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1. The actions of the n‐alcohols from pentanol to dodecanol on nicotinic acetylcholine receptor (nAChR) channels were investigated by recording single ACh‐activated channel activity from inside‐out membrane patches isolated from cultured rat myotubes. Alcohols were applied to the cytoplasmic side of the membrane; aqueous concentrations ranged from 11.7 mM‐pentanol to 0.02 mM‐dodecanol. 2. The intermediate‐chain alcohols (pentanol to octanol) caused channel currents to fluctuate between the fully open and closed state level so that openings occurred in bursts interrupted by brief gaps. Closed time distributions were fitted well with two exponential components, the fast component representing the closures within a burst. The number of gaps within a burst was dependent on alcohol concentration whereas gap duration was independent of concentration but increased with increasing chain length of the alcohol up to octanol. 3. Nonanol and decanol reduced the mean duration of bursts of openings but did not cause an increase in the number of short closed intervals within a burst. Beyond decanol there was a decline in the ability of the n‐alcohols to affect channel function. A saturated solution of undecanol (0.07 mM) reduced the mean open time by 33 +/‐ 17%, whereas a saturated solution of dodecanol had no significant effect. 4. The current integral per burst was reduced by all the n‐alcohols between pentanol and undecanol. The IC50S were as follows: hexanol, 0.53 +/‐ 0.14 mM; heptanol, 0.097 +/‐ 0.02 mM; octanol, 0.04 mM and nonanol, 0.16 +/‐ 0.035 mM. 5. The results were analysed in terms of an open channel block model with a long‐lived closed‐blocked state beyond the blocked state. Over the range of concentrations tested this describes the effects of all the n‐alcohols (C5 to C12) on channel gating reasonably well. 6. Blocking rate constants (k+B) for pentanol through to nonanol were calculated to be between 2.8 and 5.7 X 10(6) M‐1 S‐1. These values are based on the assumption that the concentration of the alcohols at their site(s) of action was equal to the aqueous concentration applied to the membrane. 7. Equilibrium dissociation constants (KD), calculated from the blocking and unblocking rate constants (KD = k‐B/k+B), decreased with increasing chain length from 8 mM for pentanol to 0.15 mM for octanol. The standard free energy per methylene group for adsorption to the site of action was calculated to be about ‐3.3 kJ mol‐1.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The electrical properties of squid giant axons were examined by means of admittance bridges at frequencies from 0.5 to 300 kHz. A simple equivalent circuit was used to estimate the membrane capacity. 2. The calculated membrane capacities decreased monotonically over the whole frequency range. 3. At 100 kHz and higher frequencies the membrane capacity was independent of potential. 4. At frequencies greater than 20 kHz, exposure of the axons to saturated or 0.9 saturated solutions of n‐pentane (275‐306 micrometer) reduced the capacity per unit area by 0.1‐0.15 micro F cm‐2. 5. At 1 kHz the effect of the saturated pentane solutions depended on the membrane potential. In axons having potentials between ‐60 and zero mV the pentane solutions lowered the capacity, whereas for potentials between ‐160 and ‐60 mV they produced little or no change. 6. Saturated solutions of n‐hexane, n‐heptane and n‐octane exhibited qualitatively similar, but quantitatively smaller influences on the membrane capacity, the changes declining as the chain length increased. 7. Under voltage clamp, the peak inward and steady‐state outward currents were partially suppressed by the hydrocarbons. Saturated solutions of n‐pentane usually reduced the former (reversibly) by 60‐80% and the latter by 20‐40%. Solutions of n‐hexane, n‐heptane and n‐octane appeared to have successively less effect. Except in deteriorating axons, none of the hydrocarbons produced any consistent changes in the passive membrane resistance, the resting potential or in the reversal potential of the transient inward current. 8. Both the changes in the clamp currents and in the membrane capacity were largely, though not usually completely, reversible. In the hydrocarbon solution the axons deteriorated more rapidly than normal. 9. The responses of axons of Doryteuthis plei to the hydrocarbons were very similar to those of Loligo forbesi with the exception that for the former all observed changes were some five times faster. 10. The time courses of the peak inward and steady‐state outward currents on exposure of the axons to n‐pentane resembled the time course of the change in membrane capacity at 100 kHz. 11. The simplest interpretation of the high frequency capacity results is suggested to be that, as for lipid bilayers, the membranes become thicker through adsorption of the hydrocarbon.

1. Sodium and potassium currents have been recorded in intracellularly perfused squid giant axons before, during and after exposure to solutions of n‐pentane in artificial sea water. 2. The currents were fitted with equations similar to those proposed by Hodgkin & Huxley (1952) and the changes in the parameters of these equations in the presence of pentane were calculated. 3. In the range of membrane potential ‐40 to 40 mV, the time constants for activation (tau m) and inactivation (tau h) of the Na current, and for activation (tau n) of the K current were all reduced by the pentane. 4. The curve of the steady‐state inactivation parameter (h infinity) for the Na current against membrane potential was shifted by the pentane in a hyperpolarizing direction (at h infinity = 0.5 this shift was approx. ‐15 mV in 275 microM‐pentane) and the slope at all potentials was reduced. 5. The curve of the steady‐state activation parameter (m infinity) for the Na current against membrane potential was also shifted by the pentane in a hyperpolarizing direction (in 153 microM‐pentane, 10 mV at m infinity = 0.5). 6. The maximum Na and K conductances gNa and gK were lowered by the pentane, though not usually completely reversibly. 7. The changes in position and slope of the steady‐state inactivation curve have been tentatively accounted for in terms of an increase in membrane thickness.

1. The anaesthetic effects of aqueous solutions of the n‐alkanes pentane to nonane on the propagated action potential of squid axons have been investigated for a range of axon diameters. 2. By the use of small axons (approx. 200 microns diameter) to minimize effects due to long diffusion times and alkane depletion it was found that n‐pentane and n‐hexane caused a rapid reversible inhibition of the impulse, while higher homologues had progressively less effect, n‐nonane being apparently inert. 3. The rate of action potential decline due to the n‐alkanes was found to be strongly dependent on axon diameter. For n‐hexane, n‐heptane and n‐octane the rate of decline was inversely proportional to the square of the axon diameter. 4. The mechanisms which may underly the increased sensitivity of small axons to impulse blockade by n‐alkanes are discussed. A quantitative comparison is made between the effects of n‐hexane, n‐heptane and n‐octane on the action potential. It is argued that this supports the idea of a real decline in anaesthetic potency on ascending the homologous series, rather than an effect due to long diffusion times and solution depletion.

The effects of the n‐alkanes propane to hexane, cyclopropane, cyclopentane and cyclohexane and carbon tetrachloride on the ionic currents and electrical capacity of the squid giant axon membrane have been examined. Both the peak inward and steady‐state outward currents were reduced reversibly by each substance, though propane at 1 atm had very little effect. The membrane capacity at 100 kHz was reduced by all substances except propane at 1 atm. Na currents were recorded in intracellularly perfused axons before and during exposure to the hydrocarbons and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the curves of the steady‐state activation and inactivation parameters (m infinity and h infinity) against membrane potential, changes in the peak heights of the activation and inactivation time constants (tau m and tau h) and reductions in the maximum Na conductance (gNa) have been tabulated. The effects of the various hydrocarbons and carbon tetrachloride on the parameters of the Hodgkin‐Huxley equations suggest that the suppression of the Na current by these substances originates from several different phenomena. The underlying physico‐chemical events are considered in the light of the observed capacity changes and of information on artificial pore‐containing membranes.

The effects of several n‐alkanols and n‐alkyl oxyethylene alcohols, methyl octanoate, glycerol 1‐monooctanoate and dioctanoyl phosphatidylcholine on the ionic currents and electrical capacity of the squid giant axon membrane have been examined. The peak inward current in voltage‐clamped axons was reduced reversibly by each substance. For n‐pentanol to n‐decanol the concentrations required to suppress the peak inward current by 50% were determined. From these data, it was estimated that the standard free energy per CH2 for adsorption to the site of action was ‐3.04 kJ mole‐1, as compared with ‐3.11 kJ mole‐1 for adsorption into phospholipid bilayers or an n‐alkane/aqueous solution interface. The membrane capacity at 100 kHz was not greatly by any of the test substances at concentrations which reduced the inward current by 50%. Na currents under voltage clamp were recorded in intracellularly perfused axons before, during and sometimes after exposure to the test substances and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the curves of the steady‐state activation and inactivation parameters (m infinity and h infinity) against membrane potential, changes in the peak heights of the activation and inactivation time constants (tau m and tau h) and reductions in the maximum Na conductance (gNa) have been tabulated. All of the test substances shifted the voltage dependence of the steady‐state activation in the depolarizing direction and lowered the peak time constants for both activation and inactivation. The origins of these effects, and of the differences in the present results from those of the hydrocarbons (Haydon & Urban, 1983), have been discussed in terms of the physico‐chemical properties of the two groups of substances and with reference to their effects on artificial membranes.

The effects of diethyl ether, methoxyflurane, halothane, dichloromethane and chloroform on the ionic currents and electrical capacity of the squid giant axon have been examined. The peak inward current in voltage‐clamped axons was reduced reversibly by each substance. Sodium currents under voltage clamp were recorded in intracellularly perfused axons before, during, and sometimes after exposure to the test substances, and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the dependence of the steady‐state activation and inactivation parameters (m infinity and h infinity) on membrane potential, reductions in the peak heights of the activation and inactivation time constants (tau m and tau h) and decreases in the maximum Na conductance (gNa) have been tabulated. For each of the anaesthetics the steady‐state inactivation curve was shifted in the hyperpolarizing direction though less markedly than for the hydrocarbons. The steady‐state activation curve was in each instance shifted in the depolarizing direction, as for the alcohols and other surface active substances. In common with both the hydrocarbons and the surface active substances the peak time constants were invariably reduced. The membrane capacity at 100 kHz was affected significantly only by methoxyflurane, where decreases of ca. 9% were observed for 3 mM solutions. The extent to which the results can be accounted for in terms of the perturbation of membrane lipid has been discussed.

The admittance of the squid giant axon membrane has been measured, using an intracellular electrode, at frequencies up to 40 MHz. The existence of a radio frequency dispersion, previously detected with extracellular electrodes (Cole, 1976) and attributed to the Schwann cell layer, has been confirmed and followed to higher frequencies. For a comparable method of analysis, membrane parameters similar to those given by Cole (1976) have been calculated. The radio frequency dispersion has a centre frequency at approximately 1.8 MHz, and the properties of a parallel combination of a 28 nF cm‐2 capacity and a 3.3 omega cm2 resistance. When the axon membrane capacity is calculated, taking into account the radio frequency dispersion, as described above, the capacity remains frequency dependent throughout the range studied. If it is assumed that at high frequencies the axolemma capacity becomes constant at approximately the value for a lipid bilayer, a radio frequency dispersion is found which cannot be accounted for in terms of a simple equivalent circuit with two passive components, but appears to arise from a network with a distribution of relaxation times. This result could be consistent with the morphology of the Schwann cell layer. The radio frequency dispersion referred to in (4) can be described reasonably well by a circuit with two dispersions having centre frequencies of 250 kHz and 3.2 MHz respectively. The corresponding axolemma capacity (100‐500 kHz) would be approximately 0.6 microF cm‐2. It is argued that between 50 and 100 kHz the geometrical capacity arising from the non‐polar regions of the membrane is a major contributor to the axon membrane capacity, and that capacity variations arising from compositional changes in the lipid bilayer are best monitored in this frequency range.

The actions of members of the homologous series of alkyl cations CH3 (CH2)n‐1 N+ (CH3)3 (Cn TMA) on the sodium current in giant axons of Loligo forbesi have been investigated. The substances tested correspond to n = 6, 8, 10, 12, 14 and 16. These cations only produced significant sodium current suppression when applied inside the axon. Actions on first‐pulse sodium currents and use‐dependent effects were separately studied. The shorter members of the series (C6TMA and C8TMA) produced suppression of first‐pulse sodium currents without causing significant use dependence. The first‐pulse suppression arose partly from a positive shift along the voltage axis of the steady‐state activation parameter (m infinity) and partly from a reduction in the maximum sodium conductance (gNa). C12TMA and C14TMA produced little first‐pulse suppression but caused clear use dependence. C10TMA showed intermediate properties while C16TMA was inactive. The use‐dependent actions have been quantitatively investigated using a double‐pulse protocol. The results are consistent with a model in which the cations enter a blocking site on the ion‐channel via the intra‐axonal aqueous phase. The cations appear able to bind to inactivated sodium channels at significant rates. The possible molecular locations of the sites responsible for m infinity shifts and use dependence are discussed. It is argued that the existence of two separate sites may help to explain certain distinctions between the actions of neutral general anaesthetics and clinical local anaesthetics on the sodium channel.

The effects of octyltrimethylammonium ions (OTMA+), octyl sulphate ions (OS‐) and octanoic acid (OA) on the sodium current of the voltage‐clamped squid giant axon have been investigated using intracellular and extracellular application of the test substances. OTMA+ applied externally at concentrations of 0.8‐5.0 mM produces a small reversible increase in the peak inward sodium current in both intact and CsF‐perfused axons. Intracellular application of OTMA+ at 0.8 mM to CsF‐perfused axons causes a reversible 50% suppression of peak inward sodium current. The inhibition of peak inward current by internal OTMA+ arises largely from a shift of the steady‐state activation parameter (m infinity) in the depolarizing direction along the voltage axis. There is little use dependence of the current suppression by OTMA+ OA applied either internally or externally is more effective at suppressing peak inward sodium current at pH 6.0 than at pH 7.4. At pH 6.0 external application of 5 mM‐OA to perfused axons causes approximately 60% suppression. This is associated with a depolarizing shift of m infinity of about 13 mV and a hyperpolarizing shift of the steady‐state inactivation (h infinity) curve of about 4 mV. The effects of internal and external OA are broadly similar except that the h infinity shift is not seen with internal application. OS‐ at concentrations above 2.0 mM produces complete irreversible loss of sodium current. At 2.0 mM, OS‐ produces 10% current suppression and a small depolarizing shift of the m infinity curve. Internal and external applications of OS‐ differ little except that external OS‐ causes a 25% increase in the time constant of activation (tau m). The possible origins of these effects are discussed. It is proposed that the shift of m infinity caused by internal OTMA+ is due to a diminution of the lipid dipole potential at the internal surface of the membrane caused by OTMA+ adsorption. This effect could also account for the m infinity shift caused by OA. The results showing that OA produces shifts of opposite sign in the voltage dependence of m infinity and h infinity are discussed with respect to their implications for models of sodium channel gating.

The effects of methyl butyl ketone, methyl heptyl ketone and methyl pentanoate on the sodium current of the squid giant axon have been examined. The peak inward current in intact axons was reduced reversibly by each substance. Sodium currents were recorded in intracellularly perfused axons before and during exposure to the test substances and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the voltage dependence of the steady‐state activation and inactivation parameters (m infinity and h infinity), reductions in the peak heights of the activation and inactivation time constants (tau m and tau h) and changes in the maximum sodium conductance (gNa) caused by these substances have been tabulated and compared with the effects of methyl octanoate (Haydon & Urban, 1983b). Each compound shifted the voltage dependence of the steady‐state inactivation parameter in the hyperpolarizing direction and that of the steady‐state activation parameter in the depolarizing direction. The shifts produced by the ketones are compared with those produced by methyl pentanoate and by methyl octanoate. The possible role of an interaction between the carbonyl oxygen of the test substance and the sodium channel protein in producing the h infinity shift is discussed. The peak time constants are reduced and the voltage dependences of tau m and tau h are shifted in a direction commensurate with the shifts in steady‐state properties. The maximum sodium conductance is not much affected either by the ketones or by methyl pentanoate. Large reductions in peak inward current coupled with little effect on gNa have been reported for the n‐alkanols and other surface‐active compounds (Haydon & Urban, 1983b). This lack of a large effect on gNa indicates that whatever direct interaction does take place between the test substance and the channel protein, it does not result in a blockage of the channel.

A number of small organic molecules with general anaesthetic action have been examined for their effects on the voltage‐dependent potassium current of the squid giant axon. They include representatives of the three classes of anaesthetics examined in previous studies on the sodium current (Haydon & Urban, 1983a, b, c), i.e. the non‐polar molecules n‐pentane, cyclopentane and CCl4, several n‐alkanols and the inhalation anaesthetics chloroform, halothane, diethyl ether and methoxyflurane. Potassium currents under voltage clamp were recorded in intact and in intracellularly perfused axons before, during and after exposure to the test substances, and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the curves of the steady‐state activation against membrane potential and reductions in the potassium conductance at 60 or 70 mV membrane potential have been tabulated. On the same intact axons, all the anaesthetics with the exception of methoxyflurane reduced potassium currents less than sodium currents by about a factor of two or more. For the n‐alkanols, butanol to decanol, the concentrations required to reduce the potassium current at 60 mV membrane potential by 50% were determined. For n‐butanol to n‐heptanol, the standard free energy per CH2 for adsorption to the site of action was estimated to be ‐2.91 kJ mol‐1 as compared with ‐3.04 kJ mol‐1 for reduction of the sodium current. The magnitude of the free energy decreased for alkanols with longer chain lengths. At anaesthetic concentrations that reduce the sodium current by 50%, the hydrophobic substances n‐pentane and cyclopentane reduced the maximal sodium conductance, gNa, and the potassium conductance at 70 mV, gK70, equally by about a third, while the n‐alkanols reduced both parameters by less than 10%. By contrast, diethyl ether and methoxyflurane were more effective in reducing the maximal potassium conductance. All of the test substances examined, except n‐pentane and n‐hexane, shifted the voltage dependence of the potassium steady‐state activation in the depolarizing direction. A broad qualitative correlation was found between the shifts in the activation curves for sodium and potassium currents but, quantitatively, the agreement between the two shifts was poor. In n‐decanol and methoxyflurane solutions, the voltage‐clamped potassium currents exhibited pronounced inactivation‐like behaviour. These currents can be fitted by the Hodgkin‐Huxley formalism if an inactivation term analogous to the sodium current inactivation is added.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The effects of some neutral clinical and experimental general anaesthetics on the resting potential of normal squid axons and squid axons exposed to tetrodotoxin and 3,4‐diaminopyridine have been studied. 2. Depolarizations of 1‐4 mV were produced by all the anaesthetics at ‘clinical’ concentrations in the normal axon. Larger depolarizations (5‐11 mV) were produced by the same anaesthetic concentrations in axons exposed to tetrodotoxin and 3,4‐diaminopyridine. 3. The conductance of axons exposed to tetrodotoxin and either tetraethyl‐ammonium or 3,4‐diaminopyridine in zero Na+, 430 mM‐K+ artificial sea water was examined by voltage clamp and AC bridge techniques. 4. The evidence that this conductance is due predominantly to K+ is discussed. 5. Pre‐pulse protocols under voltage clamp have been used to show that part of this conductance arises from the incompletely blocked delayed rectifier. 6. Substantial reductions in this conductance are produced by anaesthetics at ‘clinical’ concentrations. 7. It is concluded that there is a component of the K+ conductance of the resting squid axon other than the Hodgkin‐Huxley delayed rectifier which is extremely sensitive to anaesthetics and which to an appreciable extent determines the resting potential.

1. The effects of ‘clinical’ concentrations of some general anaesthetics on the minimum stimulus required to produce an action potential in the squid giant axon have been examined as a function of time from exposure to the anaesthetic. The resting potential in these experiments was also monitored. 2. The minimum stimulus varied with time in different ways for different anaesthetics. For chloroform, diethyl ether, n‐pentanol, halothane and cyclopropane the stimulus initially declined, reached a minimum after about 3 min and then recovered to near‐normal values at 10‐15 min. For n‐pentane, cyclopentane and, to a lesser extent methoxyflurane, the stimulus often declined to such low values that the axon exhibited spontaneous action potentials which persisted until the anaesthetic was removed. For one substance, the experimental local anaesthetic diheptanoyl phosphatidylcholine, the stimulus increased considerably over the 10‐15 min required to reach the steady state. In all instances the axons reverted to normal behaviour after removal of the anaesthetic although the time course by which they did so was more variable than for the initial exposure. 3. For all anaesthetics the resting potential changed in the positive direction monotonically by ca. 1‐5 mV and reached a steady state in approximately 3 min. On removal of the anaesthetic the resting potential returned to normal, also monotonically. 4. The voltage‐gated Na+ and K+ currents were significantly affected even at the low anaesthetic concentrations used. Estimates of the changes in the Hodgkin‐Huxley parameters were obtained partly by direct experiment and partly from results previously obtained for higher anaesthetic concentrations. 5. The time dependencies of the minimum stimuli have been accounted for semi‐quantitatively in terms of the resting potential changes and the voltage shifts in the Na+ current steady‐state activation, and the time dependencies respectively of these two parameters. 6. Quantitative calculations of the resting potential changes for comparison with experiment have been made based on the changes in K+ conductance determined in the preceding paper (Haydon, Requena & Simon, 1988) and changes in the Hodgkin‐Huxley parameters of the Na+ and delayed‐rectifier K+ currents. 7. Calculations of the minimum stimulus in the steady state have been made from the experimental resting potential changes and from the anaesthetic‐affected Hodgkin‐Huxley parameters. Good agreement with the experimental stimuli was found, especially in the prediction of high and low values.