The interaction of hydrophobic ions with lipid bilayer membranes

Summary Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and Läuger (J. Membrane Biol. 5:225, 1971). This model visualizes as three distinct steps the interfacial adsorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions adsorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon adsorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon adsorption, of a highly ordered water structure which surrounds the hydrophobic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead be attributed to a more complex effect equivalent to a reduction of membrane fluidity.Research performed while on sabbatical leave April-September, 1974.     

Influence of salts on electrostatic interactions between poliovirus and membrane filters.

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

Influence of salts on electrostatic interactions between poliovirus and membrane filters

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

Theory of electrostatic effects in soft biological interfaces using atomic force microscopy.

We calculated the electrostatic force between a planar interface, such as a planar-supported lipid bilayer membrane, and the tip of a stylus on which another lipid bilayer or some other biomacromolecular system might be deposited. We considered styli with rounded tips as well as conical tips. To take into account the effect of dynamical hydrogen-bonded structures in the aqueous phase, we used a theory of nonlocal electrostatics. We used the Derjaguin approximation and identified the systems for which its use is valid. We pointed out where our approach differs from previous calculations and to what extent the latter are inadequate. We found that 1) the nonlocal interactions have significant effects over distances of 10-15 A from the polar zone and that, at the surface of this zone, the effect on the calculated force can be some orders of magnitude; 2) the lipid dipoles and charges are located a distance L from the hydrophobic layer in the aqueous medium and this can have consequences that may not be appreciated if it is ignored; 3) dipoles, located in the aqueous region, can give rise to forces even though the polar layer is unchanged, and if this is ignored the interpretation of force data can be erroneous if an attempt is made to rationalize an observed force with a knowledge of an uncharged surface; 4) the shape of the stylus tip can be very important, and a failure to take this into account can result in incorrect conclusions, a point made by other workers; and 5) when L is nonzero, the presence of charges and dipoles can yield a force that can be nonmonotonic as a function of ionic concentration.     

Protons @ interfaces: implications for biological energy conversion

The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from "pulsed" experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the "sources" and "sinks", the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.     

The interaction of hydrophobic ions with lipid bilayer membranes.

Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and L?uger (J. Membrane Biol. 5:225, 1971). This medel visualizes as three distinct steps the interfacial absorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions absorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon absorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon absorption, of a highly ordered water structure which surrounds the hydrophic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead by attributed to a more complex effect equivalent to a reduction of membrane fluidity.     

Protons @ interfaces: Implications for biological energy conversion

The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from “pulsed” experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the “sources” and “sinks”, the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.     

Counterion-mediated membrane penetration: Cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids

Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that transport of charge across a membrane should be formally associated with an extremely high Born energy barrier. We studied partitioning of several derivatives of the CPP penetratin in a water-octanol two-phase system in presence of natural phospholipids to explore if solvation by ion-pairing to hydrophobic counter-ions may serve as a mechanism for cell internalisation. We demonstrate that anionic lipids can aid peptide partitioning into octanol. Particularly efficient partitioning into octanol is observed with an arginine-rich penetratin compared to a lysine-rich derivative. Substituting tryptophans for phenylalanines results in poor partitioning into octanol, due to decreased overall peptide hydrophobicity. Partitioning into octanol is dependent of phospholipid type and the peptides induced structural changes in the lipid assemblies found in octanol. Attachment of carboxyfluorescein as a model cargo was found to enhance peptide partitioning into octanol. We discuss our results with respect to theoretical electrostatic energies, empirical hydrophobicity scales and in terms of implications for CPP uptake mechanisms. An important improvement of the theoretical transfer energies is obtained when, instead of singular ions, the insertion of ion-paired dipolar species is considered.     

Counterion-mediated membrane penetration: cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids

Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that transport of charge across a membrane should be formally associated with an extremely high Born energy barrier. We studied partitioning of several derivatives of the CPP penetratin in a water-octanol two-phase system in presence of natural phospholipids to explore if solvation by ion-pairing to hydrophobic counter-ions may serve as a mechanism for cell internalisation. We demonstrate that anionic lipids can aid peptide partitioning into octanol. Particularly efficient partitioning into octanol is observed with an arginine-rich penetratin compared to a lysine-rich derivative. Substituting tryptophans for phenylalanines results in poor partitioning into octanol, due to decreased overall peptide hydrophobicity. Partitioning into octanol is dependent of phospholipid type and the peptides induced structural changes in the lipid assemblies found in octanol. Attachment of carboxyfluorescein as a model cargo was found to enhance peptide partitioning into octanol. We discuss our results with respect to theoretical electrostatic energies, empirical hydrophobicity scales and in terms of implications for CPP uptake mechanisms. An important improvement of the theoretical transfer energies is obtained when, instead of singular ions, the insertion of ion-paired dipolar species is considered.     

Electrical noise from lipid bilayer membranes in the presence of hydrophobic ions

In the presence of the hydrophobic ion dipicrylamine, lipid bilayer membranes exhibit a characteristic type of noise spectrum which is different from other forms of noise described so far. The spectral density of current noise measured in zero voltage increases in proportion to the square of frequency at low frequencies and becomes constant at high frequencies. The observed form of the noise spectrum can be interpreted on the basis of a transport model for hydrophobic ions in which it is assumed that the ions are adsorbed in potential-energy minima at either membrane surface and are able to cross the central energy barrier by thermal activation. Accordingly, current-noise results from random fluctuations in the number of ions jumping over the barrier from right to left and from left to right. On the basis of this model the rate constant ki for the translocation of the hydrophobic ion across the barrier, as well as the mean surface concentration Nt of adsorbed ions may be calculated from the observed spectral intensity of current noise. The values of ki obtained in this way closely agree with the results of previous relaxation experiments. A similar, although less quantitative, agreement is also found for the surface concentration Nt.     

The role of proteins in a dipole model for steady-state ionic transport through biological membranes.

The steady-state current-voltage characteristics of biological membranes are analyzed for means of an application of the electrodiffusion theory to the passage of ions through "dielectric pores", with orientable dipoles at the pore-water interfaces. A detailed evaluation of the electrostatic potential barrier shows, indeed, that the ions have practically no chance to penetrate into the phospholipid bilayer, but that they can cross the membrane through local protein inclusions, of high dielectric constant. A "gating mechanism" can be provided, moreover, by a change of the potential barrier, resulting from a dipole reorientation at the pore-water interface. Dipole-dipole interactions are opposed to the orienting effect of an applied field, but they can be neglected when the separation between the dipoles exceeds a certain critical value. The high polarizability of the pore material leads to an amplification of the effect of an applied field on the orientable dipoles. It is therefore possible to achieve a satisfactory agreement with the experimental results of Gilbert and Ehrenstein (Biophys. J., 9: 447, 1969) for the squid axon, and, in particular, to account for the width of the negative resistance regions with a relatively small value for the length of the orientable dipoles.     

Model Hydrophobic Ion Exchange Membrane

Two models of hydrophobic ion exchange membranes were examined theoretically with regard to the characteristics of cellulose acetate-nitrate membranes saturated with hydrophobic solvents. The first model, consisting of fixed negative sites dispersed in a homogeneous medium of low dielectric constant, was shown to be invalid for the experimental membranes. The second model, consisting of fixed negative sites in an aqueous channel surrounded by a medium of low dielectric constant, explains many properties of the cellulose acetate-nitrate hydrophobic membranes and was analyzed in some detail. Organic cations can enter the membranes through the hydrophobic phase as well as through the aqueous channels. The mechanism of counterion movement in such a model is assumed to consist of exchange of vacancies and or double-occupied sites positions. The presence of the medium of low dielectric constant around the aqueous channel increases the “self”-energy of the ions in the channel and the electrostatic interaction between a fixed site and a counterion in the membrane. Both these factors can account for the marked dependence of ion mobility in the aqueous channels on the dielectric constant of the surrounding medium. The model predicts membrane preference for monovalent counterions over divalent ones.     

Probing biological interfaces by tracing proton passage across them.

The properties of water at the surface, especially at an electrically charged one, differ essentially from those in the bulk phase. Here we survey the traits of surface water as inferred from proton pulse experiments with membrane enzymes. In such experiments, protons that are ejected (or captured) by light-triggered enzymes are traced on their way between the membrane surface and the bulk aqueous phase. In several laboratories it has been shown that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with increase in their electric charge, it was suggested that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. In terms of ordinary electrostatics, the barrier could be ascribed to dielectric saturation of water at a charged surface. In terms of nonlocal electrostatics, the barrier could result from the dielectric overscreening in the surface water layers. It is discussed how the interfacial potential barrier can affect the reactions at interface, especially those coupled with biological energy conversion and membrane transport.     

Comparison of the TIM and TOM channel activities of the mitochondrial protein import complexes

Water-filled channels are central to the process of translocating proteins since they provide aqueous pathways through the hydrophobic environment of membranes. The Tom and Tim complexes translocate precursors across the mitochondrial outer and inner membranes, respectively, and contain channels referred to as TOM and TIM (previously called PSC and MCC). In this study, little differences were revealed from a direct comparison of the single channel properties of the TOM and TIM channels of yeast mitochondria. As they perform similar functions in translocating proteins across membranes, it is not surprising that both channels are high conductance, voltage-dependent channels that are slightly cation selective. Reconstituted TIM and TOM channel activities are not modified by deletion of the outer membrane channel VDAC, but are similarly affected by signal sequence peptides.     

Chemical and solvent effects on the interaction of tetraphenylborate with lipid bilayer membranes

Alkali metal salts of tetraphenylboron dissociate in aqueous solution to yield the hydrophobic anion, BPh₄^?, which is strongly adsorbed at the surfaces of lipid bilayer membranes. Upon application of a transmembrane voltage pulse these anions cross the membrane without appreciable desorption, thereby exhibiting a transient electrical conductance. The relaxation time of this transient is governed by the height of the central potential barrier which the anions must surmount in crossing the membrane. Because of discrete charge effects, the barrier height and hence the observed relaxation time increase markedly with increasing surface density of adsorbed BPh₄^?. Since adsorbed BPh₄^? are in partition equilibrium with the same species dissolved in the aqueous phase, measurement of the relaxation time for BPh₄^? membrane conductance can be used to assay the aqueous-phase concentration of the hydrophobic anion. In this way we have observed the precipitation of KBPh₄ in water, obtaining a solubility concentration product of 1.0·10^?7 mol²·dm^?6 for the precipitation reaction at 25°C. This result is larger by a factor of two than the most directly comparable published values from other sources. In additional experiments we have reduced the polarity of the aqueous phases bathing the membrane by adding varying amount of ethylene glycol to the water. Using the same conductance relaxation assay, we have determined that partitioning of BPh₄^? into the membrane/solution interfaces is lessened as the polarity of the bathing solutions is reduced. This result is attributed to a lowering of the chemical potential of the BPh₄^? in the less polar medium.     

Electrogenicity of the sodium transport pathway in the Na,K-ATPase probed by charge-pulse experiments.

A charge-pulse technique was designed to measure charge movements in the Na-transport mode of the Na,K-ATPase in membrane fragments adsorbed to a planar lipid bilayer with high time resolution. 1) Na+ transport was measured as a function of membrane potential, and 2) voltage-dependent extracellular ion binding and release were analyzed as a function of Na+ concentration and membrane potential. The results could be fitted and explained on the basis of a Post-Albers cycle by simulations with a mathematical model. The minimal reaction sequence explaining the electrogenicity of the pump consists of the following steps: (Na3)E1-P <--> P-E2(Na3) <--> P-E2(Na2) <--> P-E2(Na) <--> P-E2. The conformational change, E1 to E2, is electrogenic (beta 0 < or = 0.1) and the rate-limiting step of forward Na+ transport with a rate constant of 25 s-1 (T = 20 degrees C). The first ion release step, P-E2(Na3) <--> P-E2(Na2), is the major charge translocating process (delta 0 = 0.65). It is probably accompanied by a protein relaxation in which the access structure between aqueous phase and binding site reduces the dielectric distance. The release of the subsequent Na+ ions has a significantly lower dielectric coefficient (delta1 = delta 2 = 0.2). Compared with other partial reactions, the ion release rates are fast (1400 s-1, 700 s-1, and 4000 s-1). On the basis of these findings, a refined electrostatic model of the transport cycle is proposed.     

Modification of ion transport in lipid bilayer membranes in the presence of 2,4-dichlorophenoxyacetic acid. II. Suppression of tetraphenylborate conductance and changes of interfacial potentials.

It has been shown that the blocking of negatively charged tetraphenylborate ion transport in phosphatidylcholine (PC)-cholesterol membranes by the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is dominated by suppression of TPhB- diffusion across the membrane interior, rather than by the decrease of adsorption of TPhB- ions at the membrane surface. The blocking effect can be associated with the decrease of electric potential inside the membrane with respect to that of the aqueous medium, this decreases being proportional to the concentration of 2,4-D in the aqueous solution. It has been estimated that 25 - 30% of the total 2,4-D-induced change of the potential difference is between the plane of absorption of TPhB- and the aqueous solution, and the remaining fraction is between the membrane interior and the absorption plane. The results of this study support the dipolar hypothesis of 2,4-D action in lipid membranes. These conclusions are further supported by measurements changes of electric potential difference across air/water and air/lipid monolayer/water interfaces. It has been found that the electric potential of the nonpolar side of the interface decreases in the presence of neutral molecules of 2,4-D and that this effect becomes more prominent in presence of electrolyte. We have confirmed that PC-cholesterol monolayer cannot be considered as a model for half of the bilayer membrane because of the disagreement between the changes of the interfacial potential difference of PC-cholesterol monolayers and those determined from studied of transport of positive and negative ions across bilayer membranes. In contract, we have found close agreement between the 2,4-D-induced changes of electric potential of the lipid hydrocarbon region in glycerolmonooleate (GMO) membranes and GMO monolayers. We suggest that the action of 2,4-D in lipid membranes is not associated with the changes of orientation of dipoles of lipids constituting the membranes, but rather with a layer of 2,4-D molecules absorbed at the nonpolar/polar membrane boundary.     

Permeation of ions across the potassium channel: brownian dynamics studies

The physical mechanisms underlying the transport of ions across a model potassium channel are described. The shape of the model channel corresponds closely to that deduced from crystallography. From electrostatic calculations, we show that an ion permeating the channel, in the absence of any residual charges, encounters an insurmountable energy barrier arising from induced surface charges. Carbonyl groups along the selectivity filter, helix dipoles near the oval chamber, and mouth dipoles near the channel entrances together transform the energy barrier into a deep energy well. Two ions are attracted to this well, and their presence in the channel permits ions to diffuse across it under the influence of an electric field. Using Brownian dynamics simulations, we determine the magnitude of currents flowing across the channel under various conditions. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength causes a steady decrease in the channel conductance. The current also decreases systematically when the effective dielectric constant of the channel is lowered. The conductance with the optimal choice of dipoles reproduces the experimental value when the dielectric constant of the channel is assumed to be 60. The current-voltage relationship obtained with symmetrical solutions is linear when the applied potential is less than approximately 100 mV but deviates from Ohm's law at a higher applied potential. The reversal potentials obtained with asymmetrical solutions are in agreement with those predicted by the Nernst equation. The conductance exhibits the saturation property observed experimentally. We discuss the implications of these findings for the transport of ions across the potassium channels and membrane channels in general.