Electrogenic and selective transport of biogenic amines across lipid bilayer membranes mediated by a neutral ionophore (AS701)

The neutral noncyclic imide and ether containing ionophore (AS701), a selective carrier for Li⁺ among alkali cations, was found to be capable of mediating the transport of NH₄⁺ and of biogenic amines (catechols and indoles) across lipid bilayer membranes also. Ionophore-mediated electrical properties of planar lipid bilayers were studied under experimental conditions where the positively-charged amine species was dominant. The ionophore was found to act as a selective carrier of the biogenic amines, mediating their electrogenic transport across the membrane, forming 2:1 carrier-amine permeant complexes, carrying a net-charge of +1. Selectively among the amines corresponding to the following sequence: tryptamine (35) > Li⁺ (1) > serotonin (0.60) > dopamine (0.19) > norepinephrine (0.13) > epinephrine (0.05) > NH₄⁺ (0.05). The molecular factors involved in determining these selectivities are assessed.     

Monocarboxylic acid permeation through lipid bilayer membranes

Summary The membrane permeability coefficients for the homologous monocarboxylic acids, formic through hexanoic, as well as benzoic and salicylic, were determined for egg phosphatidylcholine-decane planar bilayer membranes. The permeabilities of formic, acetic and propionic acid were also determined for solvent-free phosphatidylethanolamine bilayers. Permeability coefficients were calculated from tracer fluxes measured under otherwise symmetrical conditions, and precautions were taken to ensure that the values were not underestimated due to unstirred layer effects. The relation between the nonionic (HA) permeability (P ^m ) and the hexadecane/water partition coefficient (K _p ) was: log ^m =0.90 log K_p+0.87 (correlation coefficient=0.996). Formic acid was excluded from the analysis because its permeability was sixfold higher than predicted by the other acids. The permeabilities for solvent-free membranes were similar to those for decanecontaining membranes. The exceptionally high permeability of formic acid and the high correlation of the other permeabilities to the hexadecane/water partition coefficient is a pattern that conforms with other nonelectrolyte permeabilities through bilayers. Similarly, the mean incremental free energy change per methylene group (G-CH₂-) was –764 cal mol^–1, similar to other homologous solutes in other membrane systems. However, much less negative G values (–120, to –400 cal mol^–1) were previously reported for fatty acids permeating bilayers and biological membranes. These values are due primarily to unstirred layer effects, metabolism and binding to membranes and other cell components.     

Lithium-selective permeation through lipid bilayer membranes mediated by a di-imide ionophore with nonsymmetrical imide substituents (ETH1810)

Summary The neutral, noncyclic Li^–-selective ionophore ETH1810, which is a di-imide, differs structurally from previous similar ionophores by removal of the intramolecular symmetry of the N-imide substituents. Properties of this ionophore, as a potential carrier of lithium, were probed through studies of ionophore-induced changes in electrical properties of lipid bilayer membranes. ETH1810 was found capable of transporting lithium and other monovalent cations, across lipid bilayer membranes, forming 21 ionophore: ion membrane-permeating species. It was found to be 10-fold more potent than ETH1644, which was the previous best ionophore of this type. The selectivity sequence among alkali cations was found to be: Li⁺(1)>Na⁺ (0,009)>K⁺ (0.004)>Cs⁺(0.0035). Among the physiological alkali cations, it constitutes a 40 (vs. Na⁺) to 160% (vs. K⁺) improvement over ETH1644. ETH1810 was also found to be capable of acting as a carrier of biogenic amines and related molecules, with the following selectivity sequence: tryptamine (20)>phenylethylamine (7.8)>tyramine (4.3)>serotonin (2.5)>Li⁺ (1)>NH ₄ ⁺ (0.013)>dopamine (0.012). It was found that protons, at physiological concentrations, do not interfere with the lithium transport mediated by ETH1810. The relationship between the improvements in ionic selectivity and potencyvs. the differences in structural features is discussed.     

Transport of Na+ by monensin across bimolecular lipid membranes

Transmembrane ²²Na fluxes across bimolecular lipid membranes are measured under two different experimental conditions: (a) the pH is the same in the two bulk aqueous solutions on either side of the membrane while the concentrations of Na⁺ are different; (b) the concentrations of Na⁺ are identical but pH of the two solutions are different. In this latter case, the transport of Na⁺ occurs in the opposite direction to the difference of the proton concentration. In both cases, the electrical charge flux is negligible. A transport model is proposed to account for the experimental data.     

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.     

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.     

A hydroxide ion carrier in planar phospholipid bilayer membranes: (C6F5)2Hg (dipentafluorophenylmercury)

Although a number of molecules are known to function as current-carrying proton carriers across lipid bilayer membranes, no such hydroxide ion carriers have been found to date. We report that (C₆F₅)₂ Hg, which can function as a chloride ion carrier, can also carry a hydroxide ion. In 100 mM Na₂SO₄ solutions, membranes treated with (C₆F₅)₂Hg are almost ideally selective for H⁺/OH⁻ between pH 6.0 and 9.5. Membrane conductance varies linearly with [OH⁻] over this pH range and with the square of the (C₆F₅)₂Hg concentration. The presumed current-carrying species is the dimer [(C₆F₅)₂Hg]₂OH⁻, which, along with the neutral molecule (C₆F₅)₂Hg, shuttles back and forth within the bilayer. In 0.2 M NaCl at pH 9.5, the OH⁻ and Cl⁻ conductances are approximately equal. Thus, the carrier displays an approximately 10⁴-fold preference for OH⁻ over Cl⁻.     

Water permeation through the lipid bilayer membrane. Test of the liquid hydrocarbon model

According to the liquid hydrocarbon model, the lipid bilayer is viewed simply as a thin slice of bulk hydrocarbon liquid. This allows the water permeability of the bilayer to be calculated from bulk properties. In this paper the prediction of the liquid hydrocarbon model is compared with the known water permeability coefficient of the glycerol monoolein/n-hexadecane bilayer (Fettiplace, R. (1978) Biochim. Biophys. Acta 513, 1–10). As the alkyl chain of glycerol monoolein is equivalent to 8-heptadecene, the water permeability coefficient of 8-heptadecene/n-hexadecane mixtures was measured for temperatures between 20 and 35°C. The mole fraction of n-hexadecane in the bulk liquid was chosen at each temperature to match the known mole fraction of n-hexadecane in the bilayer (White, S. (1976) Nature 262, 421–422). The predicted water permeability coefficient agrees with the measured value at 32°C but is 40% above the measured value at 20°C. The apparent activation energy predicted by the liquid hydrocarbon model is 9.0 ± 0.3 kcal/mol, while the measured value is 14.2 ± 1.0 kcal/mol. The failure of the liquid hydrocarbon model probably results from a different molecular organization of the hydrocarbon chains in the bilayer and in the bulk liquid.     

A quartz cell for studying planar lipid bilayer membranes

A quartz chamber is proposed for use in experiments with planar lipid bilayer membranes. Membranes are formed in a hole made on the lateral wall of a fused quartz test tube, immersed in an electrolyte solution. The quartz cell is easy to clean, chemically inert and easily made. Membranes formed in this chamber had specific resistances higher than 10⁸ Ω·cm² and excellent mechanical stability.     

Proton/hydroxide conductance through lipid bilayer membranes

Summary A simple method of measuring proton/hydroxide conductance (G _H/OH) through planar lipid bilayer membranes is described. First the total conductance (G _m ) is measured electrically. Then the H⁺/OH^– transference number (T _H/OH) is estimated from the diffusion potential (V _m ) produced by a transmembrane pH gradient. The pH gradient is produced by a pair of buffered solutions which have identical concentrations of all ions except H⁺ and OH^–. Thus,V _m is due entirely to H⁺/OH^– diffusion andG _H/OH can be calculated from the relations,V _m =T _H/OH E _H/OH andG _H/OH=T _H/OH G _m , whereE _H/OH is the equilibrium potential for H⁺ and OH^–. In bilayers made from bacterial phosphatidylethanolamine (PE) inn-decane,G _H/OH is nearly independent of pH, ranging from about 10^–9 S cm^–2 at pH 1.6 to 10^–8 S cm^–2 at pH 10.5. BecauseG _H/OH is nearly independent of pH, the calculated permeability coefficients to H⁺ and/or OH^– are extremely pH dependent, which partly explains the wide range of values reported for phospholipid vesicles and biological membranes.G _H/OH appears to be independent of the membrane surface charge, because titrating either the phosphate or the amino group of PE has little effect onG _H/OH.G _H/OH is reduced about 10-fold when the water activity is reduced 33% by replacement with glycerol. Although the mechanism of H⁺/OH^– conductance is not known, the relation betweenG _H/OH and water activity suggests that several water molecules are involved in the H⁺/OH^– transport process.     

Cation transport properties of a synthetic Ca2+-selective peptide ionophore in phospholipid and sarcoplasmic reticulum vesicles

Transport by the synthetic cyclic peptide ionophore CYCLEX-2E (Deber, C.M., Young, M.E.M., and Tom-Kun, J. (1980) Biochemistry 19, 6194–6198), which in contrast to Ca²⁺ ionophore A23187 contains no ionizable protons, has been studied with respect to Ca²⁺ and Na⁺ transport, and the involvement of exchanged, or counter-transported ions during the transport process. CYCLEX-2E was found to equilibrate Na⁺ and Ca²⁺ gradients across phospholipid vesicle membranes. Experiments using the indicator dye Arsenazo III established that calcium ions were indeed reaching the aqueous intravesicular compartments. Absence of metal cations in the external buffer slowed, but did not eliminate, the efflux of Ca²⁺ from phosphatidylcholine vesicles. As an example of its activity in a biological membrane, CYCLEX-2E was shown to be capable of producing Ca²⁺ efflux from sarcoplasmic reticulum vesicles which had been loaded with Ca²⁺ in an ATP-dependent manner. The overall results suggest that in transport by synthetic peptide ionophores typified by CYCLEX-2E, electroneutrality is achieved either through (a) peptide-mediated compensating (but not coupled) fluxes of other cations, or where this is not an option, by (b) transmembrane diffusion of permeant ions such as H⁺, OH^?, or Cl^?.     

A new method of the measurement of the electrically neutral fluxes of cations through lipid bilayer membranes induced by Men+/nH+-exchangers

Electrically neutral ionophores (nigericin, monencin) incorporated into a planar bilayer lipid membrane (BLM) bring about hydrogen ion gradient formation in the unstirred layers of BLM if a metal ion gradient on the membrane is prepared. Under these conditions a diffusion potential of a hydrogen ion is generated after addition of a protonophore. Cation selectivity of nigericin, monencin and A23187 has been studied by means of electrical potential measurements in the presence of a protonophore and Men+/nH+-exchangers mentioned above. The data on cation selectivity are in a good agreement with the well known results of the direct measurements of metal ion fluxes. This shows that the effect of generation of the potential on BLM in the presence of a protonophore and a Men+/nH+-exchanger can be used for the estimation of electrically neutral ion fluxes through BLM.     

Selective transport of Li+ across lipid bilayer membranes mediated by an ionophore of novel design (ETH1644)

Summary The neutral noncyclic, lithium-selective ionophore ETH1644, which is structurally different from previously available ionophores of this type, is a selective carrier of Li^– in lipid bilayer membranes of various lipid composition. The ionophore forms a 21 carrier/cation complex, and the rate-limiting step in the overall transport process is the diffusion of the carrier/ion complex across the membrane.The selectivity sequence for lithiumvs. other ions normally found in biological systems is: Li⁺ (1)>Na⁺ (0.017)K⁺ (0.017) >Cl^– (0.001), Ca²⁺ and Mg²⁺ are impermeant. At neutral pH protons do not interfere with the Li⁺-carrying ability of this ionophore. On the basis of structural differences and supported by conductance data, it is argued that the improved selectivity of Li⁺ over the other alkali cations is due more to a decrease in the affinities of the ionophore for the latter cations that to an increase of its affinity to Li⁺. This ionophore can also act as a carrier of biogenic amines (catecholes, indoles and derivatives), with the structure of the permeant species and mechanism of permeation similar to that observed with the alkali cations. The selectivity sequence is: tryptamine (18.1)>phenylethylamine (11.6)> tyramine (2.4)>Li⁺(1)>serotonin (0.34)>epinephrine (0.09) >dopamine (0.05)>norepinephrine (0.02), showing the ionophore to be more selective to Li⁺ than to any of the neurotransmitters studies.     

Pressure variation of the lateral diffusion in lipid bilayer membranes

A pressure-induced decrease of the lateral diffusion in pure and cholesterol containing phosphatidylcholine bilayer membranes has been determined by the excimer formation technique using pyrene as probe molecule. The experimental results at pressures up to 150 bars are described satisfactorily by the free volume theory of a molecular transport in liquids. A pressure increase of extrapolated 575 bars decreases the lateral diffusion of lipids by a factor of two in pure dipalmitoylphosphatidylcholine membranes. Higher pressures are necessary to induce the same effect in cholesterol containing membranes. This result is interpreted by the condensing effect of cholesterol in fluid bilayer membranes.     

Effects of membrane composition and lipid structure on the photopolymerization of lipid diacetylenes in bilayer membranes

Molecules analogous to biological and synthetic lipids have been prepared with conjugated diacetylene moieties in the long alkyl chain. These lipid diacetylenes form bilayer structures when suspended in aqueous buffers. Ultraviolet light (254 nm) exposure initiates the polymerization of the diacetylenes in the lipid bilayer to give a fully conjugated, highly colored product. The reaction is topotactic, and its efficiency depends on the correct alignment of the monomeric units. Thus, the lipid diacetylenes are photopolymerizable if the hydrocarbon chains are in a regular lattice found at temperatures below the lipid transition temperature; polymerization is inhibited above this transition. The photopolymerization of a diacetylenic glycerophosphocholine in lipid bilayer membranes was observed in two-component mixtures with a nonpolymerizable lipid, either dioleoylphosphatidylcholine or distearoylphosphatidylcholine. The photochemical and thermochemical characteristics suggest that the diacetylenic glycerophosphocholine exists largely in separate domains in the mixed bilayers. Lipid diacetylenes analogous to a dialkyldimethylammonium salt and to a dialkyl phosphate have a plane of symmetry, which suggests that both chains penetrate equally into the bilayer. The photopolymerization of these symmetrical synthetic species is more than 10³-times more efficient than that of the diacetylenic glycerophosphocholine. These differences are interpretable in terms of the expected conformational preference of the lipid molecules.     

Pulse-length dependence of the electrical breakdown in lipid bilayer membranes

Charge-pulse experiments were performed on artificial lipid bilayer membranes with charging times in the range between 10 ns and 10 μs. If the membranes are charged to voltages in the order of 100 mV, the membrane voltage at the end of the charge pulse is a linear function of the injected charge. However, if the membranes are charged to voltages in the range of 1 V, this relationship no longer holds and a reversible high conductance state occurs. This state is defined as an electrical breakdown and it does not allow the membranes to charge to higher voltages than the breakdown voltage, $\text{V}{}_{\mathrm{<mtext>c</mtext>}}$. Between charging times of 300 ns and 5 μs at 25°C and between 100 ns and 2 μs at 40°C, $\text{V}{}_{\mathrm{<mtext>c</mtext>}}$ showed a strong dependence on the charging time of the membrane and decreased from 1.2 to 0.5 V (25°C) and from 1 to 0.4 V (40°C). For other charging times below and above these ranges, the breakdown voltage seemed to be constant. The results indicate that the breakdown phenomenon occurs in less than 10 ns.The pulse-length dependence of the breakdown voltage is consistent with the interpretation of the electrical breakdown mechanism in terms of the electromechanical model. However, it seems possible that below a charging time of the membrane of 300 ns (25°C) and 100 ns (40°C) other processes (such as the Born energy) become possible.     

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.     

Inorganic mercury (Hg2+) transport through lipid bilayer membranes

Summary Diffusion of inorganic mercury (Hg²⁺) through planar lipid bilayer membranes was studied as a function of chloride concentration and pH. Membranes were made from egg lecithin plus cholesterol in tetradecane. Tracer (²⁰³Hg) flux and conductance measurements were used to estimate the permeabilities to ionic and nonionic forms of Hg. At pH 7.0 and [Cl^–] ranging from 10–1000mm, only the dichloride complex of mercury (HgCl₂) crosses the membrane at a significant rate. However, several other Hg complexes (HgOHCl, HgCl ₃ ^– and HgCl ₄ ^2– ) contribute to diffusion through the aqueous unstirred layer adjacent to the membrane. The relation between the total mercury flux (J _Hg), Hg concentrations, and permeabilities is: 1/J _Hg=1/P ^ul[Hg ^t ]+1/P ^m [HgCl₂], where [Hg ^t ] is the total concentration of all forms of Hg,P ^ul is the unstirred layer permeability, andP ^m is the membrane permeability to HgCl₂. By fitting this equation to the data we find thatP ^m =1.3×10^–2 cm sec^–1. At Cl^– concentrations ranging from 1–100mm, diffusion of Hg ^t through the unstirred layer is rate limiting. At Cl^– concentrations ranging from 500–1000mm, the membrane permeability to HgCl₂ becomes rate limiting because HgCl₂ comprises only about 1% of the total Hg. Under all conditions, chemical reactions among Hg²⁺, Cl^– and/or OH^– near the membrane surface play an important role in the transport process. Other important metals, e.g., Zn²⁺, Cd²⁺, Ag⁺ and CH₃Hg⁺, form neutral chloride complexes under physiological conditions. Thus, it is likely that chloride can facilitate the diffusion of a variety of metals through lipid bilayer and biological membranes.     

Synthetic copoly(Lys/Phe) and poly(Lys) translocate through lipid bilayer membranes

Several membrane-transporting peptides (MTP) containing basic amino acid residues such as Lys and Arg that carry macromolecules such as DNA and proteins across cell plasma membranes by an unknown mechanism have been actively studied. On the basis of these results, we have been investigating the translocation ability of synthetic polypeptides, copoly(Lys/Phe) and poly(Lys), through negatively charged phospholipid (soybean phospholipid (SBPL)) bilayer membranes by zeta potential analysis, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, an electrophysiology technique, and confocal laser scanning microscopy (CLSM). The binding of these polypeptides to the membrane, which is the first step for translocation across the membrane, resulted in the conformational transition of the polypeptide from a random coil form or helix-poor form to a helix-rich form. The fluorescence studies demonstrated that the time-dependent decrease in the fluorescence intensities of the FITC-labeled polypeptides bound to the SBPL liposome reflected translocation of the polypeptide across the lipid bilayer with the low dielectric constant. Both the rate constant and the efficiency of the polypeptide translocation across the lipid bilayer were greater for copoly(Lys/Phe) than for poly(Lys). These results suggest that the random incorporation of the hydrophobic Phe residue into the positively charged Lys chain results in a lowering of the potential barrier for passage of the polypeptide in the hydrophobic core portion of the lipid bilayer. We presented the first direct observation that the positively charged polypeptides, copoly(Lys/Phe) (MW: 41,500) and poly(Lys) (MW: 23,400), could translocate across the lipid bilayer membrane.     

Permeability of small nonelectrolytes through lipid bilayer membranes

Summary Diffusion of small nonelectrolytes through planar lipid bilayer membranes (egg phosphatidylcholine-decane) was examined by correlating the permeability coefficients of 22 solutes with their partition coefficients between water and four organic solvents. High correlations were observed with hexadecane and olive oil (r=0.95 and 0.93), but not octanol and ether (r=0.75 and 0.74). Permeabilities of the seven smallest molecules (mol wt <50) (water, hydrofluoric acid, hydrochloric acid, ammonia, methylamine, formic acid and formamide) were 2- to 15-fold higher than the values predicted by the permeabilities of the larger molecules (50相似文献