The inner membrane barrier of lipid membranes experienced by the valinomycin/Rb+ complex: charge pulse experiments at high membrane voltages.

The kinetic analysis of charge pulse experiments at planar lipid membranes in the presence of macrocyclic ion carriers has been limited so far to the low voltage range, where, under certain simplifying conditions, an analytical solution is available. In the present study, initial voltages of up to 300 mV were applied to the membrane, and the voltage decay through the conductive pathways of the membrane was followed as a function of time. The system of differential equations derived from the transport model was solved numerically and was compared with the experimental data. The generalized kinetic analysis of charge pulse experiments and of steady-state current-voltage curves was used to study the voltage dependence of the individual transport steps and to obtain information on the shape of the inner membrane barrier. The data were found to be consistent with a comparatively broad inner barrier such as a trapezoidal barrier or an image force barrier. The inner barrier was found to sense 70-76% of the voltage applied to the membrane. As a consequence, 24-30% of the voltage acts on the two interfacial barriers between membrane and water. The data refer to membranes formed from monoolein, monoeicosenoin, or monoerucin in n-decane.     

Transport mechanism of hydrophobic ions through lipid bilayer membranes

Summary Evidence is presented that the transport of lipid-soluble ions through bilayer membranes occurs in three distinct steps: (1) adsorption to the membranesolution interface; (2) passage over an activation barrier to the opposite interface; and (3) desorption into the aqueous solution. Support for this mechanism comes from a consideration of the potential energy of the ion, which has a minimum in the interface. The formal analysis of the model shows that the rate constants of the individual transport steps can be determined from the relaxation of the electric current after a sudden change in the voltage. Such relaxation experiments have been carried out with dipicrylamine and tetraphenylborate as permeable ions. In both cases the rate-determining step is the jump from the adsorption site into the aqueous phase. Furthermore, it has been found that with increasing ion concentration the membrane conductance goes through a maximum. In accordance with the model recently developed by L. J. Bruner, this behavior is explained by a saturation of the interface, which leads to a blocking of the conductance at high concentrations.     

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.     

Noise analysis and relaxation experiments of transport of hydrophobic anions across lipid membranes at equilibrium and nonequilibrium

Under equilibrium and nonequilibnum steady-stale conditions the spectral intensity of current noise S_J(f) generated by the transport of hydrophobic unions across lipid bilayer membranes was investigated. The experimental results were compared with different reaction models S_J(f) showed a characteristic increase proportional to f² between frequency-independent tails at low and high frequencies. This gradient was found to be independent of applied voltage which indicates the contribution of a single voltage-dependent reaction step of ion translocation across the membrane From the shape of S_J(f) at low frequencies the rate constant of ion desorption from the membrane into the aqueous phase could be estimated. Unambiguous evidence for the application of a general model, which includes the coupling of slow ion diffusion in the aqueous phase to ion adsorption/desorption at the membrane interface, could not be obtained from the low-frequency shape of S_J(f). The shot noise of this ion transport determines the amplitude of S_J(f) at high frequencies which decreases with increasing voltage applied. Analysis of voltage-jump current-relaxation experiments and of current noise carried cut on one membrane yielded significant differences of the derived ion partition coefficient. This deviation is qualitatively described on the basis of incomplete reaction steps.     

Transport kinetics of hydrophobic ions in lipid bilayer membranes Charge-pulse relaxation studies

A modified version of the charge-pulse relaxation technique with improved time resolution was applied to the study of transport kinetics of hydrophobic ions (tetraphenylborate, dipicrylamine) through lipid bilayer membranes. Besides a better time resolution the charge-pulse method has the additional advantage that the perturbation of the membrane can be kept small (voltage amplitudes between 1 and 10 mV). The results of the analysis support the model proposed earlier, according to which the overall transport takes place in three consecutive steps, adsorption of the ion from water to the interface, translocation to the opposite interface, and desorption into the aqueous phase. The translocation rate constant K_i and the partition coefficient γ of the hydrophobic ion between water and the membrane were measured for lecithins with different mono-unsaturated fatty acid residues. Increasing the chain length of the fatty acid from C₁₆ to C₂₄ resulted in a decrease of k_i by a factor of about 9 in the case of tetraphenylborate and by a factor of about 17 in the case of dipicrylamine.     

Kinetics of transport of hydrophobic ions through lipid membranes including diffusion polarization in the aqueous phase

Previous interpretations of the kinetics of transport of hydrophobic ions through membranes have been based on one of three limiting assumptions. Either diffusion in the aqueous phase was taken to be rapid, or ionic motion was constrained to the membrane or a steady state was presumed to be established within the membrane. We present a general treatment of the coupled diffusion process through both the aqueous phase and the membrane; our theory contains the previous results as limiting cases. It is applied to voltage jump-current relaxation experiments on black lipid membranes in the presence of dipicrylamine or sodium tetraphenylborate. We have attempted to establish the rate of desorption from the membrane. For the system phosphatidylserine/tetraphenylborate, the rate of desorption and the rate of translocation were found to be comparable.     

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.     

Charge pulse studies of transport phenomena in bilayer membranes. I. Steady-state measurements of actin- and valinomycin-mediated transport in glycerol monooleate bilayers.

A charge pulse technique has been applied to studies of transport phenomena in bilayer membranes. The membrane capacitance can be rapidly charged (in less than a microsecond). The charge then decays through the membrane's conductive mechanism-no current flows through the solution or external circuitry. The resulting voltage decay is thus a manifestation of membrane and boundary layer phenomena only. There are a number of advantages to this approach over conventional voltage or current-clamp techniques: the rise-time of the voltage perturbation is not limited by the time constant deriving from the membrane capacitance and solution resistance, thus permitting study of extremely rapid rate processes; the membrane is exposed to high voltage for relatively short times and thus can be subjected to higher voltages without breakdown; the steady-state current-voltage behavior of the membrane can be deduced from a single charge pulse experiment; the charge (and therefore the integral of the ion flux through the membrane) is monitored allowing detection of rate processes too rapid to follow directly. In this paper we present what is primarily a steady-state analysis of actin (non-, mon-, din-, trin-)-mediated transport of ammonium ion and valinomycin-mediated transport of cesium and potassium ions through glycerol monooleate bilayers. We introduce the concept of the "intercept discrepancy", a method for measuring charge lost through extremely rapid rate processes. Directly observable pre-steady-state phenomena are also discussed but will be the main subject of part II.     

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 a Transmembrane Protein on the Permeability of Small Molecules Across Lipid Membranes

The influence of the nonchannel conformation of the transmembrane protein gramicidin A on the permeability coefficients of neutral and ionized α-X-p-methyl-hippuric acid analogues (XMHA) (X = H, OCH₃, CN, OH, COOH, and CONH₂) across egg-lecithin membranes has been investigated in vesicle efflux experiments. Although 10 mol% gramicidin A increases lipid chain ordering, it enhances the transport of neutral XMHA analogues up to 8-fold, with more hydrophilic permeants exhibiting the greatest increase. Substituent contributions to the free energies of transfer of both neutral and anionic XMHA analogues from water into the bilayer barrier domain were calculated. Linear free-energy relationships were established between these values and those for solute partitioning from water into decadiene, chlorobutane, butyl ether, and octanol to assess barrier hydrophobicity. The barrier domain is similar for both neutral and ionized permeants and substantially more hydrophobic than octanol, thus establishing its location as being beyond the hydrated headgroup region and eliminating transient water pores as the transport pathway for these permeants, as the hydrated interface or water pores would be expected to be more hydrophilic than octanol. The addition of 10 mol% gramicidin A alters the barrier domain from a decadiene-like solvent to one possessing a greater hydrogen-bond accepting capacity. The permeability coefficients for ionized XMHAs increase with Na⁺ or K⁺ concentration, exhibiting saturability at high ion concentrations. This behavior can be quantitatively rationalized by Gouy-Chapman theory, though ion-pairing cannot be conclusively ruled out. The finding that transmembrane proteins alter barrier selectivity, favoring polar permeant transport, constitutes an important step toward understanding permeability in biomembranes. Received: 12 July 1999/Revised: 20 October 1999     

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

Summary 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 at 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 constantk _i for the translocation of the hydrophobic ion across the barrier, as well as the mean surface concentrationN _t of adsorbed ions may be caluculated from the observed spectral intensity of current noise. The values ofk _i 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 concentrationN _t .     

Influence of lipids on the hydrophobic barrier within the pore of the TWIK-1 K2P channel

Several recent ion channel structures have revealed large side portals, or ‘fenestrations’ at the interface between their transmembrane helices that potentially expose the ion conduction pathway to the lipid core of the bilayer. In a recent study we demonstrated that functional activity of the TWIK-1 K2P channel is influenced by the presence of hydrophobic residues deep within the inner pore. These residues are located near the fenestrations in the TWIK-1 structure and promote dewetting of the pore by forming a hydrophobic barrier to ion conduction. During our previous MD simulations, lipid tails were observed to enter these fenestrations. In this addendum to that study, we investigate lipid contribution to the dewetting process. Our results demonstrate that lipid tails from both the upper and lower leaflets can occupy the fenestrations and partially penetrate into the pore. The lipid tails do not sterically occlude the pore, but there is an inverse correlation between the presence of water within the hydrophobic barrier and the number of lipids tails within the lining of the pore. However, dewetting still occurs in the absence of lipids tails, and pore hydration appears to be determined primarily by those side-chains lining the narrowest part of the pore cavity.     

Significance of composition and structure of the enterobacterial outer membrane to the transport of desired and undesired substances and its inhibition

The enterobacterial outer membrane forms a bilayer. Its outer monolyer consists of lipopolysaccharides and proteins, its inner monolayer of phospholipids and proteins. It thus represents an efficient penetration barrier against hydrophobic and anionic compounds (such as detergents or hydrophobic antibiotics) and against higher molecular substances (such as proteolytic, lipolytic, and murolylic enzymes). Some of the proteins (“porins”) form channels through the outer membrane through which neutral and cationic hydrophylic compounds up to a molecular weight of about 800 can pass. Besides the porins additional transport systems have been described. They play an important part in providing the bacteria with substances necessary for their growth, i.e., phosphate, iron ions, and others. Organic polycations are able to generate more or less severe disorganizations in the outer membrane through which they can pass the bilayer (“self-promoted pathway”). Some of these polycations represent efficient antibiotics (polymyxin B, nourseothricin). Bacteria are able to protect themselves against the harmful action of these substances by changing the composition of the outer membrane.     

Kinetic criteria of solid state mechanisms in biology

Kinetic criteria for solid state physical mechanisms of electron and ion transport in biological systems are summarized, and the mechanisms are discussed. A reaction which is rate-limited by electron or ion transport across a particle or membrane in accord with Ohm's law will show first order kinetics, with an hyperbolic relationship between rate constant and the sum of substrate plus product. Larger initial substrate concentrations produce smaller rate constants, thus giving the appearance of substrate inhibition. Examples are cytochrome oxidase and peroxidase, and pyruvate carboxylase. Ohmic transport mechanisms may be caused by electron conduction or superconduction through protein, by electron conduction through water, or by conduction of ions through membranes. A reaction which is rate-limited by charge transport across an activation energy barrier at an interface in accord with a logarithmic voltage-current law will show reaction kinetics conforming to the Elovich equation, and will have the appearance of a pair of simultaneous first order processes. Examples include decay of photogenerated free radicals in eye melanin particles and in photosynthetic particles of bacteria, and sodium and potassium ion transport across cell surfaces. The logarithmic voltage-current law may be regarded as an empirical relationship describing behavior of interfaces, justified by extensive experimental data on many types of interfaces, or it may be derived theoretically for individual cases from statistical mechanical and/or solid state physical considerations. Dedicated to Prof. N. Rashevsky and to his enlightened editorial policy, especially to his policy of publishing that which is new, even when he disagrees with it.     

Influence of lipids on the hydrophobic barrier within the pore of the TWIK-1 K2P channel

Several recent ion channel structures have revealed large side portals, or ‘fenestrations’ at the interface between their transmembrane helices that potentially expose the ion conduction pathway to the lipid core of the bilayer. In a recent study we demonstrated that functional activity of the TWIK-1 K2P channel is influenced by the presence of hydrophobic residues deep within the inner pore. These residues are located near the fenestrations in the TWIK-1 structure and promote dewetting of the pore by forming a hydrophobic barrier to ion conduction. During our previous MD simulations, lipid tails were observed to enter these fenestrations. In this addendum to that study, we investigate lipid contribution to the dewetting process. Our results demonstrate that lipid tails from both the upper and lower leaflets can occupy the fenestrations and partially penetrate into the pore. The lipid tails do not sterically occlude the pore, but there is an inverse correlation between the presence of water within the hydrophobic barrier and the number of lipids tails within the lining of the pore. However, dewetting still occurs in the absence of lipids tails, and pore hydration appears to be determined primarily by those side-chains lining the narrowest part of the pore cavity.     

Modification of ion transport in lipid bilayer membranes in the presence of 2,4-dichlorophenoxyacetic acid. I. Enhancement of cationic conductance and changes of the kinetics of nonactin-mediated transport of potassium.

We have found that herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has the ability to increase the rate of transport of positive ions of several kinds, and to inhibit transport of negatively charged tetraphenylborate ions in lipid bilayer membranes. It has been found that only the neutral form of 2,4-D is transport active, whereas the ionized from of 2,4-D does not modify transport of ions, and does not by itself permeate through lipid membranes. The results suggest that the enhancement of transport of positively charged ions such as tetraphenylarsonium + and nonactin-K+ is dominated by the increase of the ion translocation rate constant. It has been shown that the enhancement of nonactin-mediated transport of K+ by 2,4-D can be accounted for by a simple carrier model. We have observed that a 2,4-D concentration above 3 X 10(-4) M the potassium ion transport in phosphatidylcholine-cholesterol as well as in cholesterol-free glycerolmonooleate membranes is enhanced to such a degree that, depending upon the concentration of potassium ions, it becomes limited by the rate of recombination of K+ with nonactin, and/or by backdiffusion of unloaded nonactin molecules. Furthermore, the effect of 2,4-D is enhanced by ionic strength of aqueous solution. From the changes of kinetic parameters of nonactin-K+ transport, as well as from the changes of membranes conductance due to tetraphenylarsonium + ions, we have estimated the changes of the electrical potential of the membrane interior. We have found that the potential of the interior of the membrane becomes more negative in the presence of 2,4-D, and that its change is proportional to the aqueous concentration of 2,4-D. The effect of 2,4-D on ion transport has been attributed to a layer of 2,4-D molecules absorbed within the interfacial region, and having a dipole moment directed toward the aqueous medium. The results of kinetic studied of nonactin-K+ transport suggest that this layer is located on the hydrocarbon side of the interface.     

Measurement of the glucose permeation rate across phospholipid bilayers using small unilamellar vesicles Effect of membrane composition and temperature

Small unilamellar vesicles were used to measure the permeability of saturated phosphatidylcholine bilayers to glucose. The presented method circumvents most of the common restrictions of classical permeability experiments. Increasing the fatty acid chain length of the lipids reduced the permeation rate significantly. Raising the temperature above that of the lipid phase transition drastically increased membrane permeability. Arrhenius plots demonstrated the activation energy to be independent of membrane composition and the phase-state of the lipids. The permeation process is discussed in terms of a constant energy to disrupt all hydrogen bonds between permeant and aqueous solvent prior to penetrating the membrane. The magnitude of the permeability coefficient is partly determined by a unfavourable change in entropy of activation on crossing the water/lipid interface. All results indicate that the penetration of the dehydrated permeant into the hydrophobic barrier is the rate-limiting step in the permeation of glucose.     

Measurement of the glucose permeation rate across phospholipid bilayers using small unilamellar vesicles. Effect of membrane composition and temperature

Small unilamellar vesicles were used to measure the permeability of saturated phosphatidylcholine bilayers to glucose. The presented method circumvents most of the common restriction of classical permeability experiments. Increasing the fatty acid chain length of the lipids reduced the permeation rate significantly. Raising the temperature above that of the lipid phase transition drastically increased membrane permeability. Arrhenius plots demonstrated the activation energy to be independent of membrane composition and the phase-state of the lipids. The permeation process is discussed in terms of a constant energy to disrupt all hydrogen bonds between permeant and aqueous solvent prior to penetrating the membrane. The magnitude of the permeability coefficient is partly determined by a unfavourable change in entropy of activation on crossing the water/lipid interface. All results indicate that the penetration of the dehydrated permeant into the hydrophobic barrier is the rate-limiting step in the permeation of glucose.     

How do protons cross the membrane-solution interface? Kinetic studies on bilayer membranes exposed to the protonophore S-13 (5-chloro-3-tert-butyl-2′-chloro-4′ nitrosalicylanilide)

Summary A simple carrier model describes adequately the transport of protons across lipid bilayer membranes by the weak acid S-13. We determined the adsorption coefficients of the anionic, A^–, and neutral, HA, forms of the weak acid and the rate constants for the movement of A^– and HA across the membrane by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. These measurements agree with the results of voltage clamp and charge pulse kinetic experiments. We considered three mechanisms by which protons can cross the membranesolution interface. An anion adsorbed to the interface can be protonated by (i) a H⁺ ion in the aqueous phase (protolysis), (ii) a buffer molecule in the aqueous phase or (iii) water molecules (hydrolysis). We demonstrated that the first reaction cannot provide the required flux of protons: the rate at which H⁺ must combine with the adsorbed anions is greater than the rate at which diffusion-limited reactions occur in the bulk aqueous phase. We also ruled out the possibility that the buffer is the main source of protons: the rate at which buffers must combine with the adsorbed anions is greater than the diffusion-limited rate when we reduced the concentration of polyanionic buffer adjacent to the membrane-solution interface by using membranes with a negative surface charge. A simple analysis demonstrates that a hydrolysis reaction can account for the kinetic data. Experiments at acid pH demonstrate that the transfer of H⁺ from the membrane to the aqueous phase is limited by the rate at which OH combines with adsorbed HA and that the diffusion coefficient of OH^– in the water adjacent to the bilayer has a value characteristic of bulk water. Our experimental results demonstrate that protons are capable of moving rapidly across the membrane-solution interface, which argues against some mechanisms of local chemiosmosis.     

Valinomycin-mediated ion transport through neutral lipid membranes: Influence of hydrocarbon chain length and temperature

Summary Stationary electrical conductance experiments together with nonstationary relaxation experiments allow a quantitative determination of rate constants describing carrier-mediated ion transport. Valinomycin-induced ion transport across neutral lipid membranes was studied. The dependence of the transport parameters on the chain length of the lipid molecules, on the kind of alkali ion, and on the temperature was determined. The relaxation time the current following a voltage jump shows a marked increase with decreasing temperature or with increasing chain length of the lipid molecules. This variation of is interpreted on the basis of a varying membrane fluidity. It is shown that under favorable circumstances the equilibrium constant of complex formation in the aqueous phase may be obtained from membrane experiments. Furthermore, the kinetics of exchange of valinomycin between membrane and water was studied. We found a marked influence of the totus surrounding the black film on the kinetics as well as on the total amount of valinomycin molecules in the membrane. The problem of location of the free carrier molecules inside the membrane is discussed.