Photodynamic activation of ion transport through lipid membranes and its correlation with an increased dielectric constant of the membrane

Illumination of biological membranes with visible light in the presence of membrane-active sensitizers (e.g. rose bengal) is known to inactivate transport proteins such as ion channels and ion pumps. In some cases, however, illumination gives rise to an activation of transport. This is shown here for ion channels formed by alamethicin in lipid membranes, and for porin channels, which were isolated from the outer membrane of E. coli (OmpC) and from the outer membrane of mitochondria (VDAC) and were reconstituted in lipid membranes. An activation (in the form of an increased conductance) was also observed in the presence of the cation carriers valinomycin and nonactin. The activation phenomena were only present, if the membranes were made from lipids containing unsaturated double bonds. Activation was reduced in the presence of the antioxidant vitamin E.We suggest that the activation of the different transport systems has a common physical basis, namely an increase of the dielectric constant, epsilon(m), of the membrane interior by the presence of polar oxidation products of photodynamically induced lipid peroxidation. Experimental evidence for an enhanced dielectric constant was obtained from the finding of a light-induced increase of the membrane capacitance in the presence of rose bengal.     

Pulse radiolysis at planar lipid membranes doped with ion carriers or pore formers

The effect of 14 MeV electrons on ion transport through planar lipid membranes was investigated. The membranes were formed in the presence of well defined ion carriers or pore forming substances. In the presence of the ion carriers valinomycin or nonactin or in the presence of the pore formers nystatin or amphotericin B, irradiation produced a transient increase of the membrane conductance followed by a long lasting decrease. The effects are interpreted on the basis of a time-dependent chemical modification of the membrane structure caused by exposure to high energy radiation. The pore former gramicidin A shows an exponential inactivation with increasing dose. At pH 3 and in the presence of oxygen the pore is highly sensitive to radiation ($\text{D}{}_{37}\text{≈ 10}\text{Gy}$) whereas at pH 9.5 a considerably lower radiation sensitivity ($\text{D}{}_{37}\text{≈ 1000}\text{Gy}$), was found. In the absence of oxygen, gramicidin A is virtually insensitive to irradiation. This is considered an evidence that the inactivation of this ion channel is primarily caused by the perhydroxyl radical HO₂.     

The induction by protons of ion channels through lipid bilayer membranes

The appearance of ion channels was induced in phospholipid bilayers by acidification of the bulk solution on one side of the bilayer. by addition of HCl. acetic acid or by hydrolytic production of protons using purified acetylcholinesierase. Further acidification below an apparent critical pH range led to restoration of a low conductance state similar to that seen at neutral pH. Such experiments were performed with a heterogeneous soybean lecithin extract, with homogeneous synthetic di-phytanoylphosphatidylcholine, and with a mixture of cholesterol and synthetic dioleoylphosphatdylcholine. It is proposed that the physical mechanism for this phenomenon involves fluctuations of lipid order induced by fluctuations in protnation of phospholipid head groups within a critical pH range; these, in turn, create conductive defect in the two-dimensional lattice of the lipid bilayer.     

Influence of sodium concentration on changes of membrane capacitance associated with the electrogenic ion transport by the Na,K-ATPase

Electrogenic ion transport by the Na,K-ATPase was investigated in a model system of protein-containing membrane fragments adsorbed to a lipid bilayer. Transient Na⁺ currents were induced by photorelease of ATP from inactive caged ATP. This process was accompanied by a capacitance change of the membrane system. Two methods were applied to measure capacitances in the frequency range 1 to 6000 Hz. The frequency dependent capacitance increment, ΔC, was of sigmoidal shape and decreased at high frequencies. The midpoint frequency, f ₀, depended on the ionic strength of the buffer. At 150 mm NaCl f ₀ was about 200 Hz and decreased to 12 Hz at high ionic strength (1 M). At low frequencies (f≪f ₀) the capacitance increment became frequency independent. It was, however, dependent on Na⁺ concentration and on the membrane potential which was generated by the charge transferred. A simple model is presented to analyze the experimental data quantitatively as a function of two parameters, the capacitance of the adsorbed membrane fragments, C _P, and the potential of maximum capacitance increment, ψ ₀. Below 5 mm Na⁺ a negative capacitance change was detected which may be assigned to electrogenic Na⁺ binding to cytoplasmic sites. It could be shown that the results obtained by experiments with the presented alternating current method contain the information which is determined by current-relaxation experiments with cell membranes. Received: 3 November 1997 / Revised version: 19 February 1998 / Accepted: 21 February 1998     

A 1H-NMR investigation of the effects of ethanol and general anaesthetics on ion channels and membrane fusion using unilamellar phospholipid membranes

Using ¹H-NMR of small unilamellar vesicles in the presence of the lanthanide probe ion Pr³⁺, the effects of ethanol, diethyl ether and chloroform on various mechanisms of channel-mediated transport were studied. The mechanisms include channel formation by the polypeptide Alamethicin 30 and vesicular lysis at the gel to liquid-crystal phase transition of the lipid. Channel stabilisation and membrane fusion induced by sub-critical micelle concentrations of Triton X-100 were also investigated. The observation that ethanol and diethyl ether increase membrane permeability and fusion while chloroform inhibits them suggests a common locus of action on the properties and structure of channel-associated water. This conclusion is discussed in terms of current theories of general anaesthesia.     

Theoretical evaluation of cell membrane ion channel activation by applied magnetic fields

This letter re-examines a recently published calculation of the forces exerted on a membrane ion channel by a cation passing through in the presence of an externally applied magnetic field. We show here, in contradiction to the originally published calculation, that the forces generated due to the Lorentz force of the magnetic field on the cation are negligible compared with the forces required to activate an ion channel protein conformation change associated with the gating of the channel. Received: 11 August 1998 / Revised version: 25 October 1998 / Accepted: 11 November 1998     

Electrochemical study of ion channel behavior in incorporated poly L-glutamate bilayer lipid membranes

The lipid layer membranes were fabricated on the glassy carbon electrode (GC) and demonstrated to be bilayer lipid membranes by impedance spectroscopy. The formation of incorporated poly L-glutamate bilayer lipid membrane was achieved. The ion channel behavior of the incorporated poly L-glutamate membrane was determined. When the stimulus calcium cations were added into the electrolyte, the ion channel was opened immediately and exhibited distinct channel current. Otherwise, the ion channel was closed. The cyclic voltammogram at the GC electrode coated with incorporated poly L-glutamate DMPC film response to calcium ion is very fast compared with that at the GC electrode coated only with DMPC film. Ion channel current is not dependent on the time but on the concentration of calcium. The mechanism of the ion channel formation was investigated.     

Kinetics of ion transport in lipid membranes induced by lysine-valinomycin and derivatives

Summary Lysine-valinomycin and two N-acyl derivatives are compared with respect to their potency to transport Rb⁺ ions across thin lipid membranes. Lysine-valinomycin acts as a neutral ion carrier only above a pH of about 7 of the aqueous solutions, while at lower pH the molecules seem to be positively charged due to a protonation of the -NH₂ group of the lysine residue.A kinetic analysis based on voltage jump relaxation experiments and on the nonlinearity of the current-voltage characteristics showed that the conductance increment per carrier molecule for uncharged lysine-valinomycin is similar to that of natural valinomycin. The attachment of a rather bulky side group such as the dansyl or para-nitrobenzyloxycarbonyl group reduced by approximately one order of magnitude.Some of the relaxation data of the valinomycin analogues were influenced by an unspedfic relaxation of the pure lipid membrane. This structural relaxation represents a limitation to the possibility of analyzing specific transport systems in thin lipid membranes by the voltage jump or charge pulse techniques. It is shown that the time dependence of this structural relaxation — which was first published by Sargent (1975) — is at variance with a three capacitor equivalent circuit of the membrane, which was suggested by Coster and Smith (1974) on the basis of a.c. measurements. A modified equivalent circuit has been found to represent a satisfactory analogue for the current relaxation in the presence of valinomycin. It turned out, however, that such an equivalent circuit provides little insight into the molecular mechanism of transport.     

A simple method for the determination of the pore radius of ion channels in planar lipid bilayer membranes

Abstract A new method of pore size determination is presented. The results of applying this simple method to ion channels formed by staphylococcal α-toxin and its N-terminal fragment as well as to cholera toxin channels are shown. The advantages and the difficulties of this method are discussed. It was found that (i) the mobility of ions in solutions depends only on the percentage of concentration of added non-electrolytes and practically not on their chemical nature (sugars or polyglycols) and molecular size; (ii) the proportional change of both ion channel conductance and bulk solution conductivity by low M . non-electrolytes may be used as an indication of a diffusion mechanism of ion transport through channels; (iii) the slope of the dependence of the ion channel conductance on the bulk conductivity of solutions containing different concentrations of non-electrolyte is a good measure of channel permeability for non-electrolytes.     

A lipid dependence in the formation of twin ion channels

A gramicidin A derivative with a polyether linkage between both ethanolamine termini was synthesized and its ion channel properties were studied. The compound showed a duplication in the state of conductance for alkali cations in thick DOPC bilayer membranes, which is interpreted as the occurrence of twin-channels. In thinner DMPC membranes mono-channels were dominant. The influence of hydrophobic coupling on the mono channel/twin channel equilibrium is discussed.     

Some considerations on the ion transport properties of the rod disc membrane

The ion transport properties of the disc membranes in rod outer segments are discussed on the basis of available data. The properties of an air-water interface film of spectroscopically intact and chemically regenerable rhodopsin are presented, and results of studies of ion binding to these films are reported.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

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.     

Formulation of a coupled mechanism between solute diffusion,phosphatase-kinase reactions and membrane potentials for the primary active transport of phosphorylated substrates through biological membranes

Coupled interrelations occurring between a phosphatase/kinase reaction sequence acting in unstirred layers and on both sides of a charged biomembrane pore structure are presented as a plausible kinetic model for the primary active transport of phosphorylated molecules. Simulations conducted at the cell level and with credible numerical values demonstrate that the enzymes positions strongly regulate the membrane permeability for the transported substrate. Depending on both the enzymes positions (more or less far from the membrane) and the membrane charges, the membrane may appear either impervious, either permeable or able to actively transport a phosphorylated substrate. Globally all happens as if, in function of the enzymes positions, a permanent pore may be regulated, changing from a more closed to a more open conformation.     

Modification of ion transport in lipid bilayer membranes by the insecticides DDT and DDE

In order to elucidate the mechanism of action of organochlorine insecticides on the ion transport in biological membranes, we have studied the effect of DDT and its analog DDE on the structural parameters of phosphatidylethanolamine (PE) planar bilayers. DDT and DDE increase the conductance induced by the hydrophobic ions tetraphenylarsonium (TPhAs⁺) and tetraphenylborate (TPhB^?) in lipid bilayers. Neither DDT nor DDE alters the surface potential of PE monolayers. On the other hand, these organochlorine compounds increase only slightly the electric capacitance of the bilayers. These results are compatible with the hypothesis that these insecticides increase the fluidity of the membrane.     

Efficiency,Na+/K+ selectivity and temperature dependence of ion transport through lipid membranes by (221)C10-Cryptand,an ionizable mobile carrier

Summary The kinetics of Na⁺ and K⁺ transport across the membrane of large unilamellar vesicles (LUV) were determined at two pH's when transport was induced by (221)C₁₀-cryptand (diaza-1,10-decyl-5-pentaoxa-4,7,13,16,21-bicyclo [8.8.5.] tricosane) at various temperatures, and by nonactin at 25°C and (222)C₁₀-cryptand at 20 and 25°C. The rate of Na⁺ and K⁺ transport by (221)C₁₀ saturated with the cation and carrier concentrations. Transport was noncooperative and exhibited selectivity for Na⁺ with respect to K⁺. The apparent affinity of (221)C₁₀ for Na⁺ was higher and less pH-dependent than that for K⁺, and seven times higher than that of (222)C₁₀ for K⁺ ions (20.5vs. 1.7 kcal·mole^–). The efficiency of (221)C₁₀ transport of Na⁺ was pH-and carrier concentration-dependent, and was similar to that of nonactin; its activation energy was similar to that for (222)C₁₀ transport of K⁺ (35.5 and 29.7 kcal · mole^–1, respectively). The reaction orders in cationn(S) and in carrierm(M), respectively, increased and decreased as the temperature rose, and were both independent of carrier or cation concentrations; in most cases they varied slightly with the pH.n(S) varied with the cation at pH 8.7 and with the carrier for Na⁺ transport only, whilem(M) always depended on the type of cation and carrier. Results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes.     

Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd²⁺, La³⁺ or Tb³⁺ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.     

Temperature-jump and voltage-jump experiments at planar lipid membranes support an aggregational (micellar) model of the gramicidin A ion channel

Summary The kinetics of formation and dissociation of channels formed by gramicidin A and two analogues in planar lipid membranes was studied using a laser temperature-jump technique developed earlier [Brock, W., Stark, G., Jordan, P.C. (1981),Biophys. Chem. 13:329–348]. The time course of the electric current was found to agree with a single exponential term plus a linear drift. In case of gramicidin A the relaxation time was identical to that reported for V-jump experiments [Bamberg, E., Läuger, P. (1973),J. Membrane Biol. 11:177–194], which were interpreted on the basis of a dimerization reaction. The same results were obtained for gramicidin A and for chemically dimerized malonyl-bis-desformylgramicidin. It is therefore suggested that the dimerization represents a parallel association of two dimers to a tetramer. There is evidence that the tetramer, contrary to the presently favored dimer hypothesis, is the smallest conductance unit of an active gramicidin channel. An additional V-jump-induced relaxation process of considerably larger time constant is interpreted as a further aggregation of gramicidin dimers.Abbreviations GA gramicidin A - OPG O-pyromellitylgramicidin A - MBDG malonyl-bis-desformylgramicidin     

Carrier-mediated ion transport through black membranes of lipid mixtures and its coupling to Ca++-induced phase separation

Summary Voltage jump-current relaxation experiments have been performed with valinomycin-doped membranes of mixtures of 1,2-dipentadecylmethylidene-glycero-3-phosphorylcholine (PC) and charged-phosphatidic acid (PA). Both relaxation processes predicted by a simple carrier model could be resolved which allowed the calculation of the rate constants of the Rb⁺ transport. The dependence of the rate constants on the membrane composition indicates that (i) the lipids in the mixed membranes are homogeneously distributed and that (ii) no major difference exists between the composition of the membrane and that of the torus. The analysis of the stationary conductance data, however, shows that the valinomycin content of the mixed membranes depends strongly on their lipid composition. Addition of Ca⁺⁺ ions to a 11 mixture induces a phase separation into PA domains of very low conductivity and PC-enriched regions of high conductivity. Half saturation is reached atc _ca=5×10^–4 m. At 10^–2 m Ca⁺⁺ in the aqueous phase, the rate constants clearly indicate that all PA molecules are electrically passivated and only pure PC domains contribute to the membrane current. A detailed picture is thus derived of the coupling of a model transport system to the externally triggered membrane reorganization.     

Effects of pH and diethylpyrocarbonate on the conductance states of planar lipid bilayers containing haemocyanin

The addition of haemocyanin from Megathura crenulata to the aqueous phase bathing a bilayer lipid membrane resulted in the formation of ionic channels. With an applied voltage biased negative with respect to the haemocyanin-containing side, a single conductance state was observed above pH 7.0. Below pH 7.0 several conductance states were manifested, and the maximum conductance observed for a single channel decreased with decreasing pH. Extensive treatment of the haemocyanin with diethylpyrocarbonate, which reacts primarily with histidine residues, completely prevented the formation of ionic channels; however, milder treatment produced a chemically modified haemocyanin that was capable of forming ionic channels with modified conductance properties. Each channel conductance was typically much lower than that of the channels formed from unmodified haemocyanin, and there was now substantial variation in conductance from channel to channel. Following the use of hydroxylamine to remove the carbethoxy groups from the modified haemocyanin, it formed ionic channels that were restored to the original unit channel conductance.     

Effects of biofouling on ion transport through cation exchange membranes and microbial fuel cell performance

This study examines the effects of biofouling on the electrochemical properties of cation exchange membranes (CEMs), such as membrane electrical resistance (MER), specific proton conductivity (SC), and ion transport number (t₊), in addition to on microbial fuel cell (MFC) performance. CEM biofouling using a 15.5 ± 4.6 μm biofilm was found to slightly increase the MER from 15.65 Ω cm² (fresh Nafion) to 19.1 Ω cm², whereas an increase of almost two times was achieved when the electrolyte was changed from deionized water to an anolyte containing a high cation concentration supporting bacterial growth. The simple physical cleaning of CEMs had little effect on the Coulombic efficiency (CE), whereas replacing a biofouled CEM with new one resulted in considerable increase of up to 59.3%, compared to 45.1% for a biofouled membrane. These results clearly suggest the internal resistance increase of MFC was mainly caused by the sulfonate functional groups of CEM being occupied with cations contained in the anolyte, rather than biofouling itself.