Properties of the conductance induced in lecithin bilayer membranes by alamethicin

Current-voltage relations have been measured across lecithin bilayers doped with alamethicin molecules. The results show that there are two aspects of the induced conductances, a voltage-dependent and a voltage-independent conductance. Both have been characterized as a function of alamethicin and KCl concentration. The two aspects of the conductances do not show the same changes with those two variables. The voltage-independent conductance is affected very little by changes in KCl concentration, and its dependance on alamethicin concentration reveals that it is produced by two or three alamethicin molecules. The voltage-dependent conductance is shifted by the changes in KCl concentration only when the concentrations are greater than or equal to 100 mM; below 100 mM KCl the slope of the log conductance-voltage curve is also reduced. The effect of changing alamethicin concentration reveals that nine or ten molecules are involved for KCl concentrations larger than 100 mM; if the KCl concentration is less than 100 mM, the effect of changing the alamethicin concentration is reduced. Time-dependent measurements have also been performed; only one time constant was found and it is strongly voltage-dependent. Also a very slow voltage-dependent absorption process is found. These results can be explained if it is assumed that pores are formed of a mixture of charged and uncharged alamethicin molecules when a voltage is applied and that uncharged alamethicin can also form pores without applying a voltage, once the absorption process has been started by previously applied voltages. The voltage dependence of the time constant seems to indicate that the voltage-dependent pore formation is produced by aggregates of charged alamethicin rather than independent molecules.     

Intrinsic rectification of ion flux in alamethicin channels: studies with an alamethicin dimer.

Covalent dimers of alamethicin form conducting structures with gating properties that permit measurement of current-voltage (I-V) relationships during the lifetime of a single channel. These I-V curves demonstrate that the alamethicin channel is a rectifier that passes current preferentially, with voltages of the same sign as that of the voltage that induced opening of the channel. The degree of rectification depends on the salt concentration; single-channel I-V relationships become almost linear in 3 M potassium chloride. These properties may be qualitatively understood by using Poisson-Nernst-Planck theory and a modeled structure of the alamethicin pore.     

Inactivation of the alamethicin-induced conductance caused by quaternary ammonium ions and local anesthetics

Long alkyl chain quaternary ammonium ions (QA), the local anesthetics (LA) tetracaine and lidocaine, imipramine, and pancuronium cause inactivation of the alamethicin-induced conductance in lipid bilayer membranes. The alamethicin-induced conductance undergoes inactivation only when these amphipathic compounds are added to the side containing alamethicin. The concentration of QA required to cause a given amount of inactivation depends on the length of the hydrocarbon chain and follows the sequence C9 greater than C10 greater than C12 greater than C16. LA and imipramine, in contrast to QA or pancuronium, are able to promote appreciable inactivation only if the pH of the alamethicin-free side is equal to or lower than the pK of these compounds. The membrane permeability to QA, LA, or imipramine is directly proportional to the alamethicin-induced conductance and is larger than the one for potassium. The observed steady state and time-course of the inactivation are well described by a model similar to that proposed by Heyer et al. (1976. J. Gen. Physiol. 67:703--729) and extended for any value of the diffuse double layer potential and for LA and imipramine. In this model QA, LA, or imipramine are able to permeate through the membrane only when the alamethicin-induced conductance is turned on. The amphipathic compounds then bind to the other membrane surface, changing the transmembrane potential and turning the conductance off. For a given concentration of QA, LA, or imipramine the extent of inactivation depends on two factors: first, the binding characteristics of these compounds to the membrane surface and second, their ability to permeate through the membrane when the alamethicin-induced conductance is turned on. The several possible mechanisms of permeation of the amphipathic molecules tested are discussed.     

Evidence for nonlinear capacitance in biomembrane channel system.

The electrophysiological properties of voltage-dependent anion channels from mitochondrial membrane have been studied in a bilayer membrane system. It was observed that the probability of opening of the membrane channel depends on externally applied voltage and the plot is a bell-shaped curve symmetric around probability axis. A scheme of conformational energy levels under varying externally applied voltage was formulated. Assuming that the probability follows Boltzmann distribution, we arrive at an expression of change in energy containing a separate term identical to the energy of a capacitor. This fact indicates the possibility of existence of an added capacitance due to the channel protein. Further it was shown that the aforesaid channel capacitor could be a function of voltage leading to nonlinearity. We have offered a general method of calculating nonlinear capacitance from the experimental data on opening probability of a membrane channel. In case of voltage-dependent anion channel the voltage dependence of the capacitor has a power 0.786. The results have been interpreted in view of the structural organization of the channel protein in the membrane. Our hypothesis is that the phenomenon of capacitor behaviour is a general one for membrane channels.     

Voltage-dependent conductance for alamethicin in phospholipid vesicles. A test for the mechanism of gating.

The ion currents induced by alamethicin were investigated in unilamellar vesicles using electron paramagnetic resonance probe techniques. The peptide induced currents were examined as a function of the membrane bound peptide concentration, and as a function of the transmembrane electrical potential. Because of the favorable partitioning of alamethicin to membranes and the large membrane area to aqueous volume in vesicle suspensions, these measurements could be carried out under conditions where all the alamethicin was membrane bound. Over the concentration range examined, the peptide induced conductances increased approximately with the fourth power of the membrane bound peptide concentration, indicating a channel molecularity of four. When the alamethicin induced currents were examined as a function of voltage, they exhibited a superlinear behavior similar to that seen in planar bilayers. Evidence for the voltage-dependent conduction of alamethicin was also observed in the time dependence of vesicle depolarization. These observations indicate that the voltage-dependent behavior of alamethicin can occur in the absence of a voltage-dependent phase partitioning. That is, a voltage-dependent conformational rearrangement for membrane bound alamethicin leads to a voltage-dependent activity.     

Alamethicin-induced current-voltage curve asymmetry in lipid bilayers.

We have examined the causes of the asymmetry of the current-voltage curve induced by addition of alamethicin to one side of a black lipid membrane. We find that the alamethicin-induced current-voltage (I-V) curve has an inherent asymmetry. If it were possible to confine all alamethicin molecules to one side of the membrane, the I-V curve would exhibit a positive branch (voltage being measured with respect to the side of the membrane trans to the alamethicin addition) of steeper logarithmic slope than the negative branch and at a lower absolute value of potential. This condition is not usually realized, however, because alamethicin can leak through the membrane, so that, except at very high alamethicin concentrations and in certain kinds of membranes, the positive branch of the current-voltage curve has the same logarithmic slope as the negative branch and appears to arise from alamethicin which diffuses from the cis to the trans side of the membrane. We develop simple quantitative models for these two cases.     

Chemisorption of proteins and their thiol derivatives onto gold surfaces: characterization based on electrochemical nonlinearity

This study was conducted to monitor the electrochemical responses of two proteins (bovine serum albumin (BSA) and gelatin) and their thiol derivatives adsorbed onto gold (Au) electrodes, which were analyzed by a "nonlinear" impedance method. A sinusoidal voltage is applied to a protein-containing aqueous solution and the waveform of the output current is analyzed by fast Fourier transformation (FFT). The intensities of the higher harmonics in the FFT varied with the species of protein and their thiol derivatives, and with time. From the higher harmonics, voltage-dependent capacitance and conductance were quantitatively evaluated to differentiate the state of adsorbed protein. Adsorption and desorption characteristics of BSA and its thiol derivative on the Au surface were continuously measured by a quartz crystal microbalance (QCM) in situ. The microscopic state of thiol-derivatized BSA adsorbed onto the Au surface was imaged by atomic force microscopy (AFM). In general, thiol-derivatized proteins were tightly adsorbed on the Au surface and showed no desorption. The present electrochemical measurements clearly differentiated adsorption characteristics of physically adsorbed (physisorbed) and chemically adsorbed (chemisorbed) proteins on Au surfaces.     

Voltage-dependent channel formation by rods of helical polypeptides

Summary The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts -helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length.In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) _n -OMe,n=1–4—which adopt -helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase.The steady-state voltage-dependent conductance depends on the 8^th–9^th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 -helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.     

Pancuronium inactivates alamethicin-induced conductance in artificial membranes.

The effect of pancuronium on alamethicin-induced currents was studied in negatively charged lipid bilayer membranes. Pancuronium induces inactivation of the alamethicin-induced current. Inactivation is only observed if this compound is added to the compartment containing alamethicin. Moreover, the process of inactivation is reduced or abolished if pancuronium is added to the alamethicin-free side of the membrane. The time needed to recover from inactivation is greatly reduced if the aqueous solution in the alamethicin-free compartment is stirred. These data suggest that pancuronium permeates through the membrane when the alamethicin-induced conductance is "turned on," binds to the other membrane surface, and changes the surface potential.     

The nature of the voltage-dependent conductance induced by alamethicin in black lipid membranes

Summary Alamethicin induces a conductance in black lipid films which increases exponentially with voltage. At low conductance the increase occurs in discrete steps which form a pattern of five levels, the second and third being most likely. The conductance of each level is directly proportional to salt concentration, inversely proportional to solution viscosity, and nearly independent of voltage.The probability distribution of the five steps is not a function of voltage, but as the voltage is increased, more levels begin to appear. These can be explained as super-positions of the original five, both in position and relative probability.This suggests that the five levels are associated with a physical entity which we call a pore. This point of view is confirmed by the following measurements. The kinetic response of the current to a voltage step is first order, and shows an exponential increase in rate of pore formation and an exponential decrease in rate of pore disappearance with voltage. If these rates are statistical, the number of pores should fluctuate about a voltage-dependent mean. High conductance current fluctuations are too large to be explained by fluctuation in the number of pores alone. But if fluctuations among the five levels are included, the magnitude of the fluctuations at high conductance is accurately predicted.Alamethicin adsorbs reversibly to the membrane surface, and the conductance at a fixed voltage depends on the ninth power of alamethicin concentration and on the fourth power of salt concentration, in the aqueous phase. In our bacterial phosphatidyl ethanolamine membranes, alamethicin added to one side of the membrane produces elevated conductance only when the voltage on that side is increased.On leave of absence from the Facultad de Ciencias, Universidad de Chile, Santiago de Chile.     

Voltage-dependent conductance induced by alamethicin-phospholipid conjugates in lipid bilayers.

Alamethicin, a linear 20-amino acid antibiotic, forms voltage-dependent channels in lipid bilayer membranes. We show here that alamethicin-phospholipid conjugates can be prepared by photolysis of unilamellar vesicles containing alamethicin and a phosphatidylcholine analogue with a carbene precursor at the end of the C-2 fatty acyl chain. This result indicates that at least a portion of the alamethicin molecule is in contact with the hydrocarbon moiety of the membrane in the absence of an applied voltage. Furthermore, the alamethicin-phospholipid photoproduct is able to induce a voltage-gated conductance similar to that of natural alamethicin. The importance of these results in terms of mechanisms for channel gating is discussed.     

The competition transport of sodium ions and protons in the cytoplasmic access channel of the Na+,K+-ATPase

The changes in capacitance and conductance of lipid bilayer membranes have been studied with adsorbed membrane fragments containing Na⁺,K⁺-ATPase. These changes have been initiated by fast release of protons from a bound form (“caged H⁺”) induced by an UV flash. The changes of the capacitance in the presence of Na⁺,K⁺-ATPase were affected by the frequency of the applied voltage, pH and the concentration of sodium ions. Addition of sodium ions altered the changes of capacitance caused by a pH jump in the medium due to caged H⁺ photolysis, and the magnitude and sign of this effect depended on the initial pH. These results are explained by competitive binding of sodium ions and protons to the ion-binding sites of the Na⁺,K⁺-ATPase at its cytoplasmic side. The pH at which the sign of the sodium ion effect changed allows the evaluation of the pK of the proton binding site, which is about 7.6.     

Alamethicin channel permeation by Ca2+, Mn2+ and Ni2+ in bovine chromaffin cells.

Alamethicin causes a concentration-dependent increase of [Ca2+]i in suspensions of bovine adrenal chromaffin cells loaded with fura-2. The basal levels of Cai2+ (234 +/- 37 nM; n = 4) increased to a maximum of 2347 +/- 791 nM (n = 3) with 100 micrograms/ml alamethicin. In the presence of 1 mM Cae2+ the increase reached a plateau within about 2-5 s. This increase was due to Ca2+ entry into chromaffin cells, since in the absence of Cae2+ alamethicin did not modify [Ca2+]i. This contrasts with ionomycin (1 microM) which produced a Cai2+ transient even in the absence of Cae2+. Mn2+ ions also entered chromaffin cells in the presence of alamethicin, as measured by the quenching of fura-2 fluorescence following excitation at 360 nm. Resting chromaffin cells had a measurable permeability to Mn2+ which was drastically increased by cell depolarization by K+ (50 mM) addition. This suggests that Mn2+ is able to permeate voltage-dependent Ca2+ channels. Ni2+ uptake into either resting or K(+)-stimulated chromaffin cells was undetectable, but addition of alamethicin induced rapid uptake of this cation. The alamethicin-induced entry of Ni2+ was decreased by 50 mM K+. Overall, the results are compatible with the formation by alamethicin of ion channels in chromaffin cell plasma membranes.     

Antimicrobial peptide magainin I from Xenopus skin forms anion-permeable channels in planar lipid bilayers.

The ionophore properties of magainin I, an antimicrobial and amphipathic peptide from the skin of Xenopus, were investigated in planar lipid bilayers. Circular dichroism studies, performed comparatively with alamethicin, in small or large unilamellar phospholipidic vesicles, point to a smaller proportion of alpha-helical conformation in membranes. A weakly voltage-dependent macroscopic conductance which is anion-selective is developed when using large aqueous peptide concentration with lipid bilayer under high voltages. Single-channel experiments revealed two main conductance levels occurring independently in separate trials. Pre-aggregates lying on the membrane surface at rest and drawn into the bilayer upon voltage application are assumed to account for this behaviour contrasting with the classical multistates displayed by alamethicin.     

Voltage-dependent lipid flip-flop induced by alamethicin.

Alamethicin appears to allow voltage-dependent lipid exchange ("flip-flop") between leaflets of a planar bilayer. In membranes with one leaflet of phosphatidyl serine and one of phosphatidyl ethanolamine, the shape of the nonactin current-voltage curve accurately reports the difference in surface potential between the two sides of the membrane. The surface potential is itself a good measure of membrane asymmetry. Alamethicin added to the bathing solutions of an asymmetric membrane does not per se reduce the membrane asymmetry, but turning on the alamethicin conductance by application of a voltage pulse does. Immediately after application of a voltage pulse, large enough to turn on the alamethicin conductance, the asymmetry of the nonactin-K+ current voltage curve decreases, in some cases, nearly to zero. During the pulse, the alamethicin conductance activates if a decrease in surface potential favors turn-on of the alamethicin conductance or inactivates if a decrease in surface potential favors turn-off of the alamethicin conductance. After the pulse, the nonactin-K+ asymmetry returns to its original value if the alamethicin conductance is not turned on. The time-course of this return allows an estimate of the diffusion constant of lipid in the planar bilayer. The value obtained is 5.1 x 10(-8) cm2/s.     

Structure of the alamethicin pore reconstructed by x-ray diffraction analysis

We reconstructed the electron density profile of the alamethicin-induced transmembrane pore by x-ray diffraction. We prepared fully hydrated multiple bilayers of alamethicin-lipid mixtures in a condition where pores were present, as established previously by neutron in-plane scattering in correlation with oriented circular dichroism. At dehydrated conditions, the interbilayer distance shortened and the interactions between bilayers caused the membrane pores to become long-ranged correlated and form a periodically ordered lattice of rhombohedral symmetry. To resolve the phase problem of diffraction, we used a brominated lipid and performed multiwavelength anomalous diffraction at the bromine K edge. The result unambiguously shows that the alamethicin pore is of the barrel-stave type consisting of eight alamethicin helices. This pore structure corresponds to the stable pores detected by neutron in-plane scattering in fully hydrated fluid bilayers at high peptide/lipid ratios, which are the conditions at which alamethicin was tested for its antibacterial activity.     

Structural and membrane modifying porperties of suzukacillin, a peptide antibiotic related to alamethicin. Part B. Pore formation in black lipid films.

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete condustance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger alpha-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of "inactivation".     

A three state model for alamethicin conductance in bilayer membranes

Alamethicin is an antibiotic which produces voltage gated channels in lipid bilayer membranes. Recently completed studies of the pressure dependence of alamethicin conductance have shown that its onset following application of a suprathreshold voltage step at a pressure of 100 MPa (1000 atm) is markedly slowed relative to that observed at ambient pressure. Furthermore, the time course of the onset of conductance becomes distinctly sigmoidal at elevated pressure, a condition which is not evident at atmospheric pressure. The decay of alamethicin conductance upon removal of suprathreshold applied voltage is also slowed by application of hydrostatic pressure, but it follows a single exponential time course at all pressures. In addition, kinetic parameters characterizing the onset and decay of conductance show distinctly different pressure dependences. These observations cannot be explained by a two state model in which alamethicin moves reversibly between nonconducting and conducting states. Therefore we re-examine critically a hypothesis made by previous workers, namely that alamethicin, in monomeric or aggregate form, moves upon application of suprathreshold voltage first from a nonconducting surface state to a nonconducting preassembly or precursor state, and then finally into a conducting state. Parameters of this three state model are related to a geometric factor which measures the degree of sigmoidal conductance response and which can be evaluated directly from experimental data. An alternative aggregation-type analysis, equivalent to that applied by Hodgkin & Huxley to the potassium conductance in squid axon, is also considered in the context of this same geometric factor. The possibility of distinguishing between these analyses on the basis of experimental data is discussed.     

Melittin and a chemically modified trichotoxin form alamethicin-type multi-state pores

The bee venom constituent, melittin, is structurally and functionally related to alamethicin. By forming solvent-free planar bilayers of small area (approx. 100 microns 2) on the tip of fire-polished glass pipettes we could observe single melittin pores in these membranes. An increase in the applied voltage induced further non-integral conductance levels. This indicates that melittin forms multi-level pores similar to those formed by alamethicin. Trichotoxin A40, an antibiotic analogue of alamethicin, also induces a voltage-dependent bilayer conductivity, but no stable pore states are resolved. However, chemical modification of the C-terminal molecule part by introduction of a dansyl group leads to a steeper voltage-dependence and pore state stabilization. Comparing structure and activity of several natural and synthetic amphiphilic polypeptides, we conclude that a lipophilic, N-terminal alpha-helical part of adequate length (dipole moment) and a large enough hydrophilic, C-terminal region are sufficient prerequisites for voltage-dependent formation of multi-state pores.     

Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part B. Pore formation in black lipid films

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete conductance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation tion starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger α-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of “inactivation”.