The influence of n-alkanols on the capacity per unit area of planar lipid bilayers.

The electrical capacities per unit area of planar lipid bilayers formed from monoolein/n-hexadecane, monoolein/ squalane (or squalene) and monoolein/triolein have been measured in the presence of a range of n-alkanols. For monoolein/n-hexadecane bilayers, the effects of the n-alkanols are complicated but can be rationalized in terms of the likely changes in lipid chain order and the influence of the n-alkanol in the Plateau-Gibbs border. Monoolein/ squalane (or squalene) and monoolein/triolein bilayers exhibit behaviour quite different from the n-hexadecane membranes. For both the squalane and triolein bilayers the shorter chain alkanols increase the capacity per unit area while the longer homologues have little effect. These results help to account for the influence of the n-alkanols on gramicidin single-channel lifetimes.

Kinetics and stability of alamethicin conducting channels in lipid bilayers.

It is already well-established that conduction in lipid bilayers containing alamethicin arises from the presence of complexes in which there are several molecules of the polypeptide. It is with the nature of these complexes that this paper is primarily concerned. While it is clear that increasing alamethicin concentration and increasing potential across the membrane favour their formation, the nature of the reactions involved has not yet been elucidated. Attempts have therefore been made to clarify the sequence of events leading to the establishment of a complex in its conducting state. It has been concluded that the most likely mechanism involves, initially, a non-field-dependent aggregation of the alamethicin, in the plane of the membrane, into non-conducting oligomers. These then appear to undergo movement normal to the membrane (which is field dependent) to form the conducting species. Temperature studies have shown that the various conducting states of the oligomer have effectively equal enthalpies, and that the activation energies for transitions between these states are all approx. 1.2kcal/mol. The corresponding rate constants are very sensitive to the lipid composition of the membrane and a variety of different systems has been examined in order to clarify the origins of this effect. The only conclusion from this part of the work is that lipid fluidity might be involved.

The adsorption of lysine vasopressin at ionized interfaces.

It has been shown by surface potential measurements that lysine vasopressin and oxytocin may be bound by ionic surfaces to very varied extents. To dodecyl sulphate and phosphatidylserine monolayers the binding is very strong and is comparable to that for biological receptors such as those in toad bladder. For dioleyl phosphate and the carboxyl group of the polypeptide alamethicin, the binding is rather weaker while, for the zwitterionic lipids phosphatidylcholine and phosphatidylethanolamine, and for the erythrocyte surface, which contains two varieties of carboxylic acid group, no interaction seems to take place. In no system does the lysyl amino group of the vasopressin appear necessary for adsorption and, in the dodecyl sulphate monolayers, the interaction is strong even when the ionization of the terminal alpha-amino group is suppressed.

The interaction of n-octanol with black lipid bilayer membranes.

The electrical capacities of black lipid films formed from monoolein + n-hexadecane and monoolein + squalane (or squalene) solutions have been measured in the presence of various concentrations of n-octanol. In addition, partition coefficients for n-octanol between n-hexadecane and 0.1 M NaCl, dielectric constants for octanol-hexadecane mixtures and the interfacial tension of films and film-forming lipid solutions against the aqueous phases have been determined. It is concluded that in "solvent-free" bilayers the octanol is unlikely to have changed the bilayer thickness by more than about 1 A. The bilayer tension, on the other hand, increases appreciably in the presence of octanol.

Surface potential changes in lipid monolayers and the 'cut-off' in anaesthetic effects of N-alkanols.

The effects of 0.09 saturated solutions of the n-alkanols n-hexanol to n-tridecanol on the surface (compensation) potential of lipid monolayers have been examined. Actions on monolayers spread from pure egg phosphatidylcholine have been compared with effects on a system containing 2:1 mole ratio of egg phosphatidylcholine and cholesterol. The mean compensation potential for the pure phospholipid system was 475 +/- 9 mV; addition of cholesterol increased the potential to 503 +/- 10 mV. All n-alkanols tested reduced the surface potential in both systems. The reduction was larger in the pure phospholipid system but the difference in effect between lipid systems declined as the n-alkanol chainlength increased, becoming negligible by n-tridecanol. These results are considered in relation to the 'cut-off' in biological activity of n-alkanols around n-tridecanol.

The effects of homologous series of anaesthetics on a resting potassium conductance of the squid giant axon.

The effects of n-alkanes (n-pentane to n-octane), n-alkanols (n-pentanol to n-undecanol) and two carboxylic esters (methyl pentanoate and methyl octanoate) on the conductance of squid giant axons in a high potassium, zero sodium bathing solution have been examined. Sodium and delayed rectifier potassium channels were as far as possible pharmacologically blocked. A substantial fraction of the measured conductance is attributed to a recently-described, voltage-independent, potassium channel. Anaesthetics block this channel but its sensitivity is markedly different from those of other squid axon ion channels.

The blockage of the electrical conductance in a pore-containing membrane by the n-alkanes.

1. In monooelein bilayers made highly conducting by the addition of a fixed amount of o-pyromellitylgramicidin, the membrane conductance has been shown to be strongly dependent on the chain length of the n-alkane with which the membrane is in equilibrium. Thus for n-hexadecane, the conductance is larger by approx. 10(4) times than it is for n-octane. This result is independent of whether the polypeptide is introduced via the aqueous or lipid phases. 2. The observed conductance variations have been accounted for in terms of a mechanism (outlined in earlier publications) which is based on the thickness and tension changes produced in bilayers by the adsorption of n-alkanes. Essentially quantitative agreement between theory and experiment is found.

The influence of n-alkanols and cholesterol on the duration and conductance of gramicidin single channels in monoolein bilayers.

The mean lifetime of gramicidin A channels in bilayers formed from monoolein and squalane was sharply reduced by the absorption of a range of n-alkanols and cholesterol. Results are shown for n-hexanol, n-octanol, n-decanol, n-dodecanol, n-tetradecanol, n-hexadecanol, n-octadecanol and cholesterol. The longer chain n-alkanols were apparently more effective than the shorter members and cholesterol was the most effective of the substances examined. The single channel conductance was also affected, though to a much lesser extent than the mean channel lifetime, the n-alkanols producing increases and cholesterol a decrease. It is suggested that membrane fluidity changes are not likely to be primarily responsible for the reductions in channel lifetimes but that the bilayer tension, which is known to be increased by n-octanol, could be significant.

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.

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.

A quantitative explanation of the effects of some alcohols on gramicidin single-channel lifetime.

The effects of n-decanol, n-hexadecanol, n-octyl(oxyethylene)3 alcohol and cholesterol on gramicidin single-channel lifetime in planar lipid bilayers have been determined. The bilayers used were formed from a solution of monoolein in squalene. Measurements have also been made of the above compounds' effects on membrane thickness (as measured by electrical capacity and optical reflectance technique) and surface tension (as derived from bulk interfacial tension and bilayer-lens contact angle measurements). The reduction in single-channel lifetime caused by the n-alkanols may be accounted for quantitatively in terms of the effects of these compounds on bilayer thickness and surface tension. The n-octyl(oxyethylene)3 alcohol caused an increase in single-channel lifetime which is also consistent with the thickness/tension theory. The reduction in channel lifetime caused by cholesterol, however, was much larger than would be predicted from its effects on bilayer thickness and surface tension.

Actions of n-alcohols on nicotinic acetylcholine receptor ion channels in cultured rat muscle cells.

The effects of the n-alcohols from pentanol to dodecanol on nAChR channel function were resolved at the single channel level. ACh-activated channel activity was recorded from isolated membrane patches using the patch clamp method. The intermediate-chain alcohols (C5-C8) had two main effects: (1) They caused channel openings to be interrupted by brief shut or blocked periods, the duration of which was dependent on chain length of the alcohol but independent of concentration. (2) They caused a reduction in the duration of bursts of openings. The long-chain alcohols (C9-C11) produced only the second effect, and there was a decline in activity beyond undecanol. Results were consistent with a mechanism of channel blockade and were analyzed in terms of an open channel block model with a long-lived closed-blocked state beyond the blocked state. Affinity for the binding site increased with chain length up to octanol. The standard free energy per methylene group for adsorption to the site was calculated to be -3.3 kJ/mol, indicating the very hydrophobic nature of the site.

Effects of anesthetics and convulsants on the resting potassium conductance in squid nerve.

The effects of some fluorinated anesthetics and convulsants on the ionic conductances of the axon membrane of the squid Loligo forbesi are described. The substances studied were the inhalation anesthetics enflurane (CF2HOCF2CClFH) and isoflurane (CF2HOCClHCF3) and the convulsants flurothyl (CF3CH2OCH2CF3) and trichlorofluormethane (CCl3F). At low concentrations (0.4-0.8 mM), none of these substances significantly affected the voltage-dependent Na and K channels of the nerve. However, at these concentrations each substance produced a depolarization of the resting potential and reduced the potassium conductance of the resting membrane. This was associated with a tendency to cause axonal hyperexcitability. The potassium conductance of the resting membrane was separated into a component arising from residual open Hodgkin-Huxley delayed rectifier K channels and another voltage-independent component (g'k). The former component was insensitive to the test substances at 0.4-0.8 mM, while g'k was inhibited by 40-80%. It is suggested that the convulsant activity of certain small fluorinated molecules, and the proconvulsant actions of certain clinical anesthetics at low concentrations, may be related to the inhibition of a resting, voltage-independent potassium conductance system in the nerve membrane.