Purification of the mitochondrial calcium uniporter from the beef heart and characterization of its properties

A low-molecular-weight component (LMC) inducing selective transport of calcium across the bilayer lipid membrane has been isolated from mitochondria of the bovine heart by the method developed in our laboratory, which excludes the use of detergents and proteolytic enzymes. It was shown that, in the presence of 10 mM CaCl2, LMC forms conduction channels in the membrane multiples of 5 pS. The specific inhibitor of mitochondrial calcium uniporter, ruthenium red, closes Ca2(+)-induced channels formed in the membrane by LMC. In the absence of calcium or in the presence of potassium ions only, the component is incapable of forming channels of conduction. It was shown using nuclear magnetic resonance that LMC is a complex consisting of lipids, amino acids, and sugars with a molecular weight of 1-2 kDa.     

Isolation of calcium-transporting lipid from the mitochondrial glycolipoprotein

Summary From the mitochondrial Ca²⁺-transporting glycolipoprotein (GLP) the lipid was isolated which induced Ca²⁺-translocation through bilayer lipid membranes. Electroconductivity of modified phospholipid membranes in the presence of CaCl₂ is increased 150-200 times. At 10-fold CaCl₂ gradient a generation of membrane potential is observed close to its theoretical value. It is shown that the lipid forms separate conductivity channels of 10 and 20 pS in the bilayer. The mode of action of GLP in the membrane is proposed It is assumed that the carbohydrate part of GLP is a selective receptor-accumulator for Ca²⁻, whereas the function of the lipid component consists in forming channels in the bilayer.     

Calcium signalling in T-lymphocytes

Calcium signalling is essential for most of the biological T-cell activities, including in Th2 lymphocytes, a T-cell subset that produce interleukin 4, 5 and 13 and which is involved in allergic diseases. T-cell receptor engagement induces the production of inositol trisphosphate that binds to its receptor, releasing intracellular Ca²⁺ stores. STIM in the endo (sarco) plasmic reticulum (ER/SR) is a Ca²⁺ sensor that perceives the depletion of intracellular Ca²⁺ stores, localizes near the cell membrane and allows the activation of ORAI, the main calcium channels at the cell membrane. However, other calcium channels at the membrane of intracellular compartments and at the cell membrane can also contribute to the TCR-driven intracellular Ca²⁺ rise. Among them, voltage-dependent calcium (Ca_v1) channels have been reported in several types of T-lymphocytes, although how they are gated in these non-excitable cells remains unsolved. We have shown that Cav1 channel expression was selectively up regulated in Th2 lymphocytes. In this review, we will discuss about the diversity of the Ca²⁺ channels responsible for Ca²⁺ homeostasis in the different cell subsets and the interactions between these molecules, which can account for the variety of the calcium responses depending upon the functions of effector T-cells.     

Influence of Ca on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca-activated K channel

The involvement of Ca²⁺-activated K⁺ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3′-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be −57 ± 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K⁺ concentration, [K⁺]_o, and were significantly enhanced by exogenous calcium. The apparent V_max of Na⁺-dependent glycine uptake was doubled in the presence of calcium, whereas the K_0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca²⁺-activated K⁺ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K⁺]_o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC₅₀ of 9.4 μM; and (3) cells treated with the Ca²⁺-activated K⁺ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca²⁺-activated K⁺ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na⁺-dependent amino acids.     

Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB-polyphosphate Ca channels in the membrane

PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca²⁺ homeostasis in this organism. E. coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca²⁺ in the cytoplasm. Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells. These transitions specifically depend on the presence of PHB(Ca²⁺polyP) complexes. PHB(Ca²⁺polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA⁻ strains had greatly reduced amounts of the complexes. This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane. In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.     

Constitutively active calcium channels in plasma membrane of T-lymphocytes

Compensated influx and efflux of calcium ions maintain the constancy of Ca²⁺ concentration in cytoplasm of quiescent cells under variable external conditions. In cell plasma membrane there exist several types of Ca²⁺ channels with different properties, regulation mechanisms, and pharmacology. Using fluorescent Ca²⁺-sensitive probes, we have shown here that in T-lymphocytes under resting conditions, Ca²⁺ influx occurs through special constitutively active Ca²⁺ channels, permeable to Ni²⁺ and Mn²⁺. These channels differ from the receptor-activated SOC channels, from Ca²⁺ channels activated by arachidonic acid, and from calmidazolium-activated channels. Ca²⁺ influx rate in quiescent cells increases with a rise in temperature (Q₁₀ =1.9). The strong dependence of the constitutively active channel activity on temperature coincided with the plasma membrane Ca²⁺-ATPase dependence, indicating that intracellular enzymes regulate the channel activity. To identify the constitutively active channel, we analyzed the effects of L-type Ca²⁺ channels, SOC channels, Ca²⁺-independent phospholipase A2, and calmodulin inhibitors. Of all inhibitors listed only dihydropyridine blocker of L-type voltage-dependent Ca²⁺ channels, isradipin, at a concentration of 1.5 μM completely suppressed calcium influx. However, the channels did not exhibit sensitivity to changes in membrane potential. Our observations testify to the existence of a new nonselective Ca²⁺ channel in T-lymphocyte plasma membrane and characterize the new channels pharmacologically. The results obtained are important for understanding the regulation mechanisms of Ca²⁺ channels in plasma membrane of non-excitable cells.     

Modeling the Calcium Signaling in Stomatal Guard Cells under the Action of Abscisic Acid

A theoretical model of calcium signaling is presented that simulates oscillations of cytoplasmic calcium concentration ([Ca²⁺]_cyt) in stomatal guard cells under the action of abscisic acid. The model is based on the kinetics of inositol 1,4,5-trisphosphate-sensitive calcium channels of endoplasmic reticulum and cyclic ADP-ribose-sensitive calcium channels of the tonoplast. The operation of two energy-dependent pumps—the Ca²⁺-ATPase of the endoplasmic reticulum and the Ca²⁺/H⁺ antiporter of the tonoplast—is also included in the model. It is shown that the removal of excessive Ca²⁺ from the cytoplasm by the tonoplast Ca²⁺/H⁺ antiporter is the main factor accounting for generation of [Ca²⁺]_cyt oscillations at a wide range of ABA concentrations (0.01–1 M). The long period of [Ca²⁺]_cyt oscillations in plant cells is explained by a slow release from inhibition of inositol 1,4,5-trisphosphate-gated calcium channels.     

Comparative Model Analysis of Calcium Exchange between the Cytosol and Stores of Mitochondria or Endoplasmic Reticulum

The objects of the study were single-compartment mathematical models corresponding to a fragment of the dendrite of a cerebellar Purkinje neuron containing the mitochondria (model 1) or a cistern of the endoplasmic reticulum, ER, (model 2) as the calcium stores. We investigated the dependence of the intracellular Ca²⁺ dynamics on geometrical sizes of calcium exchanging parts of the intracellular space and the difference between the kinetic characteristics of storing in two types of stores occupying different portions of the compartment volume. The plasma membrane of the compartment bore the ion channels, particularly those conducting excitatory synaptic current, and the calcium pump typical of this neuron type. The model equations took into account Ca²⁺ exchange between the cytosol, extracellular medium, organelle stores, non-organelle endogenous buffers, and an exogenous buffer (fluorescent dye), and also the diffusion of Са²⁺ into adjacent regions of the dendrite. In model 1, the mitochondria exchanged Са²⁺ with the cytosol via the uniporter and sodium/calcium exchanger; mitochondrial processes, such as the tricarboxylic acid cycle and aerobic cellular respiration, were also taken into account. In model 2, the ER membrane contained the calcium pump, channels of passive leak, and channels of calcium-induced and inositol-3-phosphate-dependent release of Са²⁺. Increases in the portion of the stores in the total volume of the compartment from 1 to 36% led to a proportional increase in the peak values of the cytosolic calcium concentration ([Ca²⁺] _i ); the concentration of Са²⁺ in the mitochondria ([Ca²⁺]_mit) or ER ([Ca²⁺]_ER) increased correspondingly. During generation of bell-shaped cytosolic calcium signals of equal intensity and duration, the ER (due to a greater rate of storing, as compared with that in the mitochondria) was able to uptake several times more Са²⁺ (four times at 36% filling of the volume by the organelles). It is suggested that the revealed different kinetic characteristics of Са²⁺ storing by different organelles are determined by the rates of binding to transport molecules present in the store membrane and, therefore, are defined by concentrations (surface densities) of these molecules and their saturation at certain levels of [Ca²⁺]i. It has been shown that the occupancy of the intracellular volume by organelle stores of any type is a structural factor, which is able to essentially modulate the values of Ca²⁺ concentration.     

Destabilization of the cytosolic calcium level and the death of cardiomyocytes in the presence of derivatives of long-chain fatty acids

By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20–50 μM acylcarnitines increased calcium concentration in cytoplasm ([Ca²⁺]_i) and caused cell death after a lag-period of 4–8 min. This effect was independent of extracellular calcium level and Ca²⁺ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300–500 μM had little effect on [Ca²⁺]_i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these conditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of [Ca²⁺]_i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.     

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

Voltage-gated potassium (K⁺) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K⁺ channel type segregates into at least two main functional categories—hyperpolarization-activated, inward-rectifying (K_in) and depolarization-activated, outward-rectifying (K_out) channels. Voltage-gated K⁺ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in K_in and K_out channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K⁺ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between K_in and K_out channels. Among them is a loop between TMD S5 and the pore that is very short in animal K_out, longer in plant and animal K_in and the longest in plant K_out channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.     

Downregulation of TRPV6 channel activity by cholesterol depletion in Jurkat T cell line

Transient receptor potential vanilloid 6 (TRPV6) channels are key players in calcium metabolism of healthy and cancerous cells. Nevertheless, the mechanisms controlling abundance of these channels in plasma membrane of the cells to regulate Ca²⁺ transport is still poorly understood. In this study, we provide the first evidence that TRPV6 calcium channels and Ca ²⁺ influx in Jurkat T cell line are modulated by cholesterol, a main lipid component of the plasma membrane. Using patch‐clamp technique, we found that activity of TRPV6 channels decreased by cholesterol sequestration with methyl‐β‐cyclodextrin (MβCD). Continuous measurement of intracellular Ca²⁺ revealed a reduction of Ca²⁺ influx into Jurkat cells following cholesterol depletion. Immunofluorescence and immunoelectron microscopy analyses of MβCD‐treated cells detected the lower surface expression of the TRPV6 proteins in comparison with control cells. In general, our data showed that cholesterol regulates TRPV6 channel activity and TRPV6‐mediated Ca²⁺ influx in cells, apparently affecting the localization and density of the calcium channels in the plasma membrane of Jurkat T cells.     

Simulation of the effect of an external GHz electric field on the potential energy profile of Ca2+ ions in the selectivity filter of the CaVAb channel

Ca_V channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca²⁺ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion‐selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the Ca_VAb channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca²⁺ ions in the Ca_VAb channels in presence and absence of an external field. Exposing Ca_VAb channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca²⁺ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca²⁺ ions binding in the selectivity filter may interrupt the mechanism of Ca²⁺ ion conduction, and leads to a reduction of Ca²⁺ ion permeation through the Ca_VAb channel.     

Ultrafiltration is theoretically equivalent to equilibrium dialysis but much simpler to carry out.

This study shows that if one component of a reaction at equilibrium is freely diffusible through a semipermeable membrane, and if an aliquot of this component is removed through the membrane at its equilibrium concentration, the concentration of this component in the reaction mixture remains unchanged. This is illustrated by the binding of manganese (Mn²⁺) to concanavalin A. It is also shown that, at concentrations of calcium ions near saturation levels (0.01 m CaCh₂, pH 5.2, 0.2 m NaCl), the binding of 1 mol of Mn²⁺ is extremely strong, with a dissociation constant K < 10^?7, and at least 1 additional mol of Mn²⁺/mol of concanavalin A binds less strongly. As the pH is lowered, the affinity decreases to a small extent, until at pH 1.82 approximately 0.25 mol of Mn²⁺ binds/mol of protein. A possible application of the method to measure binding as a function of ligand concentration is described.     

Surface charges on the outer side of mollusc neuron membrane

Summary The shifts of current-voltage characteristics of sodium and calcium inward currents produced by changes in the concentration of divalent cations (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) and in pH of the extracellular solution have been measured on isolated neurons of the molluscHelix pomatia intracellularly perfused with potassium-free solutions. On the basis of these shifts and using Stern's theory (O. Stern, 1924.Z. Electrochem. 30508–516), the binding constants for the ions to charged groups of the outer side of the somatic membrane and the density of the surface charges produced by these groups have been calculated. For groups located in the vicinity of sodium channels we obtainedK _Ca=90±10,K _Sr=60±10,K _Ba=25±5 andK _Mg=16±5m ^–1 at pH=7.7 and for groups located in the vicinity of calcium channelsK _Ca=67±10,K _Sr=20±5 andK _Ba=19±5m ^–1 at pH=7.0. The same groups bind H⁺ ions with apparent pK=6.2±0.2 that corresponds toK _H=1.6×10⁶ m ^–1. The density of fixed charges near the sodium channels is 0.17±0.05 e/nm² (pH=7.7) and near the calcium channels is 0.23±0.05 electrons/nm² (pH=7.0). From the comparison of the obtained values with the data about binding constants of the same ions to different negatively charged phospholipids, a suggestion is made that just the phophatidylserine is responsible for the surface potential of the outer side of the somatic membrane. It was also shown that the presence of this potential results in a change in the concentration of carrier ions near the membrane which affects the maximal values of the corresponding transmembrane currents.     

Calcium Domains around Single and Clustered IP3 Receptors and Their Modulation by Buffers

We study Ca²⁺ release through single and clustered IP₃ receptor channels on the ER membrane under presence of buffer proteins. Our computational scheme couples reaction-diffusion equations and a Markovian channel model and allows our investigating the effects of buffer proteins on local calcium concentrations and channel gating. We find transient and stationary elevations of calcium concentrations around active channels and show how they determine release amplitude. Transient calcium domains occur after closing of isolated channels and constitute an important part of the channel's feedback. They cause repeated openings (bursts) and mediate increased release due to Ca²⁺ buffering by immobile proteins. Stationary domains occur during prolonged activity of clustered channels, where the spatial proximity of IP₃Rs produces a distinct [Ca²⁺] scale (0.5-10 μM), which is smaller than channel pore concentrations (>100 μM) but larger than transient levels. While immobile buffer affects transient levels only, mobile buffers in general reduce both transient and stationary domains, giving rise to Ca²⁺ evacuation and biphasic modulation of release amplitude. Our findings explain recent experiments in oocytes and provide a general framework for the understanding of calcium signals.     

Calmodulin antagonists do not inhibit IKCa channels of human erythrocytes

Patch-clamp recordings were performed to study the effects of three calmodulin (CaM) antagonists on the gating of intermediate calcium-activated K⁺ channels (IK_Ca) of human erythrocytes. In the cell-attached configuration, both opening frequency and open probability of IK_Ca channels were not significantly different in control cells and in those incubated with calmidazolium, trifluoperazine or W7. IK_Ca channels in excised membrane patches, were normally activated by the calcium bathing the cytoplasmic side in the presence of CaM antagonists, at calcium concentrations ranging from 10⁻⁷ to 10⁻³ M. The activity of IK_Ca channels, which had been previously up-modulated by an endogenous cAMP-dependent protein kinase, was not inhibited when perfused with CaM antagonists. The results presented in this study demonstrate that calmodulin antagonists do not inhibit the activity of native IK_Ca channels of human erythrocytes. These data are in accordance with findings on the cloned IK_Ca indicating that calmodulin is constitutively associated with these channels.     

Extracellular calcium and microwave enhancement of membrane conductance in snail neurons

Summary Microwave irradiation has been shown to decrease the input resistance of snail neurons. In this study, we examined the role of extracellular calcium in triggering the microwave-induced enhancement of membrane conductance. Two sets of experiments were conducted. In the first set, nerve cells were superfused using Ringer solution with added Cd²⁺ (0.9 mM) which is a known blocker of calcium channels. In the second set, cells were superfused with low Ca²⁺ (0.7 mM) Ringer solution. Microwave irradiation was conducted at 2,450 MHz for 30 min with a specific absorption rate of 13 mW/g. It was found that 7 mM to 0.7 mM lowering of Ca²⁺ in bathing solution as well as blocking of calcium channels in neuronal membrane by means of Cd²⁺ did not influence the fall in membrane resistance induced by microwave radiation. In fact, the observed changed in membrane resistance in these experiments were nearly equal to those observed for neurons superfused by normal Ringer's. Thus, these results rule out the possible contribution of external Ca²⁺ in the observed microwave effect. Experiments with high Ca²⁺ solution also support this conclusion.     

Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P₂ and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca²⁺ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P₂ complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca²⁺ concentrations is likely to be important for Ca²⁺-regulated secretion.     

The significance of extracellular Ca<Superscript>2+</Superscript> in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M₃ cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K⁺, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca²⁺ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca²⁺-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.