Monolayer and interfacial permeation

Transport across physical-chemical interfaces is considered in connection with three particular problems of biological interfaces: the structure and properties of cell membranes, the properties of the lung surfactant, and the effects of ionic currents across excitable membranes. With regard to cell membranes, studies of monolayer permeation suggest that permselectivity on the basis of size is a property of bilayer structure and probably gives rise to the observed dependence of the permeability on partition coefficients. The permeabilities of lipid and protein monolayers are consistent with the bimolecular leaflet (BML) model of the membrane and not with mosaic models. Experiments with the lung surfactant indicate that, in addition to its surface tension-lowering properties, it is unusual in its ability to form a strong two-dimensional network, which probably contributes to alveolar stability. Finally, the results of studies of interfacial ionic transference suggest a new way of accounting for the ionic fluxes in excitable membranes during an action potential without assuming ion-selective pores or carriers. In the suggested mechanism, it is possible to account for the change in ionic selectivity and the proper phasing of the fluxes, as well as other aspects of excitation in natural membranes.     

Mechanism of the electric response of lipid bilayers to bitter substances.

In order to clarify by what mechanism the lipid bilayer membrane changes its potential under the stimulation of bitter substances, a microscopic model for the effects of the substances on the membrane is presented and studied theoretically. It is assumed that the substances are adsorbed on the membrane and change the partition coefficients of ions between the membrane and the stimulation solution, the dipole orientation in the polar head, and the diffusion constants of ions in the membrane. It is shown, based on the comparison of the calculated results with the experimental ones, that the response arises mainly from a change in the partition coefficients. Protons play an essential role in the membrane potential variation due to the change in their partition coefficients. The present model reproduces the following observed unique properties in the response of lipid bilayers to bitter substances, which cannot be accounted for by the usual channel model for the membrane potential: 1) the response of the membrane potential appears even under the condition that there is no ion gradient across the membrane, 2) the response remains even when the salt in the stimulating solution is replaced with the salt made of an impermeable cation, and 3) the direction of the polarization of the potential is not reversed, even when the ion gradient across the bilayer is reversed.     

Mechanism of anion-cation selectivity of amphotericin B channels

Summary Zero current potential and conductance of ionic channels formed by polyene antibiotic amphotericin B in a lipid bilayer were studied in various electrolyte solutions. Nonpermeant magnesium and sulphate ions were used to independently vary the concentration of monovalent anions and cations as well as to maintain the high ionic strength of the two solutions separated by the membrane. Under certain conditions the channels select very strongly for anions over cations. They are permeable to small inorganic anions. However, in the absence of these anions the channels are practically impermeable to any cation. In the presence of a permeant anion the contribution of monovalent cations to channel conductance grows with an increase in the anion concentration. The ratio of cation-to-anion permeability coefficients is independent of the membrane potential and cation concentration, but it does depend linearly on the sum of concentrations of a permeant anion in the two solutions. These results are accounted for on the assumption that a cation can enter only an anion-occupied channel to form an ionic pair at the center of the channel. The cation is also assumed to slip past the anion and then to leave the channel for the opposite solution. This model with only few parameters can quantitatively describe the concentration dependences of conductance and zero current potential under various conditions.     

Subfractionation of cell populations by partitioning in dextran-poly (ethylene glycol) aqueous phases

Partitioning of cells in dextran-poly(ethylene glycol) aqueous two-phase systems depends on the interaction between the surface properties of the cells and the physical properties of the phases. The latter can be manipulated to a considerable extent by selection of polymer concentrations and ionic composition and concentration. If salts (e.g., phopshate) are used that have an unequal affinity for the two phases, an electrostatic potential difference between the phases results and, at appropriately high polymer concentrations, the partition coefficient of cells is determined predominantly by membrane charge-associated properties. By “balancing” the magnitude of the electrostatic potential difference against that of the interfacial tension (primarily a function of polymer, but also phosphate, concentrations) one can obtain phase systems that give usable partition coefficients for most cell populations (1). In work under way in our laboratory on the effects of different chemical and enzymatic modifications on the relative surface properties of rat red blood cells of different ages, we have now found that certain phase compositions did not resolve such treated cell subpopulations while other phase compositions did. Thus not all charged phase systems in which cell populations as a whole have usable partition coefficients are equally capable of detecting or subfractionating cell subpopulations. It is therefore essential, before drawing conclusions on the nonseqarability of cell subpopulations, to test cell separability in charged phase systems of different compositions if the system initially chosen does not afford a subfractionation.     

Urinary proteases degrade epithelial sodium channels

Summary The mammalian urinary bladder epithelium accommodates volume changes by the insertion and withdrawal of cytoplasmic vesicles. Both apical membrane (which is entirely composed of fused vesicles) and the cytoplasmic vesicles contain three types of ionic conductances, one amiloride sensitive, an-other a cation-selective conductance and the third a cation conductance which seems to partition between the apical membrane and the mucosal solution. The transport properties of the apical membrane (which has been exposed to urine in vivo) differ from the cytoplasmic vesicles by possessing a lower density of amiloride-sensitive channels and a variable level of leak conductance. It was previously shown that glandular kallikrein was able to hydrolyze epithelial sodium channels into the leak conductance and that this leak conductance was further degraded into a channel which partitioned between the apical membrane and the mucosal solution. This report investigates whether kallikrein is the only urinary constituent capable of altering the apical membrane ionic permeability or whether other proteases or ionic conditions also irreversible modify apical membrane permeability.Alterations of mucosal pH, urea concentrations, calcium concentrations or osmolarity did not irreversible affect the apical membrane ionic conductances. However, urokinase and plasmin (both serine proteases found in mammalian urine) were found to cause an irreversible loss of amiloride-sensitive current, a variable change in the leak current as well as the appearance of a third conductance which was unstable in the apical membrane and appears to partition between the apical membrane and the mucosal solution. Amiloride protects the amiloride-sensitive conductance from hydrolysis but does not protect the leak pathway. Neither channel is protected by sodium. Fluctuation analysis demonstrated that the loss of amiloride-sensitive current was due to a decrease in the sodium-channel density and not a change in the single-channel current. Assuming a simple model of sequential degradation, estimates of single-channel currents and conductances for both the leak channel and unstable leak channel are determined.     

Partition of amphiphilic molecules into phospholipid vesicles and human erythrocyte ghosts: measurements by ultraviolet difference spectroscopy

Molar partition coefficients for chlorpromazine and methochlorpromazine between phospholipid vesicles or human erythrocyte ghosts and buffer are determined by ultraviolet difference spectroscopy. The partition coefficients between small unilamellar egg phosphatidylcholine vesicles and buffer at pH 7.4 are 4.4 X 10(5) for chlorpromazine and 0.8 X 10(5) for methochlorpromazine, determined with 10 microM amphiphile. An increase in the partition of chlorpromazine into vesicles is seen as the pH is increased to the pKa of chlorpromazine at 9.2. Chlorpromazine also partitions preferentially into fluid-phase phospholipid compared to solid-phase phospholipid. Molar partition coefficients between unsealed human erythrocyte ghosts and buffer at pH 8.0 with 10 microM amphiphile are determined to be 6.5 X 10(5) for chlorpromazine and 2.5 X 10(5) for methochlorpromazine. Difference spectroscopy is an equilibrium technique that does not require separation of bound from free amphiphile, as do many other methods of determining membrane-buffer partition coefficients. This method is useful for any amphiphile that has an appreciable absorbance below its critical micelle concentration and whose absorbance is sensitive to environment.     

Statistical thermodynamic analysis of peptide and protein insertion into lipid membranes.

A statistical thermodynamic approach is used to analyze the various contributions to the free energy change associated with the insertion of proteins and protein fragments into lipid bilayers. The partition coefficient that determines the equilibrium distribution of proteins between the membrane and the solution is expressed as the ratio between the partition functions of the protein in the two phases. It is shown that when all of the relevant degrees of freedom (i.e., those that change their character upon insertion into the membrane) can be treated classically, the partition coefficient is fully determined by the ratio of the configurational integrals and thus does not involve any mass-dependent factors, a conclusion that is also valid for related processes such as protein adsorption on a membrane surface or substrate binding to proteins. The partition coefficient, and hence the transfer free energy, depend only on the potential energy of the protein in the membrane. Expressing this potential as a sum of a "static" term, corresponding to the equilibrium (minimal free energy) configuration of the protein in the membrane, and a "dynamical" term representing fluctuations around the equilibrium configuration, we show that the static term contains the "solvation" and "lipid perturbation" contributions to the transfer free energy. The dynamical term is responsible for the "immobilization" free energy, reflecting the loss of translational and rotational entropy of the protein upon incorporation into the membrane. Based on a recent molecular theory of lipid-protein interactions, the lipid perturbation and immobilization contributions are then expressed in terms of the elastic deformation free energy resulting from the perturbation of the lipid environment by the foreign (protein) inclusion. The model is formulated for cylindrically shaped proteins, and numerical estimates are given for the insertion of an alpha-helical peptide into a lipid bilayer. The immobilization free energy is shown to be considerably smaller than in previous estimates of this quantity, and the origin of the difference is discussed in detail.     

Membrane Potentials at Zero Current: The Significance of a Constant Ionic Permeability Ratio

The possibility has been examined that the Goldman-Hodgkin-Katz equation for V(0), the total membrane potential at zero current, can be derived with constant permeability ratios from a thermodynamic treatment. The flux equations have been integrated under zero current conditions subject only to the restriction that the total membrane potential should be independent of internal concentration profiles, which is the requirement for the premeability ratios to be phenomenological constants, independent of solution conditions. No assumptions have been made concerning the electric potential profile. It was found that a constant permeability ratio can only be characteristic of systems satisfying certain relationships between ionic conductances and chemical potentials. From these relationships it was possible to define the permeability ratio in terms of the thermodynamic properties of the membrane quite generally and to identify the permeability ratio as the product of mobility ratio and ratio of partition coefficients. Moreover, the ionic conductance ratio at any point in the membrane has been shown to be expressable explicitly in terms of the permeability ratio and the activities of an external solution which would be in equilibrium with the point under consideration. Lastly, a number of conclusions have been reached regarding the range of applicability of the Goldman-Hodgkin-Katz equation with constant permeability ratios.     

Permeability,phase-boundary potential and conductance in a cholinergic channel without constant field.

A potassium-selective, chemically excitable channel, whose characteristics cannot be accurately described by constant-field theory, is studied by a new approach based on diffusion theory but with no need for the classical assumptions of constant field, homogeneous membrane, and equal phase-boundary potentials at both interfaces. Permeability is defined, free of these constraints, and the Goldman coefficient is demonstrated to be a special case useful only when the constraints apply. Permeability can be evaluated directly from current-voltage data, and it is found not to be a parameter in this channel, but rather a function of both the voltage and the concentration of the permeant ion. However, it becomes concentration-independent when the membrane voltage is equal to the sum of the phase-boundary potentials. That sum can therefore be determined from these data, and it is -65 mV in this channel. The permeability at that potential is a channel parameter, and equal to 8.66 X 10(-6) cm/s for this channel. A constant field is shown not to exist in this channel and the Goldman coefficient not to be a parameter but a function of potential and concentration. Although errors introduced into this coefficient by nonconstant field and unequal surface potentials partially cancel each other, the coefficient is nevertheless not a correct measure of permeability.     

Phloretin-induced changes in ion transport across lipid bilayer membranes

Phloretin, the aglucone derivative of phlorizin, increases cation conductance and decreases anion conductance in lipid bilayer membranes. In this paper we present evidence that phloretin acts almost exclusively by altering the permeability of the membrane interior and not by modifying the partition of the permanent species between the membrane and the bulk aqueous phases. We base our conclusion on an analysis of the current responses to a senylborate, and the cation complex, peptide PV-K+. These results are consistent with the hypothesis that phloretin decreases the intrinsic positive internal membrane potential but does not modify to a great extent the potential energy minima at the membrane interfaces. Phloretin increases the conductance for the nonactin-K+ complex, but above 10(-5) M the steady- state nonactin-K+ voltage-current curve changes from superlinear to sublinear. These results imply that, above 10(-5) M phloretin, the nonactin-5+ transport across the membrane becomes interfacially limited.     

A theory for the effects of neutral carriers such as the macrotetralide actin antibiotics on the electric properties of bilayer membranes

Summary To develop a quantitiative theoretical treatment for the effects of neutral macrocyclic antibiotics on the electrical properties of phospholipid bilayer membranes, this paper proceeds from the known ability of such molecules to form stoichiometric, lipid-soluble complexes with cations and deduces the electrical properties that a simple organic solvent phase would have if it were made into a membrane of the thinness of the phospholipid bilayer. In effect, we postulate that the essential barrier to ion movement across a bilayer membrane is its liquid-like hydrocarbon interior and that the neutral macrocyclic antibiotics bind monovalent cations and solubilize them in the membrane as mobile positively charged complexes. Using the Poisson-Boltzmann equation to describe the equilibrium profile of the electrical potential, it is shown that an excess of the positive complexes over all the other ions is expected in the membrane as a net space charge for appropriate conditions of membrane thickness and values of the partition coefficients of the various ionic species and without requiring the presence of fixed charges. Describing the fluxes of these complexes by the Nernst-Planck equation and neglecting the contribution to the electric current of uncomplexed ions, theoretical expressions are derived for the membrane potential in ionic mixtures, as well as for the limiting value of the membrane conductance at zero current when the membrane is interposed between identical solutions. The expressions are given in terms of the ionic activities and antibiotic concentrations in the aqueous solutions so as to be accessible to direct experimental test. Under suitable experimental conditions, the membrane potential is described by an equation recognizible as the Goldman-Hodgkin-Katz equation, in which the permeability ratios are combinations of parameters predicted from the present theory to be independently determinable from the ratio of membrane conductances in single salt solutions. Since this identity between permeability and conductance ratios is expected also for systems obeying the Independence Principle of Hodgkin and Huxley, the applicability of this principle to membranes exposed to antibiotics is discussed, and it is shown that this principle is compatible with the permeation mechanism proposed here.     

Diffusion and partition of solutes in cartilage under static load

We describe experimental apparatus, methodology and mathematical algorithms to measure diffusion and partition for typical small ionic solutes and inulin (a medium size solute) in statically loaded cartilage. The partition coefficient based on tissue water (K(H(2)O)) of Na(+) increased from 1.8 to 4.5 and for SO(4)(-2) decreased from 0.5 to 0.1, when the applied pressure was raised from zero to 22 atm K(H(2)O) of inulin decreased from 0.3 to 0.05, for an increase in pressure from zero to 11 atm. Our theoretical interpretation of the results is that the partition coefficient can be expressed as a function of fixed charge density (FCD) for both loaded and unloaded cartilage. The partition coefficient shows good agreement with the ideal Gibbs-Donnan equilibrium, particularly when FCD is based on extrafibrillar water (EFW). The diffusion coefficients, D also decreased with an increase in applied pressure; raising the pressure from 0 to 22 atm resulted in the following changes in the values of D: for Na(+) from 2.86 x 10(-6) to 1.51 x 10(-6) cm(2)/s, for SO(4)(-2) from 1.58 x 10(-6) to 7.5 x 10(-7) cm(2)/s, for leucine from 1.69 x 10(-6) to 8.30 x 10(-7) cm(2)/s and for inulin from 1.80 x 10(-7) to 3.30 x 10(-8) cm(2)/s. For the three small solutes (two charged and one neutral) the diffusion coefficient D is highly correlated with the fraction of fluid volume in the tissue. These experimental results show good agreement with the simple model of Mackie and Meares: hence solute charge does not affect the diffusion of small solutes under load. For inulin D & K show some agreement with a modified Ogston model based on two major components, viz., glycosaminoglycans (GAG) and core protein. We conclude that the changes in the partition and diffusion coefficients of small and medium size solutes in statically loaded cartilage can be interpreted as being due to the reduction in hydration and increase in FCD. The change in the latter affects the partition of small ionic solutes and the partition and diffusion of larger molecules. Our results throw light on the ionic environment of chondrocytes in loaded cartilage as well as on the transport of solutes through the matrix.     

The Excitable Membrane: A Physiochemical Model

The model of the excitable membrane assumes common channels for Na⁺ and K⁺; the two ion species interact within the pores through their electrostatic forces. The electric field varies across the membrane and with time, as a result of ionic redistribution. Ionic flow is primarily controlled by energy barriers at the two interfaces and by Ca⁺⁺ adsorption at the external interface. When the membrane is polarized, the high electric field at the external interface acting on the membrane fixed charge keeps the effective channel diameter small, so that only dihydrated ions can cross the interface. The higher energy required to partially dehydrate Na⁺ accounts for its lower permeability when polarized. Depolarized, the channel entrance can expand, permitting quadrihydrated ions to pass; the large initial Na⁺ flow is the result of the large concentration ratio across the interface. The effect at the internal interface is symmetric; Na⁺ crosses with greater difficulty when the membrane is depolarized. Na⁺ inactivation occurs when the ion distribution within the membrane has assumed its new steady-state value. Calculations based on parameters consistent with physicochemical data agree generally with a wide range of experiments. The model does not obey the two fundamental Hodgkin-Huxley (HH) postulates (independence principle, ion flow proportional to thermodynamic potential). In several instances the model predicts experimental results which are not predicted by the HH equations.     

Binding of a fluorescent dansylcadaverine-substance P analogue to negatively charged phospholipid membranes

We have investigated the binding of a new dansylcadaverine derivative of substance P (DNC-SP) with negatively charged small unilamellar vesicles composed of a mixture of phosphatidylcholine (PC) and either phosphatidylglycerol (PG) or phosphatidylserine (PS) using fluorescence spectroscopic techniques. The changes in fluorescence properties were used to obtain association isotherms at variable membrane negative charges and at different ionic strengths. The experimental association isotherms were analyzed using two binding approaches: (i) the Langmuir adsorption isotherm and the partition equilibrium model, that neglect the activity coefficients; and (ii) the partition equilibrium model combined with the Gouy-Chapman formalism that considers electrostatic effects. A consistent quantitative analysis of each DNC-SP binding curve at different lipid composition was achieved by means of the Gouy-Chapman approach using a peptide effective interfacial charge (v) value of (0.95 +/- 0.02), which is lower than the physical charge of the peptide. For PC/PG membranes, the partition equilibrium constant were 7.8 x 10(3) M(-1) (9/1, mol/mol) and 6.9 x 10(3) M(-1) (7/3, mol/mol), whereas for PC/PS membranes an average value of 6.8 x 10(3) M(-1) was estimated. These partition equilibrium constants were similar to those obtained for the interaction of DNC-SP with neutral PC membranes (4.9 x 10(3) M(-1)), as theoretically expected. We demonstrate that the v parameter is a determinant factor to obtain a unique value of the binding constant independently of the surface charge density of the vesicles. Also, the potential of fluorescent dansylated SP analogue in studies involving interactions with cell membranes is discussed.     

Evaluation of the lipophilic properties of opioids, amphetamine-like drugs, and metabolites through electrochemical studies at the interface between two immiscible solutions

For the first time, the partition coefficients of the ionized forms of several opioids, amphetamine-like drugs, and their metabolites were determined by studying their ionic transfer process across the bare interface water/organic solvent. The ionic partition coefficients of the monocationic forms of 12 compounds--heroin, 6-monoacetylmorphine (6-MAM), morphine, acetylcodeine, codeine, dihydrocodeine, methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy"), 3,4-methylenedioxyamphetamine (MDA), 3-methoxy-alpha-methyldopamine (3-OMe-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA)-were attained using electrochemical measurements, by cyclic voltammetry, at the interface between two immiscible electrolyte solutions (ITIES). Then the acquired lipophilicity values were correlated to the chemical structure of the compounds and with the metabolic pathways central to each class of drugs. Although the mechanisms of biotoxicity of this type of drugs are still unclear, the data obtained evidence that the lipophilicity of metabolites may be a contributing factor for the qualitative differences found in their activity. In addition, the partition coefficients of the ionic drugs were calculated using three available software packages: ModesLab, Dragon, and HyperChem. As shown by cross-comparison of the experimental and calculated values, HyperChem was the most reliable software for achieving the main goal. The data obtained so far seem to be correlated to the proposed metabolic pathways of the drugs and could be of great value in understanding their pharmacological and/or toxicological profiles at the molecular level. This study may also contribute to gaining an insight into the mechanisms of biotransportation of this type of compounds given that the ionic partition coefficients reflect their ability to cross the membrane barriers.     

Brownian dynamics study of a multiply-occupied cation channel: application to understanding permeation in potassium channels.

The behavior of a multiply-occupied cation-selective channel has been computed by Brownian dynamics. The length, cross-section, ion-ion repulsion force, and ionic mobility within the channel are all estimated from data and physical reasoning. The only free parameter is a partition energy at the mouth of the channel, defining the free energy of an ion in the channel compared to the bath. It is presumed that this partition energy is associated with the energetics of exchanging a bulk hydration environment for a channel hydration environment. Varying the partition energy alone, keeping all other parameters fixed, gives approximately the full range of magnitudes of single channel conductances seen experimentally for K channels. Setting the partition energy at -11 kT makes the computed channel look similar to a squid axon K channel with respect to magnitude of conductance, shape of the I-V curve, non-unity of Ussing flux ratio exponents, decrease of current and increase of conductance with extracellular ion accumulation, and saturation at high ion concentration in the bathing solution. The model includes no preferred binding sites (local free energy minima) for ions in the channel. Therefore it follows that none of the above-mentioned properties of K channels are strong evidence for the existence of such sites. The model does not show supersaturation of current at very high bathing concentrations nor any pronounced voltage-dependence of the Ussing flux ratio exponent, suggesting that these features would require additional details not included in the model presented herein.     

Diffusion and partitioning of proteins in charged agarose gels.

The effects of electrostatic interactions on the diffusion and equilibrium partitioning of fluorescein-labeled proteins in charged gels were examined using fluorescence recovery after photobleaching and gel chromatography, respectively. Measurements were made with BSA, ovalbumin, and lactalbumin in SP-Sepharose (6% sulfated agarose), in phosphate buffers at pH 7 and ionic strengths ranging from 0.01 to 1.0 M. Diffusivities in individual gel beads (D) and in the adjacent bulk solution (D infinity) were determined from the spatial Fourier transform of the digitized two-dimensional fluorescence recovery images. Equilibrium partition coefficients (phi) were measured by recirculating protein solutions through a gel chromatography column until equilibrium was reached, and using a mass balance. Diffusion in the gel beads was hindered noticeably, with D/D infinity = 0.4-0.5 in each case. There were no effects of ionic strength on BSA diffusivities, but with the smaller proteins (ovalbumin and lactalbumin) D infinity increased slightly and D decreased at the lowest ionic strength. In contrast to the modest changes in diffusivity, there were marked effects of ionic strength on the partition coefficients of these proteins. We conclude that for diffusion of globular proteins through gel membranes of like charge, electrostatic effects on the effective diffusivity (Deff = phi D) are likely to result primarily from variations in phi with only small contributions from the intramembrane diffusivity.     

How pore mouth charge distributions alter the permeability of transmembrane ionic channels.

This paper investigates the effects that surface dipole layers and surface charge layers along the pore mouth-water interface can have on the electrical properties of a transmembrane channel. Three specific molecular sources are considered: dipole layers formed by membrane phospholipids, dipole layers lining the mouth of a channel-forming protein, and charged groups in the mouth of a channel-forming protein. We find, consistent with previous work, that changing the lipid-water potential difference only influences channel conduction if the rate-limiting step takes place well inside the channel constriction. We find that either mouth dipoles or mouth charges can act as powerful ion attractors increasing either cation or anion concentration near the channel entrance to many times its bulk value, especially at low ionic strengths. The effects are sufficient to reconcile the apparently contradictory properties of high selectivity and high conductivity, observed for a number of K+ channel systems. We find that localizing the electrical sources closer to the constriction entrance substantially increases their effectiveness as ion attractors; this phenomenon is especially marked for dipolar distributions. An approximate treatment of electrolyte shielding is used to discriminate between the various mechanisms for increasing ionic concentration near the constriction entrance. Dipolar potentials are far less sensitive to ionic strength variation than potentials due to fixed charges. We suggest that the K+ channel from sarcoplasmic reticulum does not have a fixed negative charge near the constriction entrance; we suggest further that the Ca+2-activated K+ channel from transverse tubule does have such a charge.     

The kinetic mechanism of action of an uncoupler of oxidative phosphorylation

Summary The chemiosmotic hypothesis predicts that the mechanism by which weak acids uncouple oxidative phosphorylation in mitochondria is identical to the mechanism by which they transport hydrogen ions across artificial bilayer membranes. We report here the results of a kinetic study of uncoupler-mediated hydrogen ion transport across bilayer membranes. We made electrical relaxation measurements on black lipid membranes exposed to the substituted benzimidazole 5,6-dichloro-2-trifluoromethylbenzimidazole. The simplest model consistent with our experimental data allowed us to deduce values for adsorption coefficients and rate constants. Our major conclusions are that the back diffusion of the neutral species is the rate limiting step for the steady state transport of hydrogen ions, that both the neutral and charged forms of the uncoupler adsorb strongly to the interfaces, and that the reactions at the membrane-solution interfaces occur sufficiently rapidly for equilibrium to be maintained. Independent measurements of the adsorption coefficients of both the neutral and anionic forms of the weak acid and also of the permeability of the membrane to the neutral form agreed well with the values deduced from the kinetic study.