Probes of the conduction process of a voltage-gated Cl- channel from Torpedo electroplax

The open-channel conductance properties of a voltage-gated Cl- channel derived from Torpedo californica electroplax and incorporated into planar bilayers were studied by several approaches. In neutral bilayers the channel conductance saturates with Cl- activity according to a rectangular hyperbolic relation with a half-saturation activity of 75 mM and a maximum conductance of 32 pmho. The observation of identical behavior in charged membranes implies that ions permeating the channel do not sense the surface potential of the bulk membrane. The Cl-:Br- permeability ratio, measured under biionic conditions, is independent of salt concentration. SCN- ion reversibly blocks the channel. The voltage dependence of the block implies the existence of two separate blocking sites within the channel: one accessible from the cis side only (the side to which vesicles are added) and the other accessible from the trans side only. The block at each site is competitive with Cl-. The results are consistent with a single-ion Eyring model of the conduction process in which the ion must traverse three kinetic barriers as it permeates the channel and in which the channel can accommodate at most one ion at a time.     

Use of weak acids to determine the bulk diffusion limitation of H+ ion conductance through the gramicidin channel.

The addition of 2 M formic acid at pH 3.75 increased the single channel H+ ion conductance of gramicidin channels 12-fold at 200 mV. Other weak acids (acetic, lactic, oxalic) produce a similar, but smaller increase. Formic acid (and other weak acids) also blocks the K+ conductance at pH 3.75, but not at pH 6.0 when the anion form predominates. This increased H+ conductance and K+ block can be explained by formic acid (HF) binding to the mouth of the gramicidin channel (Km = 1 M) and providing a source of H+ ions. A kinetic model is derived, based on the equilibrium binding of formic acid to the channel mouth, that quantitatively predicts the conductance for different mixtures of H+, K+, and formic acid. The binding of the neutral formic acid to the mouth of the gramicidin channel is directly supported by the observation that a neutral molecule with a similar structure, formamide (and malonamide and acrylamide), blocks the K+ conductance at pH 6.0. The H+ conductance in the presence of formic acid provides a lower bound for the intrinsic conductance of the gramicidin channel when there is no diffusion limitation at the channel mouth. The 12-fold increase in conductance produced by formic acid suggests that greater than 90% of the total resistance to H+ results from diffusion limitation in the bulk solution.     

Ion conduction through MscS as determined by electrophysiology and simulation

The mechanosensitive channel of small conductance (MscS) is a membrane protein thought to act as a safety valve in bacteria, regulating the release of ions and small solutes when gated by membrane tension under challenging osmotic conditions. The influence of voltage on channel activation and the functional state depicted by the available crystal structure of MscS remain debated. Therefore, in an effort to relate electrophysiological measurements on MscS and properties of the MscS crystal conformation, we report here MscS's response to voltage and pressure as determined by patch-clamp experiments, as well as MscS electrostatics and transport properties as determined through all-atom molecular dynamics simulations of the protein embedded in a lipid bilayer, a 224,000-atom system. The experiments reveal that MscS is a slightly anion-selective channel with a conductance of approximately 1 ns, activated by pressure and inactivated in a voltage-dependent manner. On the other hand, the simulations, covering over 200 ns and including biasing electrostatic potentials, show that MscS restrained to the crystal conformation exhibits low conductance; unrestrained it increases the channel radius upon application of a large electrostatic bias and exhibits then ion conduction that matches experimentally determined conductances. The simulated conductance stems mainly from Cl- ions.     

Schistosoma mansoni: patch-clamp study of a nonselective cation channel in the outer tegumental membrane of females.

An apparent ion channel with a conductance of 295 pS is present in isolated inside-out patches of outer tegumental membrane taken from female Schistosoma mansoni. With positive voltages applied to the intracellular face of the patch, percentage open time for the channel was 0 to 50; with negative voltages applied, percentage open time was greater than 99. Step changes in applied voltage characteristically induced opening-closing activity. However, there was no maintained applied voltage at which there was a high level of sustained opening-closing activity. The 295 pS conductance was by far the most commonly occurring conductance but it appears to result from cooperativity among several channels, the unitary conductance for the channel averaging 95 pS. Alterations in the Na+ or K+ concentration ratios changed the reversal potential for this conductance but alterations in the Cl- concentration did not. From this it is concluded that this channel is selective for Na+ or K+ over Cl- and it appears to be a nonselective cation channel.     

Meningococcal PorA/C1, a channel that combines high conductance and high selectivity.

Class 1 porins (PorA/C1) from Neisseria meningitidis achieve both high selectivity and high conductance. The channel is highly selective (24:1 Na+ over Cl-), suggesting a highly negatively charged selectivity filter. The trimeric nature of PorA/C1 accounts for part of the enormous conductance in 200 mM NaCl (0.97nS). However, the currents that can be achieved exceed the simple infinite-sink calculation for a pore 0.7 nm in radius (estimated from nonelectrolyte permeability). The conductance is linear with salt activity from 20 mM to 2.0 M NaCl with no sign of saturation at low salt. Impermeant polymers reduce the conductance in a manner consistent with their ability to reduce bulk conductivity. Extrapolating from the known structure of homologous porins, the selectivity filter is likely to be small and localized. If small and highly negatively charged ( approximately 9 charges), the predicted conductance would be an order of magnitude higher than that observed. The rate at which ions reach the selectivity filter seems to limit overall ionic flux. PorA/C1 rectifies strongly, and this rectification can be accounted for by calculated differences in the voltage and concentration profiles in the access regions. Thus, it appears that the conductance of this channel is determined by the access resistance and the selectivity by a highly-conductive filter.     

Ionic selectivity, saturation, and block in a K+-selective channel from sarcoplasmic reticulum

The open-channel conductance properties of a voltage-gated channel from sarcoplasmic reticulum were studied in planar phospholipid membranes. The channel is ideally selective for K+ over Cl- and for K+ over Ca++. In symmetrical 1 M solutions, the single-channel conductance (in pmho) falls in the order: K+ (214) > NH4+ (157) > Rb+ (125) > Na+ (72) > La+ (8.1) > Cs+ (< 3). In neutral bilayers, the channel conductance saturates with ion activity according to a rectangular hyperbolic relation, with half-saturation activities of 54 mM for K+ and 34 mM for Na+. Under symmetrical salt conditions, the K+:Na+ channel conductance ratio increases with salt activity, but the permeability ratio, measured by single-channel bi-ionic potentials, is constant between 20 mM and 2.5 M salt; the permeability ratio is equal to the conductance ratio in the limit of low-salt concentration. The channel conductance varies < 5% in the voltage range -100 to +70 mV. The maximum conductance varies K+ and Na+ is only weakly temperature dependent (delta H++ = 4.6 and 5.3 kcal/mol, respectively), but that of Li+ varies strongly with temperature (delta H++ = 13 kcal/mol). The channel's K+ conductance is blocked asymmetrically by Cs+, and this block is competitive with K+. The results are consistent with an Eyring-type barriers as it permeates the channel. The data conform to Lüger's (1973. Biochem. Biophys. Acta. 311:423-441) predictions for a "pure" single-ion channel.     

Blue light modulation of ion transport in the slime mutant of Neurospora crassa

Blue light is the primary entrainment signal for a number of developmental and morphological processes in the lower eucaryote Neurospora crassa. Blue light regulates photoactivation of carotenoid synthesis, conidiation, phototropism of perithecia and circadian rhythms. Changes in the electrical properties of the plasma membrane are one of the fastest responses to blue light irradiation. To enable patch-clamp studies on light-induced ion channel activity, the wall-less slime mutant was used. Patch-clamp experiments were complemented by non-invasive ion-selective measurements of light-induced ion fluxes of slime cells using the vibrating probe technique. Blue light usually caused a decrease in conductance within 2-5 minutes at both negative and positive voltages, and a negative shift in the reversal potential in whole-cell patch-clamp measurements. Both K+ and Cl- channels contribute to the inward and outward currents, based on the effects of TEA (10 mM) and DIDS (500 microM). However, the negative shift in the reversal potential indicates that under blue light the Cl- conductance becomes dominant in the electrical properties of the slime cells due to a decrease of K+ conductance. The ion-selective probe revealed that blue light induced the following changes in the net ion fluxes within 5 minutes: 1) decrease in H+ influx; 2) increase in K+ efflux; and 3) increase in Cl- influx. Ca2+ flux was unchanged. Therefore, blue light regulates an ensemble of transport processes: H+, Cl-, and K+ transport.     

Exact continuum solution for a channel that can be occupied by two ions

The classical Nernst-Planck continuum equation is extended to the case where the channel can be occupied simultaneously by two ions. A two-dimensional partial differential equation is derived to describe the steady-state channel. This differential equation is of the form of the generalized Laplace equation, but it has the novel feature that the boundary conditions are periodic. The finite difference solution takes approximately 8 s on a large computer. The equations are solved for the special case of a cylindrical channel with a fixed charge in the center. It is assumed that the forces on the ions result entirely from the sum of the Born image potential, the fixed charge potential, the interaction potential between the two ions, and the applied voltage. Approximate simple analytical expressions are derived for these potential terms, based on the assumption that the electric field perpendicular to the channel wall is zero. The potentials include the contribution from a diffuse charge (Debye-Huckel) reaction field in the bulk solution for the monovalent cation flux was obtained for channels with a radius of 4 A and lengths of 16 and 32 A and a fixed charge valence of -1 and -1.5. For these channels, a significant fraction (up to 90%) of the total resistance is contributed by the bulk solution and results were obtained for the case where the "channel" included 8 A of bulk solution at each channel end. These results for the two-ion channel were compared with the analytical solution for a one-ion channel. The one-ion channel is a fair approximation to the two-ion channel for a fixed charge of -1, underestimating the flux at high concentrations by approximately 30%. However, for a fixed charge of -1.5, the one-ion model is a poor approximation, with the two-ion flux about seven times that of the one-ion model at high concentrations. The absolute conductance and concentration dependence of these channels (with a fixed charge of -1) mimic the behavior of the large conductance K+ channel and the acetylcholine receptor channel.     

Proton pathways and H+/Cl- stoichiometry in bacterial chloride transporters

H+/Cl- antiport behavior has recently been observed in bacterial chloride channel homologs and eukaryotic CLC-family proteins. The detailed molecular-level mechanism driving the stoichiometric exchange is unknown. In the bacterial structure, experiments and modeling studies have identified two acidic residues, E148 and E203, as key sites along the proton pathway. The E148 residue is a major component of the fast gate, and it occupies a site crucial for both H+ and Cl- transport. E203 is located on the intracellular side of the protein; it is vital for H+, but not Cl-, transport. This suggests two independent ion transit pathways for H+ and Cl- on the intracellular side of the transporter. Previously, we utilized a new pore-searching algorithm, TransPath, to predict Cl- and H+ ion pathways in the bacterial ClC channel homolog, focusing on proton access from the extracellular solution. Here we employ the TransPath method and molecular dynamics simulations to explore H+ pathways linking E148 and E203 in the presence of Cl- ions located at the experimentally observed binding sites in the pore. A conclusion is that Cl- ions are required at both the intracellular (S(int)) and central (S(cen)) binding sites in order to create an electrostatically favorable H+ pathway linking E148 and E203; this electrostatic coupling is likely related to the observed 1H+/2Cl- stoichiometry of the antiporter. In addition, we suggest that a tyrosine residue side chain (Y445), located near the Cl- ion binding site at S(cen), is involved in proton transport between E148 and E203.     

Electrostatic calculations for an ion channel. II. Kinetic behavior of the gramicidin A channel.

A theoretical model of the gramicidin A channel is presented and the kinetic behavior of the model is derived and compared with previous experimental results. The major assumption of the model is that the only interaction between ions in a multiply-occupied channel is electrostatic. The electrostatic calculations indicate in a multiply-occupied channel is electrostatic. The electrostatic calculations indicate that there will be potential wells at each end of the channel and, at high concentrations, that both wells can be occupied. The kinetics are based on two reaction steps: movement of the ion from the bulk solution to the well and movement between the two wells. The kinetics for this reaction rate approach are identical to those based on the Nernst-Planck equation in the limit where the movement between the two wells is rate limiting. The experimental results for sodium and potassium are consistent with a maximum of two ions per channel. To explain the thallium results it is necessary to allow three ions per channel. It is shown that this case is compatible with the electrostatic calculations if the presence of an anion is included. The theoretical kinetics are in reasonable quantitative agreement with the following experimental measurements: single channel conductance of sodium, potassium, and thallium; bi-ionic potential and permeability ratio between sodium-potassium and potassium-thallium; the limiting conductance of potassium and thallium at high applied voltages; current-voltage curves for sodium and potassium at low (but not high) concentrations; and the inhibition of sodium conductance by thallium. The results suggest that the potential well is located close to the channel mouth and that the conductance is partially limited by the rate going from the bulk solution to the well. For thallium, this entrance rate is probably diffusion limited.     

Effects of metabolic inhibition on ion transport by dog bronchial epithelium

Mammalian bronchial epithelium absorbs Na+ under basal conditions, but Cl- secretion can be induced. We studied the effects of several modes of metabolic inhibition on the bioelectric properties and solute permeability of dog bronchial epithelium mounted in Ussing chambers. Net Na+ absorption and short-circuit current were inhibited by approximately 75% by hypoxia or by 10(-3) M NaCN. The reduced net Na+ absorption was characterized by a decrease in absorptive flux and an increase in backflux. The latter change was proportional to an increase in permeability to [14C]mannitol, implying that solute flow through a paracellular shunt was increased. In contrast, the reduction of conductance expected from exposure to amiloride (0.94 +/- 0.15 ms/cm2 or 12%) was abolished by NaCN pretreatment. Metabolic inhibition also decreased epithelial conductance and unidirectional Cl- fluxes by approximately 25%. NaCN rapidly and reversibly inhibited the hyperpolarization of potential difference (PD) induced by low luminal bath [Cl-]. This effect was mimicked by the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid. Because the transepithelial Cl- diffusion PD reflects, in part, the depolarization of the Cl- -conductive apical cell membrane, metabolic inhibition appears to affect this path. We conclude that metabolic inhibition not only decreased net ion transport by dog bronchial epithelium but also inhibited cellular Na+- and Cl- -conductive pathways and increased paracellular permeability.     

Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells.

Receptor-dependent translocation of diphtheria toxin across the surface membrane of Vero cells was studied using patch clamp techniques. Translocation was induced by exposing cells with surface-bound toxin to low pH. Whole cell current and voltage clamp recordings showed that toxin translocation was associated with membrane depolarization and increased membrane conductance. The conductance increase was voltage independent, with a reversal potential of approximately 15 mV. This value was unaffected by changing the Cl- gradient across the membrane and microfluorometric measurements showed that the cytosolic Ca2+ concentration was only marginally elevated by the translocation. The conductance increase is thus mainly due to monovalent cations. Exposing outside-out and cell-attached patches with bound toxin to low pH induced a new type of ion channel in the membrane. The channel current was inward at negative membrane potentials and the single channel conductance was approximately 30 pS. This value is about three times larger than for receptor-independent channels induced by diphtheria toxin or toxin fragments in artificial lipid membranes.     

The sodium and chloride dependence of chloride secretion by the opercular epithelium

The effects of ion substitutions on the Cl- secretion rate and tissue conductance of isolated short-circuited opercular epithelia from sea-water-adapted Fundulus heteroclitus were investigated. Serosal Na+ substitution had the same effect on the Cl- secretion rate that serosal Cl- substitution had on the active component of the Cl- efflux. This similarity indicated a 1:1 Na-Cl requirement for active Cl- secretion across this epithelium, which supports the proposal of a coupled NaCl uptake mechanism at the serosal membrane of Cl- secretory epithelia. Mucosal Na+ and Cl- substitutions appeared to inhibit completely the active Cl- secretory flux. The reductions in the tissue conductance with mucosal ion substitutions suggested that this effect can be attributed to a blocking of the apical membrane Cl- conductance. These mucosal ion effects suggested a possible direct regulatory influence of the external salinity on the Cl- secretion rate and tissue conductance, which provide alternative explanations for observations with the teleost gill epithelium.     

Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation

Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 microm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 microm HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by 22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel.     

Exploration of the structural features defining the conduction properties of a synthetic ion channel.

The finite-difference Poisson-Boltzmann methodology was applied to a series of parallel, alpha-helical bundle models of the designed ion channel peptide Ac-(LSSLLSL)3-CONH2. This method is able to fully describe the current-voltage curves for this channel and quantitatively explains their cation selectivity and rectification. We examined a series of energy-minimized models representing different aggregation states, side-chain rotamers, and helical rotations, as well as an ensemble of structures from a molecular dynamics trajectory. Potential energies were computed for single, permeating K+ and Cl- ions at a series of positions along a central pathway through the models. A variable-electric-field Nernst-Planck electrodiffusion model was used, with two adjustable parameters representing the diffusion coefficients of K+ and Cl- to scale the individual ion current magnitudes. The ability of a given DelPhi potential profile to fit the experimental data depended strongly on the magnitude of the desolvation of the permeating ion. Below a pore radius of 3.8 A, the predicted profiles showed large energy barriers, and the experimental data could be fit only with unrealistically high values for the K+ and Cl- diffusion coefficients. For pore radii above 3.8 A, the desolvation energies were 2kT or less. The electrostatic calculations were sensitive to positioning of the Ser side chains, with the best fits associated with maximum exposure of the Ser side-chain hydroxyls to the pore. The backbone component was shown to be the major source of asymmetry in the DelPhi potential profiles. Only two of the energy-minimized structures were able to explain the experimental data, whereas an average of the dynamics structures gave excellent agreement with experimental results. Thus this method provides a promising approach to prediction of current-voltage curves from three-dimensional structures of ion channel proteins.     

A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers

A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaCl with 100 mM tetraethylammonium chloride on the cis side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at +75 mV. Channel gating is sensitive to cis pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster Cl- channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance Cl- channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate Cl- channel is an evolutionary prototype of a widely distributed class of small conductance anion channels.     

Molecular dynamics estimates of ion diffusion in model hydrophobic and KcsA potassium channels

Molecular dynamics simulations are carried out to obtain estimates of diffusion coefficients of biologically important Na+, K+, Ca2+ and Cl- ions in hydrophobic cylindrical channels with varying radii and large reservoirs. Calculations for the cylindrical channels are compared to those for the KcsA potassium channel, for which the protein structure has recently been determined from X-ray diffraction experiments. Our results show that ion diffusion is maintained at reasonably high levels even within narrow channels, and does not support the very small diffusion coefficients used in some continuum models in order to fit experimental data. The present estimates of ion diffusion coefficients are useful in the calculation of channel conductance using the Poisson-Nernst-Planck theory, or Brownian dynamics.     

Hybrid MD-Nernst Planck model of α-hemolysin conductance properties

Motivated by experiments in which an applied electric field translocates polynucleotides through an α-hemolysin protein channel causing ionic current transient blockade, a hybrid simulation model is proposed to predict the conductance properties of the open channel. Time scales corresponding to ion permeation processes are reached using the Poisson–Nernst–Planck (PNP) electro-diffusion model in which both solvent and local ion concentrations are represented as a continuum. The diffusion coefficients of the ions (K⁺ and Cl^?) input in the PNP model are, however, calculated from all-atom molecular dynamics (MD). In the MD simulations, a reduced representation of the channel is used. The channel is solvated in a 1?M KCl solution, and an external electric field is applied. The pore specific diffusion coefficients for both ionic species are reduced 5–7 times in comparison to bulk values. Significant statistical variations (17–45%) of the pore-ions diffusivities are observed. Within the statistics, the ionic diffusivities remain invariable for a range of external applied voltages between 30 and 240?mV. In the 2D-PNP calculations, the pore stem is approximated by a smooth cylinder of radius ～9?Å with two constriction blocks where the radius is reduced to ～6?Å. The electrostatic potential includes the contribution from the atomistic charges. The MD-PNP model shows that the atomic charges are responsible for the rectifying behaviour and for the slight anion selectivity of the α-hemolysin pore. Independent of the hierarchy between the anion and cation diffusivities, the anionic contribution to the total ionic current will dominate. The predictions of the MD-PNP model are in good agreement with experimental data and give confidence in the present approach of bridging time scales by combining a microscopic and macroscopic model.     

Tests of continuum theories as models of ion channels. II. Poisson-Nernst-Planck theory versus brownian dynamics

We test the validity of the mean-field approximation in Poisson-Nernst-Planck theory by contrasting its predictions with those of Brownian dynamics simulations in schematic cylindrical channels and in a realistic potassium channel. Equivalence of the two theories in bulk situations is demonstrated in a control study. In simple cylindrical channels, considerable differences are found between the two theories with regard to the concentration profiles in the channel and its conductance properties. These differences are at a maximum in narrow channels with a radius smaller than the Debye length and diminish with increasing radius. Convergence occurs when the channel radius is over 2 Debye lengths. These tests unequivocally demonstrate that the mean-field approximation in the Poisson-Nernst-Planck theory breaks down in narrow ion channels that have radii smaller than the Debye length.     

An analytical model of ionic movements in airway epithelial cells.

A new mathematical model of ion movements in airway epithelia is presented, which allows predictions of ion fluxes, membrane potentials and ion concentrations. The model includes sodium and chloride channels in the apical membrane, a Na/K pump and a cotransport system for Cl- with stoichiometry Na+:K+:2Cl- in the basolateral membrane. Potassium channels in the basolateral membrane are used to regulate cell volume. Membrane potentials, ion fluxes and intracellular ion concentration are calculated as functions of apical ion permeabilities, the maximum pump current and the cotransport parameters. The major predictions of the model are: (1) Cl- concentration in the cell is determined entirely by the intracellular concentration of negatively charged impermeable ions and the osmotic conditions; (2) changes in intracellular Na+ and K+ concentrations are inversely related; (3) cotransport provides the major driving force for Cl- flux, increases intracellular Na+ concentration, decreases intracellular K+ concentration and hyperpolarizes the cell interior; (4) the maximum rate of the Na/K pump, by contrast, has little effect on Na+ or Cl- transepithelial fluxes and a much less pronounced effect on cell membrane polarization; (5) an increase in apical Na+ permeability causes an increase in intracellular Na+ concentration and a significant increase in Na+ flux; (6) an increase in apical Cl- permeability decreases intracellular Na+ concentration and Na+ flux; (7) assuming Na+ and Cl- permeabilities equal to those measured in human nasal epithelia, the model predicts that under short circuit conditions, Na+ absorption is much higher than Cl- secretion, in agreement with experimental measurements.