Conductance In Lipid Bilayers Research Articles

Functional assembly of gap junction conductance in lipid bilayers: Demonstration that the major 27 kd protein forms the junctional channel

Gap junctions isolated from rat liver were incorporated into planar lipid bilayers. A channel activity that was directly dependent on voltage was recorded. Changes of pH and (Ca 2+) had no direct effect on channel activity; however, they modulated the voltage-dependent gating of the gap junction channels differently. Single-channel fluctuations showed large scatter with peak amplitudes of 140 and 280 picoSiemmens in 0.1 M NaCl. The major protein of gap junctions (M r of 27 kd) was also reconstituted into bilayers, giving channel properties similar to those of intact gap junctions. Polyclonal antibodies specific for this protein caused inhibition of the junctional conductance in bilayers. These data provide direct evidence that the 27 kd protein is the molecular species responsible for gap junction communication between cells.

Alamethicin induced conductances in lipid bilayers: Part II. Decomposition of the observed (compound) process

The formalism required for deriving probabilities of the states of a single (elementary) pore, formed by the action of the polypeptide alamethicin in a lipid bilayer membrane, from the observed states of a single (elementary) pore from the observed probabilities of the compound states of the ensemble of pores is developed. Although the analysis has been developed in the context of the evaluation of the statistical properties of pore formation in lipid bilayers, alamethicin being the pore former, the treatment is in fact generally applicable to all situations where the modelling is most readily cast in terms of the behaviour of a single elementary system whilst the experiment deals with ensembles of such systems. The application of the formalism for the steady state is illustrated in the succeeding paper; empirical extensions to the rate processes are also illustrated.

Alamethicin induced conductances in lipid bilayers: Part III. Derivation of the properties of the elementary process from the (compound) observed process

The formalism for the derivation of the probabilities of the states of a single (elementary) pore from the observed probabilities of the compound states of the ensemble of pores, developed in the preceding paper, is extended to cover the transition probabilities between states of the elementary pore. These procedures have been applied to the voltage controlled pore formation in lipid bilayers under the action of alamethicin. Drift in this system is shown to be due to the dependence of the trigger rate for pore creation/destruction on age. The properties of the elementary process are independent of age; the elementary process exhibits an upper reflecting state (in a statistical sense).

Membrane actions of male contraceptive gossypol tautomers

The role of different gossypol tautomers in the interaction of this molecule with membranes has been investigated using the isolated hemiacetal moiety of gossypol and the pH dependency of the keto-enol tautomeric equilibrium. Our results indicate that: (a) the actions of the hemiacetal tautomer cannot explain the effects of gossypol on mitochondrial oxidative phosphorylation, lipid membrane interfacial potentials, and proton conductance of lipid bilayers; (b) the enolate forms of gossypol are the species that bind to the membrane interface and decrease the electrostatic interfacial potential; and (c) the uncharged (keto and/or enol) species in equilibrium with the enolate forms of gossypol give the molecule the ability to carry protons across biological membranes.

The measurement and interpretation of fluctuations in electrochemical systems: The electrocrystallisation of silver and the voltage-gated alamethicin induced channel conductance in lipid bilayers

The analyses of fluctuations due to the voltage-gated alamethicin induced channel conductance in lipid bilayers and due to the electrolystallisation of silver are developed further. For the first system it is shown that it is possible to decompose the experimentally observed compound system (many pores) into the behaviour of the elementary system (a single pore) together with a time-dependent trigger rate; the elementary system is confined by an upper and lower reflecting state and the edge and bulk energies of the pore are shown to be independent of time. For the second system it is shown that lattice formation takes place at the edges of circular growth centres randomly nucleated in space on the substrate electrode. Measurements of quantities related to the second moment of the time dependent reaction rate (together with measurements of the mean rate) lead to independent estimates of the rates of nucleation and of crystal growth, of the mean lifetime of growth centres and of the form of the mean current-time transient due to the growth of single centres. At low overpotentials and long times nucleation follows a Poisson distribution; at short times and at higher overpotentials nucleation has the character of a delayed renewal process. Other complex and as yet unexplained changes with time of the rates of nucleation (and of crystal growth) are observed at long times at higher overpotentials.

Dipole moment of alamethicin as related to voltage-dependent conductance in lipid bilayers

The dipole moment of alamethicin, which produces voltage-dependent conductance in lipid-bilayer membranes, was measured in mixed solvents of ethanol and dioxane. The value of the dipole moment was found to increase from 40 to 75 DU (Debye units), as the concentration of ethanol increased from 0 (pure dioxane) to 40%. The relaxation frequency of alamethicin also changes from 10 to 40 MHz, depending upon the concentration of ethanol in mixed solvents. The length of alamethicin was calculated by using the relaxation time and was found to range from approximately 40 to 20 A. The dipole moment was independently calculated from voltage-dependent conductance and compared with the measured value. The calculated value was found to be larger than the value of direct measurements, indicating that several alamethicin molecules are required to form a conducting pore and that their dipole moments are oriented parallel to each other.

Structure-activity relationships of the B fragment of diphtheria toxin: The lipid-binding domains

Two different lipid-associating domains have previously been identified in diphtheria toxin fragment B: one of the surface type in the N-terminus of B and one of the transverse type in its middle region. We have now determined about 85% of the primary structure of fragment B and show, here, that the middle part of fragment B contains a highly hydrophobic region of 72 amino acid residues (polarity index: 29%) which includes the transverse lipid-associating domain. That this domain is actually involved in a process of membrane penetration is suggested by lipid bilayer conductance measurements of the CNBr peptides of fragment B and trypsin treatment of fragment B-multilamellar liposome complexes.

Alamethicin-induced conductances in lipid bilayers: I. Data analysis and simple steady-state model.

Conductance flucutations induced by low concentrations of alamethicin in planar lipid bilayer membranes have been examined with a novel computer-aided analysis. A precise test is applied to show that the system is Markovian (except for the zeroth state) and an estimate is given for the (small) number of transitions between nonadjacent states: the system closely conforms to a birth-and-death process. The complete set of rate parameters governing the steady-state distribution is derived, and it is shown that these parameters can be used to reconstruct exactly the experimental relative frequency distribution. It is also shown that the electrochemical free energies of the conductance states vary quadratically with state number for low-lying states; the free energies of activation of both the birth and death processes are linearly related to the free-energy differences between states but the transfer coefficient is close to unity. A detailed model based on the nucleation of a two-dimensional pore accounts for these observations provided both birth and death take place via an intermediate expansion of the pore lumen. This model requires two energy parameters, the "edge" and "bulk" energies of the pore, together with a trigger rate of the initial process which is a sufficient description for the steady-state behavior of this voltage-controlled system.

Effects of unstirred layers on the steady-state zero-current conductance of bilayer membranes mediated by neutral carriers of ions.

Some effects of diffusion polarization and chemical reactions on the steady-state zero-current conductance of lipid bilayers mediated by neutral carriers of ions have been studied theoretically and experimentally. Assuming that ion permeation across the interfaces occurs via a heterogeneous reaction between ions in the solution and carriers in the membrane, the relationship between the conductance and the aqueous concentration of carriers is shown to be linear only in a limited range of sufficiently low concentrations. At higher carrier concentrations, which for the most strongly bound cations are within the range of the experimentally accessible values, the conductance is expected to become limited by diffusion of the carried ion in the unstirred layers and therefore reach an upper limiting value independent of the membrane properties. This expectation has been successfully verified for glyceryl-monooleate membranes in the presence of the ions K+, Rb+ and NH+4 and carriers such as valinomycin and trinactin. The experimental results support, at least for the present system, the generally accepted view that complexation between ions and the macrocyclic antibiotics occurs at the membrane surface; it is shown, in fact, that for a different mechanism, such as that by which the complexes would form in the aqueous solutions and cross the interfaces as lipid-soluble ions, the same type of saturation would be expected to be observable only for unrealistically high values of the rate constants of the ion-carrier association. A previously proposed criterion to distinguish between these two mechanisms, based on the dependence of the conductance on the ion concentration, is discussed from the viewpoint of this more comprehensive model.

Conductance and voltage-current relationship of lipid bilayers in the presence of TTFB

The aim of this work was to discover the mechanism of lipid bilayer conductance in the presence of one of the most effective uncouplers of oxidative phosphorylation, TTFB. Current clamp data and dependences of membrane conductance on pH and on uncoupler concentration were used for the determination of the charge-transfer-limiting-step. It was proved that the uncoupler dimer-anion takes partin the charge transfer through the inner hydrophobic layer of the membrane. In the presence of Cu 2+ ions in aqueous solutions, lipid bilayers with TTFB show N-shaped steady state voltage-current characteristics. It was shown that changes in pH and in uncoupler concentration in unstirred aqueous layers near the membrane, may be a reason for the development of this shape of the voltage-current curves.

The gramicidin A transmembrane channel: a proposed pi(L,D) helix.

A lipophilic, left-handed helical structure is proposed for gramicidin A in which the C-O bonds alternately point toward the amino and carboxyl ends; it is a hybrid of the 4.3(14) and 4.4(16) helices. The C-O groups pointing toward the carboxyl end form part of 16-membered hydrogen-bonded rings, whereas the C-O moieties pointing toward the amino end form 14-membered hydrogenbonded rings. The proposed structure is based on conformational analysis combined with requirements for the gramicidin A transmembrane channel. Two helices combine to form the channel. The alternating C-O directions allow hydrogen-bonded dimerization by the unique possibilities of head-to-head and tail-to-tail attachment. The formyl group at the amino end allows for a favorable head-to-head attachment with no loss of structural continuity. Unpublished studies. by M. C. Goodall on the lipid bilayer conductance of deformyl gramicidin A strongly argue for head-to-head attachment. Such hydrogen-bonded association is not possible with previously described helices, as the C-O groups all point in the same direction. In relation to possible pi((L,D)) helices in mammalian systems, it should be noted that glycines would fill the role of D residues. The conformation can undergo ion-induced relaxations, which provide approximate tetrahedral coordination for the ion, with facile shifting of coordinations. The ready exchange of coordinations provides the mechanism for movement of the ion along the channel. Conceivably, such transmembrane channels could have application as models for ion transport across biological membranes-an application which may be as great as, or greater than, that of carriers such as valinomycin and nonactin. Specifically, biogenic amines and drugs containing aromatic groups could control access to the channel by interactions with the two tryptophan residues at the ethanolamine end and with the negative region provided by the three oxygens.