Characterization of different reactive lysines in bovine heart mitochondrial porin

Incubation of mitochondrial outer membrane porin with citraconic anhydride prior to treatment with fluorescein isothiocyanate (FITC) resulted in the labeling of a set of lysines located at a boundary between the water phase and lipid phase. The elution pattern of porin from the cation exchanger has been considered as indicative for the location of lysines. Electrical measurements after reconstitution of the modified protein in lipid bilayer membranes revealed that certain specific lysine residues are more susceptible to alterations. The innermost positive residues were only slightly influenced, while the outermost lysines exhibited a substantial change in channel properties. These results suggest the presence of critical charged residues in mitochondrial outer membrane porin that may be responsible for both the channel's selectivity and its voltage dependence.     

Protoplasmic streaming of an internodal cell of Nitella flexilis; its correlation with electric stimulus

The sudden cessation or sudden decrease in velocity of the protoplasmic streaming of Nitella flexilis is observed whenever an action potential is elicited. The action potential can be generated by an electric stimulus after its refractory period, whether the flow is at a complete standstill or on the way to recovery. The membrane potential is generally decreased more or less when the rate of flow is decreased on application of salts or other agents. There is, however, no parallelism between these two. The membrane potential decreases proportionally with applied voltage of subthreshold intensity, while the rate of flow does not change appreciably. Only on application of a superthreshold voltage does the flow stop suddenly. In one case the rate of flow decreased to half without appreciable decrease in membrane potential. In another case it continued flowing at about one-half rate, although the membrane potential was almost zero. The Q₁₀ of the rate of flow is about 2, while it is 1.1 to 1.5 for the membrane potential. The sudden cessation of the protoplasmic streaming is supposed to be caused by the temporary formation of certain interlinkages among contractile protein networks in the endoplasm during excitation at the cathodal half of Nitella.     

A thermodynamic analysis of steady state diffusion of counterions and water through a membrane with immobile sites

Summary This paper presents a derivation, in steady state condition, of the voltage current relationship across a membrane, with charged immobile sites separating two aqueous solutions, where only two species of counterions of the same valence, to which the membrane is permeable, are present. The concentration of the sites is assumed uniform and high enough to exclude the ions of the same sign (coions) from the membrane phase. The mutual interactions between the permeant species (counterion 1 and 2 and water) and their interaction with, the membrane component are taken into account in the equations of flow. The concentration profiles of the mobile species, the partial fluxes and the conductance are expressed as functions of the voltage, the current density and of the boundary concentrations.The authors wish to express their thanks to Prof. A.Borsellino for helpful discussions, and to Prof. R.Schlögl, whose criticisms and benevolent comments on a first draft of the paper encouraged the pursuing of the work.     

Properties of lipid bilayer membranes separating two aqueous phases: The effects of Fe+3 on electrical properties

Summary Ferric ion has been found to alter the electrical properties of lecithincholesterol-decane bilayer membranes. Within minutes after the addition of microgram quantities of FeCl₃ to the ambient aqueous phase, the resistance of the membrane falls by a factor of 10⁵ to 10⁶. No change in capacitance is observed. The resistance change is obtained with membranes made from synthetic lecithin (fully saturated fatty acids) as well as by those formed from egg lecithin. The conductance of the modified membrane exhibits both time and voltage dependent behavior; the time dependence of the current is similar to that of an inductance, and the voltage dependence of the current is exponential. Concomitant with the resistance change, the modified membrane becomes permselective, passing chloride almost to the complete exclusion of sodium. Anion selectivity can be converted to cation selectivity by the subsequent addition of certain chelating agents. Area-conductance measurements show the resistance change occurs in the thin film. The addition of a reducing agent causes the effect of the ferric ion to be reversed, and the conductance returns to that characteristic of unmodified membranes. When ferric ion is added to only one side of the membrane, the system rectifies with current ratios of up to 201. It is concluded that the alteration of membrane properties owes its origin to the hydrolysis of membrane-bound ferric ion. The interaction of ferric ion with aqueous dispersions of lecithin has been investigated by several techniques, and evidence is presented that the dispersions bind charged species of iron and that this charge diminishes under conditions where iron hydrolysis occurs.     

Transient polarization currents in the squid giant axon.

It has been repeatedly noted that the change of conformation of the molecules that serve as the ion-selective channels for sodium and potassium conductance in the nerve membrane will be accompanied by a change in the dipole moment of the molecule. This time-dependent change of dipole moment will produce transient currents in the membrane. The canonical form for these currents is determined with conventional statistical mechanics formalism. It is pointed out that the voltage dependence of the conductance channel conductance determines the free energy of the system to within a factor that is an unknown function of the voltage. Since the dipole currents do not depend on this unknown function, they are completely determined 0y the observed properties of the conductance system. The predicted properties of these dipole currents, their time constants and strengths, are calculated. By using the observed properties of gating currents, the density of the sodium channels is computed. The predicted properties of the dipole currents are found to compare satisfactorily with the observed properties of gating currents.     

Voltage- and time dependence of apical membrane conductance during current clamp inNecturus gallbladder epithelium

Summary The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances ofNecturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (V _mc) results in a marked decrease in apical membrane fractional resistance (fR _a), whereas hyperpolarization ofV _mc results in either no change infR _a or a small increase, and b) the voltage-dependent changes infR _a are essentially complete within 500 msec. Exposure of the tissue to 5mm TEA⁺ on the mucosal side caused no significant change in baselineV _mc (–69±2 mV) and yet virtually abolished the voltage dependence offR _a. A possible interpretation of these results is that two types of K⁺ channels exist in the apical membrane, with different voltage dependencies and TEA⁺ sensitivities. Acidification or Ba²⁺ addition to the mucosal solution also reduced the voltage-dependent changes infR _a. The time courses of the changes infR _a and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (±200 A/cm²). No secondary change infR _a was observed with mucosa-to-serosa currents, but a slow TEA⁺-sensitive decrease infR _a (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (<500 msec) voltage-dependent decrease infR _a is due to a fall in apical membrane resistance. The later decrease infR _a is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimatingfR _a than the current-induced effects on the lateral intercellular spaces. In principle, TEA⁺ can be used to prevent the nonlinear behavior ofR _a during measurements of the voltage divider or membrane resistance ratio.     

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by ΔΦ, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of ΔΦ on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of ΔΦ. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of ΔΦ.     

The Spatial Variation of Membrane Potential Near a Small Source of Current in a Spherical Cell

A theoretical analysis is presented of the change in membrane potential produced by current supplied by a microelectrode inserted just under the membrane of a spherical cell. The results of the analysis are presented in tabular and graphic form for three wave forms of current: steady, step function, and sinusoidal. As expected from physical reasoning, we find that the membrane potential is nonuniform, that there is a steep rise in membrane potential near the current microelectrode, and that this rise is of particular importance when the membrane resistance is low, or the membrane potential is changing rapidly. The effect of this steep rise in potential on the interpretation of voltage measurements from spherical cells is discussed and practical suggestions for minimizing these effects are made: in particular, it is pointed out that if the current and voltage electrodes are separated by 60°, the change in membrane potential produced by application of current is close to that which would occur if there were no spatial variation of potential. We thus suggest that investigations of the electrical properties of spherical cells using two microelectrodes can best be made when the electrodes are separated by 60°.     

The cyclic nucleotide gated channel of olfactory receptor neurons

Olfactory transduction proceeds through a G-protein coupled cascade that produces the ubiquitous second messenger cyclic AMP. The cyclic AMP causes a chance in membrane potential by acting directly on an ion channel that allows cations to flow into the cell. This ion channel is one of a new family of ion channels that are activated by intracellular cyclic nucleotides. However, even though they are activated by binding a ligand their amino acid structure shows that they share a common ancestry with voltage activated channels, especially voltage dependent Ca²⁺ channels. In olfactory neurons these channels perform a critical role in the transduction of chemical information in the environment into changes in membrane electrical properties that are transmitted to higher order processing centers in the brain.     

Semiconductor theory of ion transport in thin lipid membranes: I. Potential and field distributions

In the theory as presented in this paper and the following one, we shall attempt to apply the semiconductor principles and methods to the study of ion transport in thin lipid membranes. Detailed formulations are given on the potential energy barriers at the interfaces, voltage drops in the polar and non-polar regions, and potential and field distributions in the diffuse double layer and within a charged membrane. These results will be used mainly as the boundary conditions for the solution of ion flow as to be given in the following paper. The analysis clearly indicates that the ion transport is interface-limited and is profoundly influenced by the presence of surface charges. An explanation of Na⁺ extrusion in nerve membrane is given based on the field distribution analysis. The theory also suggests that the “membrane potential” depends mainly on surface charges but not necessarily on ion permeation through the membrane.     

Parameter sensitivity of distributed transfer properties of neuronal dendrites: a passive cable approximation

Geometry and membrane properties of the dendrites crucially determine input–output relations in neurons. Unlike geometry often available in detail from computer reconstruction, the membrane resistivity is fragmentarily known if at all. Moreover, it varies during ongoing activity. In this study we address the question: what is the impact of the variation in membrane resistivity on the transfer properties of dendrites? Following a standard approach of the control system theory, we derive and explore the sensitivity functions complementary to the transfer functions of the passive dendrites with arbitrary geometrical parameters (length and diameter) and boundary conditions. We use the location-dependent somatopetal current transfer ratio (the reciprocal of the somatofugal voltage) as the transfer function, and its membrane resistivity derivatives, as the sensitivity functions. In the dendrites, at every path distance from the origin, the sensitivity function in a common form relates the transfer function, membrane resistivity, characteristic input conductance of semi-infinite cable and directional somatofugal input conductances at the given internal site and origin, and the length. Plotted in membrane resistivity versus path distance coordinates, the sensitivity functions display common features: along any coordinate there are low and high ranges, in which the sensitivity, respectively, increases and decreases. The ranges and corresponding rates depend on morphology and boundary conditions in a characteristic manner. These features predict existence of the geometry-dependent range of membrane resistivity (the earlier unattended mid-conductance state), such that the dendrites with a given metrical asymmetry are most distinguished in their transfer properties and electrical states if membrane resistivity is within the range and are not otherwise.     

Development of dielectrophoresis separator with an insulating porous membrane using DC‐Offset AC Electric Fields

Our previous studies revealed that the dielectrophoresis method is effective for separating cells having different dielectric properties. The purpose of this study was to evaluate the separation characteristics of two kinds of cells by direct current (DC) voltage offset/alternating current (AC) voltage using an insulating porous membrane dielectrophoretic separator. The separation device gives dielectrophoretic (DEP) force and electrophoretic (EP) force to dispersed particles by applying the DC‐offset AC voltage. This device separates cells of different DEP properties by adopting a structure in which only the parallel plate electrodes and the insulating porous membrane are disposed in the flow path through which the cell‐suspension flows. The difference in the retention ratios of electrically homogeneous 4.5 μm or 20.0 μm diameter standard particles was a maximum of 82 points. Furthermore, the influences of the AC voltage or offset voltage on the retention ratios of mouse hybridoma 3‐2H3 cells and horse red blood cells (HRBC) were investigated. The difference in the retention ratio of the two kinds of cells was a maximum of 56 points. The separation efficiency of this device is expected to be improved by changing the device shape, number of pores, and pore placement. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1292–1300, 2016     

A Dipole Model for Negative Steady-State Resistance in Excitable Membranes

A dipole model is presented for ion flow in excitable membranes. This model considers the membrane to be composed of two distinct regions: a polar region and a nonpolar region. Further, the construction of an electrodiffusive formalism which takes explicit account of the energy of partition required by an ion for passage from external fluid to nonpolar region is presented. In the polar region a cooperative effect is considered which produces a configurational transition of the polar group dependent only on membrane voltage. A resulting change in voltage drop across the polar group is brought about by this configurational transition. This gives rise to a negative steady-state resistance for the equimolar case, in reasonable agreement with observation. The theory, in addition, is in reasonable accord with nonequimolar ion flow, and provides an explanation for such effects as the following: the intercept of the voltage-current characteristic, the ion membrane concentrations inferred from electrodiffusion theories, and the effects of polyvalent cations     

The macrokinetic behavior of an enzymatic system with an enzyme inactivated in the reaction

This study deals with the behavior of a heterogeneous multisubstrate enzymatic system under enzyme inactivation in a reaction. Electronic computer modeling data have been obtained for its macrokinetics at different modes: (1) under mixed inflow of the substrates and (2) under their spatial separation. The enzymatic membrane exhibits low sensitivity to a change in the external conditions as the substrates are intermixed on the boundary. Quite the contrary, in the case of spatial separation of the substrates, the product flow from the membrane has displayed abrupt fluctuations at different boundary conditions. This work also looks into the arrangement of the reaction zones in the membrane and their transitions under different conditions.     

Electrical properties of sheep Purkinje strands. Electrical and chemical potentials in the clefts

The impedence of sheep Purkinje strands, measured to 3-5 kHz, is interpreted with circuit models based on morphology. The strand is described as a one-dimensional electrical cable. Clefts between myocytes of the strand allow radial current to flow in parallel with current across the outer membrane. A lumped model of the clefts, in which all the cleft membrane is in series with 100 omega-cm2, fits only below 20 Hz. Two distributed models, pie and disk, fit at all frequencies with somewhat different (31%) luminal resistivities, but with similar membrane parameters. Series resistance representing the endothelial sheath is small. Simulations of voltage clamp experiments include measured linear parameters and nonlinear membrane channels, as well as radial variation of cleft concentration, membrane flux, voltage, and current. Cleft potential is drastically nonuniform when sodium current flows. Cleft potential is reasonably uniform when calcium and potassium currents flow, but the calcium and potassium concentrations change markedly, enough to turn off the calcium current, even if the calcium channel did not inactivate. We conclude that physiological current flows produce significant nonuniformities in electrochemical potentials in the clefts of this cardiac preparation.     

Osmosis and solute—Solvent drag

In 1903, George Hulett explained how solute alters water in an aqueous solution to lower the vapor pressure of its water. Hulett also explained how the same altered water causes osmosis and osmotic pressure when the solution is separated from liquid water by a membrane permeable to the water only. Hulett recognized that the solute molecules diffuse toward all boundaries of the solution containing the solute. Solute diffusion is stopped at all boundaries, at an open-unopposed surface of the solution, at a semipermeable membrane, at a container wall, or at the boundary of a solid or gaseous inclusion surrounded by solution but not dissolved in it. At each boundary of the solution, the solute molecules are reflected, they change momentum, and the change of momentum of all reflected molecules is a pressure, a solute pressure (i.e., a force on a unit area of reflecting boundary). When a boundary of the solution is open and unopposed, the solute pressure alters the internal tension in the force bonding the water in its liquid phase, namely, the hydrogen bond. All altered properties of the water in the solution are explained by the altered internal tension of the water in the solution. We acclaim Hulett's explanation of osmosis, osmotic pressure, and lowering of the vapor pressure of water in an aqueous solution. His explanation is self-evident. It is the necessary, sufficient, and inescapable explanation of all altered properties of the water in the solution relative to the same property of pure liquid water at the same externally applied pressure and the same temperature. We extend Hulett's explanation of osmosis to include the osmotic effects of solute diffusing through solvent and dragging on the solvent through which it diffuses. Therein lies the explanations of (1) the extravasation from and return of interstitial fluid to capillaries, (2) the return of luminal fluid in the proximal and distal convoluted tubules of a kidney nephron to their peritubular capillaries, (3) the return of interstitial fluid to the vasa recta, (4) return of aqueous humor to the episcleral veins, and (5) flow of phloem from source to sink in higher plants and many more examples of fluid transport and fluid exchange in animal and plant physiology. When a membrane is permeable to water only and when it separates differing aqueous solutions, the flow of water is from the solution with the lower osmotic pressure to the solution with the higher osmotic pressure.     

Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999     

A model of NaCl and water flow through paracellular pathways of renal proximal tubules.

To explain how hydrostatic pressure differences between tubule lumen and interstitium modulate isotonic reabsorption rates, we developed a model of NaCl and water flow through paracellular pathways of the proximal tubule. Structural elements of the model are a tight junction membrane, an intercellular channel whose walls transport NaCl actively at a constant rate, and a basement membrane. Equations of change were derived for the channel, boundary conditions were formulated from irreversible thermodynamics, and a pressure-area relationship typical of thin-walled tubing was assumed. The boundary value problem was solved numerically. The principal conclusions are: 1) channel NaCl concentration must remain within a few mOsm of isotonic values for reabsorption rates to be modulated by transtubular pressure differences known to affect this system: 2) basement membrane and channel wall parameters determine reabsorbate tonicity; tight junction parameters affect the sensitivity of reabsorption to transmural pressure; 3) channel NaCl concentration varies inversely with transmural pressure difference; this concentration variation controls NaCl diffusion through the tight junction; 4) modulation of NaCl diffusion through the tight junction controls the rate of isotonic reabsorption; modulation of water flow can increase sensitivity to transmural pressure; 5) no pressure-induced change in permeability of the tight junction or basement membrane is needed for pressure to modulate reabsorption; and 6) system performance is indifferent to the distribution of active transport sites, to the numerical value of the compliance function, and to the relationship between lumen and cell pressures.     

Model channel ion currents in NaCl-extended simple point charge water solution with applied-field molecular dynamics.

Using periodic boundary conditions and a constant applied field, we have simulated current flow through an 8.125-A internal diameter, rigid, atomistic channel with polar walls in a rigid membrane using explicit ions and extended simple point charge water. Channel and bath currents were computed from 10 10-ns trajectories for each of 10 different conditions of concentration and applied voltage. An electric field was applied uniformly throughout the system to all mobile atoms. On average, the resultant net electric field falls primarily across the membrane channel, as expected for two conductive baths separated by a membrane capacitance. The channel is rarely occupied by more than one ion. Current-voltage relations are concentration dependent and superlinear at high concentrations.     

Steady-state current flow through gap junctions. Effects on intracellular ion concentrations and fluid movement.

Double voltage clamp studies were performed on gap junctions contained in septal membranes of the earthworm median giant axon. The gap junctions exhibited no conductance changes in response to voltages imposed across either the septal membrane or the plasma membrane. However, the trans-septal current displayed a slow (10 s) relaxation in response to transjunctional voltage steps. The experimental evidence suggests that this relaxation is a polarization of the septum due to local accumulation/depletion of permeant ions. A theoretical analysis of this observation suggests that the applied electric field causes accumulation of impermeant anions on one side of the junction and depletion on the other, which leads to a change in concentration of permeant ions to maintain macroscopic electroneutrality. The change in concentration of permeant ions generates a transjunctional equilibrium potential that opposes junctional current flow. These results indicate that currents flowing through gap junctions can have an influence on the distribution of intracellular ions. Moreover, the theoretical analysis suggests that such currents will be accompanied by significant intracellular and intercellular water flow.