Overton's Rule Research Articles

Aquaporins and CO2 diffusion across biological membrane.

Despite the physiological significance of effective CO2 diffusion across biological membranes, the underlying mechanism behind this process is not yet resolved. Particularly debatable is the existence of CO2-permeable aquaporins. The lipophilic characteristic of CO2 should, according to Overton's rule, result in a rapid flux across lipid bilayers. However, experimental evidence of limited membrane permeability poses a challenge to this idea of free diffusion. In this review, we summarized recent progress with regard to CO2 diffusion, and discussed the physiological effects of altered aquaporin expression, the molecular mechanisms of CO2 transport via aquaporins, and the function of sterols and other membrane proteins in CO2 permeability. In addition, we highlight the existing limits in measuring CO2 permeability and end up with perspectives on resolving such argument either by determining the atomic resolution structure of CO2 permeable aquaporins or by developing new methods for measuring permeability.

Membraneless physiology of the living cell. The past and the present

Since the 1880s, the concept of compartmentalizing through membranes has taken a firm place in cell physiology and has defined the objects, methods, and goals of physiologists’ research for decades. A huge mass of biologists know about the important role of intra-membrane pumps, channels, and lipids, and various hypotheses about the origin of life often begin with explanations about how the lipid membrane occurred, without which it is impossible to imagine the origin of a living cell. Against this background, there was a dissonance of statements that there are membraneless organelles in the cell, the functions of which are rapidly expanding under our eyes. Physically, they are similar to coacervate droplets, which from time to time were used to explain the origin of life, and now the coacervates are being more and more often discussed when describing the physics of the nucleus and cytoplasm of modern cells. However, ideas about the coacervate nature of cytoplasm/protoplasm originated in the first half of the 19th Century, when the contents of cells were likened to jelly, but this approach gradually faded into the shadows. Nevertheless, limited research in this area continued and was completed in the form of a membraneless cell physiology. Now that the focus of attention has turned to membraneless compartmentalization, it’s time to remember the past. The sorption properties of proteins are the physical basis of membraneless cell because of water adsorbed by proteins changes the physical state of any biomolecular system, from supramolecular and subcellular structures to the cell as a whole. A thermodynamic aqueous phase is formed because adsorbed water does not mix with ordinary water and, in this cause, is separated from the surrounding solution in the form of a compartment. This article discusses the fundamental physical properties of such a phase – a biophase. As it turned out, the Meyer–Overton rule, which led to the idea of a lipid membrane, also applies to membraneless condensates.

Quantitation of transmembrane CO₂ flux via AQP5 and NBCe1 in a neutral‐buoyancy assay

The discoveries of CO₂-impermeable membranes in gastric glands; the CO₂-permeability of the membrane protein aquaporin 1 (AQP1), the first “gas channel”; and of “CO₂-blocking” proteins incorporated into artificial membranes show that Overton's rule—namely, membrane permeability of solute X depends uniquely on the lipid solubility of X—is not universal. Various AQPs conduct CO₂ in differing proportions, in part through their hydrophilic monomeric pores, and in part via the hydrophobic central pore of the tetramer. Rhesus (Rh) proteins also can conduct CO₂. Our laboratory has identified a 3rd class of CO₂-conducting proteins that lack a classical pore. NBCe1-A (SLC4A4) cotransports Na+ and CO3=. Using out-of-equilibrium CO₂/HCO₃⁻ solutions and electrophysiological approaches with Xenopus oocytes, we previously reported that NBCe1-A also conducts CO₂, but only in the presence of HCO₃⁻. We speculate that transient pathways within the NBCe1 molecule arise during Na+/CO3= cotransport, allowing CO₂ to pass through the protein. To elucidate the mechanism of transmembrane CO₂ flux via AQPs and NBCe1-A, we modified our Neutral Buoyancy Assay (NBA), originally developed to assess transmembrane N₂ fluxes. We inject a precise volume of N₂ gas (number of gas molecules = nGas) into a Xenopus oocyte, which we transfer to a saline-containing tube. We then increase the pressure (PNB) in the air phase above the air-water interface so that it is just sufficient to collapse the injected bubble enough to make the oocyte neutrally buoyant, 5 cm depth below the meniscus. As N₂ exits the bubble and ultimately diffuses into the extracellular fluid (ECF), the bubble shrinks, cell density increases, and the oocyte sinks. A camera/computer system detects this sinking and decreases PNB to re-establish neutral buoyancy at 5 cm depth. PNB decays exponentially over 1000 s. Calibrations allow us to compute the ΔnGas time course, and thus gas efflux. We modified the NBA to quantify gas influx by measuring bubble inflation. When we raise [N₂] in the ECF from [N₂]o = 0.56 mM (room air at 1×ATA) to [N₂]o = 2.06 mM (pre-equilibrating saline with 93% N₂/7% O₂ at 3×ATA) at constant [O₂]o = 0.26 mM, N₂ enters the cell during the NBA and nGas rises. If we further increase [N₂]o to 2.81 mM at fixed [O₂]o (pre-equilibrating saline with 95% N₂/5% O₂ at 4×ATA), ΔnGas over 1000 s increases more. To perform the inflation assay with CO₂ (the high solubility of which translates to a low tendency to inflate the bubble), we expose the oocyte to 100% CO₂/95 mM HCO₃⁻/pHo 6.66. nGas increases rapidly as CO₂ rushes into the cell, and peaks at <200 s, after which N₂ exit dominates and nGas decreases. Compared to control oocytes (injected with H2O rather than cRNA), those expressing AQP5 or NBCe1-A have a significantly greater maximal rate of nGas increase (i.e., CO₂ influx). The peak ΔnGas from AQP5 or NBCe1-A oocytes is also larger and more delayed. Thus, we identify a novel approach that confirms that both AQP5 and NBCe1-A conduct CO₂. Whereas we developed the NBA specifically to measure N₂ fluxes, here we demonstrate the adaptability of this method for measuring transmembrane fluxes of other gases.

Permselectivity limits of biomimetic desalination membranes.

Water scarcity and inadequate membrane selectivity have spurred interest in biomimetic desalination membranes, in which biological or synthetic water channels are incorporated in an amphiphilic bilayer. As low channel densities (0.1 to 10%) are required for sufficient water permeability, the amphiphilic bilayer matrix will play a critical role in separation performance. We determine selectivity limits for biomimetic membranes by studying the transport behavior of water, neutral solutes, and ions through the bilayers of lipid and block-copolymer vesicles and projecting performance for varying water channel densities. We report that defect-free biomimetic membranes would have water/salt permselectivities ~108-fold greater than current desalination membranes. In contrast, the solubility-based permeability of lipid and block-copolymer bilayers (extending Overton's rule) will result in poor rejection of hydrophobic solutes. Defect-free biomimetic membranes thus offer great potential for seawater desalination and ultrapure water production, but would perform poorly in wastewater reuse. Potential strategies to limit neutral solute permeation are discussed.

Position-Dependent Diffusion Tensors in Anisotropic Media from Simulation: Oxygen Transport in and through Membranes.

A Bayesian-based methodology is developed to estimate diffusion tensors from molecular dynamics simulations of permeants in anisotropic media, and is applied to oxygen in lipid bilayers. By a separation of variables in the Smoluchowski diffusion equation, the multidimensional diffusion is reduced to coupled one-dimensional diffusion problems that are treated by discretization. The resulting diffusivity profiles characterize the membrane transport dynamics as a function of the position across the membrane, discriminating between diffusion normal and parallel to the membrane. The methodology is first validated with neat water, neat hexadecane, and a hexadecane slab surrounded by water, the latter being a simple model for a lipid membrane. Next, a bilayer consisting of pure 1-palmitoyl 2-oleoylphosphatidylcholine (POPC), and a bilayer mimicking the lipid composition of the inner mitochondrial membrane, including cardiolipin, are investigated. We analyze the detailed time evolution of oxygen molecules, in terms of both normal diffusion through and radial diffusion inside the membrane. Diffusion is fast in the more loosely packed interleaflet region, and anisotropic, with oxygen spreading more rapidly in the membrane plane than normal to it. Visualization of the propagator shows that oxygen enters the membrane rapidly, reaching its thermodynamically favored center in about 1 ns, despite the free energy barrier at the headgroup region. Oxygen transport is quantified by computing the oxygen permeability of the membranes and the average radial diffusivity, which confirm the anisotropy of the diffusion. The position-dependent diffusion constants and free energies are used to construct compartmental models and test assumptions used in estimating permeability, including Overton's rule. In particular, a hexadecane slab surrounded by water is found to be a poor model of oxygen transport in membranes because the relevant energy barriers differ substantially.

Role of Lipid Composition in Gas Permeation across Biological Membranes

Gas molecules dissolved in an aqueous environment are pre-dominatly in their hydrophilic charged state because of their respective acid/base equilibria. Overton's rule suggests that permeation of small charged molecules across the membrane boundary is highly restricted and must occur either in their neutral form or through a dedicated transport mechanism. There has been experimental evidence to the effect that the water channel family, Aquaporins, may be responsible for the permeation of neutral gas molecules such as CO2, NH3, and O2 across biological membranes. This is in apparent contrast to the observation that the permeation rate of neutral gas molecules across pure lipid bilayers is mostly unhindered. It is hence of interest to test the hypothesis the lipid composition of biological membranes may result in a vastly different in gas permeability as composed to pure model membranes.The lipid composition of physiological membranes is highly complex and further involves asymmetry in the two leaflets. Also, a major component of the cellular lipid bilayers involved in gas permeation, such as those surrounding Red Blood Cells (RBCs), is cholesterol. Cholesterol is known to have a strong clustering effect that increases the packing of the lipids in the membrane. It is possible to study the role of lipid composition and asymmetry through molecular dynamics (MD) simulations by reconstructing atomistically the important characteristics of physiological membranes. Here we present preliminary results of the permeability of model physiological membranes based on atomistic MD simulations.

Imaging Molecular Transport Across Lipid Bilayers

The passive transport of small molecules across the plasma membrane is a key physiological process. Literature measurements of membrane permeability to small molecules have varied widely. We used confocal microscopy to image the transport of molecules into a giant unilamellar lipid vesicle (GUV). Fluorescent dyes were used to trace the transport of molecules. The GUV was immobilized on the surface of a microfluidic channel by biotin-avidin binding. This microchannel allows the rapid and uniform exchange of the solution surrounding the GUV. Using a spinning-disk confocal microscope, the entire concentration field is captured in a short exposure. We used this system to study the passive transport of carboxylic acids, which have many properties common to small-molecule drugs. The transport of these acids across cell membranes has been widely studied, but there is much variation in the reported permeabilities. By using pH-sensitive fluorescein-dextran to track the acids permeating through the GUV membrane, our results showed that more lipophilic acids cross the bilayer more quickly. A finite difference model was developed to simulate the experimental process and derive precise permeability values. The permeabilities change with the same trend as oil-water partition coefficients, demonstrating that Overton's rule applies to this class of molecules. We used the imaging technique described above to study the transport of protons across compositionally asymmetric lipid bilayers. Synthetic asymmetric GUVs were prepared via a microfluidic multiphase droplet flow technology to mimic membrane charge asymmetry. Negatively charged phosphatidylserine was added to an asolectin GUV on either the internal or external leaflet. The permeation rates of protons into and out of these GUVs were measured. The proton distribution across the asymmetric GUV membrane at equilibrium was also studied. This research can reveal how asymmetric cell membrane composition affects small molecule transport behavior in physiological processes.

A Revised View on Cellular CO2 Transport Mechanisms: Biophysics, Physiology and Genomics of Aquaporin-Facilitated CO2 Transport in Plants

It is a general opinion that the Meyer-Overton correlation determines bio-membrane transport of hydrophobic molecules. This so called “Overton rule” says that the easier it is for a chemical to dissolve in a lipid the easier and faster it will be transported into a cell. In medical science for example, this passive transport is crucial for the effective delivery of many pharmaceutical agents to intracellular targets. The prediction also concerns CO2 as a hydrophobic molecule. Certainly, the membrane diffusion of CO2 is of critical importance for bacteria, animal- and plant cells. In contrast to many other organisms, plants require CO2 and its availability at the site of CO2 fixation limits the rate of net photosynthesis. In this regard, plants provide an excellent system to study CO2 transport mechanisms. Findings will be presented, which question the applicability of Overton's rule to specific plant CO2 transport processes. It could be demonstrated that the function of specific membrane proteins, i.e. distinct aquaporins, increased CO2 transport rates. The experiments were performed on synthetic membranes and cell based systems as well as plant tissues and complete plants. Techniques from biophysics, cell biology, molecular biology and plant physiology were employed and it was found that in the analyzed systems CO2 transport rates were limited by the function of these aquaporins. The results could be interpreted in a way that supports alternative cellular CO2 transport mechanisms and a modified model of cellular surfaces. If the findings were of general validity and not specific for plants, our view on diverse transport processes in many living organisms from all kingdoms could be modified.

The Arabidopsis aquaporin PIP1;2 rules cellular CO2 uptake

The membrane CO(2) flux into Arabidopsis mesophyll cells was studied using a scanning pH microelectrode. Arabidopsis thaliana mesophyll cells were exposed to photosynthesis-triggering light intensities, which induced cellular CO(2) uptake. Data obtained on a AtPIP1;2 T-DNA insertion line indicated that under these conditions, cellular CO(2) transport was not limited by unstirred layer effects but was dependent on the expression of the aquaporin AtPIP1;2. Complementation of the AtPIP1;2 knockout restored membrane CO(2) transport levels to that of controls. The results provide new arguments for the ongoing debate about the validity of the lipid bilayer model system and the Meyer - Overton rule for cellular gas transport. In conclusion, we suggest a modified model of molecular gas transport mechanisms in living cells.

Overton's Rule Helps To Estimate the Penetration of Anti-Infectives into Patients' Cerebrospinal Fluid

In 1900, Ernst Overton found that the entry of anilin dyes through the cell membranes of living cells depended on the lipophilicity of the dyes. The brain is surrounded by barriers consisting of lipid layers that possess several inward and outward active transport systems. In the absence of meningeal inflammation, the cerebrospinal fluid (CSF) penetration of anti-infectives in humans estimated by the ratio of the area under the concentration-time curve (AUC) in CSF (AUC(CSF)) to that in serum (AUC(CSF)/AUC(S)) correlated positively with the lipid-water partition coefficient at pH 7.0 (log D) (Spearman's rank correlation coefficient r(S) = 0.40; P = 0.01) and negatively with the molecular mass (MM) (r(S) = -0.33; P = 0.04). The ratio of AUC(CSF) to the AUC of the fraction in serum that was not bound (AUC(CSF)/AUC(S,free)) strongly correlated with log D (r(S) = 0.67; P < 0.0001). In the presence of meningeal inflammation, AUC(CSF)/AUC(S) also correlated positively with log D (r(S) = 0.46; P = 0.002) and negatively with the MM (r(S) = -0.37; P = 0.01). The correlation of AUC(CSF)/AUC(S,free) with log D (r(S) = 0.66; P < 0.0001) was as strong as in the absence of meningeal inflammation. Despite these clear correlations, Overton's rule was able to explain only part of the differences in CSF penetration of the individual compounds. The site of CSF withdrawal (lumbar versus ventricular CSF), age of the patients, underlying diseases, active transport, and alterations in the pharmacokinetics by comedications also appeared to strongly influence the CSF penetration of the drugs studied.

Imaging Molecular Transport across Lipid Bilayers

Low-molecular-weight carboxylic acids have many properties common to small molecule drugs. The transport of these acids across cell membranes has been widely studied, but these studies have produced wildly varying permeability values. Recent reports have even claimed that the transport behavior of these drugs is contrary to the rule of thumb called Overton's rule, which holds that more lipophilic molecules transport across lipid membranes more quickly. We used confocal microscopy to image the transport of carboxylic acids with different lipophilicities into a giant unilamellar lipid vesicle (GUV). Fluorescein-dextran, which acts as a pH-sensitive dye, was encapsulated in the GUV to trace the transport of acid. The GUV was immobilized on the surface of a microfluidic channel by biotin-avidin binding. This microchannel allows the rapid and uniform exchange of the solution surrounding the GUV. Using a spinning-disk confocal microscope, the entire concentration field is captured in a short (<100 ms) exposure. Results show that more lipophilic acids cross the bilayer more quickly. A finite difference model was developed to simulate the experimental process and derive permeabilities. The permeabilities change with the same trend as literature oil-water partition coefficients, demonstrating that Overton's rule applies to this class of molecules.

Confocal imaging to quantify passive transport across biomimetic lipid membranes.

The ability of a molecule to pass through the plasma membrane without the aid of any active cellular mechanisms is central to that molecule's pharmaceutical characteristics. Passive transport has been understood in the context of Overton's rule, which states that more lipophilic molecules cross membrane lipid bilayers more readily. Existing techniques for measuring passive transport lack reproducibility and are hampered by the presence of an unstirred layer (USL) that dominates transport across the bilayer. This report describes assays based on spinning-disk confocal microscopy (SDCM) of giant unilamellar vesicles (GUVs) that allow for the detailed investigation of passive transport processes and mechanisms. This approach allows the concentration field to be directly observed, allowing membrane permeability to be determined easily from the transient concentration profile data. A series of molecules of increasing hydrophilicity was constructed, and the transport of these molecules into GUVs was observed. The observed permeability trend is consistent with Overton's rule. However, the values measured depart from the simple partition-diffusion proportionality model of passive transport. This technique is easy to implement and has great promise as an approach to measure membrane transport. It is optimally suited to precise quantitative measurements of the dependence of passive transport on membrane properties.

ON AXOPLASMIC PRESSURE WAVES AND THEIR POSSIBLE ROLE IN NERVE IMPULSE PROPAGATION

It is suggested that the propagation of the action potential is accompanied by an axoplasmic pressure pulse propagating in the axoplasm along the axon length. The pressure pulse stretch-modulates voltage-gated Na+(Nav) channels embedded in the axon membrane, causing their accelerated activation and inactivation and increasing peak channel conductance. As a result, the action potential propagates due to mechano-electrical activation of Nav channels by straggling ionic currents and the axoplasmic pressure pulse. The velocity of such propagation is higher than in the classical purely electrical Nav activation mechanism, and it may be close to the velocity of propagation of pressure pulses in the axoplasm. Extracellular Ca2+ions influxing during the voltage spike, or Ca2+ions released from intracellular stores, may trigger a mechanism that generates and augments the pressure pulse, thus opposing its viscous decay. The model can potentially explain a number of phenomena that are not contained within the purely electrical Hodgkin–Huxley-type framework: the Meyer–Overton rule for the effectiveness of anesthetics, as well as various mechanical, optical and thermodynamic phenomena accompanying the action potential. It is shown that the velocity of propagation of axoplasmic pressure pulses is close to the measured velocity of the nerve impulse, both in absolute magnitude and in dependence on axon diameter, degree of myelination and temperature.

Membrane Transport of Hydrogen Sulfide: No Facilitator Required

Hydrogen sulfide (H2S) has emerged as a new and important member in the group of gaseous signalling molecules. However, the molecular transport mechanism has not yet been identified. Prediction of its actual membrane permeability, PM, according to Overton's rule (1) is hampered by the fact that the partition coefficient into the organic phase is not known. Because of structural similarities with H2O, it was hypothesized that aquaporins may facilitate H2S transport across cell membranes. We tested this hypothesis by reconstituting the archeal aquaporin AfAQP from sulfide reducing bacteria Archaeoglobus fulgidus into planar membranes and by monitoring the resulting facilitation of osmotic water flow and H2S flux. To measure H2O and H2S fluxes, respectively, sodium ion dilution and buffer acidification by proton release were recorded in the immediate membrane vicinity. Both [Na+] and pH were measured by scanning ion selective microelectrodes. A lower limit of PM,H2S > 0.5 ± 0.4 cm/s was calculated by numerically solving the complete system of differential reaction diffusion equations and fitting the theoretical pH distribution to experimental pH profiles. Even though reconstitution of AfAQP significantly increased water permeability through planar lipid bilayers, PM,H2S remained unchanged. The fact that cholesterol and sphingomyelin reconstitution did not turn these membranes into a H2S barrier indicates that H2S transport through epithelial barriers, endothelial barriers and membrane rafts also occurs by simple diffusion and does not require facilitation by membrane channels (2).1. Missner, A & Pohl, P (2009) 110 years of the Meyer-Overton rule: Predicting membrane permeability of gases and other small compounds ChemPhysChem 10:1405-1414.2.Mathai, JC, Missner, A, Kugler, P, Saparov, SM, Zeidel, ML, Lee, JK, Pohl, P (2009) No facilitator required for membrane transport of hydrogen sulfide Proc.Natl. Acad. Sci. USA 106:16633-16638.

Permeation of Membranes by Ribose and Its Diastereomers

It was recently found that ribose permeates membranes an order of magnitude faster than its diastereomers arabinose and xylose (Sacerdote, M. G.; Szostak, J. W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6004). On this basis it was hypothesized that differences in membrane permeability to aldopentoses provide a mechanism for preferential delivery of ribose to primitive cells for subsequent selective incorporation into nucleotides and their polymers. However, the origins of these unusually large differences have not been well understood. We address this issue in molecular dynamics simulations combined with free energy calculations. It is found that the free energy of transferring ribose from water to the bilayer is lower by 1.5-2 kcal/mol than the barrier for transferring the other two aldopentoses. The calculated and measured permeability coefficients are in excellent agreement. The sugar structures that permeate the membrane are beta-pyranoses, with a possible contribution of the alpha-anomer for arabinose. The furanoid form of ribose is not substantially involved in permeation, even though it is non-negligibly populated in aqueous solution. The differences in free energy of transfer between ribose and arabinose or xylose are attributed, at least in part, to stronger highly cooperative, intramolecular interactions between consecutive exocyclic hydroxyl groups, which are stable in nonpolar media but rare in water. Water/hexadecane partition coefficients of the sugars obtained from separate molecular dynamics simulations correlate with the calculated permeability coefficients, in qualitative agreement with the Overton rule. The relevance of our calculations to understanding the origins of life is discussed.

110 years of the Meyer-Overton rule: predicting membrane permeability of gases and other small compounds.

The transport of gaseous compounds across biological membranes is essential in all forms of life. Although it was generally accepted that gases freely penetrate the lipid matrix of biological membranes, a number of studies challenged this doctrine as they found biological membranes to have extremely low gas-permeability values. These observations led to the identification of several membrane-embedded "gas" channels, which facilitate the transport of biological active gases, such as carbon dioxide, nitric oxide, and ammonia. However, some of these findings are in contrast to the well-established solubility-diffusion model (also known as the Meyer-Overton rule), which predicts membrane permeabilities from the molecule's oil-water partition coefficient. Herein, we discuss recently reported violations of the Meyer-Overton rule for small molecules, including carboxylic acids and gases, and show that Meyer and Overton continue to rule.

Membrane Transport of Singlet Oxygen Monitored by Dipole Potential Measurements

The efficiency of photodynamic reactions depends on 1), the penetration depth of the photosensitizer into the membrane and 2), the sidedness of the target. Molecules which are susceptible to singlet oxygen ( 1O 2) experience less damage when separated from the photosensitizer by the membrane. Since 1O 2 lifetime in the membrane environment is orders of magnitude longer than the time required for nonexcited oxygen (O 2) to cross the membrane, this observation suggests that differences between the permeabilities or membrane partition of 1O 2 and O 2 exist. We investigated this hypothesis by releasing 1O 2 at one side of a planar membrane while monitoring the kinetics of target damage at the opposite side of the same membrane. Damage to the target, represented by dipole-modifying molecules (phloretin or phlorizin), was indicated by changes in the interleaflet dipole potential difference Δ φ b . A simple analytical model allowed estimation of the 1O 2 interleaflet concentration difference from the rate at which Δ φ b changed. It confirmed that the lower limit of 1O 2 permeability is ∼2 cm/s; i.e., it roughly matches O 2 permeability as predicted by Overton's rule. Consequently, the membrane cannot act as a barrier to 1O 2 diffusion. Differences in the reaction rates at the cytoplasmic and extracellular membrane leaflets may be attributed only to 1O 2 quenchers inside the membrane.

Passive transport across bilayer lipid membranes: Overton continues to rule

According to Overton's rule, membrane permeability (PM) of a molecule increases with its hydrophobicity. Experiments with a series of carboxylic acids now suggest the opposite: the most hydrophilic acid exhibited the highest PM (1).

Quantitative visualization of passive transport across bilayer lipid membranes

The ability to predict and interpret membrane permeation coefficients is of critical importance, particularly because passive transport is crucial for the effective delivery of many pharmaceutical agents to intracellular targets. We present a method for the quantitative measurement of the permeation coefficients of protonophores by using laser confocal scanning microscopy coupled to microelectrochemistry, which is amenable to precise modeling with the finite element method. The technique delivers well defined and high mass transport rates and allows rapid visualization of the entire pH distribution on both the cis and trans side of model bilayer lipid membranes (BLMs). A homologous series of carboxylic acids was investigated as probe molecules for BLMs composed of soybean phosphatidylcholine. Significantly, the permeation coefficient decreased with acyl tail length contrary to previous work and to Overton's rule. The reasons for this difference are considered, and we suggest that the applicability of Overton's rule requires re-evaluation.

Overton's rule overturned

Overton's rule overturned