Modulation of membrane potential in algal cells by temperature gradients

The aim of the present study is to ascertain whether transmembrane temperature gradients couple with transport of electric charge in living cells ofValonia utricularis and eventually measure the thermodynamic coupling coefficient (s). Simple experimental procedures are described that allow generation of temperature gradients of predetermined sense and intensity across the cell membrane. Simultaneous measurement of the potential difference is ensured by standard electrophysiological methods. The mathematical expressions that allow quantitative treatment of experimental results are indicated in the article and are based on standard nonequilibrium thermodynamic and electrophysiological formalism. The value of the coupling coefficient between temperature gradient and flow of electric charge is indicated and concisely discussed in terms of possible mechanisms of ionic membrane transport.     

The static head method for determining the charge stoichiometry of coupled transport systems. Applications to the sodium-coupled D-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled D-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.     

The static head method for determining the charge stoichiometry of coupled transport systems Applications to the sodium-coupled d-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled d-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.     

The association between K+ and H+ distribution in skeletal muscles: implication to linked transport.

Data from the literature and results from a mathematical model of steady state fluid-electrolyte balance are used to support the observation that a relationship exists between the concentration gradients of K⁺ and H⁺ in the fluids of skeletal muscle over a range of acid-base disturbances. This relationship is shown to be consistent with the premise that the steady state electrochemical potential gradients for these ions remain constant under these conditions. Using a pump-leak model of ion transport, and the constant electric field assumption, it is also demonstrated that the steady state rates of active transport of K⁺ and H⁺ are related. These results suggest that the relations between both the steady state concentration gradients and the active transport rates for these ions are not necessarily the result of fixed biochemical mechanisms, but may come about simply from coupling through macroscopic thermodynamic processes.     

The representation of membrane admittance.

An integral representation for the membrane admittance in terms of its known current response to a voltage step function is presented. It is demonstrated that the frequency-dependent terms in the contribution to the membrane admittance by the ion-selective conductance of the nerve membrane are proportional to the static conductances. The additional information contained in the real and imaginary parts of the membrane admittance should allow the parameters of the ion conductance to be determined. Eventually, these measurements should also give information about the electric dipole displacement currents of the conductance systems themselves, and about the metabolically supported active ion transport currents that maintain the ion concentration gradients.     

Effect of electric field gradients on lipid monolayer membranes.

Externally applied nonuniform electric fields can strongly affect thermodynamic phases in a lipid monolayer when applied under conditions of temperature, pressure, and composition that are near phase boundaries. Under such conditions nonuniform applied fields can produce or suppress phase separations. Field-induced phase-separated domains have sizes that are in good agreement with calculations. Field gradients can also produce large concentration gradients in binary mixtures just above their critical points. The present work elaborates our earlier studies of these field effects using thermodynamic models of the phase behavior of two-component liquid mixtures. The calculations are of interest in connection with biological membranes that, at the growth temperature, are in a liquid state close to a phase boundary.     

Acidic lipids, H-ATPases, and mechanism of oxidative phosphorylation. Physico-chemical ideas 30 years after P. Mitchell's Nobel Prize award

Peter D. Mitchell, who was awarded the Nobel Prize in Chemistry 30 years ago, in 1978, formulated the chemiosmotic theory of oxidative phosphorylation. This review initially analyzes the major aspects of this theory, its unresolved problems, and its modifications. A new physico-chemical mechanism of energy transformation and coupling of oxidation and phosphorylation is then suggested based on recent concepts regarding proteins, including ATPases that work as molecular motors, and acidic lipids that act as hydrogen ion (H⁺) carriers. According to this proposed mechanism, the chemical energy of a redox substrate is transformed into nonequilibrium states of electron-transporting chain (ETC) coupling proteins. This leads to nonequilibrium pumping of H⁺ into the membrane. An acidic lipid, cardiolipin, binds with this H⁺ and carries it to the ATP-synthase along the membrane surface. This transport generates gradients of surface tension or electric field along the membrane surface. Hydrodynamic effects on a nanolevel lead to rotation of ATP-synthase and finally to the release of ATP into aqueous solution. This model also explains the generation of a transmembrane protonmotive force that is used for regulation of transmembrane transport, but is not necessary for the coupling of electron transport and ATP synthesis.     

Mass Transfer in the Cornea: I. Interacting Ion Flows in an Arbitrarily Charged Membrane

The formalisms of irreversible thermodynamics are used to describe multi-ionic nonconvective flow through an arbitrarily charged membrane. Interactions between oppositely charged ions are included and are measured by a single phenomenological coefficient. The consequent generalized Nernst-Planck flux equations are integrated to yield a relation between the species fluxes and the composition of the solutions bounding the membrane. It is assumed in the derivation that activity coefficient gradients within the membrane and direct interactions between ions of like charge are negligible. Some special cases are examined. To illustrate the use of the final equations, a single membrane separating solutions of differing composition is modeled, and the effect of ion-ion interactions on the membrane potential and the ion fluxes is demonstrated for several values of diffusion current density and membrane charge density.     

Activity and Stoichiometry of Na+:HCO− 3 Cotransport in Immortalized Renal Proximal Tubule Cells

The proximal tubule Na⁺-HCO⁻ ₃ cotransporter is located in the basolateral plasma membrane and moves Na⁺, HCO⁻ ₃, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO⁻ ₃ anions are transported for each Na⁺ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na⁺ or HCO⁻ ₃ concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl⁻ independent; its conductance ranged between 0.12 and 0.23 mS/cm² and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na⁺ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO⁻ ₃:1Na⁺. Received: 19 January 1996/Revised: 24 April 1996     

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.     

Mechanism of L-glutamate transport in membrane vesicles from Bacillus stearothermophilus.

In the presence of electrochemical energy, several branched-chain neutral and acidic amino acids were found to accumulate in membrane vesicles of Bacillus stearothermophilus. The membrane vesicles contained a stereo-specific transport system for the acidic amino acids L-glutamate and L-aspartate, which could not translocate their respective amines, L-glutamine and L-asparagine. The transport system was thermostable (Ti = 70 degrees C) and showed highest activities at elevated temperatures (60 to 65 degrees C). The membrane potential or pH gradient could act as the driving force for L-glutamate uptake, which indicated that the transport process of L-glutamate is electrogenic and that protons are involved in the translocation process. The electrogenic character implies that the anionic L-glutamate is cotransported with at least two monovalent cations. To determine the mechanistic stoichiometry of L-glutamate transport and the nature of the cotranslocated cations, the relationship between the components of the proton motive force and the chemical gradient of L-glutamate was investigated at different external pH values in the absence and presence of ionophores. In the presence of either a membrane potential or a pH gradient, the chemical gradient of L-glutamate was equivalent to that specific gradient at different pH values. These results cannot be explained by cotransport of L-glutamate with two protons, assuming thermodynamic equilibrium between the driving force for uptake and the chemical gradient of the substrate. To determine the character of the cotranslocated cations, L-glutamate uptake was monitored with artificial gradients. It was established that either the membrane potential, pH gradient, or chemical gradient of sodium ions could act as the driving force for L-glutamate uptake, which indicated that L-glutamate most likely is cotranslocated in symport with one proton and on sodium ion.     

Virtual intermediates in photosynthetic electron transfer.

We explore the possibility of virtual transfer in the primary charge separation of photosynthetic bacteria within the context of several types of experimental data. We show that the peak that might be expected in the virtual rate as electric fields vary the intermediate state energy is severely broadened by coupling to high-frequency modes. The Stark absorption kinetics data are thus consistent with virtual transfer in the primary charge separation. High-frequency coupling also makes the temperature dependence weak over a wide range of parameters. We demonstrate that Stark fluorescence anisotropy data, usually taken as evidence of virtual transfer, can in fact be consistent with two-step transfer. We suggest a two-pulse excitation experiment to quantify the contributions from two-step and virtual transfer. We show that virtual absorption into a charge transfer state can make a substantial contribution to the Stark absorption spectrum in a way that is not related to any derivative of the absorption spectrum.     

Recognition and processing of randomly fluctuating electric signals by Na,K-ATPase.

Previous work has shown that Na,K-ATPase of human erythrocytes can extract free energy from sinusoidal electric fields to pump cations up their respective concentration gradients. Because regularly oscillating waveform is not a feature of the transmembrane electric potential of cells, questions have been raised whether these observed effects are biologically relevant. Here we show that a random-telegraph fluctuating electric field (RTF) consisting of alternating square electric pulses with random lifetimes can also stimulate the Rb(+)-pumping mode of the Na,K-ATPase. The net RTF-stimulated, ouabain-sensitive Rb+ pumping was monitored with 86Rb+. The tracer-measured, Rb+ influx exhibited frequency and amplitude dependencies that peaked at the mean frequency of 1.0 kHz and amplitude of 20 V/cm. At 4 degrees C, the maximal pumping activity under these optimal conditions was 28 Rb+/RBC-hr, which is approximately 50% higher than that obtained with the sinusoidal electric field. These findings indicate that Na,K-ATPase can recognize an electric signal, either regularly oscillatory or randomly fluctuating, for energy coupling, with high fidelity. The use of RTF for activation also allowed a quantitative theoretical analysis of kinetics of a membrane transport model of any complexity according to the theory of electroconformational coupling (ECC) by the diagram methods. A four-state ECC model was shown to produce the amplitude and the frequency windows of the Rb(+)-pumping if the free energy of interaction of the transporter with the membrane potential was to include a nonlinear quadratic term. Kinetic constants for the ECC model have been derived.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modeling electrically active viscoelastic membranes

The membrane protein prestin is native to the cochlear outer hair cell that is crucial to the ear's amplification and frequency selectivity throughout the whole acoustic frequency range. The outer hair cell exhibits interrelated dimensional changes, force generation, and electric charge transfer. Cells transfected with prestin acquire unique active properties similar to those in the native cell that have also been useful in understanding the process. Here we propose a model describing the major electromechanical features of such active membranes. The model derived from thermodynamic principles is in the form of integral relationships between the history of voltage and membrane resultants as independent variables and the charge density and strains as dependent variables. The proposed model is applied to the analysis of an active force produced by the outer hair cell in response to a harmonic electric field. Our analysis reveals the mechanism of the outer hair cell active (isometric) force having an almost constant amplitude and phase up to 80 kHz. We found that the frequency-invariance of the force is a result of interplay between the electrical filtering associated with prestin and power law viscoelasticity of the surrounding membrane. Paradoxically, the membrane viscoelasticity boosts the force balancing the electrical filtering effect. We also consider various modes of electromechanical coupling in membrane with prestin associated with mechanical perturbations in the cell. We consider pressure or strains applied step-wise or at a constant rate and compute the time course of the resulting electric charge. The results obtained here are important for the analysis of electromechanical properties of membranes, cells, and biological materials as well as for a better understanding of the mechanism of hearing and the role of the protein prestin in this mechanism.     

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Membranes, calcium, and coupling

Every eukaryotic cell contains systems linking the extracellular space and internal membrane compartments. These systems allow cells to communicate and, ultimately they allow the nervous system to control most of the cytoplasmic activity. In skeletal muscle, this system is called "excitation-contraction coupling." While much is known of the early and late steps in coupling, the critical link between the cell (i.e., here the T system) membrane and sarcoplasmic reticulum membrane is not known. Electrical coupling cannot easily account for experimental results; here we show that the Ca2+ influx is not causally related to the excitation-contraction coupling. The most likely mechanism seems to be a variant of the "remote control model" in which a voltage change and accompanying charge movement in the T membrane activates an enzyme tethered to the cytoplasmic leaflet of the T membrane but spanning part of the T--sarcoplasmic reticulum gap.     

Chemical reactions and membranes: A macroscopic basis for active transport II. Non-linear aspects

The physical principles that material and charge are conserved provide a basis for the design of a membrane capable of performing active ion transport in which the connection between “metabolic energy” input and the ion transport process itself is electrical rather than material. Molecular interactions between components in this system are irrelevant to its function. A second model built on the same principles performs active ion transport in a statically symmetrical membrane. The basis of its operation is a weakly stable unsymmetrical concentration profile arising from an enzyme-catalyzed reaction occurring within the membrane. The function of this membrane is irreversibly terminated (“killed”) by interference either with intramembrane concentration gradients or with the reactions which maintain them. Hence, any attempt to study this system by breaking it apart destroys the basis of its function. The existence of these models reveals the logical insufficiency of the molecular biologist's approach to understanding the basis of active transport: Neither the experimental techniques for structure determination (disruption, purification, characterization, and reconstitution) nor the fundamental question of that discipline (What is the molecular connection between &*|… ?) of the molecular biologist are applicable to the study or interpretation of these model systems. While the model systems are artificial, they incorporate only widely applicable concepts of physical chemistry and biochemical kinetics. The is no reason for excluding such mechanisms in natural membrane transport systems. If they are present, then more effective strategies of investigation will be required.     

A new double-chamber model of ion channels. Beyond the Hodgkin and Huxley model

This paper proposes a new double-chamber model (DCM) of ion channels. The model ion channel consists of a series of three pores alternating with two chambers. The chambers are net negatively charged. The chamber's electric charge originates from dissociated amino acid side chains and is pH dependent. The chamber's net negative charge is compensated by cations present inside the chamber and in a diffuse electric layer outside the chamber. The pore's permeability is constant independent of time. One pore of the sodium channel and one of the potassium channel is a voltage-sensing pore. Due to the channel's structure, ions flow through the pores and chambers in a time-dependent manner. The model reproduces experimental voltage clamp and action potential data. The current flowing through a single sodium channel is less then one femtoampere. The DCM is considerably simpler then the Hodgkin and Huxley model (HHM) used to describe the electrophysiological properties of an axon. Unlike the HHM, the DCM can explain refractoriness, anode break excitation, accommodation and the effect of pH and temperature on the channels without additional parameters. In the DCM, the axon membrane shows repetitive activity depending on the channel density, sodium to potassium channel ratio and external potassium concentration. In the DCM, the action potential starts from 'hot spot areas' of higher channel densities and a higher sodium to potassium channel ratio, and then propagates through the whole axon.     

Mechanism of transport and storage of neurotransmitters

This review will focus on the bioenergetics, mechanism, and molecular basis of neurotransmitter transport. As indicated in the next section, these processes play an important role in the overall process of synaptic transmission. During the last few years, direct evidence has been obtained that these processes are coupled chemiosmotically, i.e., the accumulation of neurotransmitters is driven by ion gradients. Two types of neurotransmitter transport systems have been identified: sodium-coupled systems located in the synaptic plasma membrane of nerves (and sometimes in the plasma membrane of glial cells) and proton-coupled systems which are part of the membrane of intracellular storage organelles. From a bioenergetic point of view, the sodium-coupled systems are especially interesting, since it has recently been discovered that many systems require other ions in addition to sodium. It has now been demonstrated in several cases that, besides sodium ions, these additional ions, such as chloride and potassium, serve as additional coupling ions. These systems will be reviewed here in considerable detail with emphasis on the role of the additional ions. In the second part of the review we shall focus on neurotransmitter transport into storage organelles. Although both sodium and proton coupled systems have been reviewed in the past, there has been a shift from a kinetic and thermodynamic to a biochemical approach. In fact, a few transporters have been identified and functionally reconstituted. These developments have of course been incorporated in this review.     

Electrokinetic membrane processes in relation to properties excitable tissues. II. Some theoretical considerations

A quantitative theory is presented for the behavior of a membrane-electrolyte system subject to an electric current flow (the "membrane oscillator"). If the membrane is porous, carries "fixed charges," and separates electrolyte solutions of different conductances, it can be the site of repetitive oscillatory changes in the membrane potential, the membrane resistance, and the hydrostatic pressure difference across the membrane. These events are accompanied by a pulsating transport of bulk solutions. The theory assumes the superposition of electrochemical and hydrostatic gradients and centers round the kinetics of resistance changes within the membrane, as caused by effects from diffusion and electro-osmotic fluid streaming. The results are laid down in a set of five simple, basic expressions, which can be transformed into a pair of non-linear differential equations yielding oscillatory solutions. A graphical integration method is also outlined (Appendix II). The agreement between the theory and previous experimental observations is satisfactory. The applied electrokinetic concepts may have importance in relation to analyses of the behavior of living excitable cells or tissues.