Dipole moment of alamethicin as related to voltage-dependent conductance in lipid bilayers.

The dipole moment of alamethicin, which produces voltage-dependent conductance in lipid-bilayer membranes, was measured in mixed solvents of ethanol and dioxane. The value of the dipole moment was found to increase from 40 to 75 DU (Debye units), as the concentration of ethanol increased from 0 (pure dioxane) to 40%. The relaxation frequency of alamethicin also changes from 10 to 40 MHz, depending upon the concentration of ethanol in mixed solvents. The length of alamethicin was calculated by using the relaxation time and was found to range from approximately 40 to 20 A. The dipole moment was independently calculated from voltage-dependent conductance and compared with the measured value. The calculated value was found to be larger than the value of direct measurements, indicating that several alamethicin molecules are required to form a conducting pore and that their dipole moments are oriented parallel to each other.     

Lateral diffusion and conductance properties of a fluorescein-labelled alamethicin in planar lipid bilayers

In order to follow alamethicin diffusion within membranes under conditions of pore-formation, a fluorescein isothiocyanate (FITC) analogue was synthesized. To test the influence of the fluorescent probe addition on the pore-forming activity of the new analogue, macroscopic and single-channel experiments into planar lipid bilayers were performed. Although the apparent mean number of monomers per conducting aggregate was equivalent, the voltage-dependence of the new analogue was slightly reduced and hysteresses were broader, in agreement with the much longer duration of the open single-channels. Thus, the conducting aggregates seem to be stabilized by the introduction of the probe, presumably through the interaction of the conjugated cycles with the lipid headgroups, while the added steric hindrance may account for the slightly higher conductances of the open substates. Lateral diffusion of the labelled peptide associated with the bilayer was then investigated by the fluorescence recovery after photobleaching technique. Under applied voltage, associated with high conductance, D, the lateral diffusion coefficient, was reduced by 50% when compared to peptide at rest. These results provide new independent experimental evidence for a voltage-driven insertion of the highly mobile surface-associated peptide into the bilayer as a prominent step in pore formation.     

Mechanism of alamethicin insertion into lipid bilayers.

Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition.     

Calcium-induced inactivation of alamethicin in asymmetric lipid bilayers

This paper discusses a calcium-dependent inactivation of alamethicin- induced conductance in asymmetric lipid bilayers. The bilayers used were formed with one leaflet of phosphatidyl ethanolamine (PE) and one of phosphatidyl serine (PS). Calcium, initially confined to the neutral lipid (PE) side, can pass through the open alamethicin channel to the negative lipid (PS) side, where it can bind to the negative lipid and reduce the surface potential. Under appropriate circumstances, the voltage-dependent alamethicin conductance is thereby inactivated. We have formulated a model for this process based on the diffusion of calcium in the aqueous phases and we show that the model describes the kinetic properties of the alamethicin conductance under various circumstances. EGTA on the PS side of the membrane reduces the effects of calcium dramatically as predicted by the model.     

Kinetics and stability of alamethicin conducting channels in lipid bilayers

It is already well-established that conduction in lipid bilayers containing alamethicin arises from the presence of complexes in which there are several molecules of the polypeptide. It is with the nature of these complexes that this paper is primarily concerned. While it is clear that increasing alamethicin concentration and increasing potential across the membrane favour their formation, the nature of the reactions involved has not yet been elucidated. Attempts have therefore been made to clarify the sequence of events leading to the establishment of a complex in its conducting state. It has been concluded that the most likely mechanism involves, initially, a non-field-dependent aggregation of the alamethicin, in the plane of the membrane, into non-conducting oligomers. These then appear to undergo movement normal to the membrane (which is field dependent) to form the conducting species. Temperature studies have shown that the various conducting states of the oligomer have effectively equal enthalpies, and that the activation energies for transitions between these states are all approx. 1.2 kcal/mol. The corresponding rate constants are very sensitive to the lipid composition of the membrane and a variety of different systems has been examined in order to clarify the origins of this effect. The only conclusion from this part of the work is that lipid fluidity might be involved.     

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 effect of lanthanum on alamethicin channels in black lipid bilayers

The properties of alamethicin channels in dioleyl phosphatidylcholine bilayers were studied in 1 M LaCl3 and were compared with those in 1 M NaCl. Single-channel recordings demonstrated that the mean single-channel life-time is about 0.25 s in NaCl but only about 17 ms in LaCl3. Whereas in NaCl the conductance levels 2 and 3 are mostly populated, in LaCl3 the levels 0 and 1 are preferentially adopted. The single-level conductance are slightly smaller in LaCl3 if the higher bulk solution conductivity of LaCl3 is taken into account. Multipore experiments confirmed earlier results (Boheim, G., Irmscher, G. and Jung, G. (1978) Biochim. Biophys. Acta 507, 485--506) that the bilayer conductance is less strongly dependent on voltage in LaCl3 than in NaCl solution. Current-fluctuation analysis showed that this effect can be explained by a less strong dependence on voltage of the pore-formation rate as well as of the mean channel life-time in LaCl3. The data can be interpreted as an increased lateral diffusion mobility of the alamethicin monomers in the bilayer. This can be the result of the binding of La3+ to the polar headgroups which can induce cluster formation of the phospholipids.     

Melittin induced voltage-dependent conductance in DOPC lipid bilayers

Melittin-induced conductance was measured on planar bilayers made from dioleoylphosphatidylcholine. Upon application of a fixed voltage, the current response was monophasic and remained so even after prolonged observation times. The conductance of melittin-doped bilayers increased exponentially with voltage. In addition, an ohmic contribution appeared after some current had passed. The voltage-dependent conductance increased e-fold every 22 mV and was proportional to the fourth power of the aqueous monomeric peptide concentration, for all salt concentrations investigated (0.4-1.8 M NaCl). Discrete conductance steps could be resolved at all these salt concentrations. The amplitudes of these steps were highly variable. In each experiment, conductance was initially only observed for potentials which were positive on the side of peptide addition. As more and more current passed across the bilayer, the current-voltage curves became symmetric. The system needed some time to reach stationary current-voltage characteristics: about 50 min at pH 7 but only about 15 min at pH 8, suggesting involvement of the N-terminus (pK around 7.5) of melittin in the slow formation of a 'prepore'.     

Theory of passive proton conductance in lipid bilayers

The large permeability of lipid bilayers to protons compared to other small ions calls for a special proton transport mechanism. At the present time, only mechanisms involving transient hydrogen-bonded chains of water can account for the experimental result that the conductance is nearly independent of pH. Three models involving transient hydrogen-bonded chains are discussed, including an outline of the kinetic calculations that lead to predictions of current versus voltage drop and current versus pH differences. These calculations can be compared to experiment to determine which, if any, of these models pertains to lipid bilayers.     

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.     

Pressure effects on alamethicin conductance in bilayer membranes.

We report here the first observations of the effects of elevated hydrostatic pressure on the kinetics of bilayer membrane conductance induced by the pore-forming antibiotic, alamethicin. Bacterial phosphatidylethanolamine-squalene bilayer membranes were formed by the apposition of lipid monolayers in a vessel capable of sustaining hydrostatic pressures in the range, 0.1-100 MPa (1-1,000 atm). Principal observations were (a) the lifetimes of discrete conductance states were lengthened with increasing pressure, (b) both the onset and decay of alamethicin conductance accompanying application and removal of supra-threshold voltage pulses were slowed with increasing pressure, (c) the onset of alamethicin conductance at elevated pressure became distinctly sigmoidal, suggesting an electrically silent intermediate state of channel assembly, (d) the magnitudes of the discrete conductance levels observed did not change with pressure, and, (e) the voltage threshold for the onset of alamethicin conductance was not altered by pressure. Apparent activation volumes for both the formation and decay of conducting states were positive and of comparable magnitude, namely, approximately 100 A3/event. Observation d indicates that channel geometry and the kinetics of ion transport through open channels were not affected by pressure in the range employed. The remaining observations indicate that, while the relative positions of free-energy minima characterizing individual conducting states at a given voltage were not modified by pressure, the heights of intervening potential maxima were increased by its application.     

Electrophysiological interrogation of asymmetric droplet interface bilayers reveals surface-bound alamethicin induces lipid flip-flop

The droplet interface bilayer (DIB) method offers simple control over initial leaflet compositions in model membranes, enabling an experimental path to filling gaps in our knowledge about the interplay between compositional lipid asymmetry, membrane properties, and the behaviors of membrane-active species. Yet, the stability of lipid leaflet asymmetry in DIBs has received very little attention, particularly in the presence of peptides and ion channels that are often studied in DIBs. Herein, we demonstrate for the first time parallel, capacitance-based measurements of intramembrane potential with arrays of asymmetric DIBs assembled in a microfluidic device to characterize the stability of leaflet asymmetry over many hours in the presence and absence of membrane-active peptides. DIBs assembled from opposing monolayers of the ester (DPhPC) and ether (DOPhPC) forms of diphytanoyl-phosphatidylcholine yielded asymmetric bilayers with leaflet compositions that were stable for at least 18?h as indicated by a stable |137?mV| intramembrane potential. In contrast, the addition of surface-bound alamethicin peptides caused a gradual, concentration-dependent decrease in the magnitude of the dipole potential difference. Intermittent current-voltage measurements revealed that alamethicin in asymmetric DIBs also shifts the threshold voltage required to drive peptide insertion and ion channel formation. These outcomes take place over the course of 1 to 5?h after membrane formation, and suggest that alamethicin peptides promote lipid flip-flop, even in the un-inserted, surface-bound state, by disordering lipids in the monolayer to which they bind. Moreover, this methodology establishes the use of parallel electrophysiology for efficiently studying membrane asymmetry in arrays of DIBs.     

Interaction of alamethicin pores in DMPC bilayers

We have investigated the x-ray scattering signal of highly aligned multilayers of the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine containing pores formed by the antimicrobial peptide alamethicin as a function of the peptide/lipid ratio. We are able to obtain information on the structure factor of the pore fluid, which then yields the interaction potential between pores in the plane of the bilayers. Aside from a hard core with a radius corresponding to the geometric radius of the pore, we find a repulsive lipid-mediated interaction with a range of approximately 30 A and a contact value of 2.4 k(B)T. This result is in qualitative agreement with recent theoretical models.     

Porin of Pseudomonas aeruginosa forms low conductance ion channel in planar lipid bilayers.

Protein E1, a porin of the outer membrane of Pseudomonas aeruginosa, was reconstituted into planar lipid bilayers. Single channel conductance of the protein appeared to be 230 pS (pico siemens) in 1 M KCl-10 mM Hepes, pH7.2. This value is approximately 5 times lower than the conductance of the OmpF channel of Escherichia coli. Conductance increased linearly as the membrane potential was raised from -200 mV to +200 mV, and was nearly proportional to the KCl concentration. These results show that protein E1 is probably a genuine porin in the P. aeruginosa outer membrane supporting the earlier conclusion that protein E1 forms a small channel.     

Incorporation in lipid bilayers of a large conductance cationic channel from mitochondrial membranes.

Membranes from subcellular fractions of adrenal medulla were incorporated in phospholipid bilayers formed at the tip of microelectrodes. Current fluctuations recorded in the presence of a transmembrane potential revealed the existence of a voltage-dependent channel of large conductance. This channel is characterized by fast kinetics and four conductance levels separated by jumps of 100, 220 and 220 pS in 150 mM NaCl. It is permeant to Na+,K+, tetraethylammonium, Cl- and acetate and has some cation selectivity. Exposure to trypsin or pronase abolished the voltage-dependence. Upon subcellular fractionation, the activity was found to be associated with mitochondria. A similar activity was observed in mitochondrial fractions from other organs. By its kinetics, its selectivity and its potential-dependence, this channel differs from the voltage-dependent anion channel of outer mitochondrial membranes.