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.     

Probing alamethicin channels with water-soluble polymers. Effect on conductance of channel states.

Channel access resistance has been measured to estimate the characteristic size of a single ion channel. We compare channel conductance in the presence of nonpenetrating water-soluble polymers with that obtained for polymer-free electrolyte solution. The contribution of the access resistance to the total alamethicin channel resistance is approximately 10% for first three open channel levels. The open alamethicin channel radii inferred for these first three levels from the access resistance are 6.3, 10.3, and 11.4 A. The dependence of channel conductance on polymer molecular weight also allows evaluation of the channel dimensions from polymer exclusion. Despite varying conductance, it was shown that steric radii of the alamethicin channel at different conductance levels remain approximately unchanged. These results support a model of the alamethicin channel as an array of closely packed parallel pores of nearly uniform diameter.     

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.     

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.     

Ion conductance channels of bilayer from sarcoma-45 phospholipids

Properties of black bilayer membranes formed from sarcoma-45 phospholipids differed from those of normal cell phospholipids bilayers. Two types of Ca2+-channels were discovered: those multiple to 35 pA and those multiple to 90 pA. The study of bilayers from phospholipids of nuclei, mitochondria, microsomal fractions showed that Ca2+-channels were localized in the microsomal fraction of transformed cells. The relationship between DB18C6 diacetylderivatives membrane activity and their effect to growth of sarcoma-45 in vivo was discovered: the Ca2+-channel blocker diacetyl-DB18C6 inhibited the transformed cells growth.     

"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.     

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.     

Thickness dependence of monoglyceride bilayer membrane conductance.

We have studied the conductance properties of unmodified monoglyceride membranes as a function of monoglyceride chain length. As membrane thickness decreases from 31 to 20 nm, the steepness of the current-voltage (I-V) curve increases from 80 mV per e-fold current increase to 52 mV per e-fold current increase. The zero-voltage conductance increases more than 1,000-fold and the apparent activation energy of conductance decreases from 18.4 to 14.2 kcal/mol. We have analyzed our results using both the Nernst-Planck equation and absolute rate theory. Both approaches are consistent with our results and give consistent values for the parameters describing the I-V curves. We conclude that both the surface ion concentration and the distance from the surface of the membrane at which the energy of an ion rises appreciably above its value in solution (position of the barrier) are invariant with thickness.     

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.     

Two purified fractions of alamethicin have different conductance properties

The properties of two purified alamethicin fractions, Fraction 4 and Fraction 6, have been studied in phosphatidylethanolamine (PE) membranes and phosphatidylserine (PS) membranes. Membranes doped with Fraction 4 show well-defined single channel conductance (mean lifetime about 20 ms). The autocorrelation function of the current fluctuations has one relaxation time of the same order as the mean lifetime of the single channels, and the current response to a voltage pulse follows an exponential with only one time constant. The conductance of a membrane doped with Fraction 6 has a voltage-independent part and a current-voltage curve with a slope that is half the slope of the Fraction 4 current-voltage curve. In the presence of Fraction 6, PS membranes and PE membranes both have symmetrical current-voltage curves even with Fraction 6 added to only one side. We did not detect any well-defined single channel levels in the presence of Fraction 6, and autocorrelation analysis of the current due to Fraction 6 gave two characteristic correlation times: a fast time (about 5 ms) and a slow time (about 50 ms). High current level kinetics of Fraction 6 also show two time constants. A possible explanation for the differences between the two fractions is that Fraction 6 monomers have a lower dipole moment than those of Fraction 4. The difference in channel stability can be explained by a lowered tendency of the monomers to line up parallel to the field. The negative branch and voltage-independent conductance can be explained by lowered energy of insertion of monomers into the membrane, and lowered energy of interaction between the monomers and the electric field.     

Mixing of oxidized and bilayer phospholipids

Composition and phase dependence of the mixing of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), with the oxidized phospholipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) were investigated by characterizing the aggregation states of DPPC/PGPC and DOPC/PGPC using a fluorescence quenching assay, dynamic light scattering, and time-resolved fluorescence quenching in the temperature range 5–60 °C. PGPC forms 3.5 nm radii micelles of aggregation number 33. In the gel phase, DPPC and PGPC fuse to form mixed vesicles for PGPC molar fraction, X_PGPC ≤ 0.3 and coexisting vesicles and micelles at higher X_PGPC. Data suggest that liquid phase DPPC at 50 °C forms mixed vesicles with segregated or hemi fused DPPC and PGPC for X_PGPC ≤ 0.3. At 60 °C, DPPC and PGPC do not mix, but form coexisting vesicles and micelles. DOPC and PGPC do not mix in any proportion in the liquid phase. Two dissimilar aggregates of the sizes of vesicles and PGPC micelles were observed for all X_PGPC for T ≥ 22 °C. DOPC–PGPC and DPPC–PGPC mixing is non-ideal for X_PGPC > 0.3 in both gel and fluid phases resulting in exclusion of PGPC from the bilayer. Formation of mixed vesicles is favored in the gel phase but not in the liquid phase for X_PGPC ≤ 0.3. For X_PGPC ≤ 0.3, aggregation states change progressively from mixed vesicles in the gel phase to component segregated mixed vesicles in the liquid phase close to the chain melting transition temperature to separated coexisting vesicles and micelles at higher temperatures.     

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.     

Phase separation of miscible phospholipids by sonication of bilayer vesicles.

Sonication of phospholipid vesicles may result, according to their liquid or solid crystal state, in the generation of unilamellar vesicles or structural defects within their bilayers, respectively. The transition temperature Tm of the phospholipid bilayer is usually the threshold temperature delineating the physical effects of ultrasound. However, for vesicles made from a mixture of two miscible phospholipids, this threshold temperature was not found to be the intermediate Tm of the phospholipid mixture bilayers, but the Tm of the lowest melting component. This was due to a simultaneous lateral phase separation of the two phospholipids induced by the sonication as demonstrated by differential scanning calorimetry analysis.     

Adsorption of monovalent cations to bilayer membranes containing negative phospholipids.

The electrophoretic mobilities of multilamellar phosphatidylserine vesicles were measured in solutions containing monovalent cations, and the xi potentials, the electrostatic potentials at the hydrodynamic plane of shear, were calculated from the Helmholtz--Smoluchowski equation. In the presence of 0.1 M lithium, sodium, ammonium, potassium, rubidium, cesium, tetraethylammonium, and tetramethylammonium chloride, the xi potentials were -60, -62, -72, -73, -77, -80, -82, and -91 mV, respectively. Similar results were obtained with phosphatidylglycerol vesicles; different results were obtained with cardiolipin, phosphatidylinositol, and phosphatidic acid vesicles. The phosphatidylserine results are interpreted in terms of the Stern equation, a combination of the Gouy equation from the theory of the diffuse double layer, the Boltzmann relation, and the Langmuir adsorption isotherm. Evidence is presented that suggests the hydrodynamic plane of shear is 2 A from the surface of the membrane in solutions containing the alkali metal cations. With this assumption, the intrinsic association constants of the above monovalent cations with phosphatidylserine are 0.8, 0.6, 0.17, 0.15, 0.08, 0.05, 0.03, and 0 M-1, respectively. The validity of this approach was tested in two ways. First, the xi potentials of vesicles formed from mixtures of phosphatidylserine and a zwitterionic lipid, phosphatidylcholine, were measured in solutions containing different concentrations of sodium. All the data could be described by the Stern equation if the "relaxation" of the ionic atmosphere, which is predicted by classic electrostatic and hydrodynamic theory to occur at low salt concentrations and high potentials, was circumvented by using only large (diameter greater than 13 micrometers) vesicles for these measurements. Second, the fluorescent probe 2-(p-toluidinyl)naphthalene-6-sulfonate was used to estimate the potential at the surface of phosphatidylserine and phosphatidylglycerol vesicles sonicated in 0.1 M NaCl. Reasonable agreement with the predicted values of the surface potential was obtained.     

X-ray diffraction study of lipid bilayer membranes interacting with amphiphilic helical peptides: diphytanoyl phosphatidylcholine with alamethicin at low concentrations.

A variety of amphiphilic helical peptides have been shown to exhibit a transition from adsorbing parallel to a membrane surface at low concentrations to inserting perpendicularly into the membrane at high concentrations. Furthermore, this transition has been correlated to the peptides' cytolytic activities. X-ray lamellar diffraction of diphytanoyl phosphatidylcholine-alamethicin mixtures revealed the changes of the bilayer structure with alamethicin concentration. In particular, the bilayer thickness decreases with increasing peptide concentration in proportion to the peptide-lipid molar ratio from as low as 1:150 to 1:47; the latter is near the threshold of the critical concentration for insertion. From the decreases of the bilayer thickness, one can calculate the cross sectional expansions of the lipid chains. For all of the peptide concentrations studied, the area expansion of the chain region for each adsorbed peptide is a constant 280 +/- 20 A2, which is approximately the cross sectional area of an adsorbed alamethicin. This implies that the peptide is adsorbed at the interface of the hydrocarbon region, separating the lipid headgroups laterally. Interestingly, the chain disorder caused by a peptide adsorption tends to spread over a large area, as much as 100 A in diameter. The theoretical basis of the long range nature of bilayer deformation is discussed.