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.     

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.     

Effects of uranyl ions on lipid bilayer membranes

Uranyl ions (UO₂²⁺) stabilize black lipid membranes (BLM's) as inferred from the doubling of the breakdown voltage and from a considerable increase in the lifetime of the BLM's. These effects are observed also in BLM's made of mono-olein and of oxidized cholesterol. The lytic effect of lysolecithin is significantly reduced in the presence of UO₂²⁺. Uranyl ions adsorb to the interface of BLM's made of phosphatidylcholine (PC) with a dissociation constant of about 3 : 10^?6 M and thereby charge the interface of the membrane and attain almost stoichiometric binding of one molecule of uranyl ion per one molecule of PC at 1 M ionic strength and 20 μM of UO₂²⁺. The membrane conductance induced by ionophores is considerably reduced by UO₂²⁺ and it is inferred by various tests that this is due to the charging of the interface and not to changes in membrane fluidity.     

Ion flux across lipid bilayer membranes with charged surfaces

Summary In this paper we derive expressions for the ion flux across lipid bilayer membranes with charged surfaces treating the membrane as a continuous phase interposed between two electrolyte solutions and calculating the ion flux with the Nernst-Planck equations. The theoretical results are compared with experiments of Seufert and Hashimoto on lipid bilayer membranes with charged surface active agents added to the membranes. If the charge of both membrane surfaces has the same sign the flux of the gegenions is greatly increased whereas the flux of the coions decreases to a small amount. For oppositely charged membrane surfaces the membrane behaves like a np semiconductor and typical rectification voltage-current characteristics are obtained.     

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.     

Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.

We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes.     

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.     

On the transport noise of hydrophobic ions in lipid bilayer membranes

The effect of transit time on the electrical transport noise of a closed one-barrier model at equilibrium as proposed by Kolb and Läuger [6] is studied using the master-equation approach. A transit time is the time for an ion to cross the energy barrier (membrane interior) when the energy of the ion reaches the barrier height. Both the time correlation function and the noise power spectrum are obtained as functions of the transit time of the ions. Possible effects of transit time on the time correlation function of transport of dipicrylamine ions in lipid bilayers as reported by Bruner and Hall [13] and on the noise power spectrum as reported by Kolb and Läuger [6] are discussed.     

Transport of protons and hydrochloric acid through lipid bilayer membranes

Transport of protons and hydrochloric acid through lipid bilayer membranes was studied by a combination of electrical conductance and pH electrode techniques. In the presence of large pH gradients, proton transport occurs primarily by diffusion of molecular HCl. The permeability of egg phosphatidylcholine/decane bilayers to HCl is about 3 cm . s-1, seven to nine order of magnitude higher than the permeability to H+, OH- or Cl-. The HCl permeability of phosphatidylserine or egg phosphatidylcholine/cholesterol (1 : 1) bilayers is about 50% lower than the permeability of egg phosphatidylcholine bilayers. Diffusion of molecular HCl may be an important process in tissues exposed to high HCl concentrations, e.g., gastric mucosa. However, at neutral pH the diffusion of molecular HCl is too slow to contribute significantly to net movements of H+ or Cl-.     

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 .     

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.     

Transport of auxin (indoleacetic acid) through lipid bilayer membranes

Summary Diffusion of auxin (indole-3-acetic acid) through planar lipid bilayer membranes was studied as a function of pH and auxin concentration. Membranes were made of egg or soybean lecithin or phosphatidyl serine inn-decane (25–35 mg/ml). Tracer and electrical techniques were used to estimate the permeabilities to nonionized (HA) and ionized (A^–) auxin. The auxin tracer flux is unstirred layer limited at low pH and membrane limited at high pH, i.e., when [A^–][HA]. The tracer flux is not affected by the transmembrane voltage and is much higher than the flux predicted from the membrane conductance. Thus, only nonionized auxin crosses the membrane at a significant rate. Auxin transport shows saturation kinetics, but this is due entirely to unstirred layer effects rather than to the existence of an auxin carrier in the membrane. A rapid interconversion of A^– and HA at the membrane surface allows A^– to facilitate the auxin flux through the unstirred layer. Thus, the total flux is higher than that expected for the simple diffusion of HA alone. The relation between flux (J _A), concentrations and permeabilities is: 1/J _A=1/P ^UL([A^–]+[HA])+1/P _HA ^M [HA]. By fitting this equation to our data we find thatP ^UL=6.9×10^–4 cm/sec andP _HA ^M =3.3×10^–3 cm/sec for egg lecithin-decane bilayers. Similar membrane permeabilities were observed with phosphatidyl serine or soybean lipids. Thus, auxin permeability is not affected by a net surface charge on the membrane. Our model describing diffusion and reaction in the unstirred layers can explain the anomolous relationship between pH and weak acid (or weak base) uptake observed in many plant cells.     

Non-mediated zero voltage conductance of hydrophobic ions through bilayer lipid membranes

A charge pulse technique applied to the study of charge transfer at metal-solution interfaces has been used to determine the capacity and the conductance of a membrane bilayer at both zero time and zero voltage. The transport of hydrophobic ion across a glycerol-monooleate bilayer (tetraphenyl borate, picrate, dipicrylamine and tetraphenyl arsonium) has been investigated by this method. A theoretical approach to the problem has been proposed based on one analogous to that used for the compact double layer at metallic electrodes.     

Effect of polymyxin B on the conductivity of negatively charged bilayer lipid membranes

Effect of polymyxin B on the planar bilayer lipid membranes (BLM) formed from synthetic phosphatidic acid has been studied. The addition of cholesterol to phospholipid in molar ratio 1 : 2 was followed by an increase of BLM conductance from 2 x 10(-8) to 3 x 10(-7) Ohm-1 cm-2. It was suggested that the observed increase of conductance was due to the fluidity of the membrane matrix in the presence of cholesterol. It was shown that 10(-6)--10(-5) M polymyxin slightly affected the conductance of BLM from phosphatidic acid. It was found that polymyxin increased conductance of negatively charged BLM modified by palmitic acid from 10(-8) to 10(-6) Ohm-1 cm-2.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: Detection by antibiotic membrane probes

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylocholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmertically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Non-mediated zero voltage conductance of hydrophobic ions through bilayer lipid membranes.

A charge pulse technique applied to the study of charge transfer at metal-solution interfaces has been used to determine the capacity and the conductance of a membrane bilayer at both zero time and zero voltage. The transport of hydrophobic ions across a glycerol-monooleate bilayer (tetraphenyl borate, picrate, dipicrylamine and tetraphenyl arsonium) has been investigated by this method. A theoretical approach to the problem has been proposed based on one analogous to that used for the compact double layer at metallic electrodes.     

Effect of 3-phenylindole on lipophilic ion and carrier-mediated ion transport across bilayer lipid membranes

The physical effects of 3-phenylindole, an antimicrobial compound which interacts with phospholipids, on ion transport across phosphatidylcholine-cholesterol bilayers have been investigated using three lipophilic ions and one ion-carrier complex. It was found that 3-phenylindole increased membrane electrical conductance of positively charged membrane probes and decreased electrical conductance of negatively charged probes. The enhancement of conductance detected by nonactin-K+ complex and tetraphenylarsonium+ was several orders of magnitude, whereas the suppression of conductance due to tetraphenylborate- and dipicrylamine- was less than a factor of ten. Presence of 3-phenylindole in aqueous phase slightly decreased adsorption of tetraphenylborate- and dipicrylamine- at the membrane surface. From the voltage dependence of the steady-state conductance it was shown that 3-phenylindole induced kinetic limitation of membrane transport of potassium mediated by nonactin. No such limitation was found in the case of tetraphenylarsonium+ transport. These results are shown to be consistent with the present concept of ion diffusion in membranes and the assumption that 3-phenylindole decreases the electric potential in the membrane interior. The asymmetry of the effect of 3-phenylindole on the magnitude of conductance changes for positively and negatively charged membrane permeable ions is also discussed as a reflection of the discreteness of both the absorbed 3-phenylindole and lipid dipoles.     

Transport methods for probing the barrier domain of lipid bilayer membranes.

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.