Fusion of neurohypophyseal membranes in vitro

Freeze cleaving electron microscopy has shown that fusion of isolated secretory vesicles from bovine neurohypophyses was induced by Ca²⁺ in micromolar concentrations. Mg²⁺ and Sr²⁺ were ineffective. Mg²⁺ inhibited Ca²⁺-induced fusion.In suspensions containing secretory vesicles as well as sheets of cell membrane, release of vasopressin parallel to intervesicular fusion of secretory vesicles with sheets of cell membrane was observed after exposure to Ca²⁺. Mg²⁺ and Sr²⁺ were ineffective in replacing Ca²⁺ as trigger for fusion or vasopressin release.Intervesicular fusion and exocytotic profiles were observed when isolated neurohypophyses or neurosecretosome were exposed to cold.     

Effects of divalent cations and glucose on mitotic-like events in fused interphase-metaphase cells

The effects of Ca²⁺, Mg²⁺ and glucose on the mitotic-like events of prophasing and telophasing were studied in Sendai virus-fused interphase-metaphase (I-M) Chinese hamster binucleate cells. At normal extracellular ion concentrations and neutral pH, about 80–90% of I-M binucleates show prophasing (nuclear envelope dissolution and chromatin condensation) of the I nucleus and 10–15% show telophasing (nuclear envelope reformation and chromatin decondensation) of the M nucleus. To study the effects of cellular divalent cations, cells, depleted of about 77 % of exchangeable cell Ca²⁺ as determined by ⁴⁵Ca²⁺ studies, were incubated in different concentrations of Ca²⁺ or Mg²⁺ for 30 min prior to cell fusion. We found that relatively high concentrations of Ca²⁺ or Mg²⁺ (0.84 mM) were essential for prophasing and that in the presence of 10-fold less Ca²⁺ or Mg²⁺ (0.084 mM) the majority of binucleates showed telophasing. In contrast to a differential effect of divalent cations on the nuclear changes, we found that glucose metabolism was required for both prophasing and telophasing. Additionally, interruption of glucose metabolism in the M cell, but not in the I cell, prior to cell fusion depressed the prophasing frequency about 70%. Although we do not know how divalent cations and glucose function in prophasing and telophasing, we will discuss evidence which suggests that the effects are not mediated through secondary effects on membrane potential, by changes in intracellular concentrations of Na⁺ or K⁺, by simple osmotic changes, or through inhibition of protein synthesis.     

Insulin restores expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats

The effects of extracellular Mg²⁺ on both dynamic changes of [Ca²⁺]_i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca²⁺ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca²⁺]_i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca²⁺]_i (150 nM) for several hours. The initial [Ca²⁺]_i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca²⁺]_i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg²⁺ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg²⁺, caused an increase in basal [Mg²⁺]_i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg²⁺, 1 M thapsigargin induces, in 10 mM Mg²⁺, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca²⁺]_i and a lower Ca²⁺ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca²⁺]_i which was inhibited by high extracellular and intracellular Mg²⁺.     

(Ca2+ + Mg2+)-ATPase of density-separated human red cells. Effects of calcium and a soluble cytoplasmic activator (Calmodulin)

The effect of calcium and a soluble cytoplasmic activator on (Ca²⁺ + Mg²⁺)-ATPase of density-separated human red cells was investigated. At all calcium concentrations tested, dense (old) lysed cells and their isolated membranes displayed lower activities as compared to the light (young) cells and their membranes. Isolated membranes from all density red cell fractions showed two distinct (Ca²⁺ + Mg²⁺)-ATPase activities; one at low calcium and another at moderate calcium concentrations. At high calcium concentration, (Ca²⁺ + Mg²⁺)-ATPase activity of isolated membranes was low in all cell fractions. In contrast to the isolated membranes, lysed cells from all density fractions had a maximum (Ca²⁺ + Mg²⁺)-ATPase activity only at a low concentration of calcium, while moderate and high calcium concentrations produced low activity. Upon isolation of membranes, a substantial loss of (Ca²⁺ + Mg²⁺)-ATPase activity took place from all density cell fractions. Upon membrane isolation, the relative loss of (Ca²⁺ + Mg²⁺)-ATPase activity at low Ca²⁺ concentration was greater in older cells. The extent of stimulation of (Ca²⁺ + Mg²⁺)-ATPase by the activator at low calcium concentration was 3–4-fold greater in older cell membranes than in the young ones.These data suggest that the lower (Ca²⁺ + Mg²⁺)-ATPase activity in old cells could be accounted for by a selective loss of (Ca²⁺ + Mg²⁺)-ATPase activity at low Ca²⁺ concentration presumably due to reduced affinity of old cell membranes to activator protein.     

Marcroscopic and single-channel studies of two Ca2+ channel types in oocytes of the ascidianCiona intestinalis

Summary Whole-cell and single-channel patch-clamp experiments were performed on unfertilized oocytes of the ascidianCiona intestinalis to investigate the properties of two voltage-dependent Ca²⁺ currents found in this cell. The peak of the low threshold current (channel I) occurred at –20 mV, the peak of the high-threshold current (channel II) at +20 mV. The two currents could be distinguished by voltage dependence, kinetics of inactivation and ion selectivity. During large depolarizing voltage pulses, a transient outward current was recorded which appeared to be due to potassium efflux through channel II. When the external concentrations of Ca²⁺ and Mg²⁺ were reduced sufficiently, large inward Na currents flowed through both channels I and II. Using divalent-free solutions in cell-attached patch recordings, single-channel currents representing Na influx through channels I and II were recorded. The two types of unitary events could be distinguished on the basis of open time (channel I longer) and conductance (channel I smaller). Blocking events during changel I openings were recorded when micromolar concentrations of Ca²⁺ or Mg²⁺ were added to the patch pipette solutions. Slopes of the blocking rate constantvs. concentration gave binding constants of 6.4×10⁶ m ^–1 sec^–1 for Mg²⁺ and 4.5×10⁸ m ^–1 sec^–1 for Ca²⁺. The Ca²⁺ block was somewhat relieved at negative potentials, whereas the Mg²⁺ block was not, suggesting that Ca²⁺, but not Mg²⁺, can exit from the binding site toward the cell interior.     

Increase in Serum Ca2+/Mg2+ Ratio Promotes Proliferation of Prostate Cancer Cells by Activating TRPM7 Channels

TRPM7 is a novel magnesium-nucleotide-regulated metal current (MagNuM) channel that is regulated by serum Mg²⁺ concentrations. Changes in Mg²⁺ concentration have been shown to alter cell proliferation in various cells; however, the mechanism and the ion channel(s) involved have not yet been identified. Here we demonstrate that TRPM7 is expressed in control and prostate cancer cells. Supplementation of intracellular Mg-ATP or addition of external 2-aminoethoxydiphenyl borate inhibited MagNuM currents. Furthermore, silencing of TRPM7 inhibited whereas overexpression of TRPM7 increased endogenous MagNuM currents, suggesting that these currents are dependent on TRPM7. Importantly, although an increase in the serum Ca²⁺/Mg²⁺ ratio facilitated Ca²⁺ influx in both control and prostate cancer cells, a significantly higher Ca²⁺ influx was observed in prostate cancer cells. TRPM7 expression was also increased in cancer cells, but its expression was not dependent on the Ca²⁺/Mg²⁺ ratio per se. Additionally, an increase in the extracellular Ca²⁺/Mg²⁺ ratio led to a significant increase in cell proliferation of prostate cancer cells when compared with control cells. Consistent with these results, age-matched prostate cancer patients also showed a subsequent increase in the Ca²⁺/Mg²⁺ ratio and TRPM7 expression. Altogether, we provide evidence that the TRPM7 channel has an important role in prostate cancer and have identified that the Ca²⁺/Mg²⁺ ratio could be essential for the initiation/progression of prostate cancer.     

Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER-plasma membrane tethering

The extended synaptotagmins (E-Syts) are ER proteins that act as Ca²⁺-regulated tethers between the ER and the plasma membrane (PM) and have a putative role in lipid transport between the two membranes. Ca²⁺ regulation of their tethering function, as well as the interplay of their different domains in such function, remains poorly understood. By exposing semi-intact cells to buffers of variable Ca²⁺ concentrations, we found that binding of E-Syt1 to the PI(4,5)P₂-rich PM critically requires its C2C and C2E domains and that the EC₅₀ of such binding is in the low micromolar Ca²⁺ range. Accordingly, E-Syt1 accumulation at ER-PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca²⁺ via its influx from the extracellular medium, such as store-operated Ca²⁺ entry in fibroblasts and membrane depolarization in β-cells. We also show that in spite of their very different physiological functions, membrane tethering by E-Syt1 (ER to PM) and by synaptotagmin (secretory vesicles to PM) undergo a similar regulation by plasma membrane lipids and cytosolic Ca²⁺.     

Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferation

Abstract. Bone tissue in the adult is continuously being remodelled, and overall bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation. Adequate osteoblastic proliferation is essential for both appropriate formation and for regulation of resorption, and thereby the maintenance of bone remodelling equilibrium. Objectives: Here, we have investigated the roles of melastatin‐like transient receptor potential 6 and 7 (TRPM6, TRPM7), which are calcium (Ca²⁺) and magnesium (Mg²⁺) conducting channels, during proliferation of human osteoblasts. Results: Genetic expression of TRPM6 and TRPM7 was shown in human osteoblast‐like MG‐63, SaOS and U2‐OS cells, and reduction of extracellular Mg²⁺ or Ca²⁺ led to a decrease of cell proliferation. Concomitant reduction of both ions further accentuated reduction of cell proliferation. Expression of TRPM7 channels was increased under conditions of reduced extracellular Mg²⁺ and Ca²⁺ levels whereas expression of TRPM6 was not modified, suggesting compensatory mechanisms afforded by TRPM7 in order to maintain intracellular ion homeostasis. Pre‐incubation of cells in reduced extracellular Mg²⁺ conditions led to activation of Ca²⁺ and Mg²⁺ influx. Reduction of TRPM7 expression by specific siRNA prevented latter influx and inhibited cell proliferation. Conclusions: Our results indicate that extracellular Mg²⁺ and Ca²⁺ deficiency reduces the proliferation of human osteoblastic cells. Expression and activity of TRPM7 is modulated by extracellular Mg²⁺ and Ca²⁺ availability, indicating that TRPM7 channels are involved in intracellular ion homeostasis and proliferation of osteoblasts.     

Activation of Ca2+/Mg2+ ATPase in heart sarcolemma upon electrical stimulation

Electrical stimulation of the rat heart sarcolemmal membranes with a square wave current was found to increase Ca²⁺-ATPase activity. This activation of the enzyme was dependent upon the voltage of the electric current, frequency of stimulation and duration of stimulation of the sarcolemmal membranes. The increase in ca²⁺-ATPase was reversible upon terminating the electrical stimulation. The activation of sarcolemmal Ca²⁺-ATPase due to electrical stimulation was markedly depressed when the reaction was carried out at high pH (7.8 to 8.2), low pH (6.6 to 7.0), high temperatures (45 to 50°C) and low temperatures (17 to 25°C) of the incubation medium. Ca²⁺-antagonists, verapamil and D-600, unlike other types of inhibitors such as propranolol and ouabain, were found to reduce the activation of sarcolemmal Ca²⁺-ATPase by electrical stimulation. These results support the view that Ca²⁺/Mg²⁺ ATPase may be involved in the gating mechanism for opening Ca²⁺-channels in the sarcolemmal membrane upon excitation of the cardiac muscle.     

Thermoreception of Paramecium: Different Ca2+ Channels Were Activated by Heating and Cooling

A Paramecium cell responded to heat and cold stimuli, exhibiting increased frequency of directional changes in its swimming behavior. The increase in the frequency of directional changes was maintained during heating, but was transient during cooling. Although variations were large, as expected with this type of electrophysiological recording, results consistently showed a sustained depolarization of deciliated cells in response to heating. Depolarizations were also consistently observed upon cooling. However, these depolarizations were transient and not continuous throughout the cooling period. These depolarizations were lost or became small in Ca²⁺-free solutions. In a voltage-clamped cell, heating induced a continuous inward current and cooling induced a transient inward current under conditions where K⁺ currents were suppressed. The heat-induced inward current was not affected significantly by replacing extracellular Ca²⁺ with equimolar concentrations of Ba²⁺, Sr²⁺, Mg²⁺, or Mn²⁺, and was lost upon replacing with equimolar concentration of Ni²⁺. On the other hand, the cold-induced inward current was not affected significantly by Ba²⁺, or Sr²⁺, however the decay of the inward current was slowed and was lost or became small upon replacing with equimolar concentrations of Mg²⁺, Mn²⁺, or Ni²⁺. These results indicate that Paramecium cells have heat-activated Ca²⁺ channels and cold-activated Ca²⁺ channels and that the cold-activated Ca²⁺ channel is different from the heat-activated Ca²⁺ channel in the ion selectivity and the calcium-dependent inactivation. Received: 9 September 1998/Revised: 22 January 1999     

Divalent regulation and intersubunit interactions of human Connexin26 (Cx26) hemichannels

Control of plasma membrane connexin hemichannel opening is indispensable, and is achieved by physiological extracellular divalent ion concentrations. Here, we explore the differences between regulation by Ca²⁺ and Mg²⁺ of human connexin26 (hCx26) hemichannels and the role of a specific interaction in regulation by Ca²⁺. To effect hemichannel closure, the apparent affinity of Ca²⁺ (0.33 mM) is higher than for Mg²⁺ (1.8 mM). Hemichannel closure is accelerated by physiological Ca²⁺ concentrations, but non-physiological concentrations of extracellular Mg²⁺ are required for this effect. Our recent report provided evidence that extracellular Ca²⁺ facilitates hCx26 hemichannel closing by disrupting a salt bridge interaction between positions D50 and K61 that stabilizes the open state. New evidence from mutant cycle analysis indicates that D50 also interacts with Q48. We find that the D50-Q48 interaction contributes to stabilization of the open state, but that it is relatively insensitive to disruption by extracellular Ca²⁺ compared with the D50-K61 interaction.     

Polyamine uptake in carrot cell cultures

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca²⁺. The effects of Ca²⁺ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca²⁺ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca²⁺ concentration range between 10 micromolar and 1 millimolar. La³⁺ nullified the stimulatory effect of 10 micromolar Ca²⁺ on putrescine uptake and that of 1 millimolar Ca²⁺ on spermidine uptake. La³⁺ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule.     

Ca2+- and phospholipid-binding properties ofTorpedo electric organ calelectrin

The Ca²⁺-regulated lipid-binding properties of the H- and L-forms of calelectrin present in the electric organ ofTorpedo marmorata have been compared. Binding of H-calelectrin required Ca²⁺ in millimolar concentrations, whereas that of L-calelectrin occurred in the micromolar range. Dissociation of H-calelectrin previously bound to lipids in the presence of 2 mM Ca²⁺ took place only when the Ca²⁺ concentration was reduced to micromolar concentrations. Binding was most effective to acidic phospholipids such as phosphatidylserine. Both forms of calelectrin promoted the aggregation of membrane vesicles in the presence of Ca²⁺, Mg²⁺, Na⁺ and K⁺ inhibited the Ca²⁺-induced binding to phospholipid, decreasing in effectiveness in that order. Binding was also inhibited by high pH. The surface activity and hdyrophobicity index showed that H-calelectrin is a hydrophilic molecule. It may represent a less active, more highly phosphorylated ‘down-regulated’ form of L-calelectrin. The role of calcium in H-calelectrin binding to lipid appeared to be consistent with the formation of a ternary complex of the protein, an acidic lipid and Ca²⁺, rather than with a direct interaction of lipid with hydrophobic sequences in H-calelectrin whose accessibility is Ca²⁺-regulated.     

Intracellular uptake and α-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells

Summary Intracellular uptake of A23187 and the increased release of amylase and lactate dehydrogenase (LDH) accompanying ionophore uptake was studied using dissociated acinar cells prepared from mouse pancreas. Easily detected changes in the fluorescence excitation spectrum of A23187 upon transfer of the ionophore from a Tris-buffered Ringer's to cell membranes were used to monitor A23187 uptake. Uptake was rapid in the absence of extracellular Ca²⁺ and Mg²⁺ (t1/2=1 min) and much slower in the presence of Ca²⁺ or Mg²⁺ (t1/2=20 min). Cell-associated ionophore was largely intracellular as indicated by fluorescence microscopy, lack of spectral sensitivity to changes in extracellular Ca²⁺ and Mg²⁺, and by equivalent interaction of ionophore with membranes of whole and sonicated cells.A23187 (10 m) increased amylase release 200% in the presence of extracellular Ca²⁺ and Mg²⁺. In the absence of Ca²⁺ (but in the presence of Mg²⁺) A23187 did not increase amylase release. A23187 (10 m) also produced Ca²⁺-dependent cell damage, as judged by increased LDH release, increased permeability to trypan blue, and by disruption of cell morphology. The cell damaging and amylase releasing properties of A23187 were distinguished by their time course and dose-response relationship. A23187 (1 m) increased amylase release 140% without increasing LDH release or permeability to trypan blue.     

The role of magnesium in regulating CCK-8-evoked secretory responses in the exocrine rat pancreas

This study investigates the effect of magnesium (Mg²⁺) on the secretory responses and the mobilization of calcium (Ca²⁺) and Mg²⁺ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10^–10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg²⁺ [Mg²⁺]_o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg²⁺]_o Similar effects of perturbation of [Mg²⁺]_o on amylase secretion and ⁴⁵Ca²⁺ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca²⁺ concentration [Ca²⁺]_i in zero and normal [Mg²⁺]_o compared to a much reduced response in elevated [Mg²⁺]_o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB₂ cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca²⁺]_i. In magfura-2-loaded acinar cells CCK-8 (10^–8 M) stimulated an initial transient rise in intracellular free Mg²⁺ concentration [Mg²⁺]_i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na⁺ was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg²⁺ resulted in an elevation in [Mg²⁺]_i. Upon stimulation with CCK-8, [Mg²⁺]_i. decreased only slightly compared with the response obtained in normal [Mg²⁺]_o. CCK-8 caused a net efflux of Mg²⁺ in pancreatic segments; this effect was abolished when extracellular sodium [Na⁺]_o was replaced with either NMDG or choline. The results indicate that Mg²⁺ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca²⁺ mobilization. Moreover, the movement of Mg²⁺ in pancreatic acinar cells is dependent upon extracellular Na⁺.     

Mg2+-sensitive alterations in Ca2+ regulation associated with cell transformation

The intracellular Ca²⁺ content of nontransformed Balb/c3T3 cells is two to three times higher than that of a spontaneously transformed derivative. Depriving either cell type of extracellular Mg²⁺ causes a 2- to 3-fold increase in their Ca²⁺ content over a 24-hr period. Restoring Mg²⁺ to the medium decreases the Ca²⁺ content of the cells to their original values in about the same time. The increase in Ca²⁺ content is not blocked by cycloheximide suggesting that normal rates of protein synthesis are not required to produce this effect. Mg²⁺ deprivation also decreases the initial rate of Ca²⁺ efflux from the transformed cells and increases the size of the slowly exchanging fraction of Ca²⁺ to the levels found in the nontransformed cells. Since Mg²⁺ deprivation normalizes the appearance and growth behavior of the transformed cells, the possible intermediary role of Ca²⁺ in this normalization was studied. Large changes in extracellular Ca²⁺ produced large changes in the Ca²⁺ content of the transformed cells with little change in appearance or thymidine incorporation rate. Ca²⁺ deprivation did inhibit thymidine incorporation in early passage nontransformed cells; however with repeated passage, this effect decreased, as did the Ca²⁺ content of these cells. The possible role of Mg²⁺ in regulating cellular Ca²⁺ content and distribution is discussed, as is the relation of Ca²⁺ content and distribution to the development of the transformed state.     

The ionic components of the current pulses generated by developing fucoid eggs.

Using a newly developed, extracellular vibrating electrode, we studied the ionic composition of the current pulses which traverse the developing Pelvetia embryo. External Na⁺, Mg²⁺, or SO₄^2?, are not needed for the first 20 min of pulsing. In fact, lowering external Na⁺ or Mg²⁺ (or K⁺) actually stimulates pulsing. Since tracer studies show that Ca²⁺ entry is speeded by Na⁺, Mg²⁺, or K⁺ reduction, these findings suggest that Ca²⁺ entry triggers pulsing. A sevenfold reduction in external Cl^? raises pulse amplitudes by 60%. Moreover, Cl^? is the only major ion with an equilibrium potential near the pulse reversal potential. These facts suggest that Cl^? efflux carries much of the “inward” current. We propose a model for pulsing in which increased Ca²⁺ within the growing tip opens Cl^? channels. The resulting Cl^? efflux slightly depolarizes the membrane and thus drives a balancing amount of K⁺ out. Thus, the pulses release KCl and serve to relieve excess turgor pressure. By letting Ca²⁺ into the growing tip, they should also strengthen the transcytoplasmic electrical field which is postulated to pull growth components toward this tip.     

Influence of calcium and magnesium containing fixatives of the ultrastructure of parathyroids

The effect of Ca²⁺ and Mg²⁺ on feline parathyroid cells during perfusion fixation with glutaraldehyde and subsequent immersion in OsO₄ was investigated. Both Ca²⁺ and Mg²⁺ may exert a stabilizing or destabilizing effect on cell membranes and on elements of the cytoskeleton. The effect depends (1) on the ion concentration, (2) on the buffer concentration and (3) on the fixative. Stabilization due to Ca²⁺ or Mg²⁺ during glutaraldehyde fixation is not altered during subsequent osmication but both cations may cause destabilization during osmication in tissue prefixed without cations. Ca²⁺ and Mg²⁺ also reduce cell volume in combination with low osmolar buffer but they prevent cells from excessive shrinkage due to high osmolar buffers. Ca²⁺ and Mg²⁺ alone or in combination reduce swelling of RER, extraction of cellular material and loss of subcellular compartments, such as secretory granules, under optimal conditions. Ca²⁺, however, provokes formation of dark (shrunken) and light (swollen) cells accompanied by loss of subcellular components when used in low concentration during osmication. Low concentrations of Mg²⁺ added to glutaraldehyde exert similar effects. Stabilization of membranes is assumed to be due to the binding capacity of Ca²⁺ and Mg²⁺ to both phospholipids and proteins. The influence of Ca²⁺ and Mg²⁺ to changes in cell volume is considered likely to be the result of ionic interaction in the cytoplasmic gel, the maintenance of cell volume being a matter of equilibrium between the swelling pressure of the cytoplasmic gel and osmotic pressure of the fixative solution.     

Ionic modulation of electrically induced fusion of mammalian cells

Summary During the last few years, a new technique has been developed for the electrofusion of mammalian cells. No previous treatment of the culture is needed, for the contact between cells is spontaneous. Short DC electric pulses are applied directly to a culture growing in monolayers on a culture dish. When the cell density is high enough, contacts occur between cells giving the so-called contact inhibition. In the present study, a systematic investigation of the modulation of the extent of the fusion by the ionic content of the bathing medium during the pulsation is described. An increase in the content in monovalent ions decreases the fusion yield. But this decrease is modulated by the nature of the ion; Li⁺, a potent water structure maker, is less effective than Na⁺ or K⁺. Ca²⁺, when present in the millimolar range, leads to the lysis of the cells. Mg²⁺, when present at concentrations smaller than 4mm, promotes the fusion but prevents it at larger concentrations. Microelectrophoresis measurements show that the electric surface charge is not strongly affected by these changes in ionic content. Our observations are relevant of a modulation of the cell-cell interactions by the ionic content of the bathing medium.     

MagFRET: The First Genetically Encoded Fluorescent Mg2+ Sensor

Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg²⁺, MagFRET-1. This sensor is based on the high-affinity Mg²⁺ binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg²⁺-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg²⁺ affinity (K _d = 148 µM) with a 50% increase in emission ratio upon Mg²⁺ binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg²⁺ affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg²⁺ concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg²⁺ concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca²⁺, this affinity is sufficiently attenuated (K _d of 10 µM) to make the sensor insensitive to known Ca²⁺ stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg²⁺ imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg²⁺ sensors could prove very useful in understanding intracellular Mg²⁺ homeostasis and signaling.