Permeability and electrical properties of planar lipid membranes from thylakoid lipids.

Electrical measurements were carried out on planar lipid membranes from thylakoid lipids. The specific capacitance of membranes formed from decane-containing monogalactosyldiacylglycerol (MGDG), which accounts for 57% of the total lipid content of thylakoids, showed that it adopted a bilayer structure. Solvent-free bilayers of MGDG were not formed, with very rare exceptions, indicating that decane is required to stabilize the planar conformation. However, this cone-shaped lipid produces bilayer structures in combination with other cylindrical thylakoid lipids even in the absence of organic solvent. We compared the properties of solvent-free and decane-containing bilayers from MGDG, soybean lecithin, and the quaternary mixture of lipids similar to that found in vivo. The conductance of decane-MGDG was 26 times higher than that of decane-lecithin. The flux through the decane-lecithin bilayer was found to be slightly dependent on pH, whereas the decane-MGDG membrane was not. The specific conductance of bilayers formed from the quaternary mixture of lipids was 5 to 10 times larger than lecithin (with alkane or not). Further experiments with bilayers made in the presence of a KCl gradient showed that decane-MGDG, decane-MGDG/DGDG/SQDG/PG, and solvent-free MGDG/DGDG/SQDG/PG were cation-selective. The permeability coefficient for potassium ranged from 4.9 to 8.3 x 10(-11) cm s-1. The permeability coefficient for protons in galactolipids, however, was determined to be about six orders of magnitude higher than the value for potassium ions. The HCl permeation mechanism through the lipid membranes was determined from diffusion potentials measured in HCl gradients. Our results suggest that HCl was not transported as neutral molecules. The data is discussed with regard to the function of galactolipids in the ion transport through thylakoid membranes.     

Transport of protons and hydrochloric acid through lipid bilayer membranes

Transport of protons and hydrochloric acid through lipid bilayer membranes was studied by a combination of electrical conductance and pH electrode techniques. In the presence of large pH gradients, proton transport occurs primarily by diffusion of molecular HCl. The permeability of egg phosphatidylcholine/decane bilayers to HCl is about 3 cm . s-1, seven to nine order of magnitude higher than the permeability to H+, OH- or Cl-. The HCl permeability of phosphatidylserine or egg phosphatidylcholine/cholesterol (1 : 1) bilayers is about 50% lower than the permeability of egg phosphatidylcholine bilayers. Diffusion of molecular HCl may be an important process in tissues exposed to high HCl concentrations, e.g., gastric mucosa. However, at neutral pH the diffusion of molecular HCl is too slow to contribute significantly to net movements of H+ or Cl-.     

Cadmium and thallous ion permeabilities through lipid bilayer membranes

Cadmium (Cd2+) and thallous ion (Tl+) permeabilities were measured in planar (Mueller-Rudin) lipid bilayer membranes made from diphytanoylphosphatidylcholine in decane. Permeabilities of the electroneutral Cl- complexes, measured with tracers (109Cd and 204Tl), were about 10(-8) cm X s-1 for CdCl2 and 10(-6) cm X s-1 for TlCl. Electrical conductance measurements showed that permeabilities to Cd2+ and Tl+ were approx. 10(-11) cm X s-1, similar to the Na+ permeability. The low permeabilities to both Cd2+ and CdCl2 are consistent with biological studies which suggest that Cd transport and toxicity are protein mediated and correlated with Cd2+, not CdCl2, concentration. However, the low bilayer permeability to Tl+ raises questions about recent reports that Tl+ is a lipid permeable cation in biological membranes and liposomes. An alternative explanation for the lipid permeable behavior of Tl+ is presented, based on the diffusion of TlCl and other complexes of Tl+ with inorganic and organic anions.     

Weak acid permeability through lipid bilayer membranes. Role of chemical reactions in the unstirred layer

The premeabilities of planar lipid bilayer (egg phosphatidylcholine- decane) membranes to butyric and formic acids were measured by tracer and pH electrode techniques. The purposes of the study were (a) to establish criteria for the applicability of each method and (b) to resolve a discrepancy between previously published permeabilities determined using the different techniques. Tracer fluxes of butyric acid were measured at several concentrations and pH's. Under symmetrical conditions the one-way flux of butyric acid(J) is described by 1/J = 1/Pul ([HA] + [A-]) + 1/Pm([HA]), where Pul and Pm are the unstirred layer and membrane permeability coefficients. Pm determined in this manner is 950 x 10(4) cm s-1. Published values for the butyric acid permeability for egg phosphatidylcholine-decane bilayers are 11.5 x 10(-4) (Wolosin and Ginsburg, 1975) and 640 x 10(-4) cm s-1 (Orbach and Finkelstein, 1980). Wolosin and Ginsburg measured net fluxes from a solution of pH = Pka into an unbuffered solution containing a pH electrode. Orbach and Finkelstein measured tracers fluxes under symmetrical conditions at pH 7.4. We reproduced the results of Wolosin and Ginsburg and showed that their apparently low Pm was caused by unstirred layer effects in their poorly buffered solutions. The permeability to formic acid (pKa = 3.75) measured by both tracer and pH electrode techniques was approximately 10(-2) cm s-1. However, if pm greater than Pul, the pH electrode technique cannot be used for measuring the permeabilities of weak acids with pKa's greater than approximately 4.     

Properties of lecithin-dodecaprenol macrovesicular bilayer membranes

The ionic transport properties, capacitance and breakdown voltage of bilayer macrovesicles made from lecithin, dodecaprenol and their mixtures have been studied. The electrical measurements showed that polyprenol in lipid bilayers increases membrane permeability and elasticity, and decreases membrane thickness. Some physiological implications of these findings are indicated.     

Opioid peptide interactions with lipid bilayer membranes.

The interaction of the delta-opioid receptor selective peptides, cyclic [D-Pen2, D-Pen5]-enkephalin [DPDPE] and its acyclic analog, DPDPE(SH)2, with neutral phospholipid bilayer membranes was examined by permeability and calorimetry measurements. The permeabilities were accomplished by entrapping either peptide inside of unilamellar liposomes (composed of a mixture of a molar ratio 65:25:10 phosphatidylcholine/phosphatidylethanolamine/cholesterol) then monitoring the peptide efflux through the bilayer. The initial permeability of DPDPE (first 12 h) averaged over four experiments was (0.91 +/- 0.47).10(-12) cm s-1. In contrast the average permeability of the acylic DPDPE(SH)2 was (4.26 +/- 0.23).10(-12) cm s-1. The effect of these peptides on the phase transition, Tm, of 1,2-dipalmitoylphosphatidylcholine (DPPC) bilayers was examined by high sensitivity differential scanning calorimetry. The Tm, the calorimetric enthalpy, and the van 't Hoff enthalpy of DPPC were not significantly altered by the presence of DPDPE, whereas the calorimetric data for DPPC with DPDPE(SH)2 showed a small, yet significant, increase (0.2 degrees C) in the Tm with a 30% decrease in the cooperative unit. Both the permeability and calorimetry data reveal a stronger peptide-membrane interaction in the case of the more flexible acyclic peptide.     

Magnetic anisotropy of egg lecithin membranes.

Magnetic realignment and rotational diffusion of cylindrical egg lecithin vesicles were measured under a phase contrast microscope. The anisotropy of magnetic susceptibility times membrane thickness was calculated from the data for several thin-walled vesicles. The resulting values were assigned to discrete numbers of bilayers. The difference between the susceptibilities parallel and perpendicular to the long axes of the lecithin molecules is deduced to be X parallel - X perpendicular = -(0.28 +/- 0.02) . 10(-8) cgs at 23 degrees C, if a bilayer thickness of 60 A is assumed.     

Planar bilayer membranes from pure lipids.

Planar lipid bilayer membranes are formed from mixtures of pure lipids in the absence of non-biological solvents. The solventless bilayers are characterized by a large specific capacitance (586-957 nF/cm2) comparable to that of cell membranes but considerably greater than that of conventional lipid/decane bilayers. Hydrocarbon solvents, such as n-alkanes or squalene, thicken the bilayer. Membrane dielectric thickness is used as an indicator of bilayer lipid composition. For membranes made from pure monoglyceride/triglyceride mixtures the thickness of the solventless lipid bilayer is independent of both the chain length (11-22 carbons) and mol fraction (0.1-0.9) of triglyceride in the bulk mixture. In contrast, the thickness of the bilayer (2.0-3.3 nm) depends strongly upon the length (16-24 carbons) of the monoglyceride component. Molecular volume considerations lead to the conclusion that the bulk lipid mixture disproportionates to yield bilayer membranes composed of nearly pure monoglyceride. The dielectric thickness of the monoglyceride bilayer is consistent with the notion that the lipid fatty acyl chains are fluid.     

Optical and electrical properties of thin monoolein lipid bilayers

Summary Monoolein lipid bilayers were formed using a monolayer transfer technique and from dispersions of monoolein in squalene, triolein, 1-chlorodecane and 1-bromodecane. Measurements of optical reflectance and electrical capacitance were used to determine the thickness and dielectric constant of the bilayers. The thickness of the hydrocarbon region of the five bilayer systems ranged from 2.5 to 3.0 nm. Two of the bilayer systems (made from 1-chlorodecane and 1-bromodecane solvents) had a high dielectric constant (2.8 to 2.9) whereas the other bilayer systems had dielectric constants close to that of pure hydrocarbons (2.2). The charge-pulse technique was used to study the transport kinetics of three lipophilic ions and two ion carrier complexes in the bilayers. For the low dielectric constant bilayers, the transport of the lipophilic ions tetraphenylborate, tetraphenylarsonium and dipicrylamine was governed mainly by the thickness of the hydrocarbon region of the bilayer whereas the transport of the ion-carrier complexes proline valinomycin-K⁺ and valinomycin-Rb⁺ was nearly independent of thickness. This is consistent with previous studies on thicker monoolein bilayers. The transport of lipophilic anions across bilayers with a high dielectric constant was 20 to 50 times greater than expected on the basis of thickness alone. This agrees qualitatively with predictions based on Born charging energy calculations. High dielectric constant bilayers were three times more permeable to the proline valinomycin-K⁺ complex than were low dielectric constant bilayers but were just as permeable as low dielectric constant bilayers to the valinomycin-Rb⁺ complex.     

Mechanism of proton permeation through chloroplast lipid membranes.

Electrical measurements were carried out to investigate the contribution of chloroplast lipids to the passive proton permeability of both the thylakoid and inner-envelope membranes. Permeability coefficient and conductance to protons were measured for solvent-free bilayers made from monogalactosyldiglyceride:digalactosyldiglycerid: sulfoquinovosyldiglyceride:phosphatidylglycerol (2:1:0.5:0.5, w/w) in the presence of a pH gradient of 7.4/8.1. The permeability coefficient for protons in glycolipids was 5.5 +/- 1.1 x 10(-4) cm s-1 (n = 14). To determine whether this high H+ permeability could be explained by the presence of lipid contaminants such as weak acids, we investigated the effects of (a) bovine serum albumin, which can remove some amphiphilic molecules such as free fatty acids, (b) 6-ketocholestanol, which increases the membrane dipole potential, (c) oleic acid, and (d) chlorodecane, which increases the dielectric constant of the lipid bilayer. Our results show that free fatty acids are inefficient protonophores, as compared with carbonylcyanide-m-chlorphenythydrazone, and that the hypothesis of a weak acid mechanism is not valid with glycolipid bilayers. In the presence of deuterium oxide the H+ conductane was reduced significantly, indicating that proton transport through the glycolipid matrix could occur directly by a hydrogen bond process. The passive transport of H+ through the glycolipid matrix is discussed with regard to the activity of the thylakoid ATP synthase and the inner-envelope H(+)-ATPase.     

Water permeability of asymmetric planar lipid bilayers: leaflets of different composition offer independent and additive resistances to permeation

To understand how plasma membranes may limit water flux, we have modeled the apical membrane of MDCK type 1 cells. Previous experiments demonstrated that liposomes designed to mimic the inner and outer leaflet of this membrane exhibited 18-fold lower water permeation for outer leaflet lipids than inner leaflet lipids (Hill, W.G., and M.L. Zeidel. 2000. J. Biol. Chem. 275:30176-30185), confirming that the outer leaflet is the primary barrier to permeation. If leaflets in a bilayer resist permeation independently, the following equation estimates single leaflet permeabilities: 1/P(AB) = 1/P(A) + 1/P(B) (Eq. l), where P(AB) is the permeability of a bilayer composed of leaflets A and B, P(A) is the permeability of leaflet A, and P(B) is the permeability of leaflet B. Using for the MDCK leaflet-specific liposomes gives an estimated value for the osmotic water permeability (P(f)) of 4.6 x 10(-4) cm/s (at 25 degrees C) that correlated well with experimentally measured values in intact cells. We have now constructed both symmetric and asymmetric planar lipid bilayers that model the MDCK apical membrane. Water permeability across these bilayers was monitored in the immediate membrane vicinity using a Na+-sensitive scanning microelectrode and an osmotic gradient induced by addition of urea. The near-membrane concentration distribution of solute was used to calculate the velocity of water flow (Pohl, P., S.M. Saparov, and Y.N. Antonenko. 1997. Biophys. J. 72:1711-1718). At 36 degrees C, P(f) was 3.44 +/- 0.35 x 10(-3) cm/s for symmetrical inner leaflet membranes and 3.40 +/- 0.34 x 10(-4) cm/s for symmetrical exofacial membranes. From, the estimated permeability of an asymmetric membrane is 6.2 x 10(-4) cm/s. Water permeability measured for the asymmetric planar bilayer was 6.7 +/- 0.7 x 10(-4) cm/s, which is within 10% of the calculated value. Direct experimental measurement of P(f) for an asymmetric planar membrane confirms that leaflets in a bilayer offer independent and additive resistances to water permeation and validates the use of.     

The effect of dimethylsulfoxide and thiourea on the water diffusion permeability of the E. coli membrane

It is shown that diffusive water permeability of E. coli cell membranes at 4-24 degrees C is in the range from 16.6 to 35.0 microM.s-1, that is close to erythrocyte membrane water permeability, but higher than that of lipid bilayers. Cryoprotectants (DMSO and thiourea) at the concentration of 1.0 M considerably decrease water bacterial membrane permeability. 1.5- and 2-fold, respectively. The obtained results are discussed in relation to two possible water transport ways through pores of the protein nature or lipid bilayer damages.     

Permeation of halide anions through phospholipid bilayers occurs by the solubility-diffusion mechanism.

Two alternative mechanisms are frequently used to describe ionic permeation of lipid bilayers. In the first, ions partition into the hydrophobic phase and then diffuse across (the solubility-diffusion mechanism). The second mechanism assumes that ions traverse the bilayer through transient hydrophilic defects caused by thermal fluctuations (the pore mechanism). The theoretical predictions made by both models were tested for halide anions by measuring the permeability coefficients for chloride, bromide, and iodide as a function of bilayer thickness, ionic radius, and sign of charge. To vary the bilayer thickness systematically, liposomes were prepared from monounsaturated phosphatidylcholines (PC) with chain lengths between 16 and 24 carbon atoms. The fluorescent dye MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide) served as an indicator for halide concentration inside the liposomes and was used to follow the kinetics of halide flux across the bilayer membranes. The observed permeability coefficients ranged from 10(-9) to 10(-7) cm/s and increased as the bilayer thickness was reduced. Bromide was found to permeate approximately six times faster than chloride through bilayers of identical thickness, and iodide permeated three to four times faster than bromide. The dependence of the halide permeability coefficients on bilayer thickness and on ionic size were consistent with permeation of hydrated ions by a solubility-diffusion mechanism rather than through transient pores. Halide permeation therefore differs from that of a monovalent cation such as potassium, which has been accounted for by a combination of the two mechanisms depending on bilayer thickness.     

The 2004 Biophysical Society-Avanti Award in Lipids address: roles of bilayer structure and elastic properties in peptide localization in membranes

This review details how bilayer structural/elastic properties impact three distinct areas of biological significance. First, the partitioning of melittin into bilayers and melittin-induced bilayer leakage depended strongly on bilayer composition. The incorporation of cholesterol into phosphatidylcholine bilayers decreased melittin-induced leakage from 73 to 3%, and bilayers composed of lipopolysaccharide (LPS), the main lipid on the surface of Gram-negative bacteria, also had low (3%) melittin-induced leakage. Second, transbilayer peptides of different hydrophobic lengths were largely excluded from bilayer microdomains (“rafts”) enriched in sphingomyelin (SM) and cholesterol, even when the length of the transbilayer peptide domain matched the hydrocarbon thickness of the raft bilayer. This is likely due to the large area compressibility modulus of SM:cholesterol bilayers. Third, the major water barrier of skin, the extracellular lamellae of the stratum corneum, was found to contain tightly packed asymmetric lipid bilayers with cholesterol located preferentially on one side of the bilayer and a unique skin ceramide containing an unsaturated acyl chain on the opposite side. We argue that, in each of these three areas, key factors are differences in lipid hydrocarbon chain packing for different lipids, particularly the tight hydrocarbon chain packing caused by cholesterol’s strong interaction with saturated chains.     

Permeability of human red cells to a homologous series of aliphatic alcohols. Limitations of the continuous flow-tube method

Human red cell permeability to the homologous series of methanol, ethanol, n-propanol, n-butanol, and n-hexanol was determined in tracer efflux experiments by the continuous flow tube method, whose time resolution is 2-3 ms. Control experiments showed that unstirred layers in the cell suspension were less than 2 X 10(-4) cm, and that permeabilities less than or equal to 10(-2) cm s-1 can be determined with the method. Alcohol permeability varied with the chain length (25 degrees C): Pmeth 3.7 X 10(-3) cm s-1, Peth 2.1 X 10(-3) cm s-1, Pprop 6.5 X 10(-3) cm s-1, Pbut less than or equal to 61 X 10(-3) cm s-1, Phex 8.7 X 10(-3) cm s-1. The permeability for methanol, ethanol, and n- propanol was concentration independent (1-500 mM). The permeability to n-butanol and n-hexanol, however, increased above the upper limit of determination at alcohol concentrations of 100 and 25 mM, respectively. The activation energies for the permeability to methanol, n-propanol, and n-hexanol were similar, 50-63 kJ mol-1. Methanol permeability was not reduced by p-chloromercuribenzene sulfonate (PCMBS), thiourea, or phloretin, which inhibit transport of water or hydrophilic nonelectrolytes. It is concluded (a) that all the alcohols predominantly permeate the membrane lipid bilayer structure; (b) that both the distribution coefficient and the diffusion coefficient of the alcohols within the membrane determine the permeability, and (c) that the relative importance of the two factors varies with changes in the chain length.     

Structural determinants of water permeability through the lipid membrane

Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility.     

Permeability of bilayer phospholipid membranes to superoxide oxygen radicals

Lecithin monolayer liposomes (1000 A in diameter) loaded with cytochrome c were placed into the external solution, in which O2 superoxide radicals were regenerated by the xanthine-xanthine oxidase system. The penetration of superoxide radicals across the liposomal membranes was followed by cytochrome c reduction in the interval volume of the liposomes. The effects of lipid membrane modifiers and temperature on this process were investigated. The results obtained were used for calculation of the permeability coefficients of bilayer lipid membranes for O(2) (P'O(2) = (7.6 +/- 0.3) . 10(-8) cm . s-1) or HO . 2(P'HO(2) = 4.9 x 10(-4) cm . s-1). The effect of the transmembrane electric potential (concentration gradient of H+, valinomycin) on the permeability of liposomal membranes for the superoxide radical was studied. The superoxide radical was down to penetrate across the bilayer lipid membranes in an unloaded state. Using an intramolecular cholesterol-amphotericin B-complex, the superoxide radicals were shown to penetrate across the bilayer lipid membranes, predominantly via the anionic channels.     

Membrane-directed effects of the plant hormones abscisic acid, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid

This study examines two ways plant hormones might influence membrane processes, effects on overall permeability and modifications of specific ion channels. Abscisic acid (ABA) and indole-3-acetic acid (IAA) greatly enhanced erythritol permeability in mixed egg lecithin bilayers. In single component dioleoylphosphatidylcholine bilayers ABA was less effective than IAA, while 2,4-dichlorophenoxyacetate (2,4-D) did not affect either system or alter their ABA response. In Myxicola axons ABA and IAA had no effect, while 2,4-D (10 uM) caused a depolarizing shift of voltage-dependent Na+ and K+ activation by 25 +/- 4 mV and 15 +/- 3 mV, consistent with internal negative surface charge changes of -0.002 e-/A2 and -0.0007 e-/A2. We conclude that both generalized and ion channel-directed effects may link plant hormones and intracellular regulation.     

A computer simulation of functional group contributions to free energy in water and a DPPC lipid bilayer

A series of all-atom molecular dynamics simulations has been performed to evaluate the contributions of various functional groups to the free energy of solvation in water and a dipalmitoylphospatidylcholine lipid bilayer membrane and to the free energies of solute transfer (Delta(DeltaG(o))X) from water into the ordered-chain interior of the bilayer. Free energies for mutations of the alpha-H atom in p-toluic acid to six different substituents (-CH3, -Cl, -OCH3, -CN, -OH, -COOH) were calculated by a combined thermodynamic integration and perturbation method and compared to literature results from vapor pressure measurements, partition coefficients, and membrane transport experiments. Convergence of the calculated free energies was indicated by substantial declines in standard deviations for the calculated free energies with increased simulation length, by the independence of the ensemble-averaged Boltzmann factors to simulation length, and the weak dependence of hysteresis effects on simulation length over two different simulation lengths and starting from different initial configurations. Calculated values of Delta(DeltaG(o))X correlate linearly with corresponding values obtained from lipid bilayer transport experiments with a slope of 1.1 and from measurements of partition coefficients between water and hexadecane or decadiene, with slopes of 1.1 and 0.9, respectively. Van der Waals interactions between the functional group of interest and the acyl chains in the ordered chain region account for more than 95% of the overall potential energy of interaction. These results support the view that the ordered chain region within the bilayer interior is the barrier domain for transport and that solvation interactions within this region resemble those occurring in a nonpolar hydrocarbon.     

Permeability of acetic acid across gel and liquid-crystalline lipid bilayers conforms to free-surface-area theory.

Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface area), regardless of whether chain ordering is varied by changes in temperature, lipid chain length, cholesterol concentration, or bilayer phase structure, provided that temperature effects on permeant dehydration and diffusion and the chain-length effects on bilayer barrier thickness are properly taken into account.