Journal of Physiology (1991), 437, pp. 431-448 431 With 11 figures Printed in Great Britain ACTIONS OF n-ALCOHOLS ON NICOTINIC ACETYLCHOLINE RECEPTOR CHANNELS IN CULTURED RAT MYOTUBES BY R. D. MURRELL*, M. S. BRAUNt AND THE LATE D. A. HAYDON From the Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG (Received 14 June 1990) SUMMARY 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 JC50s were as follows: hexanol, 0-53 +014 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 106 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. * Present address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5426. t Present address: Max-Planck-Institut for Brain Research, Dentschordenstrasse 46. D6000, Frankfurt a.M., Germany. MS 8564 432 R. D. MURRELL, M. S. BRA UN AND D. A. HA YDON 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'. 8. It is concluded that the action of n-alcohols on nAChR channels may involve them binding to a hydrophobic site on the channel protein, blocking ion flux through the channel and stabilizing a closed conformation of the channel. INTRODUCTION There is now convincing evidence that many cationic anaesthetics block current flow through nicotinic acetylcholine receptor (nAChR) channels by binding to an identified site located in the M2 membrane-spanning region thought to be the channel pore (Imoto, Methfessel, Sakmann, Mishina, Mori, Konno, Fukuda, Kurasaki, Bujo, Fujita & Numa, 1986; Changeux, Giraudat & Dennis, 1987; Charnet, Labarca, Leonard, Vogelaar, Czyzyk, Gouin, Davidson & Lester, 1990). The individual blocking and unblocking steps, as the molecule enters and leaves the pore, have been resolved at the single-channel level and appear as discrete fluctuations between the fully open and closed channel current level (Neher & Steinbach, 1978). Kinetic analyses of single-channel records in the presence of these compounds have helped to elucidate the mechanism of blockade. A simple sequential open channel block model has been proposed whereby the blocking molecule only binds to the channel in the open state, and the channel can only finally close when the molecule has dissociated from the site (Adams, 1976; Neher & Steinbach, 1978). Neutral molecules, such as general anaesthetics, have also been shown to block ion flux through the nAChR channel (Ogden, Siegelbaum & Colquhoun, 1981; Gage & Hamill, 1981; Lechleiter & Gruner, 1984; McLarnon, Pennefather & Quastel, 1986; Foreman & Miller, 1989), although there is less evidence for a discrete general anaesthetic site on the channel protein. The question of whether general anaesthetic type molecules, which includes most small lipophilic compounds, are able to affect channel function by binding directly to the channel protein or whether they exert their effects by perturbing the lipid environment of the channel, is still unresolved (Miller, 1985; Franks & Lieb, 1987; Elliott & Haydon, 1989). The n-alcohols from pentanol to octanol have been shown to induce a biphasic decay of miniature end- plate currents at the mouse neuromuscular junction, which is consistent with a channel blocking mechanism of action (Gage, McBurney & Schneider, 1975; McLarnon et al. 1986). In this study we investigated the actions of the series of n-alcohols from pentanol to dodecanol on nAChR channels at the single-channel level, to obtain a quantitative description of their effects on channel gating and to test whether a channel blocking model fits the data. The main findings are that the intermediate-chain alcohols (pentanol to octanol) caused single ACh-activated channel currents to fluctuate between a fully open and closed state, similar to the effects of charged anaesthetics, and they also caused a concentration-dependent reduction in the duration of bursts of openings. Nonanol and decanol caused only the second of these two effects, while beyond decanol there was a decline in the ability of the n-alcohols to affect channel function. ACH RECEPTOR CHANNEL BLOCK BY n-ALCOHOLS 433 A preliminary account ofsome ofthis work has been presented to The Physiological Society (Braun, Haydon & Murrell, 1989). METHODS Rat myotubes were prepared essentially according to the method of Horn & Brodwick (1980). Cells were plated onto glass cover-slips and used for experiments after 6-13 days. Single-channel recordings were made using the inside-out configuration of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Patch electrodes were fabricated from borosilicate glass and had a resistance of 5-12 MCI. All recordings were made with the following Ringer solution in the bath and in the patch pipette (in mM): NaCl, 145; CaCl2, 1; MgCl2, 1; HEPES, 10; pH 7-3. Acetylcholine (Sigma) at a concentration of 0-25 ,UM was included in the pipette solution. The alcohols (pentanol to dodecanol), were all puriss grade (Koch-Light Laboratories). Concentrated solutions of the alcohols were made up in Ringer solution and were diluted to the desired concentration immediately prior to the experiment. The alcohols were applied via a perfusion pipette with a tip diameter of 50-100 4um at a rate of approximately 5 ml h-'. Control, test and recovery data were collected for each experiment. To collect the test data the perfusion pipette was lowered into the chamber and the inside-out membrane patch was inserted well into the mouth of the pipette. All experiments were performed at room temperature (18-23 °C) and, unless otherwise stated, membrane potential was held at -80 mV. Current records were stored on an FM tape- recorder (Racal Recorders Ltd). For analysis, records were filtered at either 2 5, 4 or 8 kHz (-3 dB, 6-pole Bessel) and were then digitized at a sampling rate of either 20 or 50 kHz. Data analysis Opening and closing transitions were determined by setting a threshold at half the amplitude of the open channel current (Colquhoun & Sigworth, 1983). From the idealized reconstruction of open and closed intervals apparent open and closed times were obtained and stored in separate data files. Multiple channel openings were excluded from the analysis. Clearly defined partial openings and closures were few in number and were not considered separately. A minimum resolvable interval (tmin) was imposed on the data; for data filtered at 3, 5 and 8 kHz, tmin was set at 120, 85, and 50 Its respectively, for both open and closed times. Values less than this were stripped from data files prior to construction of histograms of open and closed times. Probability density functions were fitted to the data using the method of maximum likelihood (Colquhoun & Sigworth, 1983). Due to the limited time resolution a conditional probability density function for t was fitted (where t is the open or closed channel lifetime), given that it is greater than tmin. An estimate of the correct number of closures, which includes those that were missed because they were less than tmin, was made by dividing the number of events included in the fitting process by the probability that an observation was greater than tmin (Colquhoun & Sakmann, 1985). In general distributions of closed times were fitted well with the sum of two exponentials, and the fast component was taken to represent the gaps within a burst and the slow component closed times between bursts. Where there were three components the intermediate component was also taken to represent gaps within bursts. The total number of gaps was calculated by multiplying the area of the fast component with the corrected number of closed events. The total number of interburst closed times was calculated likewise and the mean number of gaps per burst obtained by dividing the total number of gaps by the total number of interburst closed times plus one.