D.A. Haydon

Ion transfer across lipid membranes in the presence of gramicidin A: I. Studies of the unit conductance channel

The conductance induced by gramicidin A in lipid bilayer membranes has been shown to be made up of discrete, well-defined units. In 0.1 M NaCl, and for 100 mV applied, the integral conductance of the unit channel at 20 °C is 5.8·10−12 Ω−1. The channels are formed by transitions involving inactive gramicidin molecules already in the membrane. The precise nature of a transition is not certain, but circumstantial evidence suggests that the final conducting structure consists of at least two polypeptide molecules. From the temperature coefficient of the average duration of the channels it has been shown that the activation energy for channel closure must be ≳ 19 kcal mole−1. The frequency of occurrence and the average duration of the channels both become larger the thinner the membrane. The equilibrium between non-conducting and conducting species therefore shifts towards the conducting species as the membrane thickness decreases. With one exception, the same unit channel conductance was observed for a range of membranes having hydrocarbon thicknesses from 26 to 64 A and, from this and other evidence, it has been concluded that the conducting channel is a pore, rather than a carrier. The length of the pore has been estimated to be less than 35 A. The pore passes univalent cations but completely excludes polyvalent cations and anions. The selectivity between the univalent cations is not great, and the sequence of the various ion conductances is similar to that for the corresponding electrolytes in aqueous solution. The activation energies for the conduction of the ions are also similar to those in aqueous solution. The current-voltage relationships for the single channel tend to be curved towards the current axis at high electrolyte concentrations, linear at intermediate concentrations and curved towards the voltage axis at low concentrations. For each of the electrolytes studied the conductance of the single channel tends towards a limiting value at high concentrations. It is noted that one of the dimeric helical structures (that which contains the πL,D6 helix) proposed by Urry et al.22 could be consistent with some of the properties of the single channel.

Ion transfer across lipid membranes in the presence of gramicidin A: II. The ion selectivity

Abstract A detailed examination has been carried out of the ion selectivity of gramicidin A. Membranes of pure lipids and of various thicknesses have been used, and electrolyte concentrations have been varied over a wide range. It was first confirmed that anion permeability is low, even at very acid pH. Only in CaCl 2 was the transference number of the cation found to be less than unity (0.8). The measurement of biionic potentials was used to give the selectivity sequence for univalent cations. The results are compared with those already in the literature and with the conductivity data for the gramicidin single channel reported in a previous paper. The latter comparison suggests that a complex theoretical model is required to account for the kinetics of ion transfer. Semi-quantitative considerations of the univalent cation selectivities lead to the conclusion that there is probably a continuous file of water molecules in the gramicidin pore, and that the binding of ions by the gramicidin does not wholly determine the relative permeabilities. Aspects of the pore structure are discussed briefly in the light of the new data.

The effects of bilayer thickness and tension on gramicidin single-channel lifetime

Measurements have been made of gramicidin single-channel lifetimes in monoacylglycerol bilayers chosen so that their thickness ranged from above to below the length of the gramicidin channel. Contact angles, electrical capacities and bulk-phase interfacial tensions have also been determined for these systems. The mean channel lifetime decreased with the hydrocarbon thickness of the membrane until the latter reached 2.2 nm, after which the lifetime was relatively constant. A theoretical model has been proposed which relates the mean channel lifetime (or dissociation constant) to both the thickness and the tension of the bilayers. The analysis of the present results and of those of previous studies has led to the idea that aggregates of water molecules may play an important rôle in the dissociation of the gramicidin channel.

The unit conductance channel of alamethicin

Abstract Some properties of the unit conductance channels of alamethicin are reported. The data suggest that, in lipid membranes, alamethicin forms relatively highly conducting pores, which occur in two-dimensional aggregates and interact with each other.

Ion movements in gramicidin pores. An example of single-file transport

Experimental results on ion movement through gramicidin membrane channels are presented and discussed in terms of ion transport in the simplest single-file pore (for review see Urban, B.W. and Hladky, S.B. (1979) Biochim. Biophys. Acta 554, 410-429). Single-channel conductance and bi-ionic potential data for Na+, K+, Cs+, NH4+ and Tl+ are used to assign values to the rate constants of the model. Not all of the rate constants can be determined uniquely and simplifications are introduced to reduce the number of free parameters. The simplified model gives good quantitative fits to the experimental results for Na+, K+, Cs+ and NH4+. For Tl+, although the model accounts qualitatively for the salient features of the results, the quantitative agreement is less satisfactory. Predictions calculated from the model and the fitted rate constants are compared with independent data from blocking and tracer-flux measurements. In agreement with experiment, the model shows that only Tl+ blocks the Na+ conductance significantly. Furthermore, the exponent, n, in the tracer flux ratio rises, as observed, well above unity. The values for the rate constants suggest internal consistency of the model in that entry is always slower to singly occupied pores than to empty pores while exit is always faster from doubly as compared to singly occupied pores. The agreement between model prediction and experimental results suggests that the main features of ion transport in the gramicidin channel arise from cation-cation interaction in a single-file pore.

Surface charge, surface dipoles and membrane conductance

Abstract The conductance of lipid membranes in the presence of nonactin is changed by the adsorption of small amounts of ionic and zwitterionic surfactants. The conductance changes are, in many instances, not accounted for by the variation in surface charge or diffuse double layer potential as calculated from Gouy-Chapman theory. The changes are, however, all accurately accounted for by the variation in total potential across the membrane interface. This potential includes contributions from surface dipoles and specific adsorption, as well as any diffuse double layer effects not included in the Gouy-Chapman theory. The total potential changes were inferred from Volta or compensational potential changes at bulk oil (and monolayer)/aqueous solution interfaces. Surface charge densities were found by standard thermodynamic methods involving the use of the Gibbs equation. Electrokinetic potentials for the appropriate surfaces were also measured and, in general, agreed well with the diffuse double layer potentials calculated from the Gouy-Chapman theory.

Membrane conductance and surface potential

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

Ion Movements in Gramicidin Channels

Publisher Summary This chapter discusses gramicidin with respect to its structure and why it is believed to be a channel. The gramicidins, of which there are several, are linear 15-amino acid polypeptides produced by Bacillus hreuis. The naturally occurring gramicidin is a mixture of gramicidin A, B, and C in the approximate ratio 72 : 9 : 1. The primary structure of gramicidin A is unusual in several respects. One of the properties of gramicidin that has greatly facilitated its study is its readily measurable single-channel conductance and duration. Unlike the single-channel conductance, both the frequency of opening of channels and the mean channel open time or duration are strong functions of the membrane lipid. The chapter also discusses gramicidins with regard to its stability and kinetics of formation and decomposition in lipid membranes and in relation to its ion and water permeability.

The surface charge of cells and some other small particles as indicated by electrophoresis: I. The zeta potential-surface charge relationships

Abstract Electrokinetic measurements of single cells are frequently interpreted, via zeta potentials, in terms of surface charge densities. One of the equations which is employed for this purpose (the Gouy equation) is usually used in a form applicable only to a surface which is impenetrable to ions. That this assumption is not valid for a cell surface is immediately obvious. Various alternative equations are developed in this paper and it is shown that in all cases the true surface charge of the cell is likely to be higher than that calculated for an impenetrable surface. It is also shown that the general form of the curves relating surface charge to electrolyte concentration in simple electrolytes is, in most cases unlikely to change appreciably.

Anaesthesia by the n-alkanes. A comparative study of nerve impulse blockage and the properties of black lipid bilayer membranes.

Abstract 1. 1.|The suppression of the propagated action potential in the squid giant axon and in the frog sciatic nerve, by saturated solutions of the n-alkanes from n-pentane to n-decane, has been examined. A progressive loss inthe activity of the alkane was found as the chain length increased, n-nonane being effectively inert. The concentration dependence of the suppression of the action potentially n-pentane was also measured. 2. 2.|The effects of the alkanes on lipid bilayers were determined using black film techniques. For both phosphatidylcholine-cholesterol and monoolein-cholesterol bilayers in alkane-saturated aqueous phases, the bilayer thickness decreased by several angstrom units on passing from n-pentane to n-decane, from which it was conclude that the adsorption also decreased. The concentration dependence of the thickness and adsorption changes for n-pentane were examined. 3. 3.|A close correlation is shown to exist between the nerve results and those for a phosphaytidylcholine-cholesterol bilayer, suggesting that the site of action of the alkane is in a lipid bilayer region of the nerve mumbrane. 4. 4.|The presence of cholesterol in the bilayer, at levels apparently comparable to those in axon membranes, is essentiol for the above correlation to hold. 5. 5.|A molecular mechanism by which the alkane may inhibit the nerve impulse is proposed. The essential feature is that a thickening of the lipid part of an axon mumbrane through adsorption of alkane reduces the stability of the ionic channels formed during electrical excitation.