Alterations in the structure of the electrical double layer adjacent to a charged membrane arising from electromagnetically induced changes in the activity of membrane bound enzymes

Electromagnetic fields of very low amplitude have been reported to influence a number of cellular functions. Many of these effects have a high degree of frequency specificity. Herein it is suggested that some of these reported results could be explained by a fieldinduced alteration in the enzymic activity of integral membrane proteins. It is shown that such a field-induced transition from an initial nonequilibrium steady-state to a final nonequilibrium steady-state can lead to an alteration in the concentration profiles of those charged species in the cell's ambient electrolyte that comprise the so-called electrical double layer. Examples of variations in the concentration profiles of those ions that react with a membrane-bound enzyme, as well as nonreacting ionic species, are given. The modulation of such effects by systematic variations in extracellular pH and ionic strength is discussed.     

Synchronization modulation of Na/K pump molecules can hyperpolarize the membrane resting potential in intact fibers

Previously, we have theoretically studied the possibility of electrical rhythmic entrainment of carrier-mediated ion transporters, and experimentally realized synchronization and acceleration of the Na/K pumping rate in the cell membrane of skeletal muscle fibers by a specially designed synchronization modulation electric field. In these studies we either used cut fibers under a voltage clamp or intact fibers, but in the presence of ion channels blockers. A question remained as to whether the field-induced activation observed in the pump molecules could effectively increase the intracellular ionic concentration and the membrane potential at physiological conditions. In this paper, we studied the effects of the field on intact fibers without any channel blockers. We monitored the field-induced changes in the ionic concentration gradient across the cell membrane and the membrane potential non-invasively by using a fluorescent probe and confocal microscopic imaging techniques. The results clearly show that the entrainment of the pump molecules by the synchronization modulation electric field can effectively increase the ionic concentration gradient, and hence, hyperpolarize the membrane potential.     

Thermodynamics,the structure of integral membrane proteins,and transport

Membranes are structures whose lipid and protein components are at, or close to, equilibrium in the plane of the membrane, but are not at equilibrium across the membrane. The thermodynamic tendency of ionic and highly polar molecules to be in contact with water rather than with nonpolar media (hydrophilic interactions) is important in determining these equilibrium and nonequilibrium states. In this paper, we speculate about the structures and orientations of integral proteins in a membrane, and about how the equilibrium and nonequilibrium features of such structures and orientations might be influenced by the special mechanisms of biosynthesis, processing, and membrane insertion of these proteins. The relevance of these speculations to the mechanisms of the translocation event in membrane transport is discussed, and specific protein models of transport that have been proposed are analyzed.     

Nonequilibrium voltage fluctuations in biological membranes. I. General framework of charge transport in discrete systems and related voltage noise

This paper continues our work on the theory of nonequilibrium voltage noise generated by electric transport processes in membranes. Introducing the membrane voltage as a further variable, a system of kinetic equations linearized in voltage is derived by which generally the time-dependent behaviour of charge-transport processes under varying voltage can be discussed. Using these equations, the treatment of voltage noise can be based on the usual master equation approach to steady-state fluctuations of scalar quantities. Thus, a general theoretical approach to nonequilibrium voltage noise is presented, completing our approach to current fluctuations which had been developed some years ago. It is explicitly shown that at equilibrium the approach yields agreement with the Nyquist relation, while at nonequilibrium this relation is not valid. A further general property of voltage noise is the reduction of low-frequency noise with increasing number of transport units as a consequence of the interactions via the electric field. In a second paper, the approach will be applied for a number of special transport mechanisms, such as ionic channels, carriers or electrogenic pumps.     

Monitoring of artificial enzyme membrane systems by electric currents: I. Analytical treatment with direct current and buffered conductivity

Continuous electric fields (E) modify the transport flows and the intramembrane concentration profiles of protons or of ionic substrates or cofactors (inhibitors). These ‘mediators’ induce variations in enzyme activity, quantifiable by a generalized Damköhler group II Ψ distinguishing electrotransport reactions from diffusion reactions. For three typical reaction schemas, using only one mediator, the steady-state equations have been established. Depending on boundary conditions, the direction of electric current (for asymmetrical systems) and the value of Ψ. activations, inhibitions or activations followed by inactivations have been found. With buffered conductivity (supporting electrolyte), the limiting concentration profiles (E → ∞) are uniformly equal to the boundary values; i.e., diffusion constraints are suppressed and the regime is controlled by the reaction. The calculations give the relative activity variations for partially suppressed transport controls.     

Kinetic Theory Model for Ion Movement through Biological Membranes: I. Field-Dependent Conductances in the Presence of Solution Symmetry

A model for ion movement through specialized sites in the plasma membrane is presented and analyzed using techniques from nonequilibrium kinetic theory. It is assumed that ions traversing these specialized regions interact with membrane molecules through central conservative forces. The membrane molecules are approximated as massive spherical scattering centers so that ionic fractional energy losses per collision are much less than one. Equations for steady-state membrane ionic currents and conductances as functions of externally applied electric field strength are derived and numerically analyzed, under the restriction of identical solutions on each size of the membrane and constant electric fields within the membrane. The analysis is carried through for a number of idealized ion-membrane molecule central force interactions. For any interaction leading to a velocity-dependent ion-membrane molecule collision frequency, the membrane chord conductance is a function of the externally applied electric field. Interactions leading to a collision frequency that is an increasing (decreasing) function of ionic velocity are characterized by chord conductances that are decreasing (increasing) functions of field strength. For ion-neutral molecule interactions, the conductance is such a rapidly decreasing function of field strength that the slope conductance becomes negative for all field strengths above a certain value.     

Further studies on the ionic strength-dependent proteolytic activation of protein kinase C in rat liver plasma membrane by endogenous trypsin-like protease

cAMP and Ca2(+)-independent histone kinase was generated from rat liver plasma membrane in an ionic strength-dependent manner by the action of an endogenous trypsin-like protease (Hashimoto, E. et al. (1986) FEBS Lett. 200, 63-66). In addition to the effect of ionic strength, this proteolytic activation of protein kinase proceeded faster at alkaline pH. In an attempt to identify the activated kinase as the protease-activated form of protein kinase C (protein kinase M), the active enzyme released from plasma membrane was highly purified and characterized. Various properties including Mg2+ requirement in histone phosphorylation, substrate specificity, effects of protein kinase activators, and inhibitors and comparison of catalytic properties by peptide map analysis were compatible with those of protein kinase M reported earlier. Immunoblot analyses also supported the idea that the protein kinase subjected to proteolytic activation was protein kinase C. The subtype of protein kinase C detected in this study was identified as type III enzyme encoding alpha-type sequence from the elution profile from hydroxyapatite column. These results suggest that type III protein kinase C bound to rat liver plasma membrane has an ability to be activated by endogenous trypsin-like protease dependently on the alteration of ionic strength and pH around the plasma membrane.     

Determining the target of membrane sterols on voltage-gated potassium channels

Cholesterol, an essential lipid component of cellular plasma membranes, regulates fluidity, mechanical integrity, raft structure and may specifically interact with membrane proteins. Numerous effects on ion channels by cholesterol, including changes in current amplitude, voltage dependence and gating kinetics, have been reported. We have previously described such changes in the voltage-gated potassium channel K_v1.3 of lymphocytes by cholesterol and its analog 7-dehydrocholesterol (7DHC). In voltage-gated channels membrane depolarization induces movement of the voltage sensor domains (VSD), which is transmitted by a coupling mechanism to the pore domain (PD) to open the channel. Here, we investigated whether cholesterol effects were mediated by the VSD to the pore or the PD was the direct target. Specificity was tested by comparing K_v1.3 and K_v10.1 channels having different VSD-PD coupling mechanisms. Current recordings were performed with two-electrode voltage-clamp fluorometry, where movement of the VSDs was monitored by attaching fluorophores to external cysteine residues introduced in the channel sequence. Loading the membrane with cholesterol or 7DHC using methyl-β-cyclodextrin induced changes in the steady-state and kinetic parameters of the ionic currents while leaving fluorescence parameters mostly unaffected in both channels. Non-stationary noise analysis revealed that reduction of single channel conductance rather than that of open probability caused the observed current decrease. Furthermore, confocal laser scanning and stimulated emission depletion microscopy demonstrated significant changes in the distribution of these ion channels in response to sterol loading. Our results indicate that sterol-induced effects on ion channel gating directly target the pore and do not act via the VSD.     

The structure of the retinylidene chromophore in bathorhodopsin.

Resonance Raman data on bathorhodopsin (bovine and squid) at 95,77, and 4 degrees K support a mechanism of excitation proposed by Lewis in which both a protein conformational transition and chromophore structural alteration to a "dicisoid" configuration are required to generate the bathorhodopsin species observed in steady-state photostationary mixtures. However, these results also suggest that the molecular structure with a red-shifted chromophore absorption detected at room temperatures in 1 ps using picosecond absorption spectroscopy may not necessarily have the same chromophore conformation as the steady-state bathorhodopsin species.     

Electroporation of the photosynthetic membrane: structural changes in protein and lipid-protein domains.

A biological membrane undergoes a reversible permeability increase through structural changes in the lipid domain when exposed to high external electric fields. The present study shows the occurrence of electric field-induced changes in the conductance of the proton channel of the H(+)-ATPase as well as electric field-induced structural changes in the lipid-protein domain of photosystem (PS) II in the photosynthetic membrane. The study was carried out by analyzing the electric field-stimulated delayed luminescence (EPL), which originates from charge recombination in the protein complexes of PS I and II of photosynthetic vesicles. We established that a small fraction of the total electric field-induced conductance change was abolished by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the H(+)-ATPase. This reversible electric field-induced conductance change has characteristics of a small channel and possesses a lifetime < or = 1 ms. To detect electric field-induced changes in the lipid-protein domains of PS II, we examined the effects of phospholipase A2 (PLA2) on EPL. Higher values of EPL were observed from vesicles that were exposed in the presence of PLA2 to an electroporating electric field than to a nonelectroporating electric field. The effect of the electroporating field was a long-lived one, lasting for a period > or = 2 min. This effect was attributed to long-lived electric field-induced structural changes in the lipid-protein domains of PS II.     

Liver microsomal membrane lipid composition in marasmic-kwashiorkor

The microsomal membrane cholesterol and phospholipid content and phospholipid composition of marasmic kwashiorkor rats have been compared with those of normal rats. A Significant increase in the cholesterol/phospholipid ratio, as well as in the sphingomyelin/phosphatidyl-choline ratio was observed in the marasmic-kwashiorkor rat. These effects would tend to decrease the fluidity of the phospholipid bilayer of the endoplasmic reticulum membrane and may thus affect drug metabolism.It is well known that a change in the quality or quantity of dietary protein causes an alteration in the rates of metabolism of many xenobiotics by the mammalian liver (1–3). These metabolic alteration have been attributed mainly to changes in the levels of microsomal membrane proteins themselves, especially that of cytochrome P-450 (4–6). However, a recent report by Suzuki et al. (7) indicates that the more subtle features of drug metabolism such as interactions between NADPH-cytochrome P-450 reductase, cytochrome P-450, cytochrome b and other specific drug metabolzing enzymes in the membrane of the endoplasmic reticulum might well be affected by the fluidity of the phospholipid bilayer.There is still a high incidence of protein-energy malnutrition (PEM) diseases such as kwashiorkor in many part of the world (8). The membrane lipid composition from microsomes of marasmic-kwashiorkor rats have therefore been investigated with a view to finding out if there are any changes in these components due to protein deficiency which could contribute to the decreased metabolism of xenobiotics in this condition.     

Interactions of voltage-sensing dyes with membranes. III. Electrical properties induced by merocyanine 540

The effects of merocyanine 540 on the electrical properties of lipid bilayer membranes have been investigated. The alterations this dye was found to produce in the intrinsic conductances of these membranes were minimal, but it profoundly altered the conductances produced by extrinsic permeant species. These alterations were much larger for neutral membranes than for negatively charged ones. The dye increased the conductances mediated by positively charged permeant species and decreased those by negatively charged permeant species, suggesting that it produces a negative electrostatic potential on the membrane; it also altered the kinetics and the voltage dependencies of permeation by these charge carriers. The magnitudes of dye-mediated conductance changes were much larger for positively charged permeants than for negatively charged ones; also, changes in ionic strength altered these dye effects in opposite directions from those predicted by the Stern equation, and the dependence of the conductance alteration on dye concentration was steeper than that predicted by this equation. Finally, only very small changes in liposome zeta potentials were induced by the dye. Calculations show that a large fraction of these effects can be accounted for by the dipole potential produced by merocyanine at the membrane surface, but that additional effects of the dye must be postulated as well.     

Current-Voltage Curves of Porous Membranes in the Presence of Pore-Blocking Ions: I. Narrow Pores Containing No More Than One Moving Ion

We propose a physical model for voltage-dependent conductance changes of excitable cell membranes. It is based on competition of uni- and bivalent ions for chains of stable sites extending through the membrane. These one-dimensional pathways (pores) have different profiles of chemical potential for the two ionic species so that bivalent ions can block the passage of univalent ions at large membrane potentials. We treat the special case that each pore is either empty or, because of electrostatic repulsion, contains no more than one uni- or bivalent ion at a time. A system of linear differential equations describes the time-dependent probabilities of the various possible pore states. The states are limited by transition rate constants involving the profile of the chemical potential, the membrane voltage, the ionic concentrations in the adjacent baths, and electrostatic interactions between the ions. The steady-state solutions (Kirchhoff-Hill theorem) yield expressions for the relationship between the small signal conductance of univalent ions and the concentration of these ions in the external bathing medium (a saturation curve) and for the ionic currents and the steady-state current-voltage curve (N-shaped). From the latter curve we compute the shift of theshold potential caused by concentration changes of the external bathing medium. The model yields a number of predictions which can be tested experimentally.     

The cesium-induced delay in myoblast membrane fusion is accompanied by changes in isolated membrane lipids

We have recently demonstrated that cesium ions delay the sharp decrease in both membrane conductivity and membrane permittivity of chick embryo myoblasts seen at fusion (Santini, M.T., Bonincontro, A., Cametti, C. and Indovina, P.L. (1988) Biochim. Biophys. Acta 945, 56-64). Analysis of the conductivity dispersion data (obtained in the radiowave frequency range) indicated that cesium delays fusion by about 30 h. We suggested that cesium is affecting both active ionic transport by blocking potassium channels as well as interfering with membrane lipid and/or protein charges. In the present study, we have investigated both the possible role of membrane lipids in myoblast fusion and the possible effects of cesium on these lipids. Our data indicate that lipid changes do occur in the isolated myoblast plasma membrane of controls during myogenic differentiation especially prior to fusion and that in cesium cultures these variations do not occur. These variations are in accordance with current membrane fusion theory. Specifically, there is a decrease in bilayer-stabilizing lipids (phosphatidylcholine) and an increase in bilayer-destabilizing ones (phosphatidylethanolamine and phosphatidic acid) and cholesterol during the fusion process. In addition, although slight, during fusion there appears to be a decrease in phosphatidylinositol which is believed to be involved in the inositol phosphate second messenger system. In cesium cultures, in which fusion is greatly delayed, the same lipid changes do not take place and those that are observed seem to reflect the fusion delay.     

Computation of the Electrical Double Layer Properties of Semipermeable Membranes in Multicomponent Electrolytes

A methodology is presented for calculating of the surface potential, Donnan potential, and ion concentration profiles for semipermeable microbial membranes that is valid for an arbitrary electrolyte composition. This model for surface potential, Donnan potential, and charge density was applied to recently reported experimental data for gram-positive bacteria, including Bacillus brevis, Rhodococcus opacus, Rhodococcus erythropolis, and Corynebacterium species. These calculations show that previously unconsidered trace amounts of divalent and trivalent cations at very low concentrations (10⁻⁶ M) can have significant effects on the calculated surface and Donnan potentials, at ionic strengths of I ≤ 0.01 M, and that these effects need to be considered in accurate modeling of microbial surface. In addition, the calculated ion concentration profiles show that owing to the relatively high surface charges that can develop in microbial membranes, electrostatic effects can act to significantly concentrate divalent (factors of 5 × 10³) and trivalent (factors of 2 × 10⁴) cations within the bacterial cell wall. Comparison of the calculated concentration factors with those derived from experiments shows that a significant fraction of the uptake of metal by bacteria can be explained by the proposed electrostatic model.     

Respiration and ecological niche influence bacterial membrane lipid compositions

Bacterial membrane compositions vary widely between phyla and within related species. The types of lipids within membranes are as diverse as the selective pressures that influence bacterial lifestyles such as their mode of respiration and habitat. This study has examined the extent that respiration and habitat affect bacterial fatty acid (FA) and polar lipid (PL) compositions. To accomplish this, over 300 FA and PL profiles from 380 previously characterized species were assembled and subjected to multivariate statistical analyses in order to determine lipid to habitat/respiration associations. It was revealed that PL profiles showed a slight advantage over FA profiles for discriminating taxonomic relationships between species. FA profiles showed greater correlation with respiration and habitat than PL. This study identified that respiration did not consistently favour uniform FA or PL changes when lipid profiles were compared between examined phyla. This suggests that although phyla may adopt similar respiration methods, it does not result in consistent lipid attributes within one respiration state. Examination of FA and PL compositions were useful to identify taxonomic relationships between related species and provides insight into lipid variations influenced by the niche of its host.     

Variation of chemical compounds in wild Heliconiini reveals ecological factors involved in the evolution of chemical defenses in mimetic butterflies

Evolutionary convergence of color pattern in mimetic species is tightly linked with the evolution of chemical defenses. Yet, the evolutionary forces involved in natural variations of chemical defenses in aposematic species are still understudied. Herein, we focus on the evolution of chemical defenses in the butterfly tribe Heliconiini. These neotropical butterflies contain large concentrations of cyanogenic glucosides, cyanide‐releasing compounds acting as predator deterrent. These compounds are either de novo synthesized or sequestered from their Passiflora host plant, so that their concentrations may depend on host plant specialization and host plant availability. We sampled 375 wild Heliconiini butterflies across Central and South America, covering 43% species of this clade, and quantify individual variations in the different CGs using liquid chromatography coupled with tandem mass spectrometry. We detected new compounds and important variations in chemical defenses both within and among species. Based on the most recent and well‐studied phylogeny of Heliconiini, we show that ecological factors such as mimetic interactions and host plant specialization have a significant association with chemical profiles, but these effects are largely explained by phylogenetic relationships. Our results therefore suggest that shared ancestries largely contribute to chemical defense variation, pointing out at the interaction between historical and ecological factors in the evolution of Müllerian mimicry.     

Molecular dynamics simulations of membrane proteins under asymmetric ionic concentrations

A computational method is developed to allow molecular dynamics simulations of biomembrane systems under realistic ionic gradients and asymmetric salt concentrations while maintaining the conventional periodic boundary conditions required to minimize finite-size effects in an all-atom explicit solvent representation. The method, which consists of introducing a nonperiodic energy step acting on the ionic species at the edge of the simulation cell, is first tested with illustrative applications to a simple membrane slab model and a phospholipid membrane bilayer. The nonperiodic energy-step method is then used to calculate the reversal potential of the bacterial porin OmpF, a large cation-specific β-barrel channel, by simulating the I-V curve under an asymmetric 10:1 KCl concentration gradient. The calculated reversal potential of 28.6 mV is found to be in excellent agreement with the values of 26–27 mV measured from lipid bilayer experiments, thereby demonstrating that the method allows realistic simulations of nonequilibrium membrane transport with quantitative accuracy. As a final example, the pore domain of Kv1.2, a highly selective voltage-activated K⁺ channel, is simulated in a lipid bilayer under conditions that recreate, for the first time, the physiological K⁺ and Na⁺ concentration gradients and the electrostatic potential difference of living cells.     

Spectroscopic investigations of the potential-sensitive membrane probe RH421.

The absorbance spectra, fluorescence emission and excitation spectra, and fluorescence anisotropy of the potential-sensitive styryl dye RH421 have been investigated in aqueous solution and bound to the lipid membrane. The potential-sensitive response of the dye has been studied using a preparation of membrane fragments containing a high density of Na+, K(+)-ATPase molecules. In aqueous solution the dye is sensitive both to changes in pH and ionic strength. Evidence has been found that the dye readily aggregates in aqueous solution. Aggregation is enhanced by an increase in ionic strength. The aggregates formed display a low fluorescence intensity. At high pH values (above approx. 8) changes in the dye's fluorescence spectra are observed, which may be due to a reaction of the dye with hydroxide ions. When bound to the membrane the dye also exhibits concentration-dependent fluorescence changes. The potential-sensitive response of the dye in Na(+),K(+)-ATPase membrane fragments after addition of MgATP in the presence of Na+ ions cannot be explained by a purely electrochromic mechanism. The results are consistent with either a potential-dependent equilibrium between membrane-bound dye monomers and membrane-bound dimers, similar to that previously proposed for the dye merocyanine 540, or with a field-induced structural change of the membrane.     

Emission wavelength-dependent decay of the 9-anthroyloxy-fatty acid membrane probes.

Using the phase-modulation technique, we have measured the fluorescence decay of 2- and 12-(9-anthroyloxy)-stearic acid (2- and 12-AS) and 16-(9-anthroyloxy)-palmitic acid (16-AP) bound to egg phosphatidylcholine vesicles or dissolved in nonpolar solvents. Heterogeneity analysis demonstrates that the decay is generally not monoexponential and exhibits large component variations across it emission spectrum. The mean decay time increases (and in parallel, the steady-state polarization decreases) monotonically with increasing wavelength from values at the blue end. The decay at the red side of the emission spectrum contains an exponential term with a negative amplitude, indicating that emission occurs from intermediates created in the excited-state. This behavior is interpreted as arising from intramolecular fluorophore relaxation occurring on the time scale of the fluorescence lifetime. We believe this to be the first study of wavelength-dependent fluorescent emission which is dominated by an intramolecular relaxation process. Although the three probes exhibit qualitatively similar effects, the emission band variations are greatest for 2-AS and smallest for 16-AP. The differences among the probes are not entirely due to environmental factors as demonstrated, for example, by the emission polarization differences observed in the isotropic solvent paraffin oil. In summary, while these findings point out some of the complexities in the 9-anthroyloxy-fatty acids as membrane probes, they also indicate how these complexities might be used as a sensitive measure of lipid-probe interaction.