Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

Electric charge effects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles

The influence of electric surface charges on the polar headgroups and the hydrocarbon region of phospholipid membranes was studied by mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with charged amphiphiles. A positive surface charge was generated with dialkyldimethylammonium salts and a negative surface charge with dialkyl phosphates. The POPC:amphiphile ratio and hence the surface charge density could be varied over a large range since stable liquid-crystalline bilayers were obtained even for the pure amphiphiles in water. POPC was selectively deuterated at both methylene segments of the choline moiety and at the cis double bond of the oleic acyl chain. Additional experiments were carried out with 1,2-dipalmitoyl-rac-glycero-3-phosphocholine labeled at the C-2 position of the glycerol backbone. Deuterium, phosphorus, and nitrogen-14 nuclear magnetic resonance (NMR) spectra were recorded for liquid-crystalline bilayers with varying concentrations of amphiphiles. Although the hydrocarbon region and the glycerol backbone were not significantly influenced by the addition of amphiphiles, very large perturbations of the phosphocholine headgroup were observed. Qualitatively, these results were similar to those observed previously with other cationic and anionic molecules and suggest that the electric surface charge is the essential driving force in changing the phospholipid headgroup orientation and conformation. While the P-N dipole is approximately parallel to the membrane surface in the pure phospholipid membrane, the addition of a positively charged amphiphile or the binding of cationic molecules moves the N+ end of the dipole toward the water phase, changing the orientation of the phosphate segment by more than 30 degrees at the highest amphiphile concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Displacement currents associated with the insertion of Alzheimer disease amyloid beta-peptide into planar bilayer membranes

The role of endogenous amyloid beta-peptides as causal factors of neurodegenerative diseases is largely unknown. We have previously reported that interactions between Alzheimer's disease A beta P[1-40] peptide in solution and planar bilayer membranes made from anionic phospholipids lead to the formation of cation-selective channels. We now find and report here that the spontaneous insertion of free A beta P[1-40] across the bilayer can be detected as an increase in bilayer capacity. To this end we recorded the displacement currents across planar bilayers (50 mM KCl on both sides) in response to sudden displacements of the membrane potential, from -300 to 300 mV in 20-mV increments. To monitor the A beta P[1-40]-specific displacement currents, we added A beta P[1-40] (1-5 microM) to the solution on either side of the membrane and noted that the direction of the displacement current depended on the side with A beta P[1-40]. The size of the A beta P[1-40]-specific charge displaced during a pulse was always equal to the charge returning to the original configuration after the pulse, suggesting that the dipole molecules are confined to the membrane. As a rule, the steady-state distribution of the A beta P[1-40]-specific charges within the bilayer could be fit by a Boltzmann distribution. The potential at which the charges were found to be equally distributed (V(o)) were approximately -135 mV (peptide added to the solution in the compartment electrically connected to earth) and 135 mV (peptide added to the solution connected to the input of the amplifier). The A beta P[1-40]-specific transfer of charge reached a maximum value (Q(max)) when the electrical potential of the side containing the amyloid beta-protein was taken to either -300 or 300 mV. For a circular membrane of 25-microm radius ( approximately 2000 microm(2)), the total A beta P[1-40]-specific charge Q(max) was estimated as 55 fC, corresponding to some 170 e.c./microm(2). Regardless of the side selected for the addition of A beta P[1-40], at V(o) the charge displaced underwent an e-fold change for a approximately 27-mV change in potential. The effective valence (a) of the A beta P[1-40] dipole (i.e., the actual valence Z multiplied by the fraction of the electric field chi acting on the dipole) varied from 1 to 2 electronic charges. We also tested, with negative results, the amyloid peptide with the reverse sequence (A beta P[40-1]). These data demonstrate that A beta P[1-40] molecules can span the low dielectric domain of the bilayer, exposing charged residues (D(1), E(3), R(5), H(6), D(7), E(11), H(13), and H(14)) to the electric field. Thus the A beta P[1-40] molecules in solution must spontaneously acquire suitable conformations (beta-pleated sheet) allowing specific interactions with charged phospholipids. Interestingly, the domain from residues 676 to 704 in the APP(751) is homologous with the consensus sequence for lipid binding found in other membrane proteins regulated by anionic phospholipids.     

Interaction of phloretin with lipid monolayers: relationship between structural changes and dipole potential change.

Phloretin is known to adsorb to lipid surfaces and alters the dipole potential of lipid monolayers and bilayers. Its adsorption to biological and artificial membranes results in a change of the membrane permeability for a variety of charged and neutral compounds. In this respect phloretin represents a model substance to study the effect of dipole potentials on membrane permeability. In this investigation we studied the interaction of phloretin with monolayers formed of different lipids in the liquid-expanded and the condensed state. Phloretin integrated into the monolayers as a function of the aqueous concentration of its neutral form, indicated by an increase of the surface pressure in the presence of phloretin. Simultaneous recording of the surface potential of the monolayers allowed us to correlate the degree of phloretin integration and the phloretin-induced dipole potential change. Increasing the surface pressure decreased the phloretin-induced shift of the isotherms, but did not influence the phloretin-induced surface potential change. This means that phloretin adsorption to the lipid surface can occur without affecting the lipid packing. The surface potential effect of phloretin is accompanied by a change of the lipid dipole moment vector dependent on the lipid packing. This means that the relation between the surface potential change and the lipid packing cannot be described by a static model alone. Taking into account the deviations of the surface potential change versus molecular area isotherms of the experimental data to the theoretically predicted course, we propose a model that relates the area change to the dipole moment in a dynamic manner. By using this model the experimental data can be described much better than with a static model.     

Interactions between charged, uncharged, and zwitterionic bilayers containing phosphatidylglycerol.

Pressure vs. distance relationships have been obtained for phosphatidylglycerol bilayers, in both charged and uncharged states. Water was removed from the lipid multilayers by the application of osmotic pressures in the range of 0-2.7 x 10(9) dyn/cm2, and the distance between adjacent bilayers was obtained from Fourier analysis of lamellar x-ray diffraction data. For phosphatidylglycerol bilayers made electrically neutral either by lowering the pH or by adding equimolar concentrations of the positively charged lipid stearylamine, the pressure-distance data could be fit with a single exponential. The measured decay lengths were 1.1 A at low pH and 1.5 A with stearylamine, which are similar to decay lengths of the hydration pressure found for gel phases of other neutral bilayers. In addition, the magnitude of this repulsive pressure was proportional to the square of the Volta potential (equivalent to the dipole potential for electrically neutral bilayers) measured in monolayers in equilibrium with bilayers, in agreement with results previously found for the hydration pressure between phosphatidylcholine bilayers. For charged phosphatidylglycerol bilayers, the pressure-distance relation had two distinct regions. For bilayer separations greater than 10 A, the pressure-distance data had an exponential decay length (11 A) and a magnitude consistent with that expected for electrostatic repulsion from double-layer theory. For bilayer separations of 2-10 A, the pressure decayed much more rapidly with increasing bilayer separation (decay length less than 1 A). We interpret these data at low bilayer separations in terms of a combination of hydration repulsion and steric hindrance between the lipid head groups and the sodium ions trapped between apposing bilayers.     

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers [published erratum appears in J Gen Physiol 1989 Apr;93(4):following 760]

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.     

Local Anesthetics Affect Gramicidin A Channels via Membrane Electrostatic Potentials

The effects of local anesthetics (LAs), including aminoamides and aminoesters, on the characteristics of single gramicidin A (GA) channels in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers were studied. Aminoamides, namely lidocaine (LDC), prilocaine (PLC), mepivacaine (MPV), and bupivacaine (BPV), reduced the conductance of GA channels. Aminoesters influenced the current fluctuations induced by GA differently; procaine (PC) did not affect the fluctuations, whereas tetracaine (TTC) distinctly reduced the conductance of single GA channels. Using electrophysiological technique, we estimated the changes in the membrane boundary potential at the adsorption of LAs; LDC, PLC, MPV, BPV, and TTC substantially increased, while PC did not affect it. To elucidate which component of the membrane boundary potential, the surface or dipole potential, is responsible for the observed effects of LAs, we employed a fluorescence assay. We found that TTC led to a significant increase in the membrane dipole potential, whereas the adsorption of LDC, PLC, MPV, BPV, and PC did not produce any changes in the membrane dipole potential. We concluded that aminoamides affected the surface potential of lipid bilayers. Together, these data suggest that the effects of LAs on the conductance of single GA channels are caused by their influence on membrane electrostatic potentials; the regulation of GA pores by aminoamides is associated with the surface potential of membranes, whereas TTC modulation of channel properties is predominantly due to changes in dipole potential of lipid bilayers. These data might provide some significant implications for voltage-gated ion channels of cell membranes.     

Surface potential determination in planar lipid bilayers: a simplification of the conductance-ratio method

One of the methods available for the measurement of surface potentials of planar lipid bilayers uses the conductance ratio between a charged and a neutral bilayer doped with ionophores to calculate the surface potential of the charged bilayer. We have devised a simplification of that method which does not require the use of an electrically neutral bilayer as control. The conductance of the charged bilayer is measured before and after the addition of divalent cations (Ba(2+)) to the bathing solution. Ba(2+) ions screen fixed surface charges, decreasing the surface potential. If the membrane is negatively charged the screening has the effect of decreasing the membrane conductance to cations. The resulting conductance ratio is used to calculate the surface potential change, which is fed into an iterative computer program. The program generates pairs of surface potential values and calculates the surface charge density for the two conditions. Since the surface charge density remains constant during this procedure, there is only one pair of surface potentials that satisfies the condition of constant charge density. Applying this method to experimental data from McLaughlin et al. [McLaughlin, S.G.A., Szabo, G. and Eisenman, G., Divalent ions and the surface potential of charged phospholipid membranes, J. Gen. Physiol., 58 (1971) 667-687.] we have found very similar results. We have also successfully used this method to determine the effect of palmitic acid on the surface potential of asolectin membranes.     

Interaction of phloretin with membranes: on the mode of action of phloretin at the water-lipid interface

 The interaction of phloretin with single lipid bilayers on a spherical support and with multilamellar vesicles was studied by differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR). The results indicated that phloretin interacts with the lipid layer and changes its structural parameters. In DSC experiments, phloretin in its neutral form strongly decreased the lipid phase transition temperature and slightly reduced the cooperativity of the phase transition within the lipid layer. In NMR measurements, phloretin led to an increase of the transverse relaxation time constant but had no effect on the spin-lattice relaxation time constant. The overall dipole moment of phloretin was experimentally determined and was found to be roughly 40% lower than has been published previously. This result suggested that the size of the dipole moment of phloretin does not provide such a high contribution to the effect of phloretin on the dipole potential of monolayers and bilayers as has been published previously. To understand the discrepancy between phloretin adsorption and dipole potential change, we performed computational conformational analysis of phloretin in the gas phase. The results showed that a wide distribution of the dipole moments of phloretin conformers exists, which mainly depends on the orientation of the OH moieties. The adsorption of phloretin as determined from its binding to solid supported bilayers differed from the one determined from dipole potential measurements on black lipid membranes. The difference between the phloretin dissociation constants of both types of experiments suggested a change of its dipole moment normal to the membrane surface in a concentration-dependent manner, which was in agreement with the results of the computational conformational analysis. Received: 21 June 1999 / Revised version: 7 January 2000 / Accepted: 31 March 2000     

Barium modulates the gating of batrachotoxin-treated Na+ channels in high ionic strength solutions.

Batrachotoxin-activated rat brain Na+ channels were reconstituted in neutral planar phospholipid bilayers in high ionic strength solutions (3 M NaCl). Under these conditions, diffuse surface charges present on the channel protein are screened. Nevertheless, the addition of extracellular and/or intracellular Ba2+ caused the following alterations in the gating of Na+ channels: (a) external (or internal) Ba2+ caused a depolarizing (or hyperpolarizing) voltage shift in the gating curve (open probability versus membrane potential curve) of the channels; (b) In the concentration range of 10-120 mM, extracellular Ba2+ caused a larger voltage shift in the gating curve of Na+ channels than intracellular Ba2+; (c) voltage shifts of the gating curve of Na+ channels as a function of external or internal Ba2+ were fitted with a simple binding isotherm with the following parameters: for internal Ba2+, delta V0.5,max (maximum voltage shift) = -11.5 mV, KD = 64.7 mM; for external Ba2+, delta V0.5,max = 13.5 mV, KD = 25.8 mM; (d) the change in the open probability of the channel caused by extracellular or intracellular Ba2+ is a consequence of alterations in both the opening and closing rate constants. Extracellular and intracellular divalent cations can modify the gating kinetics of Na+ channels by a specific modulatory effect that is independent of diffuse surface potentials. External or internal divalent cations probably bind to specific charges on the Na+ channel glycoprotein that modulate channel gating.     

Peptide binding to lipid bilayers. Binding isotherms and zeta-potential of a cyclic somatostatin analogue

The binding of the cyclic somatostatin analogue SMS 201-995, (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, to neutral and negatively charged lipids was investigated with a centrifugation assay and with electrophoretic and monolayer methods. Monolayers and bilayers were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), either in pure form or in a 75/25 (mol/mol) mixture. The expansion of monolayer films demonstrated the intercalation of the peptide between the lipid molecules with a surface area requirement of 135 A2 per peptide molecule, indicating a parallel alignment of the peptide long axis with the membrane surface. Above a limiting pressure of 32.5 mN/m for POPC and 38.5 mN/m for POPG, peptide penetration was no longer possible. The peptide binding isotherm could be measured for mixed POPC/POPG bilayers up to a peptide concentration of 0.5 mM. Due to electrostatic attraction, binding between the positively charged peptide and the negatively charged membrane surface was enhanced as compared to the binding to a neutral membrane. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherm as well as the electrophoretic zeta-potential measurement could be described by the same partition equilibrium with a surface partition constant of Kp = 36 +/- 4 M-1 (at 0.1 M NaCl). About 60-70% of SMS 201-995 is probably embedded in the headgroup region with little penetration into the lipid core. The partition constant increases with increasing salt concentration or with decreasing lipid lateral pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group

The binding of the charged form of two local anesthetics, dibucaine and etidocaine, to bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was measured simultaneously with ultraviolet spectroscopy and deuterium magnetic resonance. Because of their amphiphilic molecular structure, both drugs intercalate between the lipid molecules, increasing the surface area and imparting a positive electric charge onto the membrane. The ultraviolet (UV) binding isotherms were therefore analyzed in terms of a model which specifically took into account the bilayer expansion as well as the charge-induced concentration variations near the membrane surface. By formulating a quantitative expression for the change in surface area upon drug intercalation and combining it with the Gouy-Chapman theory, the binding of charged dibucaine and etidocaine to the lipid membrane was best described by a partition equilibrium, with surface partition coefficients of 660 +/- 80 M-1 and 11 +/- 2 M-1 for dibucaine and etidocaine, respectively (pH 5.5, 0.1 M NaCl/50 mM buffer). Deuterium magnetic resonance demonstrated further that the binding of drug changed the head-group conformation of the lipid molecules. Invoking the intercalation model, a linear variation of the deuterium quadrupole splittings of the choline segments with the surface charge density was observed, suggesting that the phosphocholine head-group may act as a 'molecular electrometer' with respect to surface charges.     

Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids

The formability, current-voltage characteristics and stability of the planar lipid bilayer membranes from the synthetic phytanyl-chained glycolipids, 1, 3-di-O-phytanyl-2-O-(beta-glycosyl)glycerols (Glc(Phyt)(2), Mal(N)(Phyt)(2)) were studied. The single bilayer membranes were successfully formed from the glycolipid bearing a maltotriosyl group (Mal(3)(Phyt)(2)) by the folding method among the synthetic glycolipids examined. The membrane conductance of Mal(3)(Phyt)(2) bilayers in 100 mM KCl solution was significantly lower than that of natural phospholipid, soybean phospholipids (SBPL) bilayers, and comparable to that of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers. From the permeation measurements of lipophilic ions through Mal(3)(Phyt)(2) and DPhPC bilayers, it could be presumed that the carbonyl groups in glycerol backbone of the lipid molecule are not necessarily required for the total dipole potential barrier against cations in Mal(3)(Phyt)(2) bilayer. The stability of Mal(3)(Phyt)(2) bilayers against long-term standing and external electric field change was rather high, compared with SBPL bilayers. Furthermore, a preliminary experiment over the functional incorporation of membrane proteins was demonstrated employing the channel proteins derived from octopus retina microvilli vesicles. The channel proteins were functionally incorporated into Mal(3)(Phyt)(2) bilayers in the presence of a negatively charged glycolipid. From these observations, synthetic phytanyl-chained glycolipid bilayers are promising materials for reconstitution and transport studies of membrane proteins.     

Dipole potential as a driving force for the membrane insertion of polyacrylic acid in slightly acidic milieu

In this work, we report on the interaction of polyacrylic acid with phosphatidylcholine bilayers and monolayers in slightly acidic medium. We found that adsorption of polyacrylic acid on liposomes composed of egg lecithin at pH 4.2 results in the formation of small pores permeable for low molecular weight solutes. However, the pores were impermeable for trypsin indicating that no solubilization of liposomes occurred. The pores were permeable for both positively charged trypsin substrate N-benzoyl-l-arginine ethyl ester and negatively charged pH-indicator pyranine. Two lines of evidence were obtained confirming the involvement of the membrane dipole potential in the insertion of polyacrylic acid into lipid bilayer. (i) Addition of phloretin, a molecule which is known to decrease dipole potential of lipid bilayer, reduced the rate of a polyacrylic acid induced leakage of pyranine from liposomes. (ii) Direct measurements of air/lipid monolayer/water interface surface potential using Kelvin probe showed that adsorption of polyacrylic acid at pH 4.2 induced a decrease in both boundary and dipole potential by 37 and 62mV for ester lipid dioleoylphosphatidylcholine (DOPC). Replacement of DOPC by ether lipid 1,2-di-O-oleyl-sn-glycero-3-phosphocholine (DiOOPC) which is known to form monolayers and bilayers with only minor dipole component of membrane potential showed that addition of PAA produced similar response in the boundary potential (by 50mV) but negligible response in dipole potential of monolayer. These observations agree with our assumption that dipole potential is an important driving force for the insertion of polyacids into biological membranes.     

紫膜碎片的电二色性研究

悬浮在水中的嗜盐菌紫膜碎片,在外电场作用下产主定向排列.在20℃时,568nm的电二色性研究表明:外加电场为2kV/m时取向程度可达60%以上;大于5.5kV/m时,取向作用趋于饱和状态;饱和时简约电二色性为-0.437左右,视黄醛生色团的跃迁矩方向与电偶极矩方向形成60.9°夹角;紫膜的永久偶极短为9.2×10~(-24)C、M,剩余电极化率为3.0×10~(-27)m~2;紫膜的旋转扩散常数为0.53秒~(-1).曲线拟合分析表明,感应偶极对紫膜碎片的定向的贡献应予考虑.本文对紫膜碎片的定向机理进行了讨论.     

Free energy potential for aggregation of giant, neutral lipid bilayer vesicles by Van der Waals attraction.

Here, we report the first direct observation of Van der Waals' attraction between biomembrane capsules using measurements of the free energy reduction per unit area of membrane-membrane contact formation. In these studies, the membrane capsules were reconstituted neutral (egg phosphatidylcholine) lipid bilayers of giant (greater than 10(-3) cm diam) vesicles. Micromanipulation methods were used to select and maneuver two vesicles into proximity for contact; after adhesion was allowed to occur, the extent of contact formation was regulated through the vesicle membrane tensions that were controlled by micropipette suction. The free energy reduction per unit area of contact formation was proportional to the membrane tension multiplied by a simple function of the pipette and vesicle dimensions. The free energy potential for Van der Waals attraction between the neutral bilayers in 120 mM NaCl solutions was 1.5 X 10(-2) ergs/cm2. Also, when human serum albumin was added to the medium in the range of 0-1 mg/ml, the free energy potential for bilayer-bilayer adhesion was not affected. Using published values for equilibrium spacing between lipid bilayers in multilamellar lipid-water dispersions and the theoretical equation for van der Waals attraction between continuous dielectric layers, we calculated the value for the Hamaker coefficient of the Van der Waals attraction to be 5.8 X 10(-14) ergs.     

Surface potential determination in planar lipid bilayers: A simplification of the conductance-ratio method

One of the methods available for the measurement of surface potentials of planar lipid bilayers uses the conductance ratio between a charged and a neutral bilayer doped with ionophores to calculate the surface potential of the charged bilayer. We have devised a simplification of that method which does not require the use of an electrically neutral bilayer as control. The conductance of the charged bilayer is measured before and after the addition of divalent cations (Ba²⁺) to the bathing solution. Ba²⁺ ions screen fixed surface charges, decreasing the surface potential. If the membrane is negatively charged the screening has the effect of decreasing the membrane conductance to cations. The resulting conductance ratio is used to calculate the surface potential change, which is fed into an iterative computer program. The program generates pairs of surface potential values and calculates the surface charge density for the two conditions. Since the surface charge density remains constant during this procedure, there is only one pair of surface potentials that satisfies the condition of constant charge density.Applying this method to experimental data from McLaughlin et al. [McLaughlin, S.G.A., Szabo, G. and Eisenman, G., Divalent ions and the surface potential of charged phospholipid membranes, J. Gen. Physiol., 58 (1971) 667–687.] we have found very similar results. We have also successfully used this method to determine the effect of palmitic acid on the surface potential of asolectin membranes.     

Increased adhesion between neutral lipid bilayers: interbilayer bridges formed by tannic acid.

Tannic acid (TA) is a naturally occurring polyphenolic compound that aggregates membranes and neutral phosolipid vesicles and precipitates many proteins. This study analyzes TA binding to lipid membranes and the ensuing aggregation. The optical density of dispersions of phosphatidylcholine (PC) vesicles increased upon the addition of TA and electron micrographs showed that TA caused the vesicles to aggregate and form stacks of tightly packed disks. Solution calorimetry showed that TA bound to PC bilayers with a molar binding enthalpy of -8.3 kcal/mol and zeta potential measurements revealed that TA imparted a small negative charge to PC vesicles. Monolayer studies showed that TA bound to PC with a dissociation constant of 1.5 microM and reduced the dipole potential by up to 250 mV. Both the increase in optical density and decrease in dipole potential produced by TA could be reversed by the addition of polyvinylpyrrolidone, a compound that chelates TA by providing H-bond acceptor groups. NMR, micropipette aspiration, and x-ray diffraction experiments showed that TA incorporated into liquid crystalline PC membranes, increasing the area per lipid molecule and decreasing the bilayer thickness by 2 to 4%. 2H-NMR quadrupole splitting measurements also showed that TA associated with a PC molecule for times much less than 10(-4) s. In gel phase bilayers, TA caused the hydrocarbon chains from apposing monolayers to fully interdigitate. X-ray diffraction measurements of both gel and liquid crystalline dispersions showed that TA, at a critical concentration of about 1 mM, reduced the fluid spacing between adjacent bilayers by 8-10 A. These data place severe constraints on how TA can pack between adjacent bilayers and cause vesicles to adhere. We conclude that TA promotes vesicle aggregation by reducing the fluid spacing between bilayers by the formation of transient interbilayer bridges by inserting its digallic acid residues into the interfacial regions of adjacent bilayers and spanning the interbilayer space.     

Presence of anionic phospholipids rules the membrane localization of phenothiazine type multidrug resistance modulator

Substances able to modulate multidrug resistance (MDR), including antipsychotic phenothiazine derivatives, are mainly cationic amphiphiles. The molecular mechanism of their action can involve interactions with transporter proteins as well as with membrane lipids. The interactions between anionic phospholipids and MDR modulators can be crucial for their action. In present work we study interactions of 2-trifluoromethyl-10-(4-[methanesulfonylamid]buthyl)-phenothiazine (FPhMS) with neutral (PC) and anionic lipids (PG and PS). Using microcalorimetry, steady-state and time-resolved fluorescence spectroscopy we show that FPhMS interacts with all lipids studied and drug location in membrane depends on lipid type. The electrostatic attraction between drug and lipid headgroups presumably keeps phenothiazine derivative molecules closer to surface of negatively charged membranes with respect to neutral ones. FPhMS effects on bilayer properties are not proportional to phosphatidylserine content in lipid mixtures. Behavior of equimolar PC:PS mixtures is similar to pure PS bilayers, while 2:1 or 1:2 (mole:mole) PC:PS mixtures resemble pure PC ones.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.