Interaction of N-alkylanthracyclines with lipid bilayers: correlations between partition coefficients, lipid phase distributions and thermotropic behavior

The thermotropic behavior of multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC), or of DPPC in admixture with cardiolipin or cholesterol, in the presence of various N-alkyl derivatives of both adriamycin and adriamycin-14-valerate has been investigated by high sensitivity differential scanning calorimetry. The analogues, particularly the 14-valerate derivatives, which were most lipophilic as judged by their lipid/buffer, and to a lesser extent by their octanol/buffer, partition coefficients, were the most effective in depressing the tm of the investigated lipids; correlations, however, were not absolute. Other factors, such as the distribution of the drugs between the solid and liquid-crystalline phases of the bilayer, were also important to the observed membrane perturbations. With all anthracyclines, however, no major changes in the transition enthalpy were observed. In the case of vesicles prepared from pure DPPC, curve fitting analysis based on ideal solution theory (J.M. Sturtevant (1984) Proc. Natl. Acad. Sci. USA 81, 1398-1400) applied at relatively low drug concentrations where single peak transitions were produced, adequately described the differential scanning calorimetric results. At high drug concentrations, however, the presence of multi-peak transitions were indicative of non-ideality.     

Hydrophobic lipid additives affect membrane stability and phase behavior of N-monomethyldioleoylphosphatidylethanolamine.

The rate of formation of high-curvature intermediates or disordered cubic phases in N-methyldioleoylphosphatidylethanolamine (N-methyl-DOPE) dispersions with or without additives was studied by 31P NMR spectroscopy. In N-methyl-DOPE dispersions, both the L alpha liquid-crystalline phase and the hexagonal HII phase convert into phases of high curvature giving rise to isotropic 31P NMR resonances. Addition of the bilayer destabilizers 1,2-diolein, 1,3-diolein, or eicosane lowers the threshold temperature of the isotropic phase. The isotropic threshold temperature is strongly correlated with the L alpha-HII phase transition temperature (TH). The addition of hexagonal phase promoters does not change the rate of formation of the isotropic phase at a temperature shifted by a fixed amount below TH. However, the formation of "isotropic" phases from the additive-stabilized hexagonal phase is slow compared to that observed in pure N-methyl-DOPE lipid dispersions. Membrane leakage and fusion are promoted by the dioleins and well as by eicosane, but changes in the rates of these processes do not correlate well with the extent of formation of isotropic phases. All three additives have similar effects on phase behavior and on vesicle leakage and fusion. These similarities occur despite the fact that eicosane is believed to partition differently into the membrane than diolein. In addition to the general similarities in the effects of the two diolein isomers, 1,2-diolein is somewhat more potent in promoting the hexagonal phase and in increasing rates of leakage and fusion than is 1,3-diolein.     

Hydrophobic mismatch and lipid sorting near OmpA in mixed bilayers: atomistic and coarse-grained simulations

To understand the effects of lipid composition on membrane protein function in a mixture as complex as a biomembrane, one must know whether the lipid composition local to the protein differs from the mean lipid composition. In this study, we simulated the transmembrane domain of a β-barrel protein, OmpA, in mixtures of lipids of different tail lengths under conditions of negative hydrophobic mismatch, i.e., local bilayer thinning. We modeled the influence of OmpA on the local lipid composition both at a coarse-grained (CG) resolution using conventional molecular dynamics, and at an atomistic resolution within the semi-grand canonical ensemble using mutation moves to rapidly approach an equilibrium lateral distribution of lipids. Moderate enrichment of the shorter tail component (either DDPC in DDPC/DMPC mixtures or DMPC in DMPC/DSPC mixtures) extending 2-3 nm away from the protein surface was observed with both the atomistic and CG models. The similarity in trends suggests that the more computationally economical CG models capture the essential features of lipid sorting induced by hydrophobic mismatch.     

The surfactant peptide KL4 in lipid monolayers: phase behavior, topography, and chemical distribution

Studies of different fragments and mutants of SP-B suggest that the function related structural and compositional characteristics in SP-B are its positive charges with intermittent hydrophobic domains. KL4 ([lysine-(leucine)4]4-lysine) is a synthetic peptide based on SP-B structure and is the major constituent of Surfaxin, a potential therapeutic agent for respiratory distress syndrome in premature infants. There is, however, no clear understanding about the possible lipid-KL4 interactions behind its function, which is an inevitable knowledge to design improved therapeutic agents. To examine the phase behavior, topography, and lipid specificity of KL4/lipid systems, we aimed to study different surfactant model systems containing KL4, neutral dipalmitoylphosphatidylcholine (DPPC) and/or negatively charged dipalmitoylphosphatidylglycerol (DPPG) in the presence of Ca2+ ions. Surface pressure-area isotherms, fluorescence microscopic images, scanning force microscopy as well as time-of-flight secondary ion mass spectrometry suggest (i) that KL4 is not miscible with DPPC and therefore forms peptide aggregates in DPPC/KL4 mixtures; (ii) that KL4 specifically interacts with DPPG via electrostatic interactions and induces percolation of DPPG-rich phases; (iii) that existing DPPG-Ca2+ interactions are too strong to be overcome by KL4, the reason why the peptide remains excluded from condensed DPPG domains and passively colocalizes with DPPC in a demixed fluid phase; and (iv) that the presence of negatively charged lipid is necessary for the formation of bilayer protrusions. These results indicate that the capability of the peptide to induce the formation of a defined surface-confined reservoir depends on the lipid environment, especially on the presence of anionic lipids.     

Solute effects on the colloidal and phase behavior of lipid bilayer membranes: ethanol-dipalmitoylphosphatidylcholine mixtures.

By means of the scanning differential calorimetry, x-ray diffractometry, and the dynamic light scattering, we have systematically studied the phase and packing properties of dipalmitoylphosphatidylcholine vesicles or multibilayers in the presence of ethanol. We have also determined the partial ternary phase diagram of such dipalmitoylphosphatidylcholine/water/ethanol mixtures. The directly measured variability of the structural bilayer parameters implies that ethanol binding to the phospholipid bilayers increases the lateral as well as the transverse repulsion between the lipid molecules. This enlarges the hydrocarbon tilt (by up to 23 degrees) and molecular area (by < or = 40%). Ethanol-phospholid association also broadens the interface and, thus, promotes lipid headgroup solvation. This results in excessive swelling (by 130%) of the phosphatidylcholine bilayers in aqueous ethanol solutions. Lateral bilayer expansion, moreover, provokes a successive interdigitation of the hydrocarbon chains in the systems with bulk ethanol concentrations of 0.4-1.2 M. The hydrocarbon packing density as well as the propensity for the formation of lamellar gel phases simultaneously increase. The pretransition temperature of phosphatidylcholine bilayers is more sensitive to the addition of alcohol (initial shift: delta Tp = 22 degrees C/mol) than the subtransition temperature (delta Ts reversible 5 degrees C/mol), whereas the chain-melting phase transition temperature is even less affected (delta Tm = 1.8 degrees C/mol). After an initial decrease of 3 degrees for the bulk ethanol concentrations below 1.2 M, the Tm value increases by 2.5 degrees above this limiting concentration. The gel-phase phosphatidylcholine membranes below Tm are fully interdigitated above this limiting concentration. The chain tilt on the fringe of full chain interdigitation is zero and increases with higher ethanol concentrations. Above Tm, some of the lipid molecules are solubilized by the bound ethanol molecules. More highly concentrated ethanol solutions (> 7 M) solubilize the phosphatidylcholine bilayers with fluid chains fully and result in the formation of mixed lipid-alcohol micelles.     

Direct visualization of asymmetric behavior in supported lipid bilayers at the gel-fluid phase transition

We utilize in situ, temperature-dependent atomic force microscopy to examine the gel-fluid phase transition behavior in supported phospholipid bilayers constructed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. The primary gel-fluid phase transition at T(m) occurs through development of anisotropic cracks in the gel phase, which develop into the fluid phase. At approximately 5 degrees C above T(m), atomic force microscopy studies reveal the presence of a secondary phase transition in all three bilayers studied. The secondary phase transition occurs as a consequence of decoupling between the two leaflets of the bilayer due to enhanced stabilization of the lower leaflet with either the support or the water entrained between the support and the bilayer. Addition of the transmembrane protein gramicidin A or construction of a highly defected gel phase results in elimination of this decoupling and removal of the secondary phase transition.     

Exponential distribution of interimpulse current intervals in lipid bilayer membrane at phase transition temperature

The statistical analysis of current fluctuations in unmodified bilayer lipid membranes at the phase transition temperature was made. An exponential distribution of current fluctuations was revealed.     

Structure of polymerizable lipid bilayers. V. Synthesis, bilayer structure and properties of diacetylenic ether and ester lipids.

Four diacetylenic phosphatidylcholines (PC's) have been synthesized and the structures of bilayers of these lipids have been determined at low resolution by low-angle X-ray diffraction. The PC's all have 18-carbon chains but differ with respect to the ether/ester linkage at the sn-1 and sn-2 positions and the relative position of the diacetylene moiety: diester-PC (1): 1,2-bis(octadeca-4',6'-diynoyl)-sn-glycero-3-phosphocholine diester-PC (2): 1-(octadeca-4',6'-diynoyl)-2-(octadeca-5',7'-diynoy l)-sn- glycero-3-phosphocholine diester-PC (3): 1,2-bis(octadeca-8',10'-diynoyl)-sn-glycerol-3-phosphocholin e diether-PC (4): 1-O-(octadeca-4',6'-diynyl)-2-O-(octadeca-5",7"-din yl)-sn- glycero-3-phosphocholine Only (1) exhibits the typical bilayer profile, whereas (2), (3) and (4) show evidence of interdigitation and/or significant disorder. Only (1) polymerized effectively upon illumination with 254 nm light, turning deep blue in seconds, indicating the formation of long, well-ordered polydiacetylenic structures. Liposomes of these derivatives were tested for permeability by osmotic swelling. Polymerized liposomes of (1) underwent osmotic swelling with urea, glycerol, and acetamide more rapidly than did liposomes of stearoyl-oleoyl-PC, but the initial rates of osmotic swelling of polymerized liposomes of (1) were 3-10-times lower than those of unpolymerized liposomes of (1). Blue polymerized multilayer samples of (1) exhibited an irreversible thermochromic transition to red at approx. 40 degrees C. Differential scanning calorimetry with liposome suspensions of (1) revealed an endotherm at 28.3 degrees C with a transition enthalpy of 40 J/g. PC (1) is a potentially useful diacetylenic lipid which exhibits facile, complete polymerization and a bilayer thickness comparable to that of biomembrane lipids.     

An anion channel of sarcoplasmic reticulum incorporated into planar lipid bilayers: Single-channel behavior and conductance properties

Summary An anion channel of sarcoplasmic reticulum vesicle has been incorporated into planar lipid bilayers by means of a fusion method and its basic properties were investigated. Analysis of fusion processes suggested that one SR vesicle contained approximately one anion channel. The conductance of this channel has several substates and shows a flickering behavior. The occupation probability of each substate was voltage dependent, which induced an inward rectification of macroscopic currents. Further, the anion channel was found to have the following properties. (1) The single-channel conductance is about 200 pS at 100mm Cl^–. (2) The channel does not select among monovalent anions but SO ₄ ^2– hardly permeates through the channel. (3) SO ₄ ^2– added to thecis side (the side to which SR vesicles were added) inhibits Cl^– current competitively in a voltage-dependent manner. (4) An analysis of this voltage dependence suggests that the binding site of SO ₄ ^2– is located at about 36% of the way across the channel from thecis entrance.     

Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact until the disordering of the chains reaches a critical limit. The perturbing effect of OG is thus different from that of another nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), which produces a general disordering at all levels of the lipid bilayer. The OG-POPC interaction was also investigated with POPC monolayers, using a Langmuir trough. In the absence of lipid, the measurement of the Gibbs adsorption isotherm for pure OG solutions yielded an OG surface area of AS = 51 +/- 3 A2. On the other hand, the insertion area AI of OG in a POPC monolayer was determined by a monolayer expansion technique as AI = 58 +/- 10 A2. The similar area requirements with AS approximately AI indicate an almost complete insertion of OG into the lipid monolayer. The OG partition constant for a POPC monolayer at 32 mN/m was Kp approximately 320 M-1 and thus was larger than that for a POPC bilayer.     

A model for phase transitions in lipid bilayers and biological membranes

We wish to present an order-disorder model for the observed phase transitions in lipid bilayers and biological membranes. We show that the model may, under certain circumstances, exhibit two phase transitions, one corresponding to positional disordering of entire lipid molecules, and the other corresponding to orientational disordering in the hydrocarbon chains. We then give results of our numerical analysis of the model and compare them with experimental data. Shortcomings of the model and future directions for analyses of this type are also discussed.     

Lateral diffusion and percolation in two-phase, two-component lipid bilayers. Topology of the solid-phase domains in-plane and across the lipid bilayer.

Fluorescence recovery after photobleaching (FRAP) has recently been used to examine the percolation properties of coexisting phases in two-component, two-phase phosphatidylcholine bilayers [Vaz, W. L. C., Melo, E. C. C., & Thompson, T. E. (1989) Biophys. J. 56, 869-876]. We now report the use of FRAP to study two additional problems in similar systems. The first is the effect of solid-phase obstacles on the lateral diffusion in the fluid phase. The second is the question of whether or not, in a single bilayer, solid-phase domains in one monolayer are exactly superimposed on solid domains in the apposing monolayer. To address the first problem, the lateral diffusion of N-(7-nitrobenzoxa-2,3-diazol-4-yl)-1-palmitoyl-2-oleoylphosp hatidylethanolamine (NBD-POPE), a probe soluble only in the fluid phase when solid and fluid phases coexist, has been studied in the mixture N-lignoceroyldihydrogalactosylceramide (LigGalCer)/dipalmitoylphosphatidylcholine (DPPC). Percolation of the fluid phase occurs at a high mass fraction of solid phase. This indicates that the solid domains have a centrosymmetric shape, a characteristic which makes this a good experimental system to test theoretical simulations of diffusion in an archipelago. It is shown that agreement between theory and experiment is poor, a result that had already been observed when the obstacles were integral membrane proteins. We develop an effective-medium model for diffusion in two-phase systems which explains both our results and those obtained with integral proteins. The distinctive feature of the model is the consideration of an annular region around the obstacles where the lipids are more ordered than in the bulk fluid phase. The diffusion coefficient is then calculated by extending the free area model to two-phase systems, taking these annuli into account. The second question, the organization of the solid-phase domains across the lipid bilayer, is examined in the systems LigGalCer/DPPC and dimyristoylphosphatidylcholine (DMPC)/distearoylphosphatidylcholine (DSPC) by comparing the diffusion of a fluid-phase-soluble, gel-phase-insoluble lipid derivative which spans the two monolayers of a bilayer (NBD-membrane-spanning-phosphatidylethanolamine, NBD-msPE) with that of a probe which is restricted to a single monolayer. In LigGalCer/DPPC, 20:80, the distribution of solid domains in one of the monolayers is independent of the distribution in the apposing monolayer. In contrast, in DMPC/DSPC, 50:50, the solid domains in one monolayer are exactly superimposed upon the solid domains existing in the apposing monolayer.     

Roles of peptide-peptide charge interaction and lipid phase separation in helix-helix association in lipid bilayer

The roles of peptide-peptide charged interaction and lipid phase separation in helix-helix association in lipid bilayers were investigated using a model peptide, P(24), as a transmembrane alpha-helical peptide, and its four analogues. Fluorescence amino acids, tryptophan (P(24)W) and pyrenylalanine (P(24)Pya), were introduced into the sequence of P(24), respectively. Association of these peptides permits the resonance excitation energy transfer between tryptophan in P(24)W and pyrenylalanine in P(24)Pya or excimer formation between P(24)Pya themselves. To evaluate the effect of charged interaction on the association between alpha-helical transmembrane segments in membrane proteins, charged amino acids, glutamic acid (P(24)EW) and lysine (P(24)KPya), were introduced into P(24)W and P(24)Pya, respectively. Energy transfer experiments indicated that the charged interaction between the positive charge of lysine residue in P(24)KPya and the negative charge of glutamic acid residue in P(24)EW did not affect the aggregation of transmembrane peptides in lipid membranes. As the content ratio of sphingomyelin (SM) and cholesterol (Ch) was increased in the egg phosphatidylcholine (PC), the stronger excimer fluorescence spectra of P(24)Pya were observed, indicating that the co-existence of SM and Ch in PC liposomes, that is, the raft of SM and Ch, promotes the aggregation of the alpha-helical transmembrane peptides in lipid bilayers. Since the increase in the contents of SM and Ch leads to the decrease in the content of liquid crystalline-order phase, the moving area of transmembrane peptides might be limited in the liposomes, resulting in easy formation of the excimer in the presence of the lipid-raft.     

Bimodal distribution and fluorescence response of environment-sensitive probes in lipid bilayers

A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, and cholesterol content. Probe F, due to excited-state intramolecular proton transfer, exhibits two bands in emission that are differently sensitive to intermolecular interactions-thereby allowing us to distinguish universal (dipole-dipole) and specific (H-bonding) interactions within the bilayer. Spectroscopic, quenching, and anisotropy data suggest the presence of two forms of probe F at different locations in the bilayer: an H-bond free form located below sn(1)-carbonyls and an H-bonded form located at the polar membrane interface. We provide a quantitative analysis of the distribution of the probe between these two locations as well as the polarity of these locations, and show that both the distribution and the polarity contribute to the probe response. Moreover, analysis of literature data on other environment-sensitive probes (Prodan, Laurdan, Nile Red, NBD lipids, etc.) in lipid bilayers allows us to suggest that the bimodal distribution in the lipid bilayer is probably a general feature of low-polar molecules with polar groups capable of H-bonding interactions.     

Roles of peptide-peptide charge interaction and lipid phase separation in helix-helix association in lipid bilayer

The roles of peptide-peptide charged interaction and lipid phase separation in helix-helix association in lipid bilayers were investigated using a model peptide, P₂₄, as a transmembrane α-helical peptide, and its four analogues. Fluorescence amino acids, tryptophan (P₂₄W) and pyrenylalanine (P₂₄Pya), were introduced into the sequence of P₂₄, respectively. Association of these peptides permits the resonance excitation energy transfer between tryptophan in P₂₄W and pyrenylalanine in P₂₄Pya or excimer formation between P₂₄Pya themselves. To evaluate the effect of charged interaction on the association between α-helical transmembrane segments in membrane proteins, charged amino acids, glutamic acid (P₂₄EW) and lysine (P₂₄KPya), were introduced into P₂₄W and P₂₄Pya, respectively. Energy transfer experiments indicated that the charged interaction between the positive charge of lysine residue in P₂₄KPya and the negative charge of glutamic acid residue in P₂₄EW did not affect the aggregation of transmembrane peptides in lipid membranes. As the content ratio of sphingomyelin (SM) and cholesterol (Ch) was increased in the egg phosphatidylcholine (PC), the stronger excimer fluorescence spectra of P₂₄Pya were observed, indicating that the co-existence of SM and Ch in PC liposomes, that is, the raft of SM and Ch, promotes the aggregation of the α-helical transmembrane peptides in lipid bilayers. Since the increase in the contents of SM and Ch leads to the decrease in the content of liquid crystalline-order phase, the moving area of transmembrane peptides might be limited in the liposomes, resulting in easy formation of the excimer in the presence of the lipid-raft.     

Staphylococcus aureus alpha-toxin-induced pores: Channel-like behavior in lipid bilayers and patch clamped cells

The conductance of pores induced by Staphylococcus aureus -toxin in Lettre cells has been compared to that in bilayers composed of synthetic lipids or Lettre cell membrane constituents. Previously described characteristics of toxin-induced conductance changes in lipid bilayers, namely rectification, voltage-dependent closure, and closure at low pH or in the presence of divalent cations (Menestrina, 1986) are displayed also in bilayers prepared from Lettre cell membranes and in patch clamped Lettre cells. It is concluded that endogenous proteins do not affect the properties of -toxininduced channels significantly and that the relative lack of ion channels in Lettre cells makes them ideal for studies of pore-forming toxins by the patch clamp technique.Dr. Sviderskaya is on leave of absence from the Physiology Institute, University of St. Petersburg, RussiaWe are grateful to Dr. J.P. Arbuthnott and Dr. K. Hungerer for gifts of S. aureus -toxin, to Dr. T.B. Bolton for collaboration with patch clamped cells and to Dr. J.M. Graham for help with the preparation of Lettre cell plasma membranes. This study was supported by the Cell Surface Research Fund, Medical Research Council, Science and Engineering Research Council, UNESCO (Molecular and Cell Biology Network) and The Wellcome Trust.     

Pressure effects on the structure and phase behavior of DMPC-gramicidin lipid bilayers: a synchrotron SAXS and 2H-NMR spectroscopy study

The influence of Gramicidin D (GD) incorporation on the structure and phase behavior of aqueous dispersions of DMPC lipid bilayers has been studied using small-angle x-ray scattering (SAXS) and (2)H-NMR spectroscopy. The experiments covered a temperature range from -10 degrees C to 60 degrees C and a pressure range of 0.001-4 kbar. Pressure was used to be able to tune the lipid bilayer conformational order and phase state and because high pressure is an important feature of certain natural biotopes. The data show that, depending on the GD concentration, the structure of the temperature- and pressure-dependent lipid phases is significantly altered by the insertion of the polypeptide, and a p,T-phase diagram could be obtained for intermediate GD concentrations. Upon gramicidin insertion, a rather narrow fluid-gel coexistence regions is formed. Two gel phases are induced which are different from those of the pure lipid bilayer system and which separate at low temperatures/high pressures. For both the temperature- and pressure-induced fluid-to-gel transition, a similar pseudocritical transitional behavior is observed, which is even more pronounced upon incorporation of the peptide.     

Theory of lipid monolayer and bilayer phase transitions: Effect of headgroup interactions

Summary Headgroup and soft core interactions are added to a lipid monolayer-bilayer model and the surface pressure-area phase diagrams are calculated. The results show that quite small headgroup interactions can have biologically significant effects on the transition temperature and the phase diagram. In particular, the difference in transition temperatures of lecithins and phosphatidyl ethanolamines is easy to reproduce in the model. The phosphatidic acid systems seem to require weak transient hydrogen bonding which is also conjectured to play a role in most of the lipid systems. By a simple surface free energy argument it is shown that monolayers under a surface pressure of 50 dynes/cm should behave as bilayers, in agreement with experiment. Although the headgroup interactions are biologically very significant, in fundamental studies of the main phase transition in lipids they are secondary in importance to the hydrocarbon chain interactions (including the excluded volume interaction, the rotational isomerism, and the attractive van der Waals interaction).     

Saturation behavior of single, amiloride-sensitive Na+ channels in planar lipid bilayers.

Single epithelial Na+ channels incorporated into planar lipid bilayers were studied to determine the effects of Na concentration on its own conductance. Amiloride-sensitive Na+ channels were obtained from apical membrane vesicles made from A6 cells, a continuous epithelial cell-line derived from amphibian kidney. Single-channel conductance was found to be a saturable function of Na+ concentration, with a Michaelis constant of approximately 17 or 47 mM, for a Gmax of approximately 4 or 44 pS, respectively.