Fabrication and Evaluation of Membranes as Specific Electrodes for Calcium ions

Membranes fabricated from an inert polyvinyl chloride matrix impregnated with tributyl phosphate and with tributyl phosphate plus thenoyl trifluoroacetone were tested as electrodes specific for calcium ions. Both types of membrane exhibited a high specificity for calcium ions in the presence of sodium, magnesium, and barium ions. In the presence of perturbing ions, the usefulness of membranes is limited by both the transport of current and development of potentials by the interfering ion. An experimental method for evaluating these interfering quantities is presented.     

Liquid membrane phenomenon in the actions of digitalis

The liquid membrane phenomenon in the actions of digitalis glycosides (digitoxin, digoxin and ouabain) has been studied. Formation of liquid membranes, in series with a supporting membrane, by digitalis alone and by digitalis in association with lecithin and cholesterol has been demonstrated. The results obtained on the transport of relevant permeants, viz. sodium, potassium and calcium ions and dopamine, adrenaline, noradrenaline and serotonin, in the presence of the liquid membrane generated by digitalis in association with lecithin and cholesterol indicate that the liquid membrane barrier to transport may have a relevance with the biological actions of digitalis.     

Two selective filters in the calcium channel of the molluscan neuron somatic membrane

Modification of the calcium channel of the somatic membrane of molluscan neurons under the influence of EDTA and other Ca-binding agents was investigated. The results showed that there are two selective filters in the calcium channel of this membrane. The first is located near the outer pore of the calcium channel and it binds bivalent cations in the order:pK _Ca:pK _Sr:pK _Ba:pK _Mg=6.6:5.5:4.8:4.2. This external filter regulates selectivity of the channel relative to the charge of the cation and it conjecturally contains several carboxyl groups. The second selective filter lies inside the channel and regulates permeability for ions with a single charge. It is suggested that the structure of the inner filter closely resembles that postulated by Hille for the selective filter of the sodium channel, and that it contains one carboxyl group. The results of investigation of the effect of Ca⁺⁺, Cd⁺⁺, and H⁺ on the fast sodium current of the somatic membrane showed that it is not blocked by these ions, but the decrease observed in its amplitude is connected with a change in the membrane surface potential and a corresponding change in the juxtamembranous concentration of carrier ions. On the basis of the experimental results it is postulated that the selective filter of the fast sodium channel of the molluscan neuron somatic membrane does not contain a carboxyl group.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 15, No. 4, pp. 420–427, July–August, 1983.     

Characteristics of apple snail giant neurons capable of generating action potentials in sodium-free solutions

The ability of apple snail giant neurons to generate action potentials in solutions that lack sodium ions is associated with the input resistance of these neurons in such a way that the higher the input resistance is, the more pronounced is this ability. Neurons in which this ability is well expressed usually exhibit low resting potential values and a slow repolarization phase. When calcium ions are replaced with barium ions, the neurons retain their excitability in a sodium-free medium for a longer period of time. Raising the calcium ion concentration to 30 µmole may exert a restorative effect on neurons that have lost their excitability in a solution that originally lacked sodium ions but contained 10 µmole of calcium ions. Increasing the calcium ion concentration to 60 µmole leads to loss of excitability, which under these conditions can be restored by means of depolarizing the neuron with an outward current. The results are discussed from the point of view of the theory of ionic conductivity of the surface membrane of neurons. It is hypothesized that the ability of the surface membrane of neurons to make use of sodium or calcium ions in generation of action potentials depends upon its permeability to potassium ions.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 1, pp. 100–106, January–February, 1970.     

The effect of calcium on the properties of charged phospholipid bilayers

We have performed molecular dynamics simulations to investigate the structure and dynamics of charged bilayers as well as the distribution of counterions at the bilayer interface. For this, we have considered the negatively charged di-myristoyl-phosphatidyl-glycerol (DMPG) and di-myristoyl-phosphatidyl-serine (DMPS) bilayers as well as a protonated di-myristoyl-phosphatidyl-serine (DMPSH) bilayer. We were particularly interested in calcium ions due to their important role in biological systems. Simulations performed in the presence of calcium ions (DMPG, DMPS) or sodium ions (DMPS) were run for 45-60 ns. Simulation results for DMPG are compared with fluorescence measurements. The average areas per molecule were 47.4+/-0.5 A2 (DMPG with calcium), 47.3+/-0.5 A2 (DMPS with calcium), 51.3+/-1.0 A2 (DMPS with sodium) and 45.3+/-0.5 A2 (DMPSH). The structure of the negatively charged lipids is significantly affected by the counterions, where calcium ions have a more pronounced effect than sodium ions. Calcium ions were found to be tightly bound to the anionic groups of the lipid molecules and as such appear to constitute an integral part of the membrane interface on nanoseconds time scales. In contrast to sodium ions, calcium ions are localised in a narrow (approximately 10 A) band around the phosphate group. The interaction of calcium with the lipid molecules enhances the molecular packing of the PG and PS lipids. This observation is in good agreement with emission spectra of the membrane partitioning probe Laurdan in DMPG multilamellar vesicles that indicate an increase in the ordering of the DMPG bilayer due to the presence of calcium. Our results indicate that calcium ions, which often function as a second messengers in living cells have a pronounced effect on membrane structures, which may have implications during signal transduction events.     

Interactions of mono- and divalent counterions with alkali- and enzyme-deesterified pectins in salt-free solutions

The free fractions of monovalent and divalent counterions were determined on salt-free solutions of pectins. The effects of charge density, distribution of the carboxyl groups, polymer concentration, and the nature of the counterion were investigated by determinating the calcium and sodium activity coefficients (with specific electrodes) and by measuring the transport parameters (by conductimetry). Poor agreement for calcium ions was found with the Manning theory. The strong binding of these ions to highly charged polymers, which is ascribed to a dimerization process was demonstrated in very dilute solutions.     

Evidence against Hydrogen–Calcium Competition Model for Activation of Electrically Excitable Membranes

TO explain the voltage-dependent sodium permeability of excitable membranes, Stephens¹ proposed a model in which sodium-selective channels are normally blocked by calcium ions bound to negatively charged sites located near the outer end of the channels. The calcium ions can be displaced competitively by hydrogen ions, opening the channels to sodium. According to this model, depolarization of an excitable membrane causes an outward flow of hydrogen ions across the membrane. The consequent transient increase in hydrogen ion concentration at the outer surface of the membrane displaces calcium and opens the sodium channels. This model is particularly interesting because it is sufficiently specific to allow direct tests. Stephens shows that it is in general agreement with a variety of experimental data. To test the model further, we have determined the effect of variation in the internal and external concentration of hydrogen ions on sodium currents.     

Modelling Metal Complexes of Calixarene Esters and Phosphine Oxides Using Molecular Mechanics

This paper focuses on the molecular modelling of a number of calixarene ester and phosphine oxide metal ion complexes. Monte Carlo conformational searches, in conjunction with the Merck Molecular Force Field, were carried out using Spartan SGI Version 5.0.1. running on Silicon Graphics O2 workstations. In the case of the calix[4]arene tetraesters, the optimised models strongly suggest that the selectivity of these ligands is strongly related to the eight-fold nature of the coordination with the Na⁺ ion, while coordination with the Li⁺ ion, for example, is merely three-fold. This feature of eight-fold coordination is also observed in the models of the complexes formed by the calix[4]arene tetraphosphine oxides with calcium. However, whereas the eight-fold coordination is unique to the model of the TPOL:Ca²⁺ complex among the ions modelled, this mode of coordination occurs for TPOS with sodium and potassium, in addition to calcium. This concurs with the observation that calcium selectivity is obtained with ion selective electrodes based on TPOL but not TPOS. Though the cavity in the calix[5]arenes PPOL and PPOLx and the calix[6]arene HPOL, in their uncomplexed form, are much larger than that of the corresponding calix[4]arenes, the pattern of selectivity is the same – the ligands are selective for calcium. The models of the complexes of these larger calixarenes, such as PPOL:Ca²⁺, strongly suggest that the reason for this similarity is that four of the available phosphine oxide groups complex with the calcium ion, and the others are forced away from the cavity region for steric reasons. The resulting eight-fold coordination, is therefore, similar to that of the calix[4]arenes studied.Electronic Supplementary Material available.     

Free-standing nanogold membranes as supports for the growth of calcium phosphate crystals

Current strategies for bone tissue regeneration focus on the development of implantable matrices that mimic biological tissues. Inorganic composites are of special interest for bone substitute applications. It is necessary to create an artificial three-dimensional scaffold-like porous material with certain geometrical structure to induce bone growth. We report here the growth of calcium phosphate crystals on free-standing carboxylic acid functionalized gold nanoparticle membranes. The gold nanoparticle membrane is synthesized by the spontaneous reduction of aqueous chloroaurate ions by a diamine molecule at a liquid-liquid interface. This membrane is robust and malleable, and most importantly, the gold nanoparticles in the membrane may be functionalized with suitable ligands. In this study, the amino acids aspartic acid and cysteine together with an aromatic bifunctional molecule, anthranilic acid, were used to modify the surface of the gold nanoparticles in the membrane. The free carboxylic acid groups on the gold nanoparticles further to functionalization with these molecules were then used to bind Ca(2+) ions and reacted with phosphate ions to yield calcium phosphate. The nature of the nanogold surface modifier directed the formation of either crystalline hydroxyapatite or amorphous calcium phosphate. The nanogold membrane thus suggests potential biomedical application as biocompatible implants and grafts.     

Internal citrate ions reduce the membrane potential for contraction threshold in mammalian skeletal muscle fibers.

An effect of internal citrate ions on excitation-contraction coupling in skeletal muscle is described. The threshold for contraction was measured in rat extensor digitorum longus, (EDL), and soleus muscle fibers using a two microelectrode voltage clamp technique with either KCl-filled or K3 citrate-filled current electrodes. Contraction thresholds were stable for many minutes with KCl current electrodes. In contrast, thresholds fell progressively towards the resting membrane potential, by as much as -15 mV over a period of 10 to 20 min of voltage-clamp with citrate current electrodes. In addition, prepulse inhibition was suppressed, subthreshold activation enhanced and steady-state inactivation shifted to more negative potentials. Fibers recovered slowly from these effects when the citrate electrode was withdrawn and replaced with a KCl electrode. The changes in contraction threshold suggest that citrate ions act on the muscle activation system at an intracellular site, since the citrate permeability of the surface membrane is probably very low. An internal citrate concentration of 5 mM was calculated to result from citrate diffusion out of the microelectrode into the recording area for 20 min. 5 mM citrate added to an artificial cell lowered the free calcium concentration from 240 to 31 microM. It is suggested that citrate modifies excitation-contraction coupling either by acting upon an anion-dependent step in activation or by reducing the free calcium and/or free magnesium concentration in the myoplasm.     

The effects of cations upon the action potentials recorded from neurohaemal tissue of the stick insect

Summary The ionic requirement for the action potentials recorded from the neurohaemal tissue on the lateral branch of the median nerve inCarausius morosus has been studied using extracellular electrodes. Sodium-free, magnesium-free, or calcium-free salines produce irreversible block of the action potentials following prolonged exposure to the nerves. Reducing the sodium concentration to 4 mM has little effect on the amplitude of the action potentials, whilst increasing the sodium concentration to 100 mM reduces the amplitude by 50%. Neither tetrodotoxin nor procaine has any effect on these action potentials.Reducing the magnesium concentration to 1 mM increases the amplitude of the action potentials, whilst increasing the concentration of magnesium reduces the amplitude.The amplitude of the action potentials is linearly related to the log of the external calcium concentration, and the action potentials are blocked by both cobalt ions and lanthanum ions.It is concluded that calcium is the major charge carrier of the inward current in these neurosecretory axons which is the first report of calcium dependent action potentials in a nerve axon. Furthermore, small amounts of sodium and magnesium are necessary to maintain electrical activity. Magnesium is a competitive inhibitor of the calcium currents.We are grateful to the Science Research Council for financial support, and to Mrs. J. Birch for the printing of the electron micrographs.     

Relation of Ca2+ efflux from the heart to the concentration of Ca-channel blockers

Dependence of calcium ion efflux from the heart ventricles on the concentration of verapamil, fenigidine and nicardipine was studied in frogs, using Ca-sensitive electrodes. The effect of sodium and potassium ions was also investigated. It was established that dependence of calcium ion efflux (delta Ca2+) on the concentration of Ca-antagonists (B) may be expressed by the following formula: (Formula: see text). With cellular membrane depolarization 50 mM KCl, none of the blockers (10(-5) M) caused Ca2+ efflux from ventricular cells. Analogous phenomenon was noted in low-sodium solution (60 mM).     

Thermodynamics of ATP hydrolysis from membrane electrode measurements of metal-ion ATP and ADP complexation

Free energies for the ATP-ADP hydrolysis reaction have been recalculated on the basis of new thermodynamic formation constants for metal-ion ATP and ADP complexes as determined by potentiometric measurements with ion-selective membrane electrodes. It is shown that free-energy maps are sharply altered at metal-ion levels greater than 10^?3m because of the effect of previously unrecognized 2:1 complexes of divalent metal ions with ATP and ADP. New formation constants for ADP complexes with sodium, potassium, calcium, and magnesium are also reported.     

A model to explain ion-induced differentiation in zoospores ofPhytophthora palmivora

Zoospores ofPhytophthora palmivora undergo synchronous encystment followed by germination when exposed to sodium ions at 3–10 mM, calcium ions at 5–30 mM, and strontium ions at 0.3–10 mM. Lithium ions induce encystment at 3–10 mM but do not induce germination. Ferric, manganese, and barium ions.act as chaotropic agents, damaging the zoospore plasma membrane under both isoosmotic and hypoosmotic conditions, at concentrations as low as 30 μM, ferric ions and 1 mM, barium ions. Cesium and ammonium ions are cytotoxic and induce lysis under hypoosmotic conditions. The capacity of cations to induce zoospore differentiation can be explained in terms of the following model. It is suggested that differentiation inP. palmivora requires the entry of sodium at one or more specific sites. Access to these sites is regulated by a calcium gated monovalent ion translocator with a high specificity for sodium. Calcium not only regulates sodium ion access but also regulates the cell's osmoregulatory system. Divalent cations act by competing for the calcium binding site, monovalent cations by restricting fodium ion access.     

Changes in "calcium" action potentials in neurons of the rat spinal ganglia during replacement of divalent cations in the extracellular medium

Single neurons of rat spinal ganglia were investigated in adult rats using a voltage clamping technique and intracellular microelectrodes. Removing sodium ions from the extracellular medium and adding tetraethylammonium to it enabled the calcium component of action potentials to be recorded. It was found that progressive selective suppression of this component takes place during extracellular recording, indicating a decrease in calcium conductivity, while sodium and potassium levels are maintained. It is suggested that this disturbance is caused by excessive influx of calcium, strontium, or barium ions into the cell. The calcium component of action potentials was also found to depend on stimulation rate; this dependence differed where calcium ions were replaced by strontium or barium ions. A possible connection between this effect and the process of voltage-dependent inactivation of calcium channels is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 202–207, March–April, 1986.     

Allosteric cotransport of sodium,chloride, and calcium by the intestine of freshwater prawns

Summary The apical membrane of the intestinal epithelium of the freshwater prawn,Macrobrachium rosenbergii, has been found to possess an apparently unique allosteric carrier mechanism for the simultaneous cotransport of sodium, chloride, and calcium from mucosal solution to cytosol. Influxes of the two monovalent ions individually were sigmoidal functions of their respective luminal concentrations, and their kinetics followed the Hill equation for homotropic cooperativity between identical binding ligands. Increased influx of chloride sigmoidally stimulated enhanced influx of sodium, suggesting the occurrence of heterotropic cooperativity between the dissimilar ligands. Calcium entry displayed hyperbolic (Michaelis-Menten) kinetics, and this cation was found to act both as an allosteric activator of sodium entry on the shared carrier system by associating with a discrete divalent cation binding site, as well as functioning as a possible competitive inhibitor of the monovalent cation binding process. Chloride was neither an allosteric activator nor inhibitor, but appeared to function mainly as an affinity modifier of the allosteric protein for sodium.     

Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12

Atomic absorption spectroscopy of isolated native and EDTA-modified (lipopolysaccharide-depleted) outer membrane revealed trace amounts of potassium, manganese, and iron (1.0-7.0 nmol/mg dry weight outer membrane). Sodium, magnesium, and calcium were approximately one order of magnitude more plentiful, but EDTA-modified outer membrane was deficient in calcium. When metal-binding assays were conducted to find the binding capacity of native and EDTA-modified outer membrane, potassium bound poorly compared with sodium. However, there was no difference in the binding of these ions between the OM preparations. In contrast, reduced amounts of magnesium, calcium, manganese, and iron III bound to the EDTA-modified OM. Partitioning of intact cells in a biphasic dextran-polyethyleneglycol system indicated that the reduced lipopolysaccharide content of the EDTA-modified outer membrane increased the hydrophobicity of the cell surface. Exposure of control and EDTA-treated cells to divalent metal salt solutions before phase partitioning also increased cell surface hydrophobicity. Freeze-etching showed that sodium ions had no effect on the membrane fractures observed in control cells, but with EDTA-treated cells, this cation increased the occurrence of small outer membrane fractures (plateaus) which are characteristic of EDTA treatment. Both magnesium and manganese increased the frequency of outer membrane cleavage in control cells, whereas calcium did not. In contrast, all three divalent metallic ions increased the frequency and extent of cleavage in the outer membrane of EDTA-treated cells.     

Electrical Properties of Neurospora crassa : Effects of external cations on the intracellular potential

Glass micropipette electrodes have been employed to study the transsurface potential difference of Neurospora crassa. For mature hyphae grown in agar cultures, the internal potential is large and negative, often exceeding -200 mv. The potential is sensitive to the concentrations of extracellular potassium, sodium, hydrogen, and calcium ions, but does not vary in a manner which is readily explained by ionic diffusion potentials. With extracellular solutions containing only potassium chloride (or sulfate) and sucrose, the internal potential shifts toward zero (becomes less negative) at 45 mv per tenfold increase of potassium, over the range 0.1 to 10 mM. A similar result has been found with sodium, though the slope is only 33 mv/log unit. Calcium (1 mM) diminishes the influence of potassium and sodium by 60 to 70 per cent. As potassium or sodium is raised above 20 mM, the slope of the internal potential increases sharply to 85 to 90 mv/log unit, both in the presence and absence of calcium. With increasing hydrogen ion concentration, too, the internal potential shifts toward zero; in this case the slope is about 12 mv/pH unit at pH 9 and rises smoothly to 33 mv/pH unit at pH 3. All these phenomena are probably properties of the plasma membrane. The polysaccharide cell wall contains few fixed negative charges, has a low transverse resistance, and supports very little potential difference when separated from the plasma membrane.     

The effect of calcium on the properties of charged phospholipid bilayers

We have performed molecular dynamics simulations to investigate the structure and dynamics of charged bilayers as well as the distribution of counterions at the bilayer interface. For this, we have considered the negatively charged di-myristoyl-phosphatidyl-glycerol (DMPG) and di-myristoyl-phosphatidyl-serine (DMPS) bilayers as well as a protonated di-myristoyl-phosphatidyl-serine (DMPSH) bilayer. We were particularly interested in calcium ions due to their important role in biological systems. Simulations performed in the presence of calcium ions (DMPG, DMPS) or sodium ions (DMPS) were run for 45-60 ns. Simulation results for DMPG are compared with fluorescence measurements. The average areas per molecule were 47.4 ± 0.5 Å² (DMPG with calcium), 47.3 ± 0.5 Å² (DMPS with calcium), 51.3 ± 1.0 Å² (DMPS with sodium) and 45.3 ± 0.5 Å² (DMPSH). The structure of the negatively charged lipids is significantly affected by the counterions, where calcium ions have a more pronounced effect than sodium ions. Calcium ions were found to be tightly bound to the anionic groups of the lipid molecules and as such appear to constitute an integral part of the membrane interface on nanoseconds time scales. In contrast to sodium ions, calcium ions are localised in a narrow (∼ 10 Å) band around the phosphate group. The interaction of calcium with the lipid molecules enhances the molecular packing of the PG and PS lipids. This observation is in good agreement with emission spectra of the membrane partitioning probe Laurdan in DMPG multilamellar vesicles that indicate an increase in the ordering of the DMPG bilayer due to the presence of calcium. Our results indicate that calcium ions, which often function as a second messengers in living cells have a pronounced effect on membrane structures, which may have implications during signal transduction events.     

Development of electrochemical calcium sensors by using silicon nanowires modified with phosphotyrosine

This paper reports the electrical detection of calcium ions by using silicon nanowires (SiNWs) as channels in a chemically gated field-effect-transistor (FET) configuration. To obtain a selective and sensitive layer for calcium sensing, the SiNWs are modified with a biologically relevant amino acid phosphotyrosine (p-Tyr), which is able to complex calcium ions with high affinity. It is found that when the p-Tyr modified SiNWs are exposed to aqueous solutions containing calcium ions, their conductances increase with the increasing of calcium concentration up to 10microM. In contrast, when the SiNWs are exposed to sodium or potassium, or when they are modified with tyrosine (Tyr), no significant increase in the conductance is observed. This finding suggests that the calcium ions complexed with the phosphate group of p-Tyr can act as a positive gate voltage on the FET device comprising of n-type SiNWs, and leads to an increase in their conductances. The FET device is also sensitive to magnesium ions. However, the response is 10 times lower than that of calcium at the same concentration. The study reported here may pave the way for designing an intracellular calcium sensor which permits the monitoring of calcium concentration in real time.