Ionic Conductances of Extracellular Shunt Pathway in Rabbit Ileum : Influence of shunt on transmural sodium transport and electrical potential differences

The unidirectional influxes of Na, K, and Cl into isolated strips of rabbit ileum are comprised of movements across the mucosal membrane of the epithelial cells and ionic diffusion into an extracellular shunt pathway. A large fraction of the Na influx across the mucosal membrane alone is inhibited by Li, suggesting the participation of a carrier mechanism in the influx process. The partial ionic shunt conductances of Na, K, and Cl account for at least 82% of the total tissue conductance. The calculated shunt permeabilities (P) are (in centimeters per hour) P_K = 0.040, P_Na = 0.035, and P_Cl = 0.019, so that P_K:P_Na:P_Cl = 1.14:1.00:0.55. Diffusion potentials across the tissue resulting from isotonic replacement of NaCl in the mucosal solution with mannitol or KCl are described by the Goldman constant-field equation together with the above permeabilities of the shunt pathway. These observations are not consistent with permeation through a fixed-charge pore but can be explained by a model featuring constant ionic partition into a neutral-polar pore that traverses the tight junction. Such a pore may be lined with either fixed dipoles or fixed dipolar ions oriented such that electronegative groups influence the permselective properties of the diffusion pathway. The essential feature of both models is that electroneutrality is maintained by means of fixed membrane components and does not depend upon the presence of mobile counterions.     

Positively charged mineral surfaces promoted the accumulation of organic intermediates at the origin of metabolism

Identifying plausible mechanisms for compartmentalization and accumulation of the organic intermediates of early metabolic cycles in primitive cells has been a major challenge in theories of life’s origins. Here, we propose a mechanism, where positive membrane potentials elevate the concentration of the organic intermediates. Positive membrane potentials are generated by positively charged surfaces of protocell membranes due to accumulation of transition metals. We find that (i) positive membrane potentials comparable in magnitude to those of modern cells can increase the concentration of the organic intermediates by several orders of magnitude; (ii) generation of large membrane potentials destabilize ion distributions; (iii) violation of electroneutrality is necessary to induce nonzero membrane potentials; and (iv) violation of electroneutrality enhances osmotic pressure and diminishes reaction efficiency, resulting in an evolutionary driving force for the formation of lipid membranes, specialized ion channels, and active transport systems.     

Lipid surface charge does not influence conductance or calcium block of single sodium channels in planar bilayers.

We have studied the effects of membrane surface charge on Na+ ion permeation and Ca2+ block in single, batrachotoxin-activated Na channels from rat brain, incorporated into planar lipid bilayers. In phospholipid membranes with no net charge (phosphatidylethanolamine, PE), at low divalent cation concentrations (approximately 100 microM Mg2+), the single channel current-voltage relation was linear and the single channel conductance saturated with increasing [Na+] and ionic strength, reaching a maximum (gamma max) of 31.8 pS, with an apparent dissociation constant (K0.5) of 40.5 mM. The data could be approximated by a rectangular hyperbola. In negatively charged bilayers (70% phosphatidylserine, PS; 30% PE) slightly larger conductances were observed at each concentration, but the hyperbolic form of the conductance-concentration relation was retained (gamma max = 32.9 pS and K0.5 = 31.5 mM) without any preferential increase in conductance at lower ionic strengths. Symmetrical application of Ca2+ caused a voltage-dependent block of the single channel current, with the block being greater at negative potentials. For any given voltage and [Na+] this block was identical in neutral and negatively charged membranes. These observations suggest that both the conduction pathway and the site(s) of Ca2+ block of the rat brain Na channel protein are electrostatically isolated from the negatively charged headgroups on the membrane lipids.     

Interactions of voltage-sensing dyes with membranes. III. Electrical properties induced by merocyanine 540

The effects of merocyanine 540 on the electrical properties of lipid bilayer membranes have been investigated. The alterations this dye was found to produce in the intrinsic conductances of these membranes were minimal, but it profoundly altered the conductances produced by extrinsic permeant species. These alterations were much larger for neutral membranes than for negatively charged ones. The dye increased the conductances mediated by positively charged permeant species and decreased those by negatively charged permeant species, suggesting that it produces a negative electrostatic potential on the membrane; it also altered the kinetics and the voltage dependencies of permeation by these charge carriers. The magnitudes of dye-mediated conductance changes were much larger for positively charged permeants than for negatively charged ones; also, changes in ionic strength altered these dye effects in opposite directions from those predicted by the Stern equation, and the dependence of the conductance alteration on dye concentration was steeper than that predicted by this equation. Finally, only very small changes in liposome zeta potentials were induced by the dye. Calculations show that a large fraction of these effects can be accounted for by the dipole potential produced by merocyanine at the membrane surface, but that additional effects of the dye must be postulated as well.     

Imperfect space clamp permits electrotonic interactions between inhibitory and excitatory synaptic conductances, distorting voltage clamp recordings

The voltage clamp technique is frequently used to examine the strength and composition of synaptic input to neurons. Even accounting for imperfect voltage control of the entire cell membrane ("space clamp"), it is often assumed that currents measured at the soma are a proportional indicator of the postsynaptic conductance. Here, using NEURON simulation software to model somatic recordings from morphologically realistic neurons, we show that excitatory conductances recorded in voltage clamp mode are distorted significantly by neighboring inhibitory conductances, even when the postsynaptic membrane potential starts at the reversal potential of the inhibitory conductance. Analogous effects are observed when inhibitory postsynaptic currents are recorded at the reversal potential of the excitatory conductance. Escape potentials in poorly clamped dendrites reduce the amplitude of excitatory or inhibitory postsynaptic currents recorded at the reversal potential of the other conductance. In addition, unclamped postsynaptic inhibitory conductances linearize the recorded current-voltage relationship of excitatory inputs comprising AMPAR and NMDAR-mediated components, leading to significant underestimation of the relative contribution by NMDARs, which are particularly sensitive to small perturbations in membrane potential. Voltage clamp accuracy varies substantially between neurons and dendritic arbors of different morphology; as expected, more reliable recordings are obtained from dendrites near the soma, but up to 80% of the synaptic signal on thin, distant dendrites may be lost when postsynaptic interactions are present. These limitations of the voltage clamp technique may explain how postsynaptic effects on synaptic transmission could, in some cases, be attributed incorrectly to presynaptic mechanisms.     

The effect of prymnesin on the electric conductivity of thin lipid membranes

Summary A proteolipidic toxin, prymnesin, when added to the aqueous solutions around thin lipid membranes causes a marked increase in membrane conductance. The toxin-treated membrane is cation-permselective. The extent of cation permselectivity is dependent upon ionic strength of the aqueous solutions in a fashion similar to the dependence of cation permselectivity of a cation exchanger containing about 100mm of fixed negative sites. Dose-response relationship studies reveal a linear relation between log prymnesin concentration and log membrane conductance. The slope of the curve is around 3 if the toxin is applied to one side of the membrane and is around 7 if the toxin is applied to both sides of the membrane. The membrane treated with toxin on one side only is clearly asymmetric in its properties. These characteristics are expressed by an asymmetric current-voltage relationship, and by asymmetric sensitivity of membrane conductance to pH and to salt concentration. The conductance of the toxin-treated membrane is inversely proportional to temperature. It is suggested that aggregates of toxin moieties assemble in the membrane to form negatively charged aqueous pores. There is roughly a good correlation between the increase in membrane conductance and the increase in membrane permeability to urea if both were attributed to the formation of aqueous channels in the membrane.     

The mechanism of cation permeation in rabbit gallbladder

Summary The questions underlying ion permeation mechanisms, the types of experiments available to answer these questions, and the properties of some likely permeation models are examined, as background to experiments designed to characterize the mechanism of alkali cation permeation across rabbit gallbladder epithelium. Conductance is found to increase linearly with bathing-solution salt concentrations up to at least 400mm. In symmetrical solutions of single alkali chloride salts, the conductance sequence is K⁺>Rb⁺>Na⁺>Cs⁺∼Li⁺. The current-voltage relation is linear in symmetrical solutions and in the presence of a single-salt concentration gradient up to at least 800 mV. The anion/cation permeability ratio shows little change with concentration up to at least 300mm. Ca⁺⁺ reduces alkali chloride single-salt dilution potentials, the magnitude of the effect being interpreted as an inverse measure of cation equilibrium constants. The equilibrium-constant sequence deduced on this basis is K⁺>Rb⁺>Na⁺∼Cs⁺∼Li⁺. These results suggest (1) that the mechanism of cation permeation in the gallbladder is not the same as that in a macroscopic ion-exchange membrane; (2) that cation mobility ratios are closer to one than are equilibrium-constant ratios; (3) that the rate-limiting step for cation permeation is in the membrane interior rather than at the membrane-solution interface; and (4) that the rate-controlling membrane is one which is sufficiently thick that it obeys microscopic electroneutrality.     

Digital simulation of associated and nonassociated liquid membrane electrochemical properties.

The method and results of a digital simulation of electrochemical properties for associated and nonassociated liquid ion-exchange membranes are presented. It is assumed that the membranes is ideally permselective, sites are completely trapped, electroneutrality holds everywhere in the membrane, and the bathing solutions contain no more than two counterions, of which one is completely dissociated in the membrane. Electrochemical properties are simulated for the single counterion case and in the interference region. Concentration profiles, potentiometric responses, transient potential responses to activity steps, and current-voltage curves are given and the effects of ion-pairing and species mobilities are studied. It is found that ion-pairing increases the potentiometric selectivity toward the complexing ion over the noncomplexing ion. Transient responses to an ion activity step are shown to depend in a complex way on the ion-pair formation constant and the various mobilities. Current-voltage curves are simulated for varying degrees of ion-pairing and qualitative agreement is found with previous theoretical treatments, as well as quantitative agreement in those cases where closed-form expressions are known.     

Membrane conductance and surface potential

The specific conductances of black lipid membranes of lecithin, glycerylmonooleate and lecithin and cholesterol in aqueous solutions of KCl + nonactin have been measured. The relative conductances of the lecithin and the glycerylmonooleate membranes are accurately accounted for by the difference in their surface potentials. The large effect of cholesterol in depressing the conductance of the lecithin membranes is, however, not accounted for by changes in the surface potential and it is necessary to invoke either changes in the membrane solubility of the nonactin-K⁺ complex, or changes in the membrane viscosity (as experiences by the ionophore in transit), or both.The surface potentials which, in all the present membranes, arise solely from layers of oriented molecular dipoles, are not affected by the KCl concentration.     

Alternating current studies of charge carrier transport in lipid bilayers. Pentachlorophenol in lecithin-cholesterol membranes.

Surface and interior electrical properties of lecithin-cholesterol bilayer membranes treated with the uncoupler pentachlorophenol have been determined on the basis of a.c. measurements over a wide range of frequencies (0.02 to 1000 kHZ). The method used depends on accurately determining the resistance of the aqueous solution in series with each individual membrane by extrapolating admittance data into infinite frequency. Loss tangent vs. frequency curves are corrected by subtracting out a loss contribution which is present in untreated membranes and is due, presumably, to dielectric relaxation. The results, which are useful below 100 kHZ, can be fitted to loss tangent curves computed for a three-element equivalent circuit consisting of frequency independent conductance-capacitance pairs, arranged in series to represent surface and interior properties of membranes. Interior conductances agree with net conductances obtained from d.c. measurements. The pH and concentration dependence of surface conductance is consistent with a scheme of transport in which a fixed number of surface binding sites are filled preferentially with neutral pentachlorophenol molecules, which in turn dissociate to supply protons to the aqueous phase. Surface capacitances range from 15 to 90 times that of interior capacitance and show a systematic increase with pentachlorophenol concentration at high pH, and a decrease with concentration at low pH.     

Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.     

Ultrastructural localization of lectin-binding sites in different basement membranes

Summary In the present work we localized binding sites for the lectins WGA, RCA I, con A and SBA at the ultrastructural levels in morphologically different basement membranes. These different basement membranes included (a) thin ones, for example, tubular basement membrane of the mouse kidney which separates epithelial cell layers from mesenchymal cells and glomerular basement membrane which separates epithelial cells from other epithelial cells, (b) thick multilayered ones, for example, Reichert's membrane which is built up during the embryonic development of rodents and as an example of a pathologically thickened basement membrane, the basement membrane of the Engelbreth-Holm-Swarm (EHS) sarcoma. We were able to show that, in contrast to the thick multilayered basement membranes, the thin ones showed a strong positive SBA-binding pattern. Thick basement membranes otherwise revealed very strong labelling with the lectins WGA and RCA I. Our findings lead us to conclude that thin and thick basement membranes differ markedly in the quality and quantity of the carbohydrates which they contain.     

A theory for the effects of diffusion-reaction coupling on the carrier-mediated ionic permeability in lipid bilayers

The zero-current membrane potential and the current-voltage relations are discussed theoretically for the case in which ionic transport is mediated by carriers that form complexes with ions in the aqueous phase (‘solution complexation’ mechanism). Interest for this topic originated partly from the finding that gradients of the neutral cyclic peptide PV, cyclo (dVal-lPro-lVal-dPro)₃, commonly thought to act as a carrier via ‘solution complexation’, generate Nernstian potentials across lipid bilayers separating solutions of identical ion composition. It is shown that the general expression for the potential in a gradient of carriers reduces to the Nernst equation under any of the following conditions: slow aqueous reaction; impermeability of the membrane to the neutral carriers; high concentration of the complexing ions in solution; finite permeability of the membrane to the neutral carrier, but faster rate of movement from the membrane surface into the torus than across the middle or out of the membrane. In symmetrical solutions, the conductance is most typically characterized by a quantity that we designate by δ*, which has the dimensions of a length and is generally a complex function of ion activity. Comparing the thory with previous data on dioleoylphosphatidylcholine membranes in the presence of PV and K⁺, the order of magnitude of the rates of the aqueous reaction and of the membrane permeability to the neutral carriers is tentatively estimated.     

A physical model of nerve axon. II: Action potential and excitation currents. Voltage-clamp studies of chemical driving forces of Na+ and K+ in squid giant axon

An adsorption model of nerve axon has been extended to account for the origin of membrane currents observed under voltage-clamp conditions. Differing from the Hodgkin-Huxley model, which attributes excitation solely to a change of ionic conductances of the membrane, the present model proposes that a layer of axoplasm attached to the membrane (axon cortex) can undergo conformational changes and hence modulate selectivity for mobile ions. To test the model, a two-step voltage-clamp study was made of the chemical driving forces of Na+ and K+ ions in squid giant axon. The forces were measured by determining the instantaneous current-voltage relation when membrane current is carried by Na+ only or K+ only. The data indicate that the chemical driving force varies as a function of time and does not agree with the Nernst relation during the early phase of excitation. Implications of the observations are discussed.     

Electrical properties and molecular architecture of the channel formed by Escherichia coli hemolysin in planar lipid membranes

A 107 kDa hemolysin from Escherichia coli is able to open pores in lipid membranes. By studying its interaction with planar phospholipid bilayers we have derived some structural information on the organization of the pore. We measured the current-voltage characteristic and the ion selectivity of the channel both in neutral membranes, made of egg phosphatidylcholine (PC) and in negatively charged membranes, made of a 1:1 mixture of PC with phosphatidylserine (PS). Experiments were performed varying both the pH and the salt concentration of the bathing KCl solution. In neutral membranes the pore is ohmic and its conductance increases almost linearly with the salt concentration. The channel is cation-selective at high pH but nearly unselective at low pH. We interpret these results in terms of a minimal model based on classical electro-diffusional theories assuming that the pore is wide and bears a negative charge at its entrances. In membranes containing the acidic lipid the current-voltage curve is non-linear in such a way to suggest that the trans (but not the cis) entrance of the pore is affected by the surface potential of the membrane. Applying our model we find that the trans and cis entrances are located, respectively, about 0.5 nm and more than 5 nm apart from the plane of the membrane. We confirmed the asymmetric disposition of the channel by enzymatic digestion of preformed pores. This was effective only when the enzyme was applied on the cis side.     

Electrical potential differences across salt transporting membranes

The electrical potential differences across membranes where active transport of ions occurs has been examined using the formalism of linear non-equilibrium thermodynamics, and can be represented as the arithmetic sum of a resistive term, a term directly dependent on metabolism (i.e. electrogenic) and terms appropriate for describing a diffusion potential. The Hittorf transport number for each ion in the latter terms is the ratio of the partial conductances of the membrane to that ion to the total membrane conductance, and the conductance to an ion consists of the arithmetic sum of conductance of active and passive pathways providing these are independent. The conductances of active transport mechanisms arise from variation of the rate of transport with the electrochemical potentials against which they operate. The electrogenic term arises from imbalance between anion and cation transport. If an ion is transported by an obligatorily electrically neutral exchange for some other ion such transport gives rise to no electrogenic effect. A membrane will transport salt most efficiently if there is no imbalance between anion and cation transport, when it will not be electrogenic, but modest deviations from this condition will not degrade the efficiency of active transport markedly.     

The ionic basis of oscillatory responses of skate electroreceptors

When physiological conditions are simulated, skate electroreceptors produce small maintained oscillatory currents. Larger damped oscillations of similar time-course are observed in voltage clamp. Subtraction of leakage in voltage clamp data shows that the oscillations involve no net outward current across the lumenal surface of the epithelium. The oscillations are much faster than the late outward current generated by the lumenal membranes of the receptor cells. Treatment of the basal surface of the epithelium with tetraethyl ammonium (TEA), high K, Co, or EGTA reversibly blocks the oscillations in voltage clamp, but has little or no effect on the epithelial action potential in current clamp or on the current-voltage relation. The TEA sensitivity of the oscillations indicates that they involve a potassium conductance in the basal membranes of the receptor cells. Treatment of the basal membranes with TEA and high calcium, with strontium, or with barium causes these membranes to produce large regenerative responses. Direct stimulation of the basal membranes then elicits a lumen-positive action potential whereas stimulation of the lumenal membranes elicits a diphasic action potential. Excitability of the basal membranes is abolished by extracellular Co, Mn, or La. Modulation of the lumenal membrane calcium conductance by the basal membrane conductances probably gives rise to the oscillatory receptor currents evoked by small voltage stimuli. The slower calcium-activated late conductance in the lumenal membranes may be involved in sensory accommodation.     

Analysis of the Torus Surrounding Planar Lipid Bilayer Membranes

The characteristics and behavior of the torus (annulus) surrounding planar lipid bilayer membranes formed across a cylindrical aperture are analyzed using equations for the shape and volume of the annulus derived by the methods of variational calculus. The analysis leads to the following results: (a) Design criteria for the aperture can be established. (b) The transition region between thin film and thick annulus can be defined quantitatively and its effect on the measurement of specific capacitance determined. (c) At fixed annulus volume the diameter of the thin membrane is a function of the thin film-annulus contact angle. This suggests a new method for examining changes in free energy of the thin film, and explains why the area of thin film increases reversibly when potentials are present across the film. (d) In the absence of buoyant forces, the equations for the shape and volume of the annulus consist of incomplete elliptic integrals of the first and second kinds; however, the shape of the annulus in the transition region can be described with good accuracy by an approximate equation of greater simplicity.     

Nitrogenous cations as probes of permeation channels

Summary Nitrogenous cations may provide information-rich probes of cation-selective channels. Hence, for 52 nitrogenous cations we have used dilution potentials and biionic potentials to measure relative permeability coefficients (P's) across gallbladder epithelia of frog and rabbit, and have also determined the free-solution mobilities. MeasuredP's of most cations are uninfluenced by the presence of the neutral form. The main permeation pathway for most hydrophilic cations is across the tight junctions.P's decrease with molecular size and increase with number of donor protons available for hydrogen-bond formation. Selectivity isotherms have been constructed from variation inP's due to pH or due to differences among individual animals. Both types of variation are consistent with the pattern expected from variation in electrostatic field strength of cation-binding sites. The isotherms permitP's to be re-expressed in a way that largely eliminates effects of species differences in field strength. Remaining species differences inP's are well fitted by a model of steric restriction, provided that one takes into account the effect of hydrogen bonding on molecular size. Rabbit gallbladder behaves as if it has narrower permeation channels than frog gallbladder. After correction for these steric effects,P is found to increase with number of donor protonsn _H up to four protons, with a steeper slope in rabbit than in frog gallbladder, but is independent ofn _H from four to at least nine. Two groups of cations appear to permeate significantly via pathways other than tight junctions: oxycations, via polar pathways in epithelial cell membranes of rabbit but not frog gallbladder; and lipid-soluble cations, via membrane lipid.The results suggest that the cation-binding sites of gallbladder tight junction are acidic proton-acceptors that discriminate more sharply among proton donors than does water. Proton-rich solutes tend to be more permeant for two reasons: stronger binding energies to membrane proton-acceptor sites, and smaller effective size in a proton-acceptor environment. As deduced from comparisons of nitrogenous cation selectivity patterns, the permeation channel through gallbladder tight junction differs from nerve's sodium channel and artificial carriers and channels in its higher hydration and lower range of selectivity. Based on the steric analysis of nitrogenous cation permeation, one can correct alkali cation permeability coefficients for the effect of steric restriction.     

Channels in the mitochondrial outer membrane: Evidence from patch clamp studies

Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane ofNeurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.