Photogating of ionic currents across lipid bilayers. Hydrophobic ion conductance by an ion chain mechanism.

The photogating of hydrophobic ion currents across the lipid bilayer membrane allows the direct study of their kinetics by symmetrically forming charge within the membrane and across each interface, rather than across the membrane. We find that the photoinduced conductance continues to increase beyond the region where the tetraphenylboride charge density in the membrane exceeds the estimated porphyrin cation density. This photoconductance is proportional to the tetraphenylboride charge density raised to the second to third power. The risetime of the photogating effect increases with increasing concentration of tetraphenyl boride. The porphyrin cation mobility is increased when the tetraphenylboride anion is present, and low concentrations of tetraphenylphosphonium cation increase the dark conductivity while inhibiting the photoconductivity. The activation energy for both the porphyrin and phosphonium cation induced conductance is more positive than that of the tetraphenylboride conductance. From these results we conclude that in addition to some cancellation of space charge within the membrane, the mechanism of increased conductance involves the transport of these hydrophobic anions via an alternating anion-cation chain, analogous to the Grotthuss mechanism for excess proton conduction in water. This ion chain conductance can be viewed as an evolutionary prototype of an ion channel across the membrane. It also underscores the importance of the counter ion in the transport of large ions such as peptides across the lipid bilayer.     

The Excitable Membrane: A Physiochemical Model

The model of the excitable membrane assumes common channels for Na⁺ and K⁺; the two ion species interact within the pores through their electrostatic forces. The electric field varies across the membrane and with time, as a result of ionic redistribution. Ionic flow is primarily controlled by energy barriers at the two interfaces and by Ca⁺⁺ adsorption at the external interface. When the membrane is polarized, the high electric field at the external interface acting on the membrane fixed charge keeps the effective channel diameter small, so that only dihydrated ions can cross the interface. The higher energy required to partially dehydrate Na⁺ accounts for its lower permeability when polarized. Depolarized, the channel entrance can expand, permitting quadrihydrated ions to pass; the large initial Na⁺ flow is the result of the large concentration ratio across the interface. The effect at the internal interface is symmetric; Na⁺ crosses with greater difficulty when the membrane is depolarized. Na⁺ inactivation occurs when the ion distribution within the membrane has assumed its new steady-state value. Calculations based on parameters consistent with physicochemical data agree generally with a wide range of experiments. The model does not obey the two fundamental Hodgkin-Huxley (HH) postulates (independence principle, ion flow proportional to thermodynamic potential). In several instances the model predicts experimental results which are not predicted by the HH equations.     

Alteration of the passive electrical properties of lymphocyte membranes induced by GM1 and GM3 glycolipids.

The electrical conductivity of normal human lymphocyte suspensions has been measured in the frequency range from 10 kHz to 100 MHz, where a well-pronounced conductivity dispersion occurs, caused by the surface polarization at the interface between the cell membrane and the extracellular solution. We have investigated the alteration of the passive electrical properties of the cytoplasmatic cell membrane induced by two different gangliosides (GM1 and GM3) inserted, at various concentrations, into the outer leaflet of membrane double layer. The alterations observed in the dielectric parameters (the membrane conductivity and the membrane permittivity) derived on the basis of a 'double-shell' model, result in an overall increase of the ion permeation across the membrane and an enhanced polarizability of its hydrophilic region for both gangliosides investigated. The relevance of these alterations is discussed.     

A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.     

Effect of ionic polarizability on electrodiffusion in lipid bilayer membranes.

Ion-carrier complexes and organic ions of similar size and shape have mobilities in lipid bilayer membranes which span several orders of magnitude. In this communication, an examination is made of the hypothesis that the basis for this unusually wide range of ionic mobilities is the potential energy barrier arising from image forces which selectively act on ions according to their polarizability. Using Poisson's equation to evaluate the electrostatic interaction between an ion and its surroundings, the potential energy barrier to ion transport due to image effects is computed, with the result that the potential energy barrier height depends strongly on ionic polarizability. Theoretical membrane potential energy profile calculations are used in conjunction with Nernst-Planck electrodiffusion equation to analyze the available mobility data for several ion-carrier complexes and lipid-soluble ions in lipid bilayer membranes. The variation among the mobilities of different ions is shown to be in agreement with theoretical predictions based on ionic polarizability and size. Furthermore, the important influence exerted by image forces on ion transport in lipid bilayer membranes compared to the frictional effect of membrane viscosity is established by contrasting available data on the activation energy of ionic conductivity with that for membrane fluidity.     

STUDIES ON PERMEABILITY OF MEMBRANES : VIII. THE BEHAVIOR OF THE DRIED COLLODION MEMBRANE TOWARD BIVALENT CATIONS.

A study of the behavior of the dried collodion membrane toward the bivalent calcium ion showed that: 1. There is almost no potential difference established across a membrane separating two calcium chloride solutions of 0.1 and 0.01 N concentrations. 2. The transfer numbers of chlorine and calcium, as measured in electrical transfer experiments, are both close to 0.5. 3. A sample of membrane in equilibrium with a solution of calcium chloride has an extremely high electrical resistance, greater than is observed with solutions of the chlorides of any of the monovalent cations. 4. The total electrolyte content of a membrane in equilibrium with a solution of calcium chloride was only 20 per cent of that observed when the solution was lithium chloride and 10 per cent of that found when the solution was potassium chloride. In explaining these various results it is supposed that (1), (2) and (3) are all the result of (4), that is, of the inability of the calcium ion to penetrate any but the largest of the membrane pores. As the total quantity of electrolyte able to penetrate the membrane is very small the electrical conductivity must also be very small. Moreover, the few larger pores that are large enough to transport the hydrated calcium ion are too large to exert any appreciable effect in decreasing the mobility of the anion. Thus the membrane has no effect in modifying the potentials established across concentration chains with CaCl₂ and the transfer numbers determined experimentally are what one would expect if no membrane were present.     

The function of the NADPH oxidase of phagocytes and its relationship to other NOXs in plants, invertebrates, and mammals

The NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (NOX) of 'professional' phagocytic cells transfers electrons across the wall of the phagocytic vacuole, forming superoxide in the lumen. It is generally accepted that this system promotes microbial killing through the generation of reactive oxygen species and through the activity of myeloperoxidase. An alternative scenario exists in which the passage of electrons across the membrane alters the pH and generates a charge that drives ions into, and out of, the vacuole. It is proposed that the primary function of the oxidase is to produce these pH changes and ion fluxes, and the issues surrounding these processes are considered. The neutrophil oxidase is the prototype of a whole family of NOXs that exist throughout biology, from plants to man, which might function, at least in part, in a similar fashion. Some examples of how these other NOXs might influence ion fluxes are examined.     

A Dipole Model for Negative Steady-State Resistance in Excitable Membranes

A dipole model is presented for ion flow in excitable membranes. This model considers the membrane to be composed of two distinct regions: a polar region and a nonpolar region. Further, the construction of an electrodiffusive formalism which takes explicit account of the energy of partition required by an ion for passage from external fluid to nonpolar region is presented. In the polar region a cooperative effect is considered which produces a configurational transition of the polar group dependent only on membrane voltage. A resulting change in voltage drop across the polar group is brought about by this configurational transition. This gives rise to a negative steady-state resistance for the equimolar case, in reasonable agreement with observation. The theory, in addition, is in reasonable accord with nonequimolar ion flow, and provides an explanation for such effects as the following: the intercept of the voltage-current characteristic, the ion membrane concentrations inferred from electrodiffusion theories, and the effects of polyvalent cations     

Space charge-limited conductance in lipid bilayer membranes

Summary A mathematical treatment is given for the flux of ions of one charge sign across lipid bilayer membranes. This treatment is a generalization of a previous analysis of the membrane conductance by D. Walz, E. Bamberg and P. Läuger which was restricted to systems with negligible space charge in the membrane. The present theory includes space charge effects, and it is no longer assumed that the electric field strength in the membrane is constant. It is found that the ohmic membrane conductivity ₀ is reduced by space charges; if only ions of one charge sign are soluble in the membrane, ₀ approaches a limiting value for increasing concentration of the permeable ion in the aqueous solution. The theory also predicts the range in which the constant field approximation is valid. It is found that space charge effects become predominant when the mean concentration of the permeable ion in the membrane exceeds 5×10^–5 m. The currentvoltage characteristic of the membrane remains practically linear even in the presence of a high space charge. It is therefore concluded that the experimentally observed nonlinearity is caused mainly by the distortion of the potential energy profile of an ion due to image forces.     

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.     

The relationship between active transport and the exchange diffusion effect

Dependences of unidirectional ionic fluxes across biological membranes on the trans concentrations of the same ion, commonly described as exchange diffusion, and the association of this phenomenon with active transport, are noted. It is suggested that this effect could arise as a result of energetic coupling between the movement of ions conveyed in each direction by the pump if the latter operates near thermodynamic equilibrium and if the rate of the energizing reactions are restricted. This hypothesis is supported by an analysis in which the transport step and the energizing reactions are separated and described according to the laws of chemical kinetics. A likely cause for such restriction of the maximum rate of energy supply is shown to lie in evolutionary optimization of the efficiency of active transport if the energizing reaction is not perfectly coupled. Similar optimization will produce gross ionic fluxes large compared with the net flux, especially if the transport step approaches perfect coupling, when restriction of the rate of energy supply will cause a large exchange diffusion effect. The range of validity of the analysis is examined with particular reference to the ionic exchanges between osmoregulating animals and their surroundings.     

Electrochemical and NMR spectroscopic investigations of the influence of the probe molecule Eu(fod)3 on the permeability of lipid membranes to ions

Investigating the action of the fluorinated europium complex Eu(fod)3 on lipid membranes we found that the complex facilitates the ion transfer through the membrane. Electric measurements on planar lipid membranes showed that the membrane conductivity increases considerably by insertion of the complex into the membrane. The increase in the conductivity was only obtained if both layers of the membrane were modified with the complex. 1H NMR spectroscopic studies using DOPC liposomes gave information about the location of the modifier complex in the lipid membrane. From chemical shift effects we concluded that the complex resides in the choline head group region of the membrane and also in the membrane interior near the -C =C- lipid double bond, but not in the center of the bilayer. For understanding of the mentioned conductivity effect we assume that the europium complex induces defects of yet unknown structure in the lipid matrix which provide paths for the ion transfer through the membrane. As appropriate measurements revealed, these paths seem to conduct cations predominantly. Investigating the current voltage behavior of the modified lipid membranes in dependence on the ion concentration we obtained different shaped current-voltage curves. Calculation showed that a model with only one energy barrier inside the membrane is unable to describe these curves kinetically. However, by assuming two energy barriers--one barrier in each membrane lipid layer--the observed curve can be described satisfactorily.     

Electric field effects on membranes: gramicidin A as a test ground

Electric fields due to transmembrane potential differences or ionic gradients across the membrane are presumably crucial for many reactions across membranes or close to membranes like signal transduction, control of ion channels or the generation of neural impulses. Molecular dynamics simulations have been used to study the influence of external electric fields on a mixed gramicidin/phospholipid bilayer system. At high field strengths, formation of membrane electropores occurred both close and distal to the gramicidin. Gramicidin was found to stabilize the membrane adjacent to the protein but also at larger distances of up to 2-3 nm. As a result, membrane pore formation was found to be significantly suppressed for the mixed gramicidin/DMPC system. Moderate field strengths only weakly affected the structure and dynamics of the gramicidin. Spontaneous potassium passage events in external electric fields were observed for both the head-to-head helical conformation as well as for the double helical conformation of gramicidin A. The double-helical conformation was found to facilitate ion passage compared to the head-to-head helical dimer.     

Derivation of unstirred-layer transport number equations from the Nernst-Planck flux equations.

Since the late 1960s it has been known that the passage of current across a membrane can give rise to local changes in salt concentration in unstirred layers or regions adjacent to that membrane, which in turn give rise to the development of slow transient diffusion potentials and osmotic flows across those membranes. These effects have been successfully explained in terms of transport number discontinuities at the membrane-solution interface, the transport number of an ion reflecting the proportion of current carried by that ion. Using the standard definitions for transport numbers and the regular diffusion equations, these polarization or transport number effects have been analyzed and modeled in a number of papers. Recently, the validity of these equations has been questioned. This paper has demonstrated that, by going back to the Nernst-Planck flux equations, exactly the same resultant equations can be derived and therefore that the equations derived directly from the transport number definitions and standard diffusion equations are indeed valid.     

Theory for carrier-mediated zero-current conductance of bilayers extended to allow for nonequilibrium of interfacial reactions,spatially dependent mobilities and barrier shape

Summary A generalized form of the electrodiffusion equation, allowing for any shape of symmetrical energy barrier and any spatial dependence of the diffusion coefficient, is used to deduce theoretically the carrier-mediated conductance for thin (e.g., bilayer) membranes in the limit of low applied current. Both the Nernst-Planck and the Eyring single-barrier treatments are special cases of this more general approach, which allows for the effect of non-uniform properties of the lipid and non-uniform profiles of the forces acting within the membrane interior. Two independent mechanisms for ions to cross the membrane-solution interfaces are considered; namely, (1) the reaction at the interface between ions from solution and carriers from the membrane, and (2) the partition across the interfaces of complexes already formed in the solution. The rates of these reactions are taken into account using the rate equations of chemical kinetics; and the Poisson-Boltzmann equation is integrated in the aqueous solutions to evaluate the effect of charged polar head groups of the lipid. The analysis leads to an expression for the conductance, which, in the approximation of constant field, is an explicit function of such experimentally variable parameters as the concentrations and types of permeant ions and carriers in the aqueous phases, the total ionic strength and the nature of the polar head groups of the lipid. The functional relationship observable in an unknown membrane can, in principle, enable one to deduce such information as the mechanism of ion permeation across the interfaces, the magnitude of the surface charge, and the degree of ion-carrier complexation in the aqueous solutions.     

A two-channel electrostatic model of an ionic counterport

An alternative model is presented for an ionic counterport that depends upon electrostatic rather than steric forces. It consists of two passive ion channels, one selective for I-type ions and the other for J-type ions. The ions interact electrostatically such that the presence of one type of ion within its channel affects the motion of the second type of ion within its channel. In these circumstances it is possible to arrange that the spontaneous flow of I ions across the membrane, down their electrochemical potential gradient, pumps J ions in the opposite direction across the membrane, against their electrochemical gradient. To illustrate this type of model, a particular example of interionic coupling is described in which both types of ion interact with the electric dipole moments of some membrane-spanning alpha-helical sections of the counterport protein complex. By assuming that a group of four alpha-helices is free to rotate slightly about an axis perpendicular to the membrane, the desired form of coupling is obtained. Making simplifying assumptions, it is possible to calculate the kinetics of the model and to compare these with those expected in real counterports. Finally it is shown that, if the helix group rotation is powered by an external energy source, the pair of coupled passive ion channels can mimic a primary exchange pump such as Na+-K+ ATPase. Here both types of ion are propelled in opposite directions across the membrane and simultaneously against their electrochemical potential gradients.     

The surface activity of PVP and other polymers and their antihemolytic capacity

The polymeric cryoprotective agents polyvinylpyrrolidone, dextran, and hydroxyethyl starch do not penetrate the cell membrane and are not present in high osmotic concentrations. Thus, they can exert little of the "antifreeze" behavior generally attributed to glycerol or dimethyl sulfoxide, and must protect cells from freezing injury by some action external to the cell surface. Surface energy measurements of droplets of hemoglobin solution immersed in solutions of cryoprotective polymers indicate that these polymers lower the surface energy of the solution below that of the hemoglobin droplets and form a stable interface. In injured cells, these polymers will therefore hide membrane defects by forming an interface across which hemoglobin cannot easily pass. When freezing is slow, the polymers have little if any true cryoprotective effect but interfere with hemoglobin release as an assay of injury.     

The Role of Sarcolipin and ATP in the Transport of Phosphate Ion into the Sarcoplasmic Reticulum

In a previous study, sarcolipin (SLN) was shown to form channels selective toward chloride ion when incorporated in a mercury-supported tethered bilayer lipid membrane (tBLM). Its incorporation had only a modest permeabilizing effect on phosphate ion. In this note the resistance of a tBLM membrane incorporating sarcolipin was investigated by electrochemical impedance spectroscopy in aqueous solutions of 0.05 M sodium phosphate of pH ranging from 5.3 to 8, in the presence of ATP, adenosine monophosphate, and phenylphosphonic acid. At pH 5.3, submicromolar additions of ATP increase the conductivity of the tBLM incorporating SLN up to a maximum limiting value. The dependence of the conductivity on the ATP concentration satisfies the Michaelis-Menten equation, with an association constant of 0.1 μM. Phenylphosphonium ion and adenosine monophosphate exert an inhibitory effect on membrane permeabilization to phosphate ions by ATP if they are added before ATP, but not if they are added after it. An explanation for this behavior is provided. In conclusion, SLN acts as an ATP-induced phosphate carrier exhibiting a behavior quite similar to that of the unidentified P_i transporter described previously. No ion-channel activity is exhibited by the T18A mutant of SLN.     

Lipid-water interface mediates reversible ionophore conformational change

A new procedure of conformational analysis was used to demonstrate that the ionophore conformation is mediated by its membrane environment. In the hydrophobic lipid matrix, the ionomycin-Ca++ complex adopts a conformation well suited for translocation across the interior of the membrane whereas at the lipid-water interface, the Ca++ ion is immersed into the aqueous phase in a position favorable to its complexation or decomplexation. The translocation of Ca++ across the lipid bilayer supposes a reversible transformation of the two conformers. The conformational analysis shows how the dielectric constant discontinuity existing at the lipid-water interface mediates the reversible transformation of one structure into the other.     

Evidence for ion chain mechanism of the nonlinear charge transport of hydrophobic ions across lipid bilayers.

The conductivity across a lipid bilayer by tetraphenylborate anion is increased 10-fold on the photoformation of lipophilic porphyrin cations. The cations alone have negligible conductivity. This nonlinear photogenerated increase of ion conductivity is termed the photogating effect. Substitution of H by Cl in the para position of tetraphenylborate leads to a 100-fold enhancement of conductivity, whereas the dark conductivities for this and other substituted borates are the same. Moreover, the halo-substituted borates show a large enhancement of conductivity in the low concentration range (10(-8) M), whereas that of tetraphenylborate is small and space charge is negligible. The enhanced ion conductivity has great structural sensitivity to the structure of the anion, the cation, and the lipid, whereas the partition coefficient of all the borates and the concentration of photoformed cations are only slightly affected. The photogated ion transport has a twofold larger activation energy than transport in the dark. Time-resolved photocurrents and voltages demonstrate that the translocation rate of the porphyrin cation is also enhanced 100-fold by the Cl-borate anion but only 10-fold by the H-borate anion. For these reasons the nonlinear gating effect cannot be explained by electrostatics alone, but requires an ion chain or ion aggregate mechanism. Kinetic modeling of the photoinduced current with a mixed cation-anion ion chain can fit the data well. The photogating effect allows the direct study of ion interactions within the bilayer.