Cambridge CB2 3EG (Received 14 June 1990)

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. The definition of 'bursts' of openings was as described by Colquhoun & Sakmann (1985). A critical gap length for bursts below which a closure is classified as a gap within a burst, was calculated so that the proportion of long closures misclassified as gaps was equal to the proportion of short intervals misclassified as closures between bursts. Burst length was measured directly on the video monitor by aligning vertical cursors with the beginning and end of bursts. To measure the total charge per burst a box was fitted around the burst and the amplitudes of all the sample points in the box were summed. For those current records where the individual transitions were too fast to be resolved, for example in the presence of pentanol, the total open time per burst was calculated by dividing the current integral by the open channel current amplitude and the total closed time obtained by subtracting this from the burst length. The apparent open times will be longer than the true open times due to the presence of unresolved short gaps. To calculate the true mean open time the total open time of the whole record 434 R. D. MURRELL, M. S. BRAUN AND D. A. HA YDON was divided by the corrected number of closed times plus one. The total open time was calculated as the product of the mean apparent open time and the observed number of openings.

RESULTS Channel blockade by the n-alcohols Single-channel currents recorded from an isolated membrane patch in the presence of 0-25 ,tM-ACh occurred as either brief isolated openings or longer openings sometimes interrupted by short closures or gaps (Fig. IA). At low ACh concentrations single or bursts of openings are due to single activations of one channel. In general there were probably many channels in a patch each with a low probability of being open due to the low concentration of agonist that was used. More than 95 % of channel events were due to opening of extrajunctional-type channels as judged by the single-channel conductance of 35-50 pS (Hamill et al. 1981; Siegelbaum, Trautmann & Koenig, 1984; Jaramillo & Schuetze, 1988). When 5 mM- pentanol was applied to the cytoplasmic side of the membrane patch, channel openings occurred in bursts separated by numerous very brief closures (Fig. 1B) and the mean duration of openings within a burst was reduced (4-0 to 05 ms). The closures or gaps were so short lived that in the traces shown they are not fully resolved. In records less heavily filtered a greater proportion ofdownward transitions reached the baseline suggesting that the gaps do represent a zero conductance state. The frequency of gaps increased with concentration of pentanol; with 10 mM- pentanol the fluctuations were so rapid that the open channel conductance appeared to be reduced because the individual openings were also not fully resolved (Fig. 1 C). The effects of pentanol were reversible. In four experiments where pentanol was washed out the true mean open time returned to 94 +10 % of the control value. In general the interval between bursts of openings decreased slightly when pentanol was added. Following wash-out of pentanol there was a significant decline in the frequency of bursts. On average, burst frequency during the recovery period was only 58 + 9 % of the burst frequency prior to addition of pentanol. Aqueous solutions of hexanol, heptanol and octanol when perfused onto an inside- out membrane patch, had similar effects on single-channel currents as pentanol. They caused a concentration-dependent increase in the number of gaps within a burst of openings and a reduction in the mean open time with no effect on the single- channel conductance (Fig. 2). Histograms of apparent open times for control data and in the presence of 0 4 mM-hexanol are shown in Fig. 3A and B. Both open time distributions were fitted by the sum of two exponential components and the effect of hexanol was to cause a reduction in the time constant of the slow component from 7-2 to 1-5 ms. Distributions of closed times both in the absence and in the presence of the n- alcohols were in general fitted well by the sum of two exponential components, the fast component representing the gaps within bursts and the slow component the interburst intervals. Histograms of closed times for control data and data collected in the presence of 0-4 mM-hexanol are shown in Fig. 4 with the maximum likelihood, two exponential, fits superimposed on the histograms. The histogram inset in Fig. 4B shows the fit to the shortest events. The fast time constant (rfast) was 0-13 ms which ACH RECEPTOR CHANNEL BLOCK BY n-ALCOHOLS 435 is an estimate of the mean duration of gaps. The fraction of the area represented by the fast component (afast) increased from 5-6 % in the control to 70 % in the presence of hexanol. afast was the proportion of gaps per burst. The increase in the number of gaps per burst suggests that the intermediate-chain alcohols were acting as channel blockers (Neher & Steinbach, 1978).

Control Closed

Openr

Pentanol;>r~~~~~~~~~~~I(5 mM)

Pentanol (10 mM)

2 pAL 10 ms Fig. 1. Single-channel recordings from inside-out patches with 0-25 1tM-ACh in the pipette. Records were filtered at 2-5 kHz (-3 dB point, Bessel response). The top two traces were recorded under control conditions and the middle and lower pairs were recorded in the presence of 5 and 10 mM-pentanol, respectively. These traces show an unusually high frequency of events. Membrane potential was -80 mV. Inward membrane current is shown as a downward transition. Temperature 18-22 'C.

The sequential open channel block model (Adams, 1976; Neher & Steinbach, 1978) predicts that the number of gaps per burst (nb) should increase linearly with increasing concentration of the blocking molecule (XB) according to the following expression: nb = xB(k+B/a), where k+B is the blocking rate constant and a the closing rate constant. In this case afast plotted as a function of XB should approach an 436 R. D. MURRELL, M. S. BRA UN AND D. A. HA YDON asymptote at 100 % of the total distribution. The model also predicts that the mean duration of gaps (Tfast) should be independent of concentration and should equal the reciprocal of the unblocking rate constant (k-B). In Fig. 5, af..t and Tfast are shown plotted against concentration of hexanol. afast increased with concentration to 0-87

Hexanol (0.40 mM) roF~~~~~~~~~~~~~~~

Heptanol (0-20 mM)

Octanol (0-10 mM)

Decanol (0-14 mM)

2 pA 10 ms Fig. 2. Single-channel records from inside-out patches with 0-25 4uM-ACh in the pipette, in the presence of the n-alcohol indicated. Records were filtered at 2-5 kHz (-3 dB point, Bessel response). Membrane potential was -80 mV. at 1 mM-hexanol and then appeared to saturate close to this value. For longer-chain homologues, heptanol and octanol, afast saturated at a lower value, i.e. the maximum achievable mean number of gaps within a burst declined with increasing chain length. The results do not fit with the simple sequential channel block model and suggest that if the alcohols are blocking the channel then the channel can close with the blocking molecule still bound. For hexanol (Fig. 5) and the other intermediate-chain n-alcohols Tfast was largely independent of concentration except at high concentrations (see below) although it increased with increasing chain length up to octanol (Table 1). This increase in the ACH RECEPTOR CHANNEL BLOCK BY n-ALCOHOLS 437

A B

100 200 0

E 0 0

0 4 8 4 8 Open times (ms) Open times (ms) Fig. 3. Histograms of open times for control records (A) and in the presence of 0 4 mM- hexanol (B). All distributions were fitted well with two exponentials by the method of maximum likelihood. A, rfMt =0=28 ms (25%), rl0w=7'2 ms (75%). B, T7aft =04 ms (29%), TSIow = 15 ms (71%). Membrane potential was -80 mV.

A B

200-

1100 400 - 100

0 1~~~~~~~~~~~ ~~~~100 0~~~~~~~~~~~~~~~

0 50 0 50 Closed times (ms) Closed times (ms) Fig. 4. Histograms of closed times for control records (A) and in the presence of 0'4 mM- hexanol (B). Distributions fitted with 2-exponentials by method of maximum likelihood, shown superimposed on the histograms. A, rf,8t = 0-22 ms (5-6 %), TrI1w = 40 ms (94-4 %). B, Trfst = 0 13 ms (70%), r810W= 30 ms (30%). Inset is the same distribution on an expanded time scale to illustrate the fit to the shortest events. Bin-width is 100 ,s. Minimum resolvable duration was set at 100 ,us. Membrane potential was -80 mV. mean length of gaps can be seen when single bursts of openings obtained in the presence of the different alcohols are compared as in Fig. 6. These recordings were made in the presence of equal fractional saturation of the four n-alcohols and thus the aqueous concentrations used were lower for the longer-chain molecules. With high concentrations of heptanol and octanol (005 saturated solutions) there appeared to be an additional component to the distribution of closed times, suggesting the existence of two kinetically distinct blocked states. From one 438 R. D. MURRELL, M. S. BRAUN AND D. A. HA YDON

1.0 A 0.50 B

0.8 0_ T a 0.5 ~~~~~~~Eu0.25

0.3

0-0 1000- 0-0 1.0 2-0 3-0 0.0 1.0 2.0 3-0 [Hexanoll (mM) [Hexanoll (mM) Fig. 5. A, the area of the fast component of the distributions of closed times as a function of hexanol concentration. All closed time distributions were fitted well with the sum of two exponentials. The points with error bars represent the mean of two or three patches. The curve is drawn through the points by eye. B, the fast time constant of the distribution of closed times as a function of hexanol concentration. Membrane potential was -80 mV, ACh concentration was 0-25 /tM.

TABLE 1. Mean duration of blocked periods in the presence of the n-alcohols T!rt (ms) Pentanol < 0-1 (n = 6) Hexanol 0-18 + 0 03 (n = 9) Heptanol 0-32 + 0-04 (n = 6) Octanol 0-64+0-17 (n = 5) Trst is the time constant of the fast component of the distribution of closed times. experiment with 0-81 mm-heptanol (-80 mV) the histogram of closed time within bursts showed marked deviations from a single exponential. A double exponential function provided a much better fit with time constants of 0-12 ms (42 %) and 1 0 ms (58%). A similar result was obtained with 0-21 mM-octanol. Channel block is not dependent on voltage The n-alcohols are uncharged molecules and one would predict that their rates of binding and unbinding to the channel would not show obvious voltage-dependance regardless of where the binding site(s) was located in the membrane field. We compared single-channel currents in the presence of either 2-8 mM-hexanol or 0-81 mM-heptanol (0-05 saturated solutions) at membrane potentials of -60, -80 and - 120 mV. Neither in the presence of hexanol or heptanol was there a significant difference in the mean duration of gaps at the different membrane potentials, although burst length increased significantly at the more hyperpolarized potentials suggesting that the closing rate constant (oc) decreased with hyperpolarization as has been shown previously (Colquhoun & Sakmann, 1985). In the presence of hexanol the distributions of gaps within bursts were fitted well with a single exponential with time constants as follows: at -60 mV, r = 0-26 ms; at -80 mV, r = 0-25 ms; and at ACH RECEPTOR CHANNEL BLOCK BY n-ALCOHOLS 439 - 120 mV, r = 0-28 ms. In the presence of0-81 mM-heptanol the distributions ofgaps within bursts could only be fitted well by the sum of at least two exponential components with time constants at -80 mV, of 0412 ms (42 %) and 10 ms (58 %) and at -120 mV, of 04 ms (50 %) and 0'8 ms (50%). The mean apparent open times

Pentanol 80 mV

Hexanol

Heptanol

Octanol

2 pA 5 ms Fig. 6. Single bursts of openings in the presence of0'05 saturated solutions of the different alcohols, illustrating the increase in the duration of gaps within bursts with increasing chain length. Aqueous concentrations were as follows (in mM): pentanol, 11'7; hexanol, 2'8; heptanol, 0'81; octanol, 0'21. Records were filtered at 4 kHz (-3 dB point, Bessel response). Membrane potential was -80 mV. in the presence of hexanol were 011 ms at both -60 mV and -80 mV and 0-12 ms at - 120 mV. Thus the actions of hexanol and heptanol show no apparent voltage dependence. Long-chain alcohols Nonanol and decanol, when applied to the cytoplasmic side of the membrane, did not cause an increase in the number of gaps within a burst of openings although they did cause a reduction in the duration of bursts. An example of a current trace obtained in the presence of0-14 mM-decanol is shown in Fig. 2D. The mean apparent 440 R. D. MURRELL, M. £ BRAUN AND D. A. HA YDON open time was reduced from 5'0 to 2-0 ms. There appeared- to be a decline in the activity of long-chain alcohols beyond decanol. A saturated solution of undecanol (0 07 mM) reduced the mean apparent open time to 67 + 17 % (± S.D., n = 3) of the control and a saturated solution of dodecanol (002 mM) reduced it to 80 + 20 % (±S.D., n= 3).

1C0 C7

U) a.5 00A 0~~~~~~~

0*0 l l l l 001 0.10 1.0 10X0 100*0 n-Alcohol concentration (mM) Fig. 7. The reduction in the mean charge carried per burst: (1- [Qtest/Qcontrol]), where Q is the mean current integral per burst for either the test or control data, as a function of n-alcohol concentration for pentanol (C5) to nonanol (09). A least-squares linear regression line is drawn through each set of points.

Reduction in the total charge carried per burst An open channel block model in which the channel is unable to close with the blocking molecule bound predicts that the mean duration of bursts of openings should increase with concentration of blocking molecule and that the mean total charge carried per burst should be independent of the blocker as long as the single- channel conductance is unchanged. The total charge carried per burst was calculated by summing the amplitude of all the sample points within the burst (see Methods). All the n-alcohols caused a concentration-dependent reduction in the mean burst length and a concomitant reduction in the total charge carried per burst. The percentage reduction in the current integral per burst is shown in Fig. 7 for pentanol to nonanol. The IC50s were as follows: hexanol, 0 53 + 014 mM; heptanol, 0-097 + 0'02 mm and octanol, 004 mm. The IC50 for nonanol was 0416 + 0035, indicating a decline in potency per methylene group. These data are again inconsistent with the idea that the n-alcohols are acting as simple channel blockers and suggest that the channel can undergo a conformational change to a closed state with the blocker still bound. A channel blocking model with a long-lived closed-blocked state The simple sequential open channel block model can be extended to include an additional long-lived blocked state such that entry to this state terminates a burst of openings. The type of scheme shown in Fig. 8, where there is a direct pathway ACH RECEPTOR CHANNEL BLOCK BY n-ALCOHOLS 441 between a blocked state and a closed-blocked state, has been discussed previously (Ogden & Colquhoun, 1985). We have no evidence that the alcohols are able to bind directly to the channels in the closed conformation and therefore a pathway between a closed and closed-blocked state has not been included in the scheme. If the rate at

(Closed) (Closed) (Closed) (Open) 2k+1 k+2 p R* R AR A== A2R * kL1 2Lk2 a

k-B XB k+B

Pb [BB A2RB , A2R*B ab (Closed-blocked) (Blocked) Fig. 8. A simple kinetic scheme to describe the actions of the n-alcohols on the gating of the nAChR channel, where R is the ACh receptor, A the agonist (ACh), and B the blocking molecule (n-alcohol). The rate constants are shown adjacent to their corresponding arrows. which a blocked channel closed was independent of the nature of the blocking molecule, this model would predict that the higher the binding affinity of a blocker the greater the likelihood of the channel entering the closed-blocked state and terminating the burst rather than reopening. This would fit in with our findings that with increasing chain length of the alcohol, the duration of gaps within a burst increased and the maximum number of gaps per burst decreased. Mean open time as a function of alcohol concentration Where there is only one open state the mean length of an individual opening should be equal to the reciprocal of the sum of the rate constants leading away from this state. The model in Fig. 8 would predict that in the presence of the blocking molecule the mean open time would decrease from 1/a to l/(ac+xBk+B). A plot of the reciprocal of the mean open time versus concentration should then give a straight line with a slope of k+B and an intercept of 1/ac. Plots for hexanol, octanol, nonanol and decanol are shown in Fig. 9, with the mean open times corrected for missed events. This was possible when only either gaps or openings were short with respect to tmin, for example, with low concentrations of pentanol and hexanol where the gaps are very brief but the individual openings are much longer than tmin. With high concentrations of pentanol and hexanol, where both openings and gaps are very short so that significant numbers of both are missed, it is not possible to correct the mean open time. For this reason only low concentrations of pentanol were used to estimate k+B and for hexanol the highest concentration was excluded from the plot. For each plot a line was fitted through the points by the method of least squares and the values for k+B for the different alcohols are given in Table 2. For pentanol to nonanol k+B was between 2-8 and 5-7 x 106 M-1 s-1. Values for k+B for undecanol and dodecanol were calculated from the reduction in mean open time produced by 442 R. D. Mt.rRRELL. M. S. BRA UNX AND D. A. HA YDON saturated solutions of each. Thus, 0(070 mm-undecanol reduced the mean open time by 33 % of the control while 0 020 mM-dodecanol reduced the mean open time by 20 % of the control, which gives values for k+13 shown in Table 2. These estimates of k+B were based on the assumption that the effective concentration of the alcohols at

6.0 A 1.5 B

4.0 10 E

2.0 0.5 _ 0

0.0 0.0 0-0 0-5 1.0 1.5 0.00 0.05 0.10 0.15 0.20 0.25 [Hexanoll (mM) [Octanoll (mM)

3.0 E 2.0 0.5 c 0. c 1.0

0.0 0.0 0.000 0.25 0.50 0Q75 1.00 0.00 0.10 0.20 0.30 [Nonanoll (mM) [Decanol] (mM) Fig. 9. The effects of hexanol (A), octanol (B), nonanol (C) and decanol (D) on the mean open time. The reciprocal of the mean open time is plotted against concentration of the n-alcohol. The lines were fitted by the method of least squares. A: hexanol, slope = 2-8 x 106M-1s-1, intercept = 0-10 ms-1. B: octanol. slope = (5 72+0 53) x I06M-S-1s intercept = (0-14+0 06) ms-Q. C: nonanol, slope = (3 6+0-3) x 106 M-1S-1, intercept = (0 22 + 0 14)ms-1. D: decanol, slope = (2 0 + 0 4) x 106M-IS-1, intercept = (0-13 +007)ms-1. Mlembrane potential was -80 mV. their binding site was the same as the aqueous concentration applied to the cytoplasmic side of the membrane. Although the alcohols permeate the lipid bilayer we cannot precisely assess what the concentration was on the extracellular side of the membrane and if this was the effective concentration then our estimates of k+B may be lower than the true values. Blockage frequency plot For the simple sequential channel block model the frequency of gaps per unit open time is determined only by the concentration of the blocking molecule and the blocking rate constant. Where there is a long lived closed-blocked state beyond the blocked state the frequency of gaps is reduced because only those blocked periods followed by the channel reopening are defined as gaps. The probability of a channel ACH RECEPTOR CHANNEL BLOCK BY n-ALCOHOLS 443 in the blocked state reopening is determined by the ratio of k-B to (k_ + ab), where xb is the rate at which a blocked channel closes. The expression for the frequency of gaps (f), is therefore:

A 2400 B 500 C

c- 6000 400 C,, Cu 1600 cm 4000 300 14-o > C.) ~~0200 ca 2000 8000 0) U- 0 0 0 0.0 1.6 3-2 0-0 0-4 0.8 0.00 0.12 0-24 [Hexanol] (mM) [Heptanoll (mM) [Octanoll (mM) Fig. 10. The effects of hexanol (A), heptanol (B) and octanol (C) on the frequency of blocking events per unit open time. Lines fitted by the method of least squares and have a slope of: A, (2 35+0 26) x 06m-s-1; B, (3-2+0-13) x 106M-S-1; C, (2 1 +0(14) x 106M-'S-1. Membrane potential -80 mYe. A plot of the frequency of gaps versus concentration of blocker should therefore give a straight line with a slope of k+B(k_B/kB + ab). The frequency of gaps per unit open time was calculated by dividing the corrected total number of gaps in the record by the total open time of the whole record. Plots for hexanol, heptanol and octanol are shown in Fig. 10. In each case a line was fitted by the method of least squares. Having already calculated values for k+B, the values for kB and ab were calculated from the slope of these plots together with the mean gap duration, Tfast, which should equal the reciprocal of (kB+±b) Values for the rate constants are given in Table 2. For hexanol to octanol there was a slight increase in ab from 892 to 1540 s-', whereas kB decreased progressively with increasing chain length of the alcohol. The gaps in the presence of pentanol were too brief to obtain an accurate estimate of their mean duration and therefore kB and ab were not calculated for pentanol. The affinity of binding of alcohols to site on the channel An estimate of the equilibrium dissociation constant for pentanol was made from the ratio of total blocked time to total conducting time within a burst (Neher, 1983). This parameter is a good estimate of KD if individual openings are short with respect to burst duration, which holds for high concentrations of pentanol and hexanol but not for the longer chain alcohols. The total open time within a burst was calculated by integrating the current for each burst and dividing by the open channel current amplitude. This value was subtracted from the burst length to give the total blocked time. The advantage of this method is that it does not depend on resolving the individual opening and shutting transitions. KD for pentanol calculated in this way was 8 x 10-3 M. KDs for the other alcohols were calculated from the blocking and unblocking rate constants (KD = k_B/k+B) and are given in Table 2. These values depend heavily on correctly assessing the true effective alcohol concentration and on 444 R. D. MURRELL, M. S. BRA UN AND D. A. HA YDON

-10 -

-20-

-30 I l l 3 4 5 6 7 8 Number of methylene groups Fig. 11. The change in the free energy for binding of the n-alcohols, from pentanol to octanol, to their site of action. A least-squares regression line has been fitted to the data and the slope of the line is -33 kJ mol-1.

TABLE 2. Channel block by the n-alcohols k+B k-B aB KD X 106 M-1 s-1) (S-1) (S-') (10-3 M) Pentanol 3-7 29000 8 Hexanol 2-8 4663 892 1-66 Heptanol 5-6 1850 1490 0 33 Octanol 5-7 900 1540 015 Nonanol 3.5 Decanol 2-0 Undecanol 1P2 Dodecanol 2-0 k+B is the channel blocking rate constant, kiB is the unblocking rate constant, aB is the rate constant from the blocked state to the closed-blocked state, and KD is the equilibrium dissociation rate constant. the estimates of the open lifetimes as these are used to calculate k+B (Fig. 9) which is then used to calculate k-B. Calculating KD for hexanol in the same way as for pentanol, from the ratio of the total conducting time to the total blocked time for bursts of openings in the presence ofhigh concentrations ofthe alcohol (0-94-2-8 mM), gives a value of 1-76+0-24 mm, which is very similar to the value for hexanol of 1-66 x 10-3 M given in Table 2. The free energy change for binding of the n-alcohols to their site of action is equal to -RTlnKD, where R is the gas constant and T, the temperature on the ideal gas scale. The standard free energy per methylene group for transfer from the aqueous phase to a site of action can be estimated from the slope of the plot of -RTlnKD against the number of methylene groups in the molecule (Fig. 11). A least-squares linear regression line through the points for pentanol through to octanol has a slope of -3.30 kJ mol-1. The dissociation constants for nonanol and decanol were not obtained. There were so few gaps within bursts in the presence of nonanol and decanol that they did not ACH RECEPTOR CHANNEL BLOCK BY n-ALCOHOLS 445 form a distinct component in the distribution of closed times. From the values obtained for k/B and ab for hexanol, heptanol and octanol one could predict that the value of kB for nonanol would be approximately one fifth the value of aB and thus only between 15 and 20 % of the time would a blocked channel reopen.

DISCUSSION Resolution of the effects of the n-alcohols on ACh-activated currents at the single channel level has provided information about their mechanism of action. The n-alcohols from pentanol to octanol have been shown to have two main effects on channel gating. Firstly, they caused channel openings to be interrupted by brief closed or blocked periods. The frequency of these blocked periods was linearly dependent upon alcohol concentration, and their duration was dependent on the chain length of the homologue. Secondly, they caused a decrease in burst length with a concomitant reduction in the mean charge carried per burst. The longer-chain n- alcohols up to undecanol caused only the second of these two effects, and beyond undecanol there appeared to be a decline in the ability of the n-alcohols to affect channel function. The effects of the alcohols from pentanol to dodecanol on the nAChR channel were consistent with a model in which the alcohols bind to the channel in the open state thereby blocking ion conduction through the pore. The channel can undergo a conformational change to a closed state with the blocking molecule still bound, and the rate at which the blocked channel closes is between 5 and 10 times faster than the rate at which the unblocked channel closes. This model suggests that when an alcohol molecule is bound to the channel the open channel conformation is destabilized relative to the closed conformation. An alternative and consistent model is that at least two binding sites are involved; one, for example, in or near the channel pore producing a channel-blocking effect, and another site located elsewhere on the channel or at the channel protein-lipid interface, which when occupied causes the open state to be destabilized relative to the closed state. In either case, the closed state with the alcohol molecule bound may represent a desensitized state of the channel. There is evidence from binding studies that the n-alcohols, in the presence of an agonist, enhance the extent of desensitization of nAChR channels by binding with higher affinity to the desensitized state than to the open or resting state (Firestone, Sauter, Braswell & Miller, 1986). The channel blocking reaction The rate constants for the blocking reaction, for pentanol to nonanol, were calculated to be between 2-8 and 5.5 x 106 M-1 s-1. These are lower than values obtained for charge anaesthetics and other putative channel blockers (Adams, 1976; Neher & Steinbach, 1978), and are also a factor of 100 less than would be expected if it were a diffusion-limited process, suggesting that binding of an alcohol molecule to the channel may involve a conformational change in a portion of the channel protein. In this study alcohols were perfused onto the cytoplasmic side of the membrane whereas charged anaesthetics have been shown to block from the outside only. 446 R. D. MURRELL. M. S. BRA(UN AND D. A. HAYI)O McLarnon et al. (1986) investigated the effects of externally applied n-alcohols on the rate of decay of miniature endplate currents at the mouse neuromuscular junction and obtained values for blocking rate constants (k+B) for hexanol and octanol of 3-6 x 106 M-l s-5 and 17 x 106 M-1 s-51 respectively. The value for hexanol is similar to that we obtained suggesting either that the intermediate-chain alcohols are able to block the channel from both the inside and outside, or that they block from the lipid phase, or that these alcohols permeate the membrane so rapidly that their effective concentration on either side of the membrane is the same. The intermediate- chain alcohols are known to permeate cell membranes very rapidly (Brahm, 1983), although the extracellular concentration will depend on the balance between the rate of permeation through the membrane patch and the rate of diffusion away from the membrane surface into the pipette solution and therefore cannot be precisely assessed. One possible explanation for why lower values of k±B were obtained for the long-chain alcohols is that they permeated the membrane less rapidly and thus the effective concentrations were lower than those used to calculate k+B. Another possible explanation is that for these larger molecules access to the binding site was restricted. Nature of the binding site For the n-alcohols from pentanol to octanol there was a decrease in the equilibrium dissociation constant for binding with each additional methylene group in the molecule which strongly suggests that the binding site was hydrophobic in nature. For each methylene group in the molecule that escaped from an aqueous to a hydrophobic environment there would be a favourable free energy change and the higher the net free energy change the more stable the bound complex would be. From the values of KD for pentanol to octanol the standard free energy per methylene group for transfer from the aqueous phase to a site of action was calculated to be -3 3 kJ mol'. This is approximately equal to the free energy change associated with the incorporation of the n-alcohols from water into the alkanes, as measured by Aveyard & Mitchell (1969). It is slightly higher than the value of -305 kJ mol', calculated for the reduction in sodium current in rat dorsal root ganglion neurones (Elliott & Elliott, 1989) but it is very similar to that associated with shifting the steady-state level of inactivation of the sodium current. The fact that KD continues to decrease up to octanol suggests that the hydrophobic binding site must be at least large enough to accommodate an eight hydrocarbon chain. Location of the binding site Interpreting the actions of the alcohols by a mechanism of open channel blockade suggests that the alcohols bind to a site within the channel pore thereby sterically blocking the pore and preventing the passage of ions. From our data it is not possible to distinguish between this mechanism of action and one in which the molecule binds to an allosteric site removed from the pore which then induces a change to a closed conformation of the channel where the binding reaction is the rate-limiting process. However, there is convincing evidence (Giraudat, Dennis, Heidmann, Chang & Changeux, 1986; Imoto et al. 1986; Imoto, Busch, Sakmann, Mishina, Konno, Nakai, Bufo, Mori, Fukuda & Numa, 1988) that it is the M2 membrane spanning domain of ACH RECEPTOR CHANNEL BLOCK BY n-ALCOHOLS 447 each of the channel subunits which form the lining of the pore. This twenty amino acid segment contains no charged residues except at its two ends and although there are a number of polar residues there is one stretch, between amino acids 13 to 19, which is almost wholly hydrophobic. Point mutations of the polar residues in this region have been shown to affect the binding affinity of the charged local anaesthetic open channel blocker QX222 (Leonard, Labarca, Charnet, Davidson & Lester, 1988; Charnet et al. 1990). It would be interesting to see whether the binding affinity of the alcohols were affected by point mutations involving the replacement of one of the hydrophobic residues for a charged or polar residue. We would also like to determine whether the actions of the alcohols were mutually exclusive or whether they bind to different sites on the channel. We cannot rule out the possibility that there are at least two and possibly many sites involved in the effects of the n-alcohols on nAChRs. However, we have demonstrated the range of effects of the n-alcohols between pentanol and dodecanol on nAChR channels, from the flickering, to the reduction in burst length, to the cut- off in activity, and shown that they are consistent with the alcohols acting at a single binding site on the channel protein. Interestingly, the concentrations of the n-alcohols from pentanol to nonanol causing a 50% reduction in the charge carried per burst are very similar to the concentrations causing general anaesthesia (ED50s) in amphibians (Alifimoff, Fineston & Miller, 1989), suggesting that a similar site on a channel protein may be involved in the general anaesthetic effect of the n-alcohols.

We would like to thank the SERC for financial support and Drs J. R. Elliott, R. Fettiplace and D. Ogden for helpful comments on the manuscript.

REFERENCES ADAMS, P. R. (1976). Drug blockade of open end-plate channels. Journal of Physiology 260, 531-552. ALIFIMOFF, J. K., FINESTON, L. L. & MILLER, K. XW. (1989). Anaesthetic potencies of primary alkanols: implications for the molecular dimension of the anaesthetic site. British Journal of Pharmacology 96, 9-16. AVEYARD, R. & MITCHELL, R. WV. (1969). Distribution of n-alkanols between water and n-alkanes. Transactions of the Faraday Society 65, 2645-2653. BRAHM, J. (1983). Permeability of human red cells to a homologous series of aliphatic alcohols. Journal of General Physiology 81, 283-304. BRAUN, M. S., HAYDON, D. A. & MURRELL, R. D. (1989). The effects of n-alcohols on the nicotinic acetvlcholine receptor channel in cultured rat myotubes. Journal of Physiology 418, 37P. CHANGEUX, J.-P., GIRAtUDAT, J. & DENNIS. M. (1987). The nicotinic acetylcholine receptor: molecular architecture of a ligand-regulated ion channel. Trends in Pharmacological Science 8, 459-464. CHARNET, P., LABARCA, C., LEONARD, R. J., VOGELAAR, N. J., CZYZYK, L., GOUTN, A., DAVIDSON, N. & LESTER, H. A. (1990). An open-channel blocker interacts with adjacent turns of A-helices in the nicotinic acetylcholine receptor. Neuron 2, 87-95. COLQUHOUN, D. & SAKMANN, B. (1985). Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. Journal of Physiology 369, 501-557. COLQUIIOUN, D. & SIGWORTH, F. J. (1983). Fitting and statistical analysis of single channel data. In Sinyle Channel Recording, ed. SAKMANN, B. & NEHER, E. Plenum Press, New York. ELLIOTT, A. A. & ELLIOTT, .J. R. (1989). The role of inactivation in the effects of n-alkanols on the sodium current of cultured rat sensory neurones. Journal of Physiology 415, 19-33. .448 R. D. MURRELL, M. S. BRAUN AND D. A. HA YDON ELLIOTT, J. R. & HAYDON, D. A. (1989). The actions of neutral anaesthetics on ion conductances of nerve membrane. Biochimica et Biophysica Acta 988, 257-277. FIRESTONE, L. L., SAUTER, J. F., BRASWELL, L. M. & MILLER, K. W. (1986). Actions of general anesthetics on acetylcholine receptor membranes from Torpedo californica. Anesthesiology 64, 694-702. FOREMAN, S. A. & MILLER, K. W. (1989). Molecular sites of anesthetic action in postsynaptic nicotinic membranes. Trends in Pharmacological Science 10, 447-452. FRANKS, N. P. & LIEB, W. R. (1987). What is the molecular nature of general anaesthetic target sites? Trends in Pharmacological Science 8, 169-174. GAGE, P. W. & HAMILL, 0. P. (1981). Effects of anaesthetics on ion channels in synapses. In Neurophysiology IV, International Review ofPhysiology 25, ed. PORTER, R. University Park Press, Baltimore, MD, USA. GAGE, P. W., MCBURNEY, R. N. & SCHNEIDER, G. T. (1975). Effects of some aliphatic alcohols on the conductance change caused by a quantum of acetylcholine at the toad end-plate. Journal of Physiology 244, 409-429. GIRAUDAT, J., DENNIS, M., HEIDMANN, T., CHANG, J.-Y. & CHANGEUX, J.-P. (1986). Structure of the high affinity binding site for noncompetitive blockers of the acetylcholine receptor: Serine- 262 of the d subunit is labelled by [3H]chlorpromazine. Proceedings of the National Academy of Sciences of the USA 83, 2719-2723. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patch- clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pfluiger Archiv 391, 85-100. HORN, R. & BRODWICK, M. S. (1980). Acetylcholine-induced current in perfused rat myoballs. Journal of General Physiology 75, 297-321. IMOTO, K., BUSCH, C., SAKMANN, B., MISHINA, M., KONNO, T., NAKAI, J., BUFO, H., MORI, Y., FUKUDA, K. & NUMA, S. (1988). Rings of negatively charged amino acids determine the. acetylcholine receptor channel conductance. Nature 335, 645-648. IMOTO, K., METHFESSEL, C., SAKMANN, B., MISHINA, M., MORI, Y., KONNO, T., FUKUDA, K., KURASAKI, M., BuJo, H., FUJITA, Y. & NUMA, S. (1986). Location of a d-subunit region determining ion transport through the acetylcholine receptor channel. Nature 324, 670-674. JARAMILLO, F. & SCHUETZE, S. M. (1988). Kinetic differences between embryonic- and adult-type acetylcholine receptors in rat myotubes. Journal of Physiology 396, 267-296. LECHLEITER, J. & GRUNER, R. (1984). Halothane shortens acetylcholine receptor channel kinetics without affecting conductance. Proceedings of the National Academy of Sciences of the USA 81, 2929-2933. LEONARD, R. J., LABARCA, C. G., CHARNET, P., DAVIDSON, N. & LESTER, H. A. (1988). Evidence that the M2 membrane spanning region lines the ion channel pore of the nicotinic receptor. Science 242, 1578-1581. MCLARNON, J. G., PENNEFATHER, P. & QUASTEL, D. M. J. (1986). Mechanism of nicotinic channel blockade by anesthetics. In Molecular and Cellular Mechanisms ofAnesthetics, ed. ROTH, S. H. & MILLER, K. W., pp. 155-163. Plenum Press, New York. MILLER, K. W. (1985). The nature of the site of general anaesthesia. International Review of Neurobiology 27, 1-61. NEHER, E. (1983). The charge carried by single-channel currents of rat cultured muscle cells in the presence of local anaesthetics. Journal of Physiology 339, 663-678. NEHER, E. & STEINBACH, J. H. (1978). Local anaesthetics transiently block currents through single acetylcholine-receptor channels. Journal of Physiology 277, 153-176. OGDEN, D. C. & COLQUHOUN, D. (1985). Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction. Proceedings of the Royal Society B 225, 329-355. OGDEN, D. C., SIEGELBAUM, S. A. & COLQUHOUN, D. (1981). Block of ACh-activated ion channels by an uncharged local anaesthetic. Nature 289, 596-599. SIEGELBAUM, S. A., TRAUTMANN, A. & KOENIG, J. (1984). Single acetylcholine-activated channel currents in developing muscle cells. Developmental Biology 104, 366-379.