Kinetics of the Opening and Closing of Individual Excitability-Inducing Material Channels in a Lipid Bilayer

The kinetics of the opening and closing of individual ion-conducting channels in lipid bilayers doped with small amounts of excitability-inducing material (EIM) are determined from discrete fluctuations in ionic current. The kinetics for the approach to steady-state conductance during voltage clamp are determined for lipid bilayers containing many EIM channels. The two sets of measurements are found to be consistent, verifying that the voltage-dependent conductance of the many-channel EIM system arises from the opening and closing of individual EIM channels. The opening and closing of the channels are Poisson processes. Transition rates for these processes vary exponentially with applied potential, implying that the energy difference between the open and closed states of an EIM channel is linearly proportional to the transmembrane electric field. A model incorporating the above properties of the EIM channels predicts the observed voltage dependence of ionic conductance and conductance relaxation time, which are also characteristic of natural electrically excitable membranes.     

Chloride Transport in Porous Lipid Bilayer Membranes

This paper describes dissipative Cl_- transport in "porous" lipid bilayer membranes, i.e., cholesterol-containing membranes exposed to 1–3 x 10^-7 M amphotericin B. P_DCl (cm·s^-1), the diffusional permeability coefficient for Cl^-, estimated from unidirectional ³⁶Cl^- fluxes at zero volume flow, varied linearly with the membrane conductance (Gm, Ω^-1·cm^-2) when the contributions of unstirred layers to the resistance to tracer diffusion were relatively small with respect to the membranes; in 0.05 M NaCl, P_DCl was 1.36 x 10^-4 cm·s^-1 when Gm was 0.02 Ω^-1·cm^-2. Net chloride fluxes were measured either in the presence of imposed concentration gradients or electrical potential differences. Under both sets of conditions: the values of P_DCl computed from zero volume flow experiments described net chloride fluxes; the net chloride fluxes accounted for ~90–95% of the membrane current density; and, the chloride flux ratio conformed to the Ussing independence relationship. Thus, it is likely that Cl^- traversed aqueous pores in these anion-permselective membranes via a simple diffusion process. The zero current membrane potentials measured when the aqueous phases contained asymmetrical NaCl solutions could be expressed in terms of the Goldman-Hodgkin-Katz constant field equation, assuming that the P_DNa/P_DCl ratio was 0.05. In symmetrical salt solutions, the current-voltage properties of these membranes were linear; in asymmetrical NaCl solutions, the membranes exhibited electrical rectification consistent with constant-field theory. It seems likely that the space charge density in these porous membranes is sufficiently low that the potential gradient within the membranes is approximately linear; and, that the pores are not electrically neutral, presumably because the Debye length within the membrane phase approximates the membrane thickness.     

The Nature of the Negative Resistance in Bimolecular Lipid Membranes Containing Excitability-Inducing Material

When sufficiently small amounts of excitability-inducing material (EIM) are added to a bimolecular lipid membrane, the conductance is limited to a few discrete levels and changes abruptly from one level to another. From our study of these fluctuations, we have concluded that the EIM-doped bilayer contains ion-conducting channels capable of undergoing transitions between two states of different conductance. The difference in current between the "open" and "closed" states is directly proportional to the applied membrane potential, and corresponds to a conductance of about 3 x 10^-10 ohm^-1. The fraction of the total number of channels that is open varies from unity to zero as a function of potential. The voltage-dependent opening and closing of channels explains the negative resistance observed for bimolecular lipid membranes treated with greater amounts of EIM.     

Impedance Analysis of Ion Transport Through Supported Lipid Membranes Doped with Ionophores: A New Kinetic Approach

Kinetics of facilitated ion transport through planar bilayer membranes are normally analyzed by electrical conductance methods. The additional use of electrical relaxation techniques, such as voltage jump, is necessary to evaluate individual rate constants. Although electrochemical impedance spectroscopy is recognized as the most powerful of the available electric relaxation techniques, it has rarely been used in connection with these kinetic studies. According to the new approach presented in this work, three steps were followed. First, a kinetic model was proposed that has the distinct quality of being general, i.e., it properly describes both carrier and channel mechanisms of ion transport. Second, the state equations for steady-state and for impedance experiments were derived, exhibiting the input–output representation pertaining to the model’s structure. With the application of a method based on the similarity transformation approach, it was possible to check that the proposed mechanism is distinguishable, i.e., no other model with a different structure exhibits the same input–output behavior for any input as the original. Additionally, the method allowed us to check whether the proposed model is globally identifiable (i.e., whether there is a single set of fit parameters for the model) when analyzed in terms of its impedance response. Thus, our model does not represent a theoretical interpretation of the experimental impedance but rather constitutes the prerequisite to select this type of experiment in order to obtain optimal kinetic identification of the system. Finally, impedance measurements were performed and the results were fitted to the proposed theoretical model in order to obtain the kinetic parameters of the system. The successful application of this approach is exemplified with results obtained for valinomycin–K⁺ in lipid bilayers supported onto gold substrates, i.e., an arrangement capable of emulating biological membranes.     

Transmembrane Lipid Migration in Planar Asymmetric Bilayer Membranes

Planar asymmetric bilayer membranes, formed by apposing a monolayer of the neutral lipid glyceroldioleate (GDO) with one of the negatively charged lipid oleyl acid phosphate (OAP), were used to measure the rate of transmembrane OAP migration. The assay for this lipid flip-flop was the interaction of Ca²⁺ ions with negatively charged lipids which causes membranes to break: when Ca²⁺ is added to the compartment limited initially by the neutral lipid, flip-flop of the charged lipid eventually results in membrane breakdown. At 22 ± 2°C, in the absence of an externally applied electric field, an upper limit to the half time of OAP flip-flop was measured as 18.7 h, with a tentative lower limit of 14.4 h.     

Asymmetrical Lipid Bilayer Structure for Biological Membranes

IT is generally accepted that the matrix of cellular membranes is a bimolecular leaflet of phospholipid molecules in which the phospholipids are oriented so that their polar heads reside on the outer surfaces of the bilayer, in contact with the aqueous environment, the interior of the sandwich being composed of hydrophobic lipid chains^1–5. To this basic structure proteins cholesterol, glycolipids and other molecules are usually inserted in such a way as to confer on the bilayer the functional properties appropriate for the particular membrane.     

Voltage-Gated Lipid Ion Channels

Synthetic lipid membranes can display channel-like ion conduction events even in the absence of proteins. We show here that these events are voltage-gated with a quadratic voltage dependence as expected from electrostatic theory of capacitors. To this end, we recorded channel traces and current histograms in patch-experiments on lipid membranes. We derived a theoretical current-voltage relationship for pores in lipid membranes that describes the experimental data very well when assuming an asymmetric membrane. We determined the equilibrium constant between closed and open state and the open probability as a function of voltage. The voltage-dependence of the lipid pores is found comparable to that of protein channels. Lifetime distributions of open and closed events indicate that the channel open distribution does not follow exponential statistics but rather power law behavior for long open times.     

The Properties of Ion Channels Formed in Bilayer Lipid Membranes by Amphotericin and N-Methyl Derivative of Amphotericin under Their Action on One Side

It was found that the modification of one side of lipid membranes by amphotericin B and N?methyl derivatives of amphotericin B (methamphocin) resulted in a discrete increase in the membrane conductivity by the channel mechanism. The conditions under which amphotericin B increased the conductivity of membranes upon addition on one side of the membranes were found. The effect of amphotericin B upon addition on one side of the membranes was observed in an acidic medium (pH 3.0) and at a two-fold lower concentration of phospholipids in the membrane-forming solution. A large dispersion of the conductivity from 2 to 20 pS of single channels was revealed. The channels with the conductivity of 10 pS were most likely to occur. The histogram of distribution of the conductivity of metamphocin channels showed that the channels with the conductivity of 5 pS were most likely to occur. The selective permeability of membranes upon addition of methamphocin on one side of the membranes was predominantly anionic and did not depend on the concentration of cholesterol in the membranes. The mechanism of the amphotericin B and methamphocin action from one side of the membranes was due to the formation of semipores in the membranes, which were asymmetric in their structure. It was assumed that the selective permeability of the amphotericin and metamphocin channels was determined by the molecular structure of the hydrophilic chain that lines the inner cavity of the semipore.

     

The Rate Constants of Valinomycin-Mediated Ion Transport through Thin Lipid Membranes

Electrical relaxation experiments have been performed with phosphatidylinositol bilayer membranes in the presence of the ion carrier valinomycin. After a sudden change of the voltage a relaxation of the membrane current with a time constant of about 20 μsec is observed. Together with previous stationary conductance data, the relaxation amplitude and the relaxation time are used to evaluate the rate constants of valinomycin-mediated potassium transport across the lipid membrane. It is found that the rate constants of translocation of the free carrier S and the carrier-ion complex MS⁺ are nearly equal (2·10⁴ sec^-1) and are of the same order as the dissociation rate constant of MS⁺ in the membrane-solution interface (5·10⁴ sec^-1). The equilibrium constant of the heterogeneous association reaction M⁺ (solution) + S (membrane) → MS⁺ (membrane) is found to be ~ 1 M^-1, about 10⁶ times smaller than the association constant in ethanolic solution.     

Association of Model Neurotransmitters with Lipid Bilayer Membranes

Aimed at reproducing the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in a standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neurotransmitter adsorption to the bilayer—a process not considered in the established models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling of neurotransmitters via their membrane interaction. Experimental verification has been difficult because binding affinities of neurotransmitters to the lipid bilayer are low. We quantify this interaction with surface plasmon resonance to measure equilibrium dissociation constants in neurotransmitter membrane association. Neutron reflection measurements on artificial membranes, so-called sparsely tethered bilayer lipid membranes, reveal the structural aspects of neurotransmitters’ association with zwitterionic and anionic bilayers. We thus establish that serotonin interacts nonspecifically with the membrane at physiologically relevant concentrations, whereas γ-aminobutyric acid does not. Surface plasmon resonance shows that serotonin adsorbs with millimolar affinity, and neutron reflectometry shows that it penetrates the membrane deeply, whereas γ-aminobutyric is excluded from the bilayer.     

Permeability of Lipid Bilayer Membranes to Organic Solutes

A sensitive fluorescence technique was used to measure transport of organic solutes through lipid bilayer membranes and to relate permeability to the functional groups of the solute, lipid composition of the membrane, and pH of the medium. Indole derivatives having ethanol, acetate, or ethylamine in the 3-position, representing neutral, acidic, and basic solutes, respectively, were the primary models. The results show: (a) Neutral solute permeability is not greatly affected by changes in lipid composition but presence or absence of cholesterol in the membranes could greatly alter permeability of the dissociable substrates. (b) Indole acetate permeability was reduced by introduction of phosphatidylserine into membranes to produce a net negative charge on the membranes. (c) Permeability response of dissociable solutes to variation in pH was in the direction predicted but not always of the magnitude expected from changes in the calculated concentrations of the undissociated solute in the bulk aqueous phase. Concentration gradients of amines across the membranes caused substantial diffusion potentials, suggesting that some transport of the cationic form of the amine may occur. It is suggested that factors such as interfacial charge and hydration structure, interfacial polar forces, and lipid organization and viscosity, in addition to the expected solubility-diffusion relations, may influence solute flux.     

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.     

Oligomeric Structure of Colicin Ia Channel in Lipid Bilayer Membranes

Colicin Ia is a soluble, harpoon-shaped bacteriocin which translocates across the periplasmic space of sensitive Escherichia coli cell by parasitizing an outer membrane receptor and forms voltage-gated ion channels in the inner membrane. This process leads to cell death, which has been thought to be caused by a single colicin Ia molecule. To directly visualize the three-dimensional structure of the channel, we generated two-dimensional crystals of colicin Ia inserted in lipid-bilayer membranes and determined a ∼17 three-dimensional model by electron crystallography. Supported by velocity sedimentation, chemical cross-linking and single-particle image analysis, the three-dimensional structure is a crown-shaped oligomer enclosing a ∼35 Å-wide extrabilayer vestibule. Our study suggests that lipid insertion instigates a global conformational change in colicin Ia and that more than one molecule participates in the channel architecture with the vestibule, possibly facilitating the known large scale peptide translocation upon channel opening.Colicin Ia is a pore-forming water-soluble bacterial toxin produced by some strains of Escherichia coli to kill other competing bacteria (1, 2). It belongs to a functionally and structurally similar group of proteins that also includes colicins A (3), E1 (4), and N (5). Each of these proteins consist of three domains with distinct properties; the receptor domain (R), which binds a specific outer membrane receptor on the target cell, and the translocation domain (T) at the N terminus, responsible for traversing the outer membrane and the periplasmic space to deliver the channel-forming domain (C) at the C terminus to the bacterial inner membrane. The bundle of 10 α-helices that compose the C domain changes its conformation to form a voltage-gated ion channel in the plasma membrane. Opening of the channel produces an efflux of ions that depletes the cellular energy resources and ultimately leads to cell death.The x-ray structure of full-length, soluble colicin Ia (69 kDa) has been determined (6). The monomeric molecule is mostly α-helical, with the R domain separated from the T and C domains by a pair of unusually long (∼160 Å) α-helices thought possibly to span the periplasmic space during channel formation (6). The C domain is characterized by two hydrophobic helices (VIII and IX; residues Ala-580—Ile-612) that is surrounded by the remaining eight largely amphipathic α-helices. The same structural motif for the C domain is conserved in other members of the colicin family and is also present in the channel-forming domains of diphtheria toxin, exotoxin A, and the Bcl family of pro- and anti-apoptotic proteins (7). This pair of helices, termed the hydrophobic hairpin, is instrumental in driving the initial membrane insertion event (8) that is followed by a series of large scale pH and voltage-dependent conformational changes in the C domain, resulting in the opening of the ion channel in the plasma membrane (9, 10). In the absence of a high resolution membrane-inserted structure of a channel-forming colicin, solid-state NMR (11, 12), streptavidin binding (8) and cross-linking of site-directed cysteine mutants (9) have suggested that the initial membrane-bound intermediate exists as a two-dimensional helical array of the eight amphipathic helices (I-VII and X) spread across the membrane surface, with the hydrophobic helices (VIII and IX) embedded in the bilayer. A recent electron paramagnetic resonance study using preparations of spin-labeled ColA proteoliposomes has supported a similar umbrella model where the eight amphipathic helices reside at the air-water interface for the closed-channel state (13). Biotin-labeled cysteine mutants have also been used to determine how much of the C domain (aside from the hydrophobic hairpin) crosses the plasma membrane (14, 15) for colicin Ia. A large region of the amphipathic sequence (helices II-V; residues Leu-474—Tyr-541) has been found to cross from the cis to the trans side of the membrane in planar lipid bilayer experiments, resulting in a four-transmembrane segment molecule that is thought to form the ion channel.Because the 12–13 residue α-helices of the C domain are well short of the ∼20 residues required to span the plasma membrane, it has been proposed that conformational changes causing helix extension take place during the channel formation process. ¹³C spin diffusion NMR has indicated that whereas the overall secondary structure of the C domain is preserved, most of the helices undergo “opening,” and modulation of the tertiary structure allows for the required extension of the helices to cross the plasma membrane and form the channel (16). The internal structure of the colicin Ia channel has been investigated by examining the effect of different nonelectrolyte molecules on the single-channel conductance in planar lipid bilayer membranes (17). It was determined that the diameter at the cis entrance (equivalent to the outside of the cell) is 18 Å, and the diameter at the trans entrance (inside the membrane) is 10 Å, with a 7 Å diameter constriction located in close proximity to the trans entrance of the channel. More recent studies (18) employing the substituted cysteine accessibility method to determine what residues line the open colicin Ia channel suggest an hourglass-shaped pore with the most constricted part near the cis rather than the trans side, as opposed to the conclusion of Krasilnikov et al. (17). Both studies point to a pore constriction inside the membrane, and as pointed out by Kienker et al. 18), there exist plausible explanations to reconcile some of the differing results. The large diameter of the colicin Ia channel coupled with the studies which indicate that each colicin Ia molecule contributes four transmembrane segments in the membrane integrated state (14) suggests that the ion channel is formed by a multimer of colicin Ia molecules. However, all of the past studies directed at determining the oligomeric state of any of the colicin channels indicate a monomeric structure. The question as to how a four-transmembrane monomeric protein can form an ion channel of sufficient diameter to allow the passage of ions as large as tetraethyl ammonium (19) has remained unanswered.In this work we have subjected colicin Ia incorporated into lipid bilayer membranes to structural and biochemical investigations. We show, based on cross-linking and velocity sedimentation experiments, single-particle analysis of electron micrographs and results from electron crystallographic analysis of two-dimensional crystals of colicin Ia that the protein forms oligomers upon insertion into the bilayer. The suggested architecture of this oligomer based on the ∼17 Å resolution three-dimensional model and the biological implications, are discussed.     

Measuring Ion Channels on Solid Supported Membranes

Application of solid supported membranes (SSMs) for the functional investigation of ion channels is presented. SSM-based electrophysiology, which has been introduced previously for the investigation of active transport systems, is expanded for the analysis of ion channels. Membranes or liposomes containing ion channels are adsorbed to an SSM and a concentration gradient of a permeant ion is applied. Transient currents representing ion channel transport activity are recorded via capacitive coupling. We demonstrate the application of the technique to liposomes reconstituted with the peptide cation channel gramicidin, vesicles from native tissue containing the nicotinic acetylcholine receptor, and membranes from a recombinant cell line expressing the ionotropic P2X₂ receptor. It is shown that stable ion gradients, both inside as well as outside directed, can be applied and currents are recorded with an excellent signal/noise ratio. For the nicotinic acetylcholine receptor and the P2X₂ receptor excellent assay quality factors of Z′ = 0.55 and Z′ = 0.67, respectively, are obtained. This technique opens up new possibilities in cases where conventional electrophysiology fails like the functional characterization of ion channels from intracellular compartments. It also allows for robust fully automatic assays for drug screening.     

Coupling of Solute and Solvent Flows in Porous Lipid Bilayer Membranes

The present experiments were designed to evaluate coupling of water and nonelectrolyte flows in porous lipid bilayer membranes (i.e., in the presence of amphotericin B) in series with unstirred layers. Alterations in solute flux during osmosis, with respect to the flux in the absence of net water flow, could be related to two factors: first, changes in the diffusional component of solute flux referable to variations in solute concentrations at the membrane interfaces produced by osmotic flow through the unstirred layers; and second, coupling of solute and solvent flows within the membrane phase. Osmotic water flow in the same direction as solute flow increased substantially the net fluxes of glycerol and erythritol through the membranes, while osmotic flow in the opposite direction to glycerol flow reduced the net flux of that solute. The observed effects of osmotic water flow on the fluxes of these solutes were in reasonable agreement with predictions based on a model for coupling of solute and solvent flows within the membrane phase, and considerably in excess of the prediction for a diffusion process alone.     

Controlling and Measuring Local Composition and Properties in Lipid Bilayer Membranes

Local composition, structure, morphology, and phase are interrelated in lipid bilayer membranes. This gives us the opportunity to control one or more of these properties by manipulating others. We investigate theserelationships with combinations of simultaneous two-color widefield fluorescence imaging, three-dimensional rendering of vesicle domains, andmanipulation of the vesicle morphology via optical trapping and micropipetteaspiration. We describe methods to modulate, to measure, and to probe thelocal structure of model membranes through control of membrane curvature inliposomes.     

Platinum-supported Bilayer Lipid Membranes modified by ferrocene and its derivatives

The biferrocene-containing Schiff base complexes (1) and (2) were synthesized and characterized by elemental analyses and spectral data. The Pt-supported Bilayer Lipid Membranes (BLMs) modified by ferrocene and its derivatives were studied by cyclic voltametry (CV) and the electrochemical properties of this system are reported. The oxidation mechanism of electrocatalysis of ascorbic acid on the Pt-supported BLMs is discussed.     

Measurement of Dipole Potential in Bilayer Lipid Membranes by Dielectric Spectroscopy

Planar bilayer lipid membranes formed from egg phosphatidylcholine in aqueous media containing the lipophilic anion, dipicrylamine (DPA), were studied by dielectric spectroscopy over a frequency range of 10 Hz–10 MHz. The membranes showed dielectric relaxation due to the translocation of DPA between the membrane interfaces. Incorporating either cholesterol or 6-ketocholestanol into the membranes increased the characteristic frequency of the relaxation, which is proportional to the translocation rate constant of DPA. The results suggested that the sterol dipoles induced positive potential changes within the membrane interior. The changes of the dipole potential were 70 mV for cholesterol and 150 mV for 6-ketocholestanol when the sterol mole fraction was 0.67. The opposite effect was caused by phloretin added to the aqueous media, and the maximum dipole potential change was ?90 mV at 100 μM.