1. CHOLESTEROL/AMPHOTERICIN B CHANNELS IN LIPID BILAYERS
INTRODUCTION
Amphotericin B is a polyene macrolide antibiotic that is widely used for its antifungal activity, despite its undesirable side effects. In biological systems, amphotericin B acts by forming channels that induce ion leakage across lipid membranes. The formation of these channels is highly dependent on the presence of sterols (cholesterol or ergosterol) in the membrane. The sterol molecules facilitate the assembly of the channel by acting as ‘glue’ that links the amphotericin B monomers together. The fact that ergosterol (sterol derived from fungi) containing membranes are more sensitive to the action of amphotericin B is the basis for its use as an antifungal agent. However, most cholesterol-containing membranes are still vulnerable to the action of amphotericin B.
Here we report thermodynamic as well as channel data on amphotericin B/cholesterol mixtures in monolayers and artificial lipid bilayer membranes in hopes of better understanding the mechanism of action, and channel properties of amphotericin B.
METHODS
Surface area-surface pressure isotherms:
Lipid monolayer surface properties and depositions were carried out using Langmuir-Blodgett (LB) film balances KSV 2200 LB and KSV5000 (KSV-Chemicals, Finland). Series of mixed amphotericin B/cholesterol solutions, ranging from 0 to 1 mole fractions of amphotericin B, were individually spread on the LB subphase solution during separate runs. Surface pressure-surface area (-A) isotherms were measured at 10  0.1 and 20  0.1 ºC. The specific molecular areas of amphotericin B/cholesterol mixed monolayers were estimated by the equation: Aa = (Ae - AcNc) /Na, where Ae and Ac are extrapolated “zero pressure” areas for the mixed monolayer and cholesterol monolayer, respectively, and Nc and Na are mole fractions of cholesterol and amphotericin B, respectively.
Reconstitution of Amphotericin B in lipid bilayers:
Lipid bilayers containing pure 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Inc.) were formed on the tip of patch pipettes using the tip-dip technique. The bilayers were formed in asymmetric saline condition by the successive transfer of two lipid monolayers upon the tip of the patch pipette. The extracellular solution (bathing the cis-side of the menbrane) contained (in mM ) 125 NaCl, 5 KCl, 1.25 NaH2PO4, and 5 Tris-HCl at pH 7.4, while the intracellular solution (inside the patch pipette) contained 110 KCl, 4 NaCl, 2 NaH2CO3, 0.1 CaCl2, 1 MgCl2, and 2 MOPS at pH A 2:1 (mol/mol) mixture of 0.6 g amphotericin B and 0.12 g cholesterol dissolved in a chloroform-methanol (2:1) solvent was sonicated together with 10 g phospholipid (in hexane) and 10 l of extracellular solution in order to form liposomes. The emulsion was then carefully transferred to the surface (air-fluid interface) of the solution on the cis-side of the membrane. Incorporation of amphotericin B into the membrane was achieved by dipping the tip of the patch pipette into the emulsion. The resulting single channel current fluctuations were filtered at 5 kHz and recorded on VHS tapes for later analysis. In certain instances, these single channel current fluctuations were subsequently blocked by the addition of 100 mM tetraethylammonium chloride (TEA) to the cis-side of the membrane.
Data Analysis:
Single channel data segments of 5 – 120 sec lengths were digitized at 100 s intervals and transferred to a computer as data files. The data files were later subjected to statistical analysis using the Fetchan module of pCLAMP data analysis program (Axon Instruments) as wells as the Microcal Origin data analysis and technical graphics program. The resulting data points were compiled to generate the displayed graphs.
RESULTS
Figure 1. Mean molecular areas, at various surface pressures, of mixed monolayers of AmB/Chol spread on a subphase at 20oC. The subphase solution contained 55 mM KCl, 4 mM NaCl, 0.1 mM CaCl2, 1 mM MgCl2, and 2 mM 3-(N-morpholino)-propanesulfonic acid (MOPS) made with deionized doubly distilled water (pH 7.4). XAmB= mole fraction of amphotericin B.
CONCLUSIONS
• Amphotericin B and cholesterol form a complex with highest thermodynamic stability at 2:1 stoichiometry .
• At this ratio, amphotericin B and cholesterol readily form large amplitude ion channels of diverse conductance in lipid bilayers.
• The formation of these ion channels is dependent on the presence of cholesterol in the membrane.
• The large diversity in conductance is likely due to the varying numbers of amphotericin B monomers that assemble (in the presence of cholesterol) to make up channels of different pore sizes.
• The amplitude distribution and frequency of occurrence of a given pore size can be explained using a simple model based on the polymerization of a two-dimensional pore.
• The compound tetraethylammonium chloride inhibits amphotericinB/cholesterol channels.
Figure 2. Properties of Cholesterol-Amphotericin B Channels, and Blockage by TEA. (A) Voltage clamp studies of lipid bilayers containing amphotericin B were carried out in the absence (top) and presence (bottom) of cholesterol. Voltage was clamped at 165 mV. Traces were filtered at 5 kHz and sampled at 100 ms intervals. Openings are upward. (B) Amplitude distribution histograms of single channel data observed in the presence of cholesterol (lower trace in A) revealed a distinct closed level and multiple open levels. (C) Dwell-time distribution histograms of the different channel states (C, O1, and O2) observed in the presence of cholesterol (lower trace in A). Graphs were fitted with first-order exponential curves. The resulting value for the average dwell-time of each state is indicated by . (D) Voltage clamp studies of lipid bilayers containing a mixture of cholesterol and amphotericin B were carried out in the absence (top) and presence (bottom) of the channel blocker TEA. Voltage was clamped at 150 mV. Traces were filtered at 5 kHz and sampled at 100 ms intervals. Openings are upward. (E) Probability of the channel states in the absence (left) and presence (right) of TEA are shown. B O1 O2 A
amphotericin B
2:1 amphotericin B + cholesterol
Vhold = 165 mV
no TEA
100 mM TEA sec E
Vhold = 150 mV D
ACKNOWLEDGMENTS
This work was funded and supported by DARPA grant MDA
ABSTRACT
The polyene antibiotic amphotericin B (AmB) is known for both its antifungal action, and its ability to induce ion leakage in lipid membranes. In the presence of sterols (cholesterol or ergosterol), AmB forms ion permeable channels that facilitate movement of ions across the membrane. To better understand how AmB functions in lipid membranes, studies were carried out in monolayer and bilayer systems. Monolayers containing AmB/cholesterol mixture at 2:1 ratio showed the highest thermodynamic stability and electrical conductance. When AmB and cholesterol at this ratio were incorporated into patch phospholipid bilayers, ion channel current fluctuations with diverse conductance levels of up to 400 pS were observed. No current fluctuations were detected in the absence of cholesterol. The varying number of AmB monomers that assemble to make up channels of different pore sizes could explain the large diversity in conductance. The current-voltage relationships of the channels were linear over the range of +200 to -200 mV with a reversal potential near 0 mV. Kinetic analysis of the single channel fluctuations revealed an open time of a few milliseconds. The addition of 100 mM tetraethylammonium chloride to the cis-side of the membrane markedly inhibited the amplitude of the channel currents. Our observations for amplitude distribution and frequency of occurrence of a given pore size can be explained using a simple model based on the polymerization of a two-dimensional pore. These findings provide better understanding of AmB mechanism of action in lipid membranes.
Figure 3. Diversity of Amphotericin B channel conductances can be explained by a mathematical model. (A) Cholesterol-amphotericin B channels of diverse conductance were compiled and subjected to statistical analysis to determine the distribution of the various conductance populations. The plot was subsequently fitted with a third-order Gaussian curve to determine the maxima of the peaks. (B) Voltage-current relationship of a one of the conductance populations ( pS) was plotted in order to elucidate the voltage-current properties of the channel. (C) The pore structure of the cholesterol-amphotericin B channel can be estimated using a geometric model (left). Using a geometric formula for calculating the area inside the pore of the model (right), one can estimate the size of the pore inside the cholesterol-amphotericin B channel. (D) The relative channel conductance and opening area were plotted as a function of the number of A2C subunits (one subunit is composed of 2 amphotericin B and 1 cholesterol molecules). Calculated values are indicated by () while experimental values are indicated by (). (E) The mole fraction of x-mer were plotted as a function of the number of A2C subunits (1-mer is composed of 2 amphotericin B and 1 cholesterol molecules). Calculated values are indicated by () while experimental values are indicated by ().
S = d2[ncot(/n)–(n/2–1)]
2A+C = A2C; A2C+A2C = A4C2
Mole fraction of x-mer, Xx,
Xx = px –1(1 – p), where
p-fraction of bound sites.
119.7 Å2
49.7 Å2 d
10.4 Å2
CHOLESTEROL/AMPHOTERICIN B CHANNELS IN LIPID BILAYERS
S. Yilma1; J.J. Cannon2; T.T. Lo3; A. Samoylov1; E.E. Morrison1*; T.A. Roppel3; W.C. Neely2; V. Vodyanoy1
1Department of Anatomy, Physiology, & Pharmacology, 2Department of Chemistry, 3Department of Electrical Engineering, Auburn University, AL, USA