 45Ca show no change in calcium efflux upon bleaching. The light-activated calcium release is also eliminated if disks sonicated in the presence of 45Ca are treated with a calcium ionophore prior to bleaching. The results demonstrate that the light-released calcium comes from the disks and not from the external disk surface. Lowering temperature to 3--4 degrees C surpresses the light-stimulated release, implicating a transition after the formation of metarhodopsin I in the transport process. The resluts suggest a model for the disk in which each bleached rhodopsin functions as a "one-shot carrier" to transport a single calcium ion across the membrane.     

Apoplastic calcium executes a shut-down function on plant peroxidases: A hypothesis

Recently it was demonstrated that PO activity is switched by calcium within the typical range of apoplastic free calcium concentrations (Plieth and Vollbehr, Plant Signal Behav 2012;7: 650–660). The heat stability of POs is also dependent on calcium. Here, a scenario is put forward which assigns calcium a switch-off function under heat: Peroxidases are switched off by heat stress-triggered apoplastic calcium depletion. It is assumed that this initiates apoplastic accumulation of reactive oxygen species (ROS) and eventually triggers a self-amplifying cascade of cellular events involving plasma membrane ion transport. Calcium depletion-initiated ROS accumulation (CaDIRA) may also trigger signal percolation and the formation of systemic responses to many different stress factors in plants. This hypothesis can explain some as yet unexplained observations.     

Carbonyl cyanide m-chlorophenylhydrazone induced calcium signaling and activation of plasma membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae.

The plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is an enzyme that plays a very important role in the yeast physiology. The addition of protonophores, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), also triggers a clear in vivo activation of this enzyme. Here, we demonstrate that CCCP-induced activation of the plasma membrane H(+)-ATPase shares some similarities with the sugar-induced activation of the enzyme. Phospholipase C and protein kinase C activities are essential for this activation process while Gpa2p, a G protein involved in the glucose-induced activation of the ATPase, is not required. CCCP also induces a phospholipase C-dependent increase in intracellular calcium. Moreover, we show that the availability of extracellular calcium is required for CCCP stimulation of H(+)-ATPase, suggesting a possible connection between calcium signaling and activation of ATPase.     

Differential inhibitory effects of carbon tetrachloride on the hepatic plasma membrane, mitochondrial and endoplasmic reticular calcium transport systems: implications to hepatotoxicity

Mitochondrial, endoplasmic reticular and plasma membrane fractions were isolated by a new method from control male Fischer 344 rats and rats given CCl4 by gavage. After 1 h of CCl4 treatment, rats were in glucose and pancreatic hormone balance but plasma levels of T3 and T4 were decreased 29 and 22%, respectively. After 24 hours of CCl4 treatment, rats were: hypoglycaemic and insulin and glucagon levels were increased 33- and 35-fold, respectively; total T4 levels were decreased 62%; while total T3 levels were normalized. In liver fractions from CCl4-treated rats, 1 h after CCl4 administration: (i) calcium binding was decreased 65% in the mitochondrial fraction, 66% in the endoplasmic reticular fraction and 46% in the plasma membrane fraction; (ii) calcium uptake was decreased 59% in the mitochondrial fraction, 46% in the endoplasmic reticular fraction and 37% in the plasma membrane fraction. After 24 h of CCl4 administration: (i) calcium binding was decreased 57% in the mitochondrial fraction, 50% in the endoplasmic reticular fraction and 71% in the plasma membrane fraction; (ii). calcium uptake was decreased 55% in the mitochondrial fraction, 17% in the endoplasmic reticular fraction and 53% in the plasma membrane fraction. In vitro studies indicated the plasma membrane calcium transport system to be rapidly (within a minute) and strongly (>90%) inhibited by CCl4. We conclude that CCl4 produces a differential inhibitory effect on the hepatocyte calcium pumps that are implicated with hepatocellular damage.     

Low pH inhibits compensatory endocytosis at a step between depolarization and calcium influx

Cell function can be modulated by the insertion and removal of ion channels from the cell surface. The mechanism used to keep channels quiescent prior to delivery to the cell surface is not known. In eggs, cortical vesicle exocytosis inserts voltage-gated calcium channels into the cell surface. Calcium influx through these channels triggers compensatory endocytosis. Secretory vesicles contain high concentrations of calcium and hydrogen ions. We propose that lumenal hydrogen ions inhibit vesicular calcium channel gating prior to exocytosis, discharge of lumenal protons upon vesicle-plasma membrane fusion enables calcium channel gating. Consistent with this hypothesis we find that cortical vesicle lumens are acidic, and exocytosis releases lumenal hydrogen ions. Acidic extracellular pH reversibly blocks endocytosis, and the windows of opportunity for inhibition with a calcium-channel blocker or hydrogen ions are indistinguishable. Calcium ionophore treatment circumvents the low pH block, suggesting that calcium influx, or an upstream step, is obstructed. Inhibition of calcium influx by preventing membrane depolarization is unlikely, as elevation of the extracellular potassium concentration failed to overcome the pH block, and low extracellular pH was found to depolarize the membrane potential. We conclude that low pH inhibits endocytosis at a step between membrane depolarization and calcium influx .     

The intraacrosomal calcium pool plays a direct role in acrosomal exocytosis

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. UV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP(3)-sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP(3)-sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis.     

Ultrastructural organisation and the sites of calcium localisation in the lamprey striated muscle

The present paper examines the ultrastructure of the sarcoplasmic recitulum (SR) and the T system in the striated muscle of the lamprey. The pyroantimonate method was used to visualise the sites of intracellular calcium localisation. Characteristic for the muscle studied are the presence of numerous intricately shaped invaginations on the surface membrane of muscle fibres and peripheral contacts between SR cisternae and the sarcolemma. In addition to calcium localised in the terminal cisternae of SR and N-bands of the I-disk, as typical of vertebrate muscles, a great amount of calcium is present in the subsarcolemmal region, corresponding to the area of invaginations, and in longitudinal elements of SR.     

Trafficking and regulation of neuronal voltage-gated calcium channels

The importance of voltage-gated calcium channels is underscored by the multitude of intracellular processes that depend on calcium, notably gene regulation and neurotransmission. Given their pivotal roles in calcium (and hence, cellular) homeostasis, voltage-gated calcium channels have been the subject of intense research, much of which has focused on channel regulation. While ongoing research continues to delineate the myriad of interactions that govern calcium channel regulation, an increasing amount of work has focused on the trafficking of voltage-gated calcium channels. This includes the mechanisms by which calcium channels are targeted to the plasma membrane, and, more specifically, to their appropriate loci within a given cell. In addition, we are beginning to gain some insights into the mechanisms by which calcium channels can be removed from the plasma membrane for recycling and/or degradation. Here we highlight recent advances in our understanding of these fundamentally important mechanisms.     

Stabilizing role of calcium store-dependent plasma membrane calcium channels in action-potential firing and intracellular calcium oscillations

In many biological systems, cells display spontaneous calcium oscillations (CaOs) and repetitive action-potential firing. These phenomena have been described separately by models for intracellular inositol trisphosphate (IP3)-mediated CaOs and for plasma membrane excitability. In this study, we present an integrated model that combines an excitable membrane with an IP3-mediated intracellular calcium oscillator. The IP3 receptor is described as an endoplasmic reticulum (ER) calcium channel with open and close probabilities that depend on the cytoplasmic concentration of IP3 and Ca2+. We show that simply combining this ER model for intracellular CaOs with a model for membrane excitability of normal rat kidney (NRK) fibroblasts leads to instability of intracellular calcium dynamics. To ensure stable long-term periodic firing of action potentials and CaOs, it is essential to incorporate calcium transporters controlled by feedback of the ER store filling, for example, store-operated calcium channels in the plasma membrane. For low IP3 concentrations, our integrated NRK cell model is at rest at -70 mV. For higher IP3 concentrations, the CaOs become activated and trigger repetitive firing of action potentials. At high IP3 concentrations, the basal intracellular calcium concentration becomes elevated and the cell is depolarized near -20 mV. These predictions are in agreement with the different proliferative states of cultures of NRK fibroblasts. We postulate that the stabilizing role of calcium channels and/or other calcium transporters controlled by feedback from the ER store is essential for any cell in which calcium signaling by intracellular CaOs involves both ER and plasma membrane calcium fluxes.