The effects of some cations and anions on spin labeled cytoplasmic membranes of bacillus subtilis

The effects of CaCl₂, MgCl₂, LaCl₃ and some alkali halides on $\text{Bacillus subtilis}$ cytoplasmic membranes were studied using stearic acid spin labels. The results indicate that two mechanisms are operating when these ions interact with $\text{B. subtilis}$ membranes. At low ionic concentrations (0 to 0.1 M) there is direct cation binding to the anionic membranes which in the case of Ca²⁺, Mg²⁺ and La³⁺, confers rigidity on the membranes and reaches saturation when the number of cations present equals the number of anionic sites on the lipids. At high concentrations there is a further effect on the membranes that parallels the known organizing/disorganizing effects that the ions studied have on water structure.     

Conformational transitions of calmodulin as studied by vacuum-uv CD

CD measurements were made for calmodulin and its calcium (Ca²⁺) complexes at different ionic strengths and Ca²⁺ concentrations. Calmodulin at an ionic strength of 0.00M and in the absence of Ca²⁺ exists as an α-helical protein with a negligible amount of β-sheet. An increase in ionic strength, whether or not Ca²⁺ is present, increases α-helix at the expense of “other” (coil) structure. The changes in β-sheet and β-turns are insignificant. Binding of Ca²⁺ at low ionic strength occurs in stages with at least one folding intermediate before attaining the final stable state. Binding of Ca²⁺ at an ionic strength of 0.165M causes only a slight increase in α-helix, so that the secondary structure of the protein depends on ionic strength and is insensitive to the nature of the cation (i.e., Ca²⁺). Thus, the activation of calmodulin by Ca²⁺ must be due to a structural reorientation rather than to a major secondary structural alteration. The CD estimation of secondary structure with 4 mol Ca²⁺/calmodulin (61% α-helix, 2% antiparallel β-sheet, 2% parallel β-sheet, 21% β-turns, and 14% other) is in excellent agreement with the x-ray results.     

Interaction of alginate single-chain polyguluronate segments with mono- and divalent metal cations: a comparative molecular dynamics study

The interaction of a set of monovalent (Na⁺, K⁺) and divalent (Mg²⁺, Ca²⁺) metal cations with single-chain polyguluronate (periodic chain based on a dodecameric repeat unit, 2₁-helical conformation) is investigated using explicit-solvent molecular dynamics simulations (at 300 K and 1 bar). A total of 14 (neutralising) combinations of the different ions are considered (single type of cation or simultaneous presence of two types of cation, either in the presence or absence of chloride anions). The main observations are: (1) the chain conformation and intramolecular hydrogen bonding is insensitive to the counter-ion environment; (2) the binding of the cations is essentially non-specific for all ions considered (counter-ion atmosphere confined within a cylinder of high ionic density, but no well-defined binding sites); (3) the density and tightness of the distributions of the different cations within the counter-ion atmosphere follow the approximate sequence Ca²⁺>Mg²⁺>K⁺～Na⁺; (4) the solvent-separated binding of the cations to the carboxylate groups of the chain is frequent, and its occurrence follows the approximate sequence K⁺>Na⁺>Ca²⁺>Mg²⁺ (contact-binding events as well as the binding of a cation to multiple carboxylate groups are very infrequent); and (5) the counter-ion atmosphere typically leads to a complete screening of the chain charge within 1.0–1.2 nm of the chain axis and, for most systems, to a charge reversal at about 1.5 nm (i.e. the effective chain charge becomes positive at this distance and as high in magnitude as one-quarter of the bare chain charge, before slowly decreasing to zero). These findings agree well (in a qualitative sense) with available experimental data and predictions from simple analytical models, and provide further insight concerning the nature of alginate–cation interactions in aqueous solution.     

Use of the coulombic interactions of the lanthanide series to identify two classes of Ca2+ binding sites in mitochondria

The degree of inhibition of respiration-dependent vs respiration-independent Ca²⁺ binding by rat liver mitochondria by different members of the lanthanide family was used to establish the existence of two different classes of Ca²⁺ binding sites. The distinction is based on the differences in cation:site interactions between the two classes of sites and the members of the lanthanide series. Lanthanide inhibition of respiration-dependent Ca²⁺ uptake suggests that the binding site is specific for the calcium ion. Those members of the lanthanide family whose ionic radii are nearer that of Ca²⁺ are the best inhibitors. The inhibition of respiration-independent Ca²⁺ binding is much different, indicating non-specific cation absorption.     

Ca2+ buffer sites in intact bovine rod outer segments: Introduction to a novel optical probe to measure ionic permeabilities in suspensions of small particles

Summary The nature of the Ca²⁺ buffer sites in intact rod outer segments isolated from bovine retinas (ROS) was investigated. The predominant Ca²⁺ buffer in intact ROS was found to be negatively charged groups confined to the surface of the disk membranes. Accordingly, Ca²⁺ buffering in ROS was strongly influenced by the electrostatic surface potential. The concentration of Ca²⁺ buffer sites was about 30mm, 80% of which were located at the membrane surface in the intradiskal space. A comparison with observations in model systems suggests that phosphatidylserine is the major Ca²⁺ buffer site in ROS. Protons and alkali cations could replace Ca²⁺ as mobile counterions for the fixed negatively charged groups. At physiological ionic strength, the total number of these diffusible, but osmotically inactive, counterions was as large as the number of osmotically active cations in ROS. The surface potential is dependent on the concentration of cations in ROS and can be measured with the optical dye neutral red. Addition of cations to the external solution led to the release of the internally bound dye as the cations crossed the outer membrane. The chemical and spectral properties of the dye enable its use as a real-time indicator of cation transport across the outer envelope of small particles in suspension. In this study, the dye method is illustrated by the use of well-defined ionophores in intact ROS and in liposomes. In the companion paper this method is used to describe the cation permeabilities native to ROS.     

Ion modulation of membrane permeability: Effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents

Summary Leakage of ions (Na⁺, K⁺) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by pore-forming agent has been studied. Leakage from intact cells isinduced by protons and by divalent cations such as Cu²⁺, Cd²⁺ or Zn²⁺. Leakage from agent-modified cells—or across phospholipid bilayers modified by agent—isprevented by low concentrations of the same cations and by higher concentrations of Ca²⁺, Mn²⁺ or Ba²⁺; Mg²⁺, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca²⁺) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na⁺, K⁺, Rb⁺, NH ₄ ⁺ ) and divalent (Mg²⁺, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn²⁺ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.     

Molecular constituents of the node of Ranvier

The interaction between neurons and glial cells that results in myelin formation represents one of the most remarkable intercellular events in development. This is especially evident at the primary functional site within this structure, the node of Ranvier. Recent experiments have revealed a surprising level of complexity within this zone, with several components, including ion channels, sequestered with a very high degree of precision and sharply demarcated borders. We discuss the current state of knowledge of the cellular and molecular mechanisms responsible for the formation and maintenance of the node. In normal axons, Na⁺ channels are present at high density within the nodal gap, and voltage-dependent K⁺ channels are sequestered on the internodal side of the paranode—a region known as the juxtaparanode. Modifying the expression of certain surface adhesion molecules that have been recently identified, markedly alters this pattern. There is a special emphasis on contactin, a protein with multiple roles in the nervous system. In central nervous system (CNS) myelinated fibers, contactin is localized within both the nodal gap and paranodes, and appears to have unique functions in each zone. New experiments on contactin-null mutant mice help to define these mechanisms.     

Electron paramagnetic resonance evidence of metal-ion binding sites in Micrococcus lysodeikticus (M. luteus) F1-ATPase

Evidence is presented for the presence of divalent cation binding sites in purified F₁-ATPase from Micrococcus lysodeikticus (Micrococcus luteus). Electron paramagnetic resonance studies of native F₁-ATPase indicate that the enzyme binds Mn²⁺ and Cu²⁺. Scatchard-type plot for Mn²⁺ binding to the enzyme indicates the presence of 3–4 independent and identical sites with a dissociation constant of 18.3 · 10⁻⁶ M. Cu²⁺ binds to the enzyme at only one kind of site(s). This Cu²⁺ binding site(s) is characterized by a moderately ionic ligand field provided by the protein and by a tetragonal symmetry of nitrogen and/or oxigen ligands. Competition studies indicate that Mg²⁺ binds at these Mn²⁺ and Cu²⁺ binding sites.     

Anionic detergents as divalent cation ionophores across black lipid membranes

Summary Three ionic detergents commonly used in membrane-bound protein isolation and reconstitution experiments, SDS, cholate, and DOC, are shown to act as divalent cation ionophores when incorporated into black lipid membranes made from either oxidized cholesterol or a mixture of phosphatidylcholine and cholesterol (PC/cholesterol=51 mg). At a concentration greater than or equal to 1 m, SDS shows large selectivity differences between cations and anions and among the different cations tested (Ba²⁺, Ca²⁺, Sr²⁺, Mg²⁺, and Mn²⁺). Deoxycholate and cholate at concentrations greater than 4×10^–4 m and 10^–3 m, respectively, also act as divalent cation ionophores. The selectivity sequence measured for these two detergents is evidence for a strong ionic interaction between the divalent cation, and the anionic charged groups on the detergent. In the case of cholate, the conductance depends on the third or fourth power of the cholate concentration and shows a linear dependence on CaCl₂ concentration. The conductance for deoxycholate depends on the sixth or seventh power of the DOC concentration and is also linearly dependent on the CaCl₂ concentration. In an oxidized cholesterol black lipid membrane in the presence of 5mm CaCl₂, small concentrations of LaCl₃ (<1 m) inhibit the ionophoric activity of each of the detergents tested. Evidence is presented to show that this inhibitory effect is a nonspecific effect on oxidized cholesterol BLM's, and is not due to a direct effect of La³⁺ on detergent-mediated transport.     

Cation Permeability and Selectivity of a Root Plasma Membrane Calcium Channel

Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca²⁺-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K⁺, Na⁺, Ca²⁺ and Mg²⁺ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar K _m for the relationship between unitary conductance and Ca²⁺ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca²⁺ on K⁺ and Na⁺ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca²⁺ influx under physiological conditions. Received: 23 August 1999/Revised: 12 November 1999     

Terbium binding to rat brain acetylcholinesterase. A fluorescence probe of anionic sites

Studies are in progress to characterize the nature of ligand interactions at peripheral anionic sites on mammalian brain AChE, including the beta-anionic or "accelerator" anionic sites where enzyme activity is increased upon Ca2+ binding. Terbium was studied as a fluorescence probe of Ca2+ binding sites in partially purified AChE from whole rat brain. Scatchard analysis of Tb3+ binding in low ionic strength (2 mM) Pipes buffer revealed at least two populations of sites: high affinity sites with Kd(app) approximately 7.6 microM and low-affinity sites with a Kd(app) approximately 49.6 microM. Low-affinity binding was selectively inhibited by 50 mM NaCl; high-affinity binding was completely inhibited by 2 mM CaCl2; and all the bound Tb3+ could be displaced by 1 mM EDTA. The heterogeneity of Tb3+ binding sites is consistent with the multiple, concentration-dependent effects of Tb3+ on enzyme activity.     

Calcium Induced Modulation of Peripheral-Type Benzodiazepine Receptors in Rat Kidney Membranes

Abstract

The effects of Ca²⁺ ions on ³H-RO 5–4864 binding to the peripheral benzodiazepine receptor were examined. Preincubation of rat kidney membranes with Ca²⁺ at 37°C produced a dose-dependent inhibition of ³H-RO 5–4864 binding. No inhibition was observed in membranes preincubated at 0°C.

The effect of Ca²⁺ was competitive in nature and was fully reversed by the addition of EGTA. At 1 mM, the maximal effect was achieved with CaCl₂, whereas CoCl₂ and CdCl₂ had lesser effects. No other divalent cation salts examined decreased ³H-RO 5–4864 binding to rat kidney membranes. Collectively, these data demonstrate that the affinity of ³H-RO 5–4864 binding to rat kidney membranes is regulated by Ca²⁺ and suggest the presence of cation recognition binding sites coupled to the peripheral benzodiazepine receptor.     

The K+ channel in the plasma membrane of rye roots has a multiple ion residency pore

The permeation of K⁺ and Na⁺ through the pore of a K⁺ channel from the plasma membrane of rye roots was studied in planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers. The pore contains at least two ion-binding sites which can be occupied simultaneously. This was indicated by: (i) biphasic relationships with increasing cation concentration of both channel conductance at the zero-current (reversal) potential of the channel (E _rev) and unitary-current at a specified voltage and (ii) a decline in E _rev in the presence of equimolar Na⁺ (cis):K⁺ (trans) as the cation concentration was increased. To determine the spatial characteristics and energy profiles for K⁺ and Na⁺ permeation, unitary-current/ voltage data for the channel were fitted to a three energy-barrier, two ion-binding site (3B2S) model. The model allowed for simultaneous occupancy of binding sites and ionic repulsion within the pore, as well as surface potential effects. Results suggested that energy peaks and energy wells (ion binding sites) were situated asymmetrically within the electrical distance of the pore, the trans energy-well being closer to the center of the pore than its cis counterpart; that the energy profile for K⁺ permeation differed significantly from that of Na⁺ in having a higher cis energy peak and a deeper cis energy well; that cations repelled each other within the pore and that vestibule surface charge was negligible. The model successfully simulated various aspects of K⁺ and Na⁺ permeation including: (i) the complexities in current rectification over a wide range of contrasting ionic conditions; (ii) the biphasic relationships with increasing cation concentration of both channel conductance at E _rev and unitary-current at a specified voltage; (iii) the decline in E _rev in equimolar Na⁺ (cis):K⁺ (trans) as cation concentrations were increased and (iv) the complex relationships between mole fraction and E _rev at total cation concentrations of 100 and 300 mm.We thank Prof. O. Alvarez (Universidad de Chile, Santiago, Chile) for supplying the computer program AJUSTE and Prof. D. Sanders (University of York, UK), Prof. D. Gradmann and Dr. G. Thiel (University of Göttingen, Germany) for stimulating ideas. This work was supported by the Agricultural and Food Research Council.     

The effects of acetate anion on cellulose dissolution and reaction in imidazolium ionic liquids

Quantum mechanical calculations were carried out to determine the mechanisms for the superiority of the imidazolium acetate-based ionic liquids to the corresponding chloride-based ionic liquids. Our results indicate that the imidazolium cation can react with the acetate anion to generate a carbene, a highly reactive intermediate. The carbene produced then reacts with cellulose to facilitate its dissolution in the ionic liquid solvents in addition to the stronger hydrogen bonds formed between the acetate anion and the hydroxyl groups on cellulose. The mechanisms for the imidazolium cation and acetate anion reactions involve the initial ion pairing of the cation and anion via hydrogen bonding and electrostatic interactions. The hydrogen bond formed between the C2–H on the imidazolium cation and COO⁻ of the anion facilitates the transfer of the H⁺ to the anion to form a carbene intermediate.     

The influence of the thylakoid membrane surface properties on the distribution of ions in chloroplasts

Thylakoid membranes isolated from peas have been subjected to ionic analyses using the technique of neutron activation. This has allowed the analyses of K⁺, Na⁺, Mg²⁺, Ca²⁺ and Cl^? to be measured simultaneously on the same sample. By varying the ionic composition of the suspending medium it has been shown that these chloroplast membranes have no obvious chemical specificity for the inorganic cations studied and that the major controlling factor is the electrostatic neutralization of the surface negative charges. In agreement with the Gouy-Chapman theory and for the conditions used, divalent cations were preferentially attracted to the membrane surface. This finding, together with the ionic analysis of the unwashed thylakoids and of isolated intact chloroplasts, indicated that the major physiological surface cation is Mg²⁺ and that K⁺ is probably the main inorganic cation of the stroma. This conclusion is discussed in terms of counterion movement in response to light induced proton pumping at the thylakoid membrane.     

Predicting the disruption by of a protein-ligand interaction

The uranyl cation (UO₂²⁺) can be suspected to interfere with the binding of essential metal cations to proteins, underlying some mechanisms of toxicity. A dedicated computational screen was used to identify UO₂²⁺ binding sites within a set of nonredundant protein structures. The list of potential targets was compared to data from a small molecules interaction database to pinpoint specific examples where UO₂²⁺ should be able to bind in the vicinity of an essential cation, and would be likely to affect the function of the corresponding protein. The C‐reactive protein appeared as an interesting hit since its structure involves critical calcium ions in the binding of phosphorylcholine. Biochemical experiments confirmed the predicted binding site for UO₂²⁺ and it was demonstrated by surface plasmon resonance assays that UO₂²⁺ binding to CRP prevents the calcium‐mediated binding of phosphorylcholine. Strikingly, the apparent affinity of UO₂²⁺ for native CRP was almost 100‐fold higher than that of Ca²⁺. This result exemplifies in the case of CRP the capability of our computational tool to predict effective binding sites for UO₂²⁺ in proteins and is a first evidence of calcium substitution by the uranyl cation in a native protein.     

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study

The interaction of bovine prothrombin with Ca²⁺ and Mg²⁺ ions was investigated by following H⁺ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca²⁺ and Mg²⁺ binding is characterized by high- and low-affinity sites. M²⁺ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H⁺ release by M²⁺ is bell shaped and consistent with controlling pK_a values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca²⁺ and Mg²⁺ titrations following H⁺ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M²⁺. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca²⁺ and remains dimeric in the presence of Mg²⁺. These results and those for metal ion-dependent H⁺ release indicate that H⁺ release occurs concomitantly with association processes involving prothrombin.Abbreviations GLA -carboxyglutamic acid; fragment 1. amino terminal residues 1–156 of bovine prothrombin - MES 2-(N-morpholino) ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - PS/PC phosphatidylserine/phosphatidylcholine vesicles - ionic strength     

Cation binding to brain plasma membranes: An evaluation of the use of anionic fluorescent probes

Cation binding to brain plasma membranes has been studied using anionic sulfonate fluorescent probes. Ion affinity sequences follow the order Mg²⁺ > Ca²⁺ ? K⁺ > Cs⁺ > Na⁺ > Li⁺. The order of effectiveness, in increasing probe fluorescence, is the reverse of the affinity sequence for ions of the same charge. The affinity orders for erythrocyte membranes and dipalmitoyl lecithin are Mg²⁺ > Ca²⁺ ? Cs⁺ > K⁺ > Na⁺ > Li⁺ and Mg²⁺ > Ca²⁺ ? Li⁺ > Na⁺ > K⁺ > Cs⁺. These sequence variations are related to the differences in the nature of the ion binding sites. Heterogeneity in ion binding sites is demonstrated. Evidence is presented for the role of proteins in binding hydrophobic probes. The problem of separating specific conformational effects on ion binding from nonspecific charge neutralization effects is discussed. Pyrene excimer fluoresence rules out the possibility of extensive changes in mobility in the lipid phase on cation binding. Tetrodotoxin has been shown to inhibit Li⁺-, Na⁺-, and K⁺-induced fluorescence enancements of 1-anilino-8-naphthalene sulfonate bound to brain membranes.     

Relationships between water chemistry and respiration rate in several populations of lymnaea peregra Müller (Gastropoda: Mollusca)

Respiration rates of specimens of L. peregra obtained from waters with a wide range of ionic concentrations were measured using a constant volume respirometer. Multiple regression analysis showed that hardness of the water from which the snails were taken could significantly affect dry-wet weight relationships, oxygen consumption in relation to shell-free dry weight and oxygen consumption in relation to live wet weight. However there were also significant differences in respiration rate between populations from within water hardness categories (hard > 40 mg l^–1 Ca²⁺, medium 40 – 10 mg l^–1 Ca²⁺, soft < 10 mg l^–1 Ca²⁺). In experiments with constant anion but different cation concentrations, calcium and potassium concentrations were the most significant variables tested. There were no differences in respiration rates of animals taken from the same sites in different years.     

Nutrient uptake as a contributing explanation for deep rooting in arid and semi-arid ecosystems

Explanations for the occurrence of deep-rooted plants in arid and semi-arid ecosystems have traditionally emphasized the uptake of relatively deep soil water. However, recent hydrologic data from arid systems show that soil water potentials at depth fluctuate little over long time periods, suggesting this water may be rarely utilized or replenished. In this study, we examine the distributions of root biomass, soil moisture and nutrient contents to 10-m depths at five semi-arid and arid sites across southwestern USA. We couple these depth distributions with strontium (Sr) isotope data that show deep (>1 m) nutrient uptake is prevalent at four of the five sites. At all of the sites, the highest abundance of one or more of the measured nutrients occurred deep within the soil profile, particularly for P, Ca²⁺ and Mg²⁺. Phosphate contents were greater at depth than in the top meter of soil at three of five sites. At Jornada, for example, the 2–3 m depth increment had twice the extractable P as the top meter of soil, despite the highest concentrations of P occurring at the surface. The prevalence of such deep resource pools, and our evidence for cation uptake from them, suggest nutrient uptake as a complementary explanation for the occurrence of deep-rooted plants in arid and semi-arid systems. We propose that hydraulic redistribution of shallow surface water to deep soil layers by roots may be the mechanism through which deep soil nutrients are mobilized and taken up by plants.Electronic Supplementary Material Supplementary material is available for this article at