Control of the mitochondrial inner membrane permeability by sulfhydryl groups

The effect of some thiol alkylating agents (N-substituted maleimide derivatives) on the permeability of the mitochondrial inner membrane was investigated. Several experimental approaches were used to study the modifications of the permeability properties. Alkylation of sulfhydryl groups led to an increase in the nonspecific permeability as judged by (i) the augmentation of the rate of osmotic shrinkage of mitochondria induced by polyethylene glycol, (ii) the sensitization of succinate dehydrogenase toward oxaloacetate, (iii) the enhancement of the oxidation rate of exogenous NADH, and (iv) the increase of the sucrose permeable space. The sulfhydryl groups involved in the maintenance of the selective permeability were shown to be located in the hydrophobic core of the membrane. Energization of mitochondria provoked an unmasking of these sulfhydryl groups. When magnesium ions were present in the incubation medium, N-substituted maleimide derivatives promoted gross modifications of the intramitochondrial ionic contents. Effluxes of endogenous calcium ions, inorganic phosphate, adenine nucleotides, and NAD(P)H were established. It was concluded that sulfhydryl groups probably play a crucial role in the maintenance of the membrane integrity and thus control the mitochondrial inner membrane permeability.     

NaCl interactions with phosphatidylcholine bilayers do not alter membrane structure but induce long-range ordering of ions and water

It is generally accepted that ions interact directly with lipids in biological membranes. Decades of biophysical studies on pure lipid bilayer systems have shown that only certain types of ions, most significantly large anions and multivalent cations, can fundamentally alter the structure and dynamics of lipid bilayers. It has long been accepted that at physiological concentrations NaCl ions do not alter the physical behavior or structure of bilayers composed solely of zwitterionic phosphatidylcholine (PC) lipids. Recent X-ray scattering experiments have reaffirmed this dogma, showing that below 1 M concentration, NaCl does not significantly alter bilayer structure. However, despite this history, there is an ongoing controversy within the molecular dynamics (MD) simulation community regarding NaCl/PC interactions. In particular, the CHARMM and GROMOS force fields show dramatically different behavior, including the effect on bilayer structure, surface potential, and the ability to form stable, coordinated ion–lipid complexes. Here, using long-timescale, constant-pressure simulations under the newest version of the CHARMM force field, we find that Na⁺ and Cl⁻ associate with PC head groups in a POPC bilayer with approximately equal, though weak, affinity, and that the salt has a negligible effect on bilayer structure, consistent with earlier CHARMM results and more recent X-ray data. The results suggest that interpretation of simulations where lipids interact with charged groups of any sort, including charged proteins, must be carefully scrutinized.     

Investigation of the TEA-resistant outward current in the somatic membrane of perfused nerve cells

Outward currents remaining after addition of 20–50 mM of tetraethylammonium (TEA) ions to the extracellular or intracellular solution, were investigated in perfused isolatedHelix neurons. After this addition, the inactivated inward current carried by potassium ions, the potential-dependent and kinetic characteristics of which differ from those of potassium outward currents suppressed by TEA, is preserved in the membrane. A component dependent on the inward calcium current was found in this TEA-resistant outward current; it was abolished by replacement of the extra-cellular calcium ions by magnesium ions, by blocking of the calcium channels by extracellular cadmium ions, and by their destruction by intracellular fluoride ions. Increasing the intracellular concentration of free calcium ions by perfusing the cell with solutions containing calcium-EGTA buffer potentiated the TEA-resistant component of the outward current, whereas removal of these ions with EGTA weakened it. It is concluded that a system of outward current channels whose activation depends on the presence of calcium ions near the inner surface of the membrane is present in the somatic membrane. It is suggested that to keep these channels capable of being activated, calcium ions must bind with the structures forming their internal opening.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 460–468, September–October, 1979.     

Interaction of a membrane preparation of Na+, K+-ATPase with a spin-labeled analog of ATP]

Interaction of membrane Na+, K+-ATPase preparation from brain gray matter with spin-labelled ATP analogue, in which free iminoxyl radical is joined as a result of 2'(3')-OH ribose groups acylation, is studied. The rotatory mobility of spin-labelled ATP analogue in Na+,K+-ATPase preparation is found to change in non-linear manner during temperature variation (the break-point on the curve being at 20-23degrees C). It correlates with temperature dependence of Na+,K+-ATPase and temperature dependence of lipid viscosity in the membranes, determined by means of hydrophobic spin probes. Substitution of Mg2+ ions with paramagnetic Mn2+ ions resulted in an intense magnetic dipole-dipole interaction between a spin label and Mn2+ ion, which indicated the formation of triple complex enzyme--spin-labelled ATP--Mn2+.     

Surface charges on the outer side of mollusc neuron membrane

Summary The shifts of current-voltage characteristics of sodium and calcium inward currents produced by changes in the concentration of divalent cations (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) and in pH of the extracellular solution have been measured on isolated neurons of the molluscHelix pomatia intracellularly perfused with potassium-free solutions. On the basis of these shifts and using Stern's theory (O. Stern, 1924.Z. Electrochem. 30508–516), the binding constants for the ions to charged groups of the outer side of the somatic membrane and the density of the surface charges produced by these groups have been calculated. For groups located in the vicinity of sodium channels we obtainedK _Ca=90±10,K _Sr=60±10,K _Ba=25±5 andK _Mg=16±5m ^–1 at pH=7.7 and for groups located in the vicinity of calcium channelsK _Ca=67±10,K _Sr=20±5 andK _Ba=19±5m ^–1 at pH=7.0. The same groups bind H⁺ ions with apparent pK=6.2±0.2 that corresponds toK _H=1.6×10⁶ m ^–1. The density of fixed charges near the sodium channels is 0.17±0.05 e/nm² (pH=7.7) and near the calcium channels is 0.23±0.05 electrons/nm² (pH=7.0). From the comparison of the obtained values with the data about binding constants of the same ions to different negatively charged phospholipids, a suggestion is made that just the phophatidylserine is responsible for the surface potential of the outer side of the somatic membrane. It was also shown that the presence of this potential results in a change in the concentration of carrier ions near the membrane which affects the maximal values of the corresponding transmembrane currents.     

HgCl2 disrupts the structure of the human erythrocyte membrane and model phospholipid bilayers

The structural effects of Hg(II) ions on the erythrocyte membrane were studied through the interactions of HgCl2 with human erythrocytes and their isolated resealed membranes. Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Hg(II) induced shape changes in erythrocytes, which took the form of echinocytes and stomatocytes. This finding means that Hg(II) locates in both the outer and inner monolayers of the erythrocyte membrane. Fluorescence spectroscopy results indicate strong interactions of Hg(II) ions with phospholipid amino groups, which also affected the packing of the lipid acyl chains at the deep hydrophobic core of the membrane. HgCl2 also interacted with bilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that Hg(II) ions induced molecular disorder to both phospholipid bilayers, while fluorescence spectroscopy of dimyristoylphosphatidylcholine large unilamellar vesicles confirmed the interaction of Hg(II) ions with the lipid polar head groups. All these findings point to the important role of the phospholipid bilayers in the interaction of Hg(II) on cell membranes.     

Calcium binding to the purple membrane: A molecular dynamics study

The purple membrane (PM) is a specialized membrane patch found in halophilic archaea, containing the photoreceptor bacteriorhodopsin (bR). It is long known that calcium ions bind to the PM, but their position and role remain elusive to date. Molecular dynamics simulations in conjunction with a highly detailed model of the PM have been used to investigate the stability of calcium ions placed at three proposed cation binding sites within bR, one near the Schiff base, one in the region of the proton release group, and one near Glu9. The simulations suggest that, of the sites investigated, the binding of calcium ions was most likely at the proton release group. Binding in the region of the Schiff base, while possible, was associated with significant changes in local geometry. Calcium ions placed near Glu9 in the interior of bR (simultaneously to a Ca(2+) near the Schiff base and another one near the Glu194-Glu204 site) were not stable. The results obtained are discussed in relation to recent experimental observations and theoretical considerations.     

Effect of cupric ions on the permeability of erythrocyte membrane to non-electrolyte spin labels

The addition of cupric ions caused decreased permeability to hydrophilic molecules and increased permeability to hydrophobic molecules. These results suggest that TEMPOL penetrates the erythrocyte in a different way than TEMPO. Penetration of TEMPOL is controlled by-SH groups, while TEMPO probably diffuses through the lipid bilayer. Cupric ions increase the permeability of erythrocyte membranes to both non-electrolytes in vivo.     

Acetylcholine receptor-controlled ion flux in electroplax membrane vesicles: Identification and characterization of membrane properties that affect ion flux measurements

Summary Several intrinsic properties of acetylcholine receptor-rich membrane vesicles prepared fromElectrophorus electricus, which need to be considered in measurements of receptor-mediated ion flux, have been identified. One of these properties is a slow exchange of inorganic ions in the vesicles. The slow exchange of ions is not related to the receptor-mediated flux of ions and accounts for 30–35% of the efflux observed. A method to separate this process from the receptor-controlled flux has been developed. It has also been shown, using a light-scattering method, that aggregation-disaggregation of the vesicles can interfere with the efflux measurements, and a method to overcome this problem has been developed. The difference in the amplitude of effluxes induced by saturating amounts of carbamylcholine and gramicidin has been investigated and has been shown not to be due to a receptor-controlled process; therefore, the amplitude difference does not need to be considered in understanding the receptor-controlled process.     

Ion selectivity of aqueous leaks induced in the erythrocyte membrane by crosslinking of membrane proteins

The aqueous leak induced in the human erythrocyte membrane by crosslinking of spectrin via disulfide bridges formed in the presence of diamide (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210) was further characterized with respect to its ion selectivity by means of (a) measurements of cell volume changes or hemolysis, (b) determination of membrane potentials and (c) analysis of potential-driven ion fluxes. The leak turned out to be slightly cation-selective (PK:PCl approximately equal to 4:1). It discriminates mono- from divalent ions (PNa:PMg greater than 100:1, PCl:PSO4 greater than 10:1) and to a much lesser extent monovalent ions among each other. The selectivities for monovalent ions follow the sequence of free solution mobilities, increasing in the order Li+ less than or equal to Na+ less than K+ less than or equal to Rb+ less than Cs+ and F- less than Cl- less than Br- less than I-. Polyatomic anions also fit into that order. Quantitatively, the ratios of permeabilities of the leak are larger than those of the ion mobilities in free solution. The ion permeability of the leak is concentration-independent up to at least 150 mM. The ion milieu, however, has marked effects on leak permeability, most pronounced for chaotropic ions (guanidinium, nitrate, thiocyanate), which increase leak fluxes of charged and uncharged solutes. The results support the view that, besides geometric constraints, weak coulombic or dipolar interactions between penetrating ions and structural elements of the leak determine permselectivity.     

Hyperpolarization of a barnacle photoreceptor membrane following illumination

Membrane potential changes following illumination of a photoreceptor cell in the lateral ocellus of a barnacle (Balanus eburneus) were studied by means of intracellular recording and polarization techniques. Illumination produces a depolarizing response. When the illumination is terminated, the membrane potential temporarily becomes more negative than the resting potential prior to illumination. Although the amplitude of this postillumination hyperpolarization depends upon the intensity as well as the duration of the light pulse, the time course is fairly constant. The hyperpolarization is not associated with any significant membrane conductance increase and is abolished by 10^-5 M ouabain. It diminishes when the external Na or K ions are removed. An intracellular injection of Na ions produces a hyperpolarization similar to that following illumination. It is suggested that the postillumination hyperpolarization is produced by an electrogenic Na pump which is activated by the Na influx during illumination.     

Anion permeability of the olfactory receptive membrane

The ionic mechanism of the electropositive olfactory receptor potential was studied in the bullfrog and the swamp frog. The positive receptor potential strikingly decreased in amplitude in chloride-free solution. When the olfactory epithelium was immersed in high-KCl-Ringer's solution and then in Cl-free, high-K solution, the polarity of the positive potential could be reversed. This is supposed to be due to the exit of the increased internal chloride ion. From the above two experiments it is concluded that the positive olfactory receptor potential depends primarily upon the influx of the chloride ion through the olfactory receptive membrane. Some contribution by potassium and possibly other ions may occur. The ability of other anions to substitute for chloride was examined. It was found that only Br-, F-, and HCO2- could penetrate the olfactory receptive membrane. The sieve hypothesis in the inhibitory post-synaptic membrane (Coombs, Eccles, and Fatt, 1955) is not applicable to the olfactory receptive membrane on the basis of the size of hydrated ions, but it may be applicable on the basis of the sizes of naked ions.     

Interfacial tension of phosphatidylcholine-phosphatidylserine system in bilayer lipid membrane

The effect of pH of electrolyte solution on the interfacial tension of lipid membrane formed of phosphatidylcholine (PC, lecithin)–phosphatidylserine (PS) system was studied. In this article, three models describing the H⁺ and OH⁻ ions adsorption in the bilayer lipid surface are presented. In Model I and Model II, the surface is continuous with uniformly distributed functional groups constituting the centres of H⁺ and OH⁻ ions adsorption while in the other the surface is built of lipid molecules, free or with attached H⁺ and OH⁻ ions. In these models contribution of the individual lipid molecule forms to interfacial tension of the bilayer were assumed to be additive. In Model III the adsorption of the H⁺ and OH⁻ ions at the PC–PS bilayer surface was described in terms of the Gibbs isotherm. Theoretical equations are derived to describe this dependence in the whole pH range.     

Transport of ions across peritoneal membrane

The electrical conductance of ions across the peritoneal membrane of young buffalo (approximately 18-24 months old) has been recorded. Aqueous solutions of NaF, NaNO3, NaCl, Na2SO4, KF, KNO3, KCl, K2SO4, MgCl2, CaCl2, CrCl3, MnCl2, FeCl3, CoCl2, and CuCl2 were used. The conductance values have been found to increase with increase in concentration as well as with temperature (15 to 35 degrees C) in these cases. The slope of plots of specific conductance, kappa, versus concentration exhibits a decrease in its values at relatively higher concentrations compared to those in extremely dilute solutions. Also, such slopes keep on increasing with increase in temperature. In addition, the conductance also attains a maximum limiting value at higher concentrations in the said cases. This may be attributed to a progressive accumulation of ionic species within the membrane. The kappa values of electrolytes follow the sequence for the anions: SO4(2-)>Cl->NO3->F- while that for the cations: K+>Na+>Ca2+>Mn2+>Co2+>Cu2+>Mg2+>Cr3+>Fe3+. In addition, the diffusion of ions depends upon the charge on the membrane and its porosity. The membrane porosity in relation to the size of the hydrated species diffusing through the membrane appears to determine the above sequence. As the diffusional paths in the membrane become more difficult in aqueous solutions, the mobility of large hydrated ions gets impeded by the membrane framework and the interaction with the fixed charge groups on the membrane matrix. Consequently, the membrane pores reduce the conductance of small ions, which are much hydrated. An increase in conductance with increase in temperature may be due to the state of hydration, which implies that the energy of activation for the ionic transport across the membrane follows the sequence of crystallographic radii of ions accordingly. The Eyring's equation, kappa=(RT/Nh)exp[-DeltaH*/RT]exp[DeltaS*/R], has been found suitable for explaining the temperature dependence of conductance in the said cases. This is apparent from the linear plots of log[kappaNh/RT] versus 1/T. The results indicate that the permeation of ions through the membrane giving negative values of DeltaS* suggest that there may be formation of either covalent linkage between the penetrating ions and the membrane material or else the permeation may not be the rate-determining step. On the one hand, a high DeltaS* value associated with the high value of energy of activation, Ea, for diffusion may suggest the existence of either a large zone of activation or loosening of more chain segments of the membrane. On the other hand, low value of DeltaS* implies that converse is true in such cases, i.e., either a small zone of activation or no loosening of the membrane structure upon permeation.     

Ion passage pathways and thermodynamics of the amphotericin B membrane channel

Amphotericin B is a polyene macrolide antibiotic used to treat systemic fungal infections. Amphotericin B's chemotherapeutic action requires the formation of transmembrane channels, which are known to transmit monovalent ions. We have investigated the ion passage pathways through the pore of a realistic model structure of the channel and computed the associated thermodynamic properties. Our calculations combined the free energy computations using the Poisson equation with a continuum solvent model and the molecular simulations in which solvent molecules were present explicitly. It was found that there are no substantial structural barriers to a single sodium or chloride ion passage. Thermodynamic free energy calculations showed that the path along which the ions prefer to move is off center from the channel's central axis. In accordance with experiments, Monte Carlo molecular simulations established that sodium ions can pass through the pore. When it encounters a chloride anion in the channel, the sodium cation prefers to form a solvent-bridged pair configuration with the anion.     

Protein fouling resistant membrane prepared by amphiphilic pegylated polyethersulfone

A mild and facile grafting of poly(ether glycol) methyl ether methacrylate (PEGMA) monomers onto polyethersulfone (PES) was carried out. Then, the PES-g-PEGMA membranes with integrally anisotropic morphology were fabricated through the coupling of non-solvent induced phase inversion and surface segregation. Compared with PES control membrane, the surface hydrophilicity of PES-g-PEGMA membranes was remarkably enhanced due to the drastic enrichment of poly(ethylene glycol) (PEG) segments on the membrane surface; protein adsorption was significantly inhibited due to the hydrogen bonding interactions between hydrophilic groups and water molecules. Ultrafiltration experiments were used to assess the permeability and protein fouling resistance of the PES-g-PEGMA membranes. It was found that the PES-g-PEGMA membranes with higher surface coverage of PEG segments displayed stronger antibiofouling property. Moreover, the stable antibiofouling property for PES-g-PEGMA membranes was acquired due to covalent bonding interactions between hydrophilic PEGMA side chains and PES main chains.     

Molecular dynamics simulation of cation-phospholipid clustering in phospholipid bilayers: possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K(+), Mg(2+), and Ca(2+) ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca(2+) ions with lipids is significantly stronger than that of K(+) and Mg(2+) ions, regardless of the composition of the lipid bilayer. The binding of Ca(2+) ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K(+)- and Mg(2+)-containing systems. The Ca(2+) ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength-most notably for Ca(2+). The formation of cation-lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca(2+)-phospholipid clusters across apposed lipid bilayers can work as a "cation glue" to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.     

Contributions of secondary active transport processes to membrane potentials

Summary Equations are developed to examine the effects of secondary active transport processes on the steady-state membrane potential of symmetrical cells. It is shown that, with suitable modifications, equations of the type developed by Goldman, Hodgkin and Katz may be derived to accommodate the contributions to the membrane potential of both electroneutral and electrogenic transporters. Where the membrane potential is function of the dominant medium ions (Na, K, and Cl), other contributions can come only from an electrogenic Na pump and from neutral co- and counter-transporters if, and only if, these involve the dominant ions. Experimental approaches to measure the parameters necessary to solve the equations developed here are discussed.     

A kinetic role for ionizable sites in membrane channel proteins

Electrically charged residues in a membrane channel protein will certainly have a direct effect upon its gating and selectivity if they are near the channel pore. It is customary to regard the charged state of such residues as a fixed feature of the channel. In this paper it is argued that far from being fixed, the charged state of ionizable residues near the pore will very probably change rapidly in response to the channel opening and to ions passing through it. Calculations are presented using simple models which demonstrate that changes in the dielectric environment and changes in the distances to other charged groups resulting from channel opening can shift the effective pK values of the sites by 3 or 4 units leading to switching of its charged state. Examples are given of how this time dependent charge state of ionizable residues may play an important role in the functioning of channels. Also, by considering the influence of the electric field due to the mobile ion upon the charge state of a residue in the channel wall, it is shown that a channel lined with acid residues may very effectively block the passage of cations while allowing the passage of anions.     

Depolarized potassium currents and time delay in nerve membrane

An equation with five exponential terms is shown to fit the depolarized potassium current with different initial conditions. This equation has a molecular basis and was derived from the assumption that the potassium ions are transported under the combined forces via Newton's second law. It points out that the initial phase of the potassium current may take a different form and the usual concept that potassium conductance has a time delay may be misleading.