Effect of amiodarone on thermotolerance and Hsp104p synthesis in the yeast Saccharomyces cerevisiae

Amiodarone (AMD) is known to induce a transient increase in cytosolic Ca²⁺ level in cells of the yeast Saccharomyces cerevisiae. In the present study the effect of AMD on the thermotolerance and Hsp104p synthesis of the yeast was studied. AMD induced Hsp104p synthesis and increased survival of the yeast after a severe heat shock (50°C). The development of thermotolerance to a considerable extent depended on the presence of Hsp104p. The same effect was achieved by treatment with the classical uncoupler CCCP, which is also known to increase the cytosolic Ca²⁺ level. It is supposed that the change in intracellular Ca²⁺ concentration plays an important role in activation of the HSP104 gene expression and in increasing the thermotolerance of the yeast. The possible link between mitochondrial activity and calcium homeostasis is discussed.     

Mitochondrial retrograde regulation of HSP101 expression in Arabidopsis thaliana under heat stress and amiodarone action

Heat stress in plants elevates the potential across the inner mitochondrial membrane (mtΔψ) and activates the expression of heat shock proteins (HSPs). The treatment of Saccharomyces cerevisiae cells with amiodarone (AMD) elevated the cytosolic Ca²⁺ level ([Ca²⁺]_cyt) in parallel with (mtΔψ) increase and led to the induction of Hsp104 synthesis. The hyperpolarization was presumably due to the increase in [Ca²⁺]_cyt. In the present study the effects of AMD (0–100 μM) on cell viability, HSP expression, mtΔψ, and [Ca²⁺]_cyt were investigated using the cell culture of Arabidopsis thaliana (L.) Heynh. The treatment of cultured cells with AMD led to the elevation of [Ca²⁺]_cyt, which was accompanied by the increase in mtΔψ and by activation of HSP101 expression. The increase in [Ca²⁺]_cyt and expression of HSP101 were also observed upon the treatment with the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone, 4 μM) known to diminish mtΔψ. The results suggest that plant cell mitochondria modulate the cytosolic Ca²⁺ level by changing the potential at the inner mitochondrial membrane and, thereby, participate in the retrograde regulation of HSP101 expression.     

Bile Acid-Induced Ca<Superscript>2+</Superscript> Efflux from Liver Mitochondria as a Factor Preventing the Formation of Mitochondrial Pores

The effect of bile acids as inducers of Ca²⁺ efflux from the matrix was studied on isolated rat liver mitochondria. Mitochondria in the presence of cyclosporin A (CsA) were energized by succinate, then loaded with Ca²⁺ and after the addition of the calcium uniporter inhibitor ruthenium red were de-energized by malonate. It was shown that under these conditions hydrophobic bile acids lithocholic and chenodeoxycholic at concentrations of 10 and 30 μM respectively and hydrophilic bile acids ursodeoxycholic and cholic at a concentration of 400 μM induce Ca²⁺ efflux from the mitochondrial matrix. It is noted that the efflux of these ions is not associated with damage of the inner mitochondrial membrane by bile acids, since it is accompanied by the generation of Δψ, i.e., the formation of the diffusion potential. It is assumed that along with induction of calcium efflux from the matrix, bile acids are also capable of transporting hydrogen and potassium ions in the opposite direction, i.e., perform H⁺/Ca²⁺ and K⁺/Ca²⁺ exchange. It was found that ruthenium red added to Ca²⁺-loaded energized mitochondria prevents the return of these ions to the matrix and weakens the effect of chenodeoxycholic acid as an inducer of the CsA-sensitive mitochondrial pore and the effect of ursodeoxycholic acid as an inducer of CsA-insensitive permeability of the inner mitochondrial membrane. We conclude that in the conditions of the calcium uniporter activity decrease, Ca²⁺ efflux from the matrix induced by bile acids can be considered as one of the mechanisms reducing their effectiveness as inducers of the Ca²⁺-dependent CsA-sensitive pore and CsA-insensitive permeability transition in mitochondria.     

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.     

Contrasting Effects of Cd<Superscript>2+</Superscript> and Co<Superscript>2+</Superscript> on the Blocking/Unblocking of Human Ca<Subscript>v</Subscript>3 Channels

Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Ca_v1 and Ca_v2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Ca_v3 family) is less documented. We have studied the blocking effect of Cd²⁺, Co²⁺ and Ni²⁺ on T-currents expressed by human Ca_v3 channels: Ca_v3.1, Ca_v3.2, and Ca_v3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca²⁺ (2 mM) currents from HEK−293 cells stably expressing recombinant T-type channels. Cd²⁺ and Co²⁺ block was 2- to 3-fold more potent for Ca_v3.2 channels (EC₅₀ = 65 and 122 μM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co²⁺ and Ni²⁺ shift the voltage dependence of Ca_v3.1 and Ca_v3.3 channels activation to more positive potentials. Interestingly, block of those two Ca_v3 channels by Co²⁺ and Ni²⁺ was drastically increased at extreme negative voltages; in contrast, block due to Cd²⁺ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd²⁺ on Ca_v3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.     

Dynamic fluorescence polarization studies on lipid mobilities in phospholipid vesicles in the presence of calcium mediators

The influence of Ca²⁺ mediators (nifedipine, verapamil and prostaglandin F_2α) on fluorescence polarization of l-anilino-8-napthalene-sulphonate in dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine liposomes was studied at various temperatures to understand the dynamic behaviour of membrane lipids. We also studied the effect of change in calcium concentration on the fluorescence polarization of the dye in the liposomes. Our results show increase in polarization (indicative of stiffening of the membrane) in the presence of Ca²⁺ ions. In the case of dimyristoyl phosphatidylcholine liposomes, all 3 drugs caused decrease in fluorescence polarization (increase in fluidity of the membrane) with or without Ca²⁺ ions in the medium. Contrary to this, in the case of dipalmitoyl phosphatidylcholine liposomes, the fluidization effect is observed for all the 3 drugs in the absence of Ca²⁺ ions; in the presence of Ca²⁺ ions stiffening is observed upon addition of nifedipine and verapamil which are antagonists, and fluidization is observed upon addition of prostaglandin F_2α. The role of drug-induced fluidity changes in membranes in therapy planning is discussed in the paper.     

Synergistic action of calcium-lonophores and hyperthermia is best interpreted as thermal enhancement of calcium toxicity

It has been shown that no relation exists between [Ca²⁺]_i and hyperthermic cell killing, although heat-induced increase of [Ca²⁺]_i can be observed in some cell lines. When ionophores are used, dose-dependent rises in [Ca²⁺]_i may be found. Beyond a certain threshold of ionophore-induced increases in [Ca²⁺]_i, cells may be killed. Different threshold levels of [Ca²⁺]_i exist in different cell lines. Hyperthermia can act synergistically with calcium ionophores to potentiate cell killing. Since there is no causal relation between [Ca²⁺]_i and heat toxicity, this synergism can be explained as heat enhanced Ca²⁺ toxicity. In the current report, it is shown that both ionophore-induced Ca²⁺ toxicity (37°C) and its potentiation by heat are dependent on extracellular calcium and related to sustained increases in [Ca²⁺]_i. With ionomycin concentrations up to 15 μM, no increase in [Ca²⁺]_i was seen in cells maintained in medium without Ca²⁺. Ionomycin effects on intracellular compartments were absent, and the drug seemed to act solely on the level of the plasmamembrane. Also, the synergism of heat and ionomycin appeared to act at the plasmamembrane, because depletion of extracellular calcium completely abolished this synergistic effect. The data presented are also discussed in the light of controversies existing in the literature for the role of calcium in hyperthermic cell killing. © 1993 Wiley-Liss, Inc.     

A comparative study of the effects of Pr<Superscript>3+</Superscript> and La<Superscript>3+</Superscript> ions on calcium dependent processes in frog cardiac muscle and rat heart mitochondria

The inotropic effect of Pr³⁺ and La³⁺ ions on the heart muscle of frog Rana ridibunda, as well as the influence of the ions on respiration, swelling, and the potential (ΔΨ_mito) on the inner membrane of Ca²⁺- loaded rat heart mitochondria, energized by glutamate and malate or succinate in the presence of rotenone were studied. It was found that 2 mM Pr³⁺ in Ringer’s solution reduces the force of spontaneous contractions and those induced by electrical stimulation in the heart; it had a negative chronotropic effect, decreasing the frequency of spontaneous contractions. Pr³⁺ and La³⁺ prevented a decrease in the 2,4-dinitrophenol (DNP)- uncoupled respiration of energized rat heart mitochondria, swelling of these organelles in salt media, and a reduction in ΔΨ_mito on the inner mitochondrial membrane that were induced by Ca²⁺ ions. Retardation by Pr³⁺ and La³⁺ ions of these calcium-induced effects may suggest that in the inner mitochondrial membrane these metals inhibit the opening of the mitochondrial permeability transition pore caused by Ca²⁺ overload of mitochondria. The data we obtained are important for a better understanding of the mechanisms of the damaging action of rare-earth elements on Ca²⁺-dependent processes in the vertebrate myocardium.     

The influence of Methylprednisolone on the energy metabolism of Ehrlich ascites tumour cells

Using Ehrlich ascites tumour cells, the short-term effects of the therapeutic glucocorticoid Methylprednisolone (MP) on the cellular energy metabolism were studied. ATP-consuming processes involved in the rapid MP effects were identified indirectly from the effects of MP on cellular oxygen consumption related to the inhibition of respiration by selective inhibitors of Ca²⁺-ATPase and protein synthesis. The effects of MP on plasma membrane permeability for Ca²⁺ ions and phospholipid turnover were studied directly by using confocal laser scanning microscopy and tracerkinetic measurements, respectively. MP inhibited cellular oxygen consumption, suppressed the inhibitory effect of lanthanum but not that of cycloheximide on oxygen consumption, blocked the [Ca²⁺]_i rise in response to calcium ionophore A 23187, and decreased phospholipid turnover. MP acted instantly in a dose-dependent manner.The observed effects of MP are discussed in relation to the hypothesis that the drug has direct membrane effect affecting plasma membrane permeability and function.     

Effect of rare earth element europium on amaranthin synthesis in Amarathus caudatus seedlings

Considering the resemblances between Eu³⁺ and Ca²⁺ in their atomic radius and structures of the valence electron, the effects of Eu³⁺ on amaramthin synthesis in Amarathus caudatus seedling were studied. Eu³⁺ had both promoting and inhibiting effects on amaramthin synthesis. The optimum promoting concentration and half inhibiting concentration of Eu³⁺ to synthesis of amaranthin were 0.4 mmol/L and 2.5 mmol/L, respectively. In the dark, A₂₃₁₈₇ (ions carrier) could carry Eu³⁺ into cells through the Ca²⁺ channel. When Ca²⁺ was chelated with EGTA, the synthesis of amaranthin could be partly retrieved by Eu³⁺. Eu³⁺ treatment could also activate Ca²⁺-ATPase on plasma membrane. Moreover, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of total proteins from the plants treated by Eu³⁺ and Ca²⁺ were similar but slightly different in the contents. It suggested that the effects of Eu³⁺ and Ca²⁺ on amaranthin synthesis were similar. After being treated by Eu³⁺ or Ca²⁺, the outside Ca²⁺ could enter into cells to promote synthesis of amaranthin. The results above indicated that Eu³⁺ might replace Ca²⁺ in the calcium/calmidulin-dependent phytochrome signal transduction system and play important roles in plant development by promoting calcium transportation across plasma membrane.     

Evidence for the possible involvement of calmodulin in regulation of steady state levels of Hsp90 family members (Hsp87 and Hsp85) in response to heat shock in sorghum

Pharmacological studies, using Ca²⁺ channel blockers (LaCl₃ and verapamil) and calmodulin (CaM) antagonists (CPZ and W7), were carried out to understand the role of Ca²⁺/CaM in the regulation of heat shock-induced expression of Hsp90 (Hsp87 and Hsp85) and Hsp70 (Hsp75 and Hsp73) members in sorghum. It was observed that the expression of both Hsp87 and Hsp85 proteins was decreased in presence of Ca²⁺ channel blockers and CaM antagonists, under both control and heat stress conditions, as contrary to the steady state levels of Hsp75 and Hsp73, which were not affected significantly under similar conditions. Further, the exposure of sorghum seedlings to geldanamycin, a specific inhibitor of Hsp90, resulted in induction of Hsp87 and Hsp85 in the absence of heat shock also. This study provides the first evidence suggesting that in plants, the in vivo expression of Hsp90 (Hsp87 and Hsp85) is likely to be modulated by Ca²⁺/CaM under normal and thermal stress conditions. The likely implications of these findings are discussed.Key words: calmodulin, calmodulin-binding proteins, heat shock proteins, sorghum     

Determination of intracellular Ca2+ in cells of intact wheat roots: loading of acetoxymethyl ester of Fluo‐3 under low temperature

Loading of Ca²⁺-sensitive fluorescent probes into plant cells is an essential step to measuring activities of cytoplasmic free Ca²⁺ ions with a fluorescent imaging technique. A major barrier to the loading of the fluorescent probes into plant cells using the acetoxymethyl (AM) esters of the Ca²⁺-sensitive dyes is the presence of cell-wall associated esterases. These esterases hydrolyse the esterified form of the fluorescent probes, rendering the probes membrane-impermeable. A novel non-invasive loading protocol was described in this paper to load the Ca²⁺-sensitive fluorescent probe Fluo-3/AM ester into apical cells of intact wheat roots by incubating the roots in Fluo-3/AM ester solution at 4°C for 2 h followed by 2-h incubation in the dye-free solution at 20°C. The incubation at low temperature inhibited extracellular hydrolysis of Fluo-3/AM ester but had less effect on diffusion of membrane-permeable Fluo-3/AM ester across the plasma membrane, because hydrolysis of Fluo-3/AM ester by extracellular esterases is a chemical process (high Q₁₀), while diffusion of Fluo-3/AM across the plasma membrane is a physical process (low Q₁₀). The Fluo-3/AM ester, accumulated in the root cells during the low temperature incubation, was then cleaved by intracellular esterases during the incubation at 20°C, releasing the membrane-impermeable Ca²⁺-sensitive Fluo-3 in the cytoplasm. The root cells loaded with Fluo-3 showed strong intracellular fluorescence under confocal microscopy. The fluorescence from the root cells was sensitive to the Ca²⁺ ionophore and hydrogen peroxide, indicating that the intracellular fluorescence was due to intracellular Ca²⁺ ions.     

ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.     

Participation of Intracellular Ca2+ Stores in Ca2+ Signalling in Neurons of the Thalamic Laterodorsal Nucleus of Rats

Experiments were carried out on isolated neurons of the thalamic nucleus lateralis dorsalis (LD) from 12-day-old rats. According to the morphological characteristics, LD neurons were classified as relay thalamo-cortical units and interneurons. The concentration of free Ca²⁺ ions in the cytoplasm ([Ca²⁺] _i ) was measured by a fluorescent calcium indicator, fura-2AM. Application of 30 mM caffeine caused a transient change in the [Ca²⁺] _i in 8 of 15 and in 6 of 11 of the thalamo-cortical units and interneurons under study, respectively. After stimulation of a cell with application of 50 mM KCl, a caffeine-induced increase in the [Ca²⁺] _i was observed in all tested neurons. To study the contribution of Ca²⁺-induced Ca²⁺ release (CICR) to the calcium transient evoked by depolarization of the neuronal membrane, caffeine in a subthreshold concentration was pre-applied. After 50 mM KCl had been added to the medium following pre-application of 0.5 mM caffeine, the calcium transient amplitude in thalamo-cortical neurons increased by 51 ± 7% (n = 16). In interneurons this effect was not observed (n = 11). The data obtained allow us to hypothesize that CICR contributes to the depolarization-evoked calcium transient only in the relay (thalamo-cortical) neurons. Differences in the pattern of calcium signalling, which were detected in two types of neurons of the thalamic LD, can be a factor determining distinctions in the physiological characteristics of these neurons.     

Haloperidol modulates ion transport in <Emphasis Type="Italic">Chara corallina</Emphasis> cells

The effects of haloperidol, an antagonist of D2 dopamine receptors, on the functioning of Ca²⁺, K⁺, and Cl^? ion channels in the membrane of Chara corallina cells and on the functional properties of their cytoskeleton was studied. Haloperidol blocked Ca²⁺ channels of the plasmalemma. In addition to bringing about a decrease in the amplitude of the calcium current, exposure to haloperidol decelerated the activation and inactivation of calcium channels. The effect of haloperidol was reversible; after it was removed, the characteristics of calcium current were restored. Haloperidol did not affect Ca²⁺-activated chloride channels. Haloperidol also inhibited microfilament-dependent motion of the cytoplasm. Cytoplasmic streaming was restored after haloperidol was removed from the extracellular solution. These results suggest that the concentration of free Ca²⁺ ions in the cytoplasm increases in the presence of haloperidol, and that Ca²⁺ channels of C. corallina plasmalemma possess specific binding sites both for dopamine receptors and for their antagonists.     

Anoxia-induced elevation of cytosolic Ca<Superscript>2+</Superscript> concentration depends on different Ca<Superscript>2+</Superscript> sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, was investigated in plants differing in tolerance to hypoxia. The [Ca²⁺]_cyt was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca²⁺]_cyt in rice leaf protoplasts. A tenfold drop in the external Ca²⁺ concentration (to 0.1 mM) resulted in considerable decrease of the [Ca²⁺]_cyt shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La³⁺ and EGTA) and intracellular membrane Ca²⁺-channel antagonists (Li⁺, ruthenium red and cyclosporine A) reduced the anoxic Ca²⁺-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca²⁺]_cyt. In wheat leaf protoplasts, the amplitude of the Ca²⁺-shift little depended on the external level of Ca²⁺. Wheat root protoplasts were characterized by a small shift of [Ca²⁺]_cyt under anoxia. Plasmalemma Ca²⁺-channel blockers had little effect on the elevation of cytosolic Ca²⁺ in wheat protoplasts. Intact rice seedlings absorbed Ca²⁺ from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca²⁺. Verapamil abolished the Ca²⁺ influx in rice roots and Ca²⁺ efflux from wheat roots. Anoxia-induced [Ca²⁺]_cyt elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca²⁺ stores are important for anoxic [Ca²⁺]_cyt elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca²⁺]_cyt rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

The alteration of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl₄) solution was investigated. Rats received a single oral administration of CCl₄ (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl₄ administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity, indicating that the increased Ca²⁺ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity increased by CCl₄ administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca²⁺-Mg²⁺)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl₄-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver obtained from CCl₄-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca²⁺ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl₄ administration.     

Effects of temperature on sperm motility of burbot Lota lota: spontaneous activation and calcium dependency

Several factors regulating activation of spermatozoon motility in Eurasian burbot, Lota lota, including osmolality, calcium (Ca²⁺) ions, and temperature were investigated. Spermatozoon motility in Eurasian burbot, Lota lota was assessed at 4 and 30°C in seminal fluid, isotonic media (with and without Ca²⁺) and hypotonic media (with and without Ca²⁺). Spermatozoa were spontaneously activated in seminal fluid at 20°C and the maximum motility was recorded at 30°C, which is out of the spawning temperature range, indicating that no risk of activation occurs during routine semen handling in artificial insemination. Initiation of spermatozoon motility in L. lota is mediated by Ca²⁺ and sensitivity to Ca²⁺ is dependent on temperature.     

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.