Distribution of alamethicin in lipid membranes and water

The concentration of alamethicin in aqueous solutions was quantitated using measurements of the spot area on thin-layer chromatograms. These data were utilized to measure a partition coefficient of 17 for alamethicin in a phospholipid membrane-water system under equilibrium dialysis conditions.     

Changes in lipid mobility associated with alamethicin incorporation into membranes

The binding state of the antibiotic peptide alamethicin with phospholipid bilayers was investigated in terms of the changes induced in lipid mobility. Fluorescence anisotropy was used for the study. It was found that an increase in peptide concentration induced different changes in lipid mobility above and below a critical peptide concentration. This concentration was also critical for an increase in the cooperative binding of the peptide, as detected by circular dichroism. Above the critical peptide concentration, the mobility of both lipid regions, around the polar head and hydrocarbon chain, became restricted with an increased peptide concentration. Below the critical level, however, an increased peptide concentration induced a "wobbling" of the lipid hydrocarbon chain. These results show that an increase in the cooperative binding of the peptide is accompanied by a change in the dominant configuration of the binding peptide. When the binding peptide increases, the dominant configuration appears to shift from surface association to deep incorporation within the membrane. This shift in configuration means that in the formation of ion-conductive pores, voltage-driven insertion of the peptide is a prominent step below a critical peptide concentration.     

Oscillation phenomena in black lipid membranes induced by a single alamethicin pore.

In this paper we show how alamethicin (a small cyclic peptide of molecular weight 1691) can produce voltage oscillations in black lipid membranes and how a nonactin-alamethicin oscillator can be constructed. Alamethicin alone induces oscillations only with an applied bias current, but with nonactin and appropriate salt solutions oscillations occur with no bias current. Both kinds of oscillations can be quantitatively understood in terms of the known properties of alamethicin and nonactin and both depend on the statistical nature of the formation opores in the membrane by alamethicin.     

Simple model of ion transport through alamethicin channels in lipid membranes

The electrical characteristics of wide membrane channels such as those induced in lipid membranes by alamethicin have been analyzed using an electrodiffusion model. The channel is considered to be a water filled cylinder in which the potential energy barrier is a result of the difference in polarization energy of the ion environment when the ion is located inside as compared to outside of the channel. In addition, an electric field related to the channel structure is assumed. It is shown that without postulating any specific chemical ion-channel interaction one can reproduce experimental membrane potentials for NaCl, KCl, and CaCl₂ concentration gradients with a single set of channel parameters. The calculations also yield experimental J-V characteristics of discrete conduction states. In addition, a simple mechanism of interchannel coupling based on the above model is discussed. The model suggests a unifying approach to the problem of the origin of interionic selectivity of membrane channels induced by polyene antibiotics.     

Charge-pulse relaxation studies with lipid bilayer membranes modified by alamethicin

Charge-pulse relaxation studies with the alamethicin-lipid membrane system reveal a triphasic decay of membrane voltage. At short times (resolution time 2 microseconds), where a voltage decay due to the orientation of alamethicin dipoles from the interface into the membranes interior ("gating current") could possibly be expected, only a slow decrease with a time constant determined by the bare membrane conductance occurs. After approximately 1 ms (depending on the experimental conditions) the formation of alamethicin pores starts, leading to an increase in the voltage decay rate. When the characteristic voltage Vcpc is approached, pores close and after passing Vcpc the voltage decreases slowly again according to the bare membrane conductance. Vcpc is determined as a function of the initially applied voltage Vo, alamethicin and KCl concentration. Since the membrane voltage decreases continuously, the system does not reach the equilibrium states obtained at constant voltages. Taking the presented experimental results into account the estimate of the electrical potential at the functional membrane of photosynthesis induced by a saturating single turnover flash of deltaphio approximately 105-135 mV (Zickler, Witt and Boheim (1976) FEBS Lett. 66, 142-148) is changed to deltaphio approximately 200 mV.     

Oscillation phenomena in black lipid membranes induced by a single alamethicin pore

In this paper we show how alamethicin (a small cyclic peptide of molecular weight 1691) can produce voltage oscillations in black lipid membranes and how a nonactin-alamethicin oscillator can be constructed. Alamethicin alone induces oscillations only with an applied bias current, but with nonactin and appropriate salt solutions oscillations occur with no bias current. Both kinds of oscillations can be quantitatively understood in terms of the known properties of alamethicin and nonactin and both depend on the statistical nature of the formation of pores in the membrane by alamethicin.     

Comparison of the conformation and orientation of alamethicin and melittin in lipid membranes

The secondary structure of alamethicin in lipid membranes below and above the lipid phase transition temperature Tt is determined by Raman spectroscopy and circular dichroism (CD) measurements. In both cases structural data are obtained by fitting the experimental spectra by a superposition of the spectra of 15 reference proteins of known three-dimensional structure. According to the Raman experiments, in a lipid bilayer above Tt alamethicin is helical from residue 1 to 12, whereas below Tt the helix extends from residue 1 to 16. The remaining C-terminal part is nonhelical up to the end residue 20 both above and below Tt. A considerable lower helix content is derived from CD, namely, 38% and 46% above and below Tt, respectively, in agreement with several reported values for CD in the literature. It is shown that the commonly used set of CD spectra of water-soluble reference proteins is unsuitable to describe the CD spectra of alamethicin correctly. Therefore the secondary structure of alamethicin as derived from CD measurements is at the present state of analysis unreliable. In contrast to the case of alamethicin, the CD spectra of melittin in lipid membranes are correctly described by the reference protein spectra. The helix content of melittin is determined thereby to be 72% in lipid membranes above Tt and 75% below Tt. The data are in accord with a structure where the hydrophobic part of melittin adopts a bent helix as determined recently by Raman spectroscopy [Vogel, H., & J?hnig, F. (1986) Biophys. J. 50, 573]. The orientational order parameters of the helical parts of alamethicin and of melittin in a lipid membrane are deduced from the difference between a corresponding CD spectrum of a polypeptide in planar multibilayers and that in lipid vesicles. The presented method for determining helix order parameters is new and may be generally applicable to other membrane proteins. The orientation of the helical part of both polypeptides depends on the physical state of the lipid bilayer at maximal membrane hydration and in the ordered lipid state furthermore on the degree of membrane hydration. Under conditions where alamethicin and melittin are incorporated in an aggregated form in a fluid lipid membrane at maximal water content the helical segments are oriented preferentially parallel to the membrane normal. Cooling such lipid membranes to a temperature below Tt changes the orientation of the helical part of alamethicin as well as melittin toward the membrane plane.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

TOAC spin labels in the backbone of alamethicin: EPR studies in lipid membranes

Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.     

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.     

C-terminally shortened alamethicin on templates: influence of the linkers on conductances.

In order to test the influence of chemical modifications designed to allow covalent coupling of channel-forming peptide motifs into variable sized oligomers, a series of alamethicin derivatives was prepared. The building block encompassing the N-terminal 1-17 residues of alamethicin behaved normally in the conductance assay on planar lipid bilayers, albeit at higher concentration and with a slightly reduced voltage-dependence. A linker Ac-K-OCH(2)C(6)H(4)CH(3)p attached via the epsilon amino group of lysine to the C-terminus of alamethicin(1-17) increased membrane affinity. The latter was further enhanced in a dimer and a tetramer in which alamethicin(1-17) chains were tethered to di- or tetra-lysine linkers, respectively, but macroscopic current-voltage curves displayed much reduced voltage-dependencies and reversed hysteresis. An usual behaviour with high voltage-dependence was restored with the modified dimer of alamethicin(1-17) in which alanine separated the two consecutive lysine residues in the linker. Of special interest was the development of a 'negative resistance' branch in macroscopic current-voltage curves for low concentrations of this dimer with the more flexible linker. Single channel events displayed only one single open state with fast kinetics and whose conductance matches that of the alamethicin heptamer or octamer.     

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.     

pH modulation of transport properties of alamethicin oligomers inserted in zwitterionic-based artificial lipid membranes

Electric features of biological membranes are major determinants of the function and physiological manifestation of membrane-penetrating peptides, and such features are prone to be modulated by the properties of the surrounding aqueous medium. In this work, we demonstrate that pH plays crucial roles in modulating electric characteristics of zwitterionic-based artificial lipid membranes. The effect of pH on electrical properties of such membranes was probed by evaluating the transport properties of embedded alamethicin oligomers over a wide range of pH values (i.e., 0.65, 2.08, 2.94, 7 and 10.1). Our data strongly support the paradigm of a pH-dependent variation of the surface and membrane dipole potential which, in conjunction with possible lateral pressure effects within the lipid membrane, lead to a non-monotonic modulation of the electrical conductance of alamethicin oligomers. As expected, pH modulation of transport properties through the alamethicin oligomer is more visible for narrower pores (that is, the 1st conductive state) with slightly better cation selectivity as compared to larger oligomers.     

"Reversed" alamethicin conductance in lipid bilayers.

Alamethicin at a concentration of 2 micrograms/ml on one side of a lipid bilayer, formed at the tip of a patch clamp pipette from diphytanoyl phosphatidylcholine and cholesterol (2:1 mol ratio) in aqueous 0.5 M KCl, 5 mM Hepes, pH 7.0, exhibits an asymmetric current-voltage curve, only yielding alamethicin currents when the side to which the peptide has been added is made positive. Below room temperature, however, single alamethicin channels created in such membranes sometimes survive a sudden reversal of the polarity. These "reversed" channels are distinct from transiently observed states displayed as the channel closes after a polarity reversal. Such "reversed" channels can be monitored for periods up to several minutes, during which time we have observed them to fluctuate through more than 20 discrete conductance states. They are convenient for the study of isolated ion-conducting alamethicin aggregates because, after voltage reversal, no subsequent incorporation of additional ion-conducting aggregates takes place.     

The separation of voltage-dependent photoemfs and conductances in Rudin-Mueller membranes containing magnesium porphyrins

It is shown that the photoemf in a pigmented membrane is specific to the magnesium-porphyrin conductance channel. A null current method was devised to measure directly the voltage dependence of the photoemf and the magnesium-porphyrin conductance. Their voltage dependence is in agreement with the hypothesis that the magnesium-porphyrin cation is the majority carrier