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 field-induced 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.     

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.     

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.     

The human erythrocyte membrane skeleton may be an ionic gel

Biochemical and biophysical observations indicate that the erythrocyte membrane skeleton is composed of a swollen network of long, flexible and ionizable macromolecules located at the cytoplasmic surface of the fluid membrane lipid bilayer. We have analyzed the mechanochemical properties of the erythrocyte membrane assuming that the membrane skeleton constitutes an ionic gel (swollen ionic elastomer). Using recently established statistical thermodynamic theory for such gels, our analysis yields mathematical expressions for the mechanochemical properties of erythrocyte membranes that incorporate membrane molecular parameters to an extent not achieved previously. The erythrocyte membrane elastic shear modulus and maximum elastic extension ratio predicted by our membrane model are in quantitative agreement with reported values for these parameters. The gel theory predicts further that the membrane skeleton modulus of area compression, K _G, may be small as well as large relative to the membrane elastic shear modulus, G, depending on the environmental conditions. Our analysis shows that the ratio between these two parameters affects both the geometry and the stability of the favoured cell shapes.     

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.     

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.     

An examination of the factors involved in determining phosphatase activities in estuarine waters. 2: Sampling procedures

Sampling procedures which are significant in the assessment of phosphatase activities in water bodies have been studied. Variations in neutral phosphatase activities among sampling stations and over depth profiles were considered, as well as seasonal and diurnal variations. Large differences were found in neutral phosphatase activities between sampling stations, but the activities in water samples from within a sampling area varied by the less than 3%. Most frequently, phosphatase activity was constant throughout the water column until just above the sediment. Distinct diurnal patterns were always found, but the patterns were not consistent between either sampling stations or sampling dates. Phosphatase activity also varied seasonally. All of the above variations may reflect differences in biomass composition and concentration. The implications of such variations for setting up an adequate sampling program and for assessing data are stressed.     

Periodic responses in squid axon membrane exposed intracellularly and extracellularly to solutions containing a single species of salt

Summary A periodic membrane potential change was found to occur in squid giant axons which were internally and externally perfused with solutions of an identical composition and were hyperpolarized by passing a sustained inward current. The solution contained Co²⁺ or Mn²⁺ as the sole cation species at a concentration of 1–10mm. The amplitude of the response was roughly 100 mV. The current intensity and the ion concentration had large effects on the response. The voltage-clamp technique revealed an N-shapedI-V characteristic of the membrane system. The membrane emf of the resting and excited states was almost the same but the membrane conductance was increased in the excited states. The response was suppressed with 4-aminopyridine reversibly but unchanged with tetrodotoxin or D-600. Those unusual ionic conditions did not deprive axons of their ability to produce ordinary action potentials in physiological solutions. The experimental conditions employed and the results obtained were very close to those for some of the artificial membrane models. Applicability of the physico-chemical theories developed for these models is discussed.     

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.     

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.     

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.     

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells

The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.     

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.     

Role of membrane organization and membrane domains in endocytic lipid trafficking

Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.     

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.     

Effects of arachidonic acid and the other long-chain fatty acids on the membrane currents in the squid giant axon

Summary The effects of arachidonic acid and some other long-chain fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular application of the test substances. The effects of these acids, which are usually insoluble in solution, were examined by using -cyclodextrin as a solvent. -cyclodextrin itself had no effect on the excitable membrane. Arachidonic acid mainly suppresses the Na current but has little effect on the K current. These effects are completely reversed after washing with control solution. The concentration required to suppress the peak inward current by 50% (ED₅₀) was 0.18mm, which was 10 times larger than that of medium-chain fatty acids like 2-decenoic acid. The Hill number was 1.5 for arachidonic acid, which is almost the same value as for medium-chain fatty acids. This means that the mechanisms of the inhibition are similar in both long- and medium-chain fatty acids. When the long-chain fatty acids were compared, the efficacy of suppression of Na current was about the same value for arachidonic acid, docosatetraenoic acid and docosahexaenoic acid. The suppression effects of linoleic acid and linolenic acid on Na currents were one-third of that of arachidonic acid. Oleic acid had a small suppression effect and stearic acid had almost no effect on the Na current. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (Hodgkin, A.L., Huxley, A.F. (1952)J. Physiol (London) 117:500–544) and the change in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m ) for the Na current against membrane potential and the time constant of activation ( _m ) were shifted 10 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation ( _h ) has almost unaffected by application of these fatty acids.     

Diffusion of energetic compounds through biological membrane: Application of classical MD and COSMOmic approximations

Computational studies of the potential biological impact of several energetic compounds were performed. The most commonly used explosives were considered in the present studies: trinitrotoluene (TNT), 2,4-dinitrotoluene (2,4-DNT), 2,4-dinitroanisole (DNAN), and 5-Nitro-2,4-dihydro-3H-1,2,4-triazol-3-one (NTO). The effect of such factors as ionic strength and presence of DMSO in the water solution on the structure of the membrane were considered using the POPC lipid bilayer as an example. Molecular dynamics (MD) simulations revealed that, even on a short-time scale, the influence of those additives is noticeable, and therefore those factors should always be taken into account. The MD and the COSMOmic approaches were used to elucidate the ability of the energetic compounds to penetrate the living cell. Calculated free energy profiles and partitioning coefficients revealed distributions of the compounds in the lipid bilayer as well as an overall ability to enter the cell. MD in this case provides a better representation of the free energy profile, while the COSMOmic approach works better to predict log(K_lipw) values. The effect of the functional group was observed for the profiles that were obtained using the MD method.     

Effects of dietary eritadenine on the liver microsomal Delta6-desaturase activity and its mRNA in rats

Eritadenine, a hypocholesterolemic factor of Lentinus edodes mushroom, has a wide range of effects on lipid metabolism such as an increase in the liver microsomal phosphatidylethanolamine (PE) concentration, a decrease in the liver microsomal Delta6-desaturase activity, and an alteration of the fatty acid and molecular species profile of liver and plasma lipids. In this study, the time-dependent effects of dietary eritadenine on several variables concerning lipid metabolism were investigated in rats to clarify the sequence of metabolic changes caused by eritadenine, with special interest in the association of the liver microsomal phospholipid profile and the activity of Delta6-desaturase. The effect of dietary eritadenine on the abundance of mRNA for Delta6-desaturase was also investigated. When the time required for a half-change of variables was estimated during the first 5 days after the change from the control diet to the eritadenine-supplemented (50 mg/kg) diet, the change rates of the variables were fastest in the following order: alteration of the liver microsomal phospholipid profile>decrease in liver microsomal Delta6-desaturase activity>alteration of the fatty acid and molecular species profiles of microsomal and plasma phosphatidylcholine (PC)>decrease in the plasma cholesterol concentration. There was a significant correlation between the Delta6-desaturase activity and liver microsomal PE concentration, but not PC concentration, or the proportion of PC and PE or the PC/PE ratio. The suppression of Delta6-desaturase activity by dietary eritadenine was accompanied by a significant reduction in the abundance of mRNA for the enzyme. These results suggest that dietary eritadenine might suppress the activity of liver microsomal Delta6-desaturase by altering the microsomal phospholipid profile, as represented by an increase in PE concentration, and that the effect of eritadenine is mediated by the regulation of gene expression.