Considering the partition coefficient of alcohol between lipid and aqueous solution, (Received 3 January 1992)

Transcription

1 Journal of Physiology (1993), 463, pp With II figures Printed in Great Britain KINETIC ANALYSIS OF THE DENATURATION PROCESS BY ALCOHOLS OF SODIUM CHANNELS IN SQUID GIANT AXON BY FUMIO KUKITA AND SHIGEKI MITAKU From the Ine Marine Laboratory, National Institute for Physiological Sciences, Ine, Kyoto, , the * Laboratory of Membrane Biology, National Institute for Physiological Sciences, Myodaiji-cho, Okazaki 444 and the Faculty of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 110, Japan (Received 3 January 1992) SUMMARY 1. The effects of several aliphatic alcohols on sodium currents were examined in the intracellularly perfused squid giant axon when the same concentration of alcohol was applied on both sides of the membrane. 2. An irreversible suppression of sodium currents, accompanied by anaesthesia at high alcohol concentration, was examined in detail using four aliphatic alcohols, that is, ethanol, 1-propanol, 1-butanol and 1-pentanol. 3. This irreversible effect seemed to be attributable to the sequential denaturation of sodium channels, because the kinetics, the current-voltage relation and the sodium channel activation-voltage curve did not change after the sodium current decreased. 4. The time course of the remaining sodium conductance was measured as a function of the sum of the alcohol application time by repeating the process of applying and completely washing out alcohol. The remaining sodium conductance decayed as a function of time in a single exponential manner. This decay time constant depended strongly on the concentration of alcohol and could be assumed to be the denaturation time constant of the sodium channel. 5. The denaturation time constant decreased as the alcohol concentration increased. This time constant is proportional to the Nth power of the alcohol concentration. The N values are 4 3, 4 5, 5-8 and 7-6 for ethanol, 1-propanol, 1- butanol and 1-pentanol, respectively. This implies that alcohol molecules bind to a restricted number of specific sites in the sodium channel protein to cause the denaturation. 6. The concentration of alcohol which caused the same amount of denaturation is related to the exponential function of the carbon number of the alcohol. Considering the partition coefficient of alcohol between lipid and aqueous solution, the concentration of alcohol in the membrane which denatured half of the sodium channels in 2 h can be calculated to be 05 M for all alcohols. * To whom correspondence should be addressed. MtS 1016

2 524 F KUKITA AND S. MITAKU INTRODUCTION Recently the determination of the primary structure of the voltage-dependent sodium channel has led to studies aiming to clarify the relationships between physiological functions and the higher molecular structure of the sodium channel protein (Noda et al. 1984, 1986; Salkoff, Butler, Scavarda & Wei, 1987). However, at present the tertiary structure of the sodium channel protein is not yet clear. Furthermore, the investigation of many other membrane proteins is also in a similar state. In this situation, aspects of the structure and function relationship in the membrane proteins may be revealed by the denaturation experimental approach (Tanford, 1968). The denaturation measurements have been carried out extensively for the bacteriorhodopsin of the Halobacterium halobium membrane (Eisenbach, Caplan & Tanny, 1979; Mitaku et al. 1988), indicating that the hydrogen bonding interaction is important for the tertiary structure formation of this protein. It is also interesting to know how the physiological function of the sodium channel protein changes when the global tertiary structure of this protein is altered in the denaturation process, because nerve cells suffer two kinds of effects when treated by many chemicals: an anaesthetic effect and a denaturation effect. Aliphatic alcohols are interesting chemicals because they have been widely known as nerve anaesthetics and have additional effects other than anaesthesia (Armstrong & Binstock, 1964; Haydon & Urban, 1983; Paternostre, Pichon & Dupeyrat, 1983; Haydon, Elliot & Hendry, 1984). The latter effects might be related to the denaturing effects of these agents which have been demonstrated, in the bacteriorhodopsin, to disrupt only the tertiary structure and not the secondary structure (Mitaku et al. 1988). We examined the effects of several species of alcohol on the sodium currents in the intracellularly perfused squid giant axon, which is a very useful preparation because changing both the external and internal solutions is easy and complete and is rarely damaging. There were two types of effect of alcohol, i.e. reversible and irreversible effects. The former effect appeared quickly and at very low concentrations of alcohol. This anaesthetic effect has been widely examined. As the alcohol concentration increased and/or the application time became longer, the irreversible effect became marked (Haydon et al. 1984). However, it has been difficult to clarify the mechanism of the latter effect. It is true that if we use steady-state analysis as have previous investigators, it is difficult to discriminate between the denaturation of the sodium channel and various other types of damaging irreversible effects. Therefore, we intensively examined this effect from the kinetic viewpoint in carefully designed experimental conditions. The final results we obtained are so simple that this irreversible effect could be attributed to the denaturation of sodium channel proteins. Preliminary results have been reported elsewhere (Mitaku, Kukita & Kasai, 1990; Kukita & Mitaku, 1991). METHODS Squids (Doryteuthis bleekeri), obtained from the coast of Ine, Kyoto, Japan, were decapitated with fine scissors. The hindmost stellar giant axons ( ,um in diameter) were used for an intracellular perfusion using the modified squeezing method (Kukita, 1982). The membrane

3 ALCOHOL DENATURATION OF Na+ CHANNELS potential was measured with asbestos-filled glass-tipped capillary electrodes containing floating platinum wires (Conti, Inoue, Kukita & Stiihmer, 1984). Voltage clamping was performed as described previously (Kukita, 1988). The series resistance was decreased to less than 5 Q cm2 with the proper arrangement of the potential measuring electrode and then compensated with a series resistance compensation circuit by more than 70 %. All membrane current data were digitized with an A-D converter (Autonics 204C, Japan) using a sampling time of 10,us after passing through a low-pass 50 khz Bessel filter, and analysed with a microcomputer (NEC PC9801RA, Japan). Capacitative surges and leakage currents were removed from the membrane current records in off-line computer analysis by subtracting, after appropriate scaling, the membrane current records obtained with a hyperpolarizing voltage pulse of 40 mv. Depolarizing voltage pulses for 8 ms were applied after a conditioning voltage pulse of -100 mv for s to remove the sodium channel inactivation (Meves, 1978). Furthermore, to remove the remaining very slow sodium current inactivation, two different protocols were used. The initial resting potential of the- axon used was mv (n = 20). The holding potential value was chosen as follows. When the resting potential was less negative than -60 mv, the holding potential was -60 mv but when the resting potential was more negative than -60 mv, the holding potential was set at the value of the resting potential. In protocol 1, the membrane potential was held at this holding potential throughout the whole experiment, while in protocol 2, the membrane potential was held at this holding potential for more than 5 min before the current measurements and the holding potential was maintained through each series of measurements, stopping the voltage clamp until the next measurements. The step of measuring the sodium current during the application of alcohol to minimize unexpected damage was frequently skipped, and the sodium currents were measured before and after the alcohol application to measure the time course of the denaturation process. All voltage clamp experiments were finished before an appreciable amount of leakage current was observed. The external solution contained (mm): 450 NaCl, 10 KCl, 20 CaCl2, 30 MgCl2 and 15 Tris-Hepes (ph 80). The internal solution contained (mm): 120 KF, 80 potassium phosphate (ph 74), 10 tetraethylammonium phosphate and 614 glucose, the last of which was added to obtain a final osmolarity of 1020 mosmol l-'. In solutions containing alcohol, water was replaced with alcohol in order to maintain the molarity of other solutes. The temperature was maintained at around 10 C by circulating the external solution through a heat exchange unit. Solutions both inside and outside the axon were changed at the same time and the hydrostatic pressure for intracellular perfusion was as small as possible to minimize the possible mechanical damage to the axon (Haydon & Kimura, 1981) but large enough for rapid change of solution. Usually the level of the internal solution in the container was 5 cm above the axon except for the experiment in Fig. 1. The denaturation kinetics of the sodium channel during the anaesthesia was measured as follows. The sodium currents before and after the anaesthesia were measured. This process was repeated several times. Then the remaining sodium conductance obtained from the linear portion of the current-voltage curve was plotted as a function of the total time that the axon had been immersed in the alcohol-containing solution before the measurement, which was the cumulative time of anaesthesia. 525 RESULTS Alcohol concentrations in the membrane can be changed by the flow rate of an internal solution When 2 M ethanol was applied from the inside of the membrane, the sodium current decreased to one-third within a few minutes at the fast flow rate with a larger hydrostatic pressure at which the water level of the internal solution was 20 cm above the axon (Fig. 1A). When the flow rate was decreased by decreasing the water level to 3 cm, the sodium current was gradually restored within 10 min to more than 90 % of the original magnitude before ethanol application, even though the current might have been suppressed to some extent by the remaining ethanol

4 526 F KUKITA AND S. MITAKU in the internal solution. When 2 M ethanol was applied from both sides of the membrane, the change of flow rate did not affect the magnitude of the sodium current (Fig. IB). When the flow rate was decreased by decreasing the water level of the internal solution from 20 to 1 cm, the sodium current did not change for A 50 B ma cm-2 50 mv Fig. 1. The flow rate of an intracellular solution changes the effects of ethanol application. A, the magnitude of peak sodium currents decreased to less than one-quarter of the initial magnitiude (0) when the internal solution containing 2 M ethanol was perfused with the level of the internal solution 20 cm above the axon (@). When the flow rate was decreased by decreasing the water level from 20 to 3 cm, the sodium current returned to more than 90 % of the initial value within 10 min (E). B, the magnitude of the sodium current did not change for 18 min when the flow rate was decreased by decreasing the level of the internal solution from 20 (0) to 1 cm (E) when both the internal and external solution contained 2 M ethanol. When only the external solution was switched to solutions which did not contain ethanol, the sodium current increased to about 3 times the initial value in 10 min (@). 18 min. This result shows that the increase in the sodium current after decreasing the hydrostatic pressure in Fig. IA is not attributable to mechanical effects (Haydon & Kimura, 1981). It was also observed that the axons perfused in the solution without ethanol on both sides were not as sensitive to hydrostatic pressure change. When only the external solution was switched to the solution which did not contain ethanol, the sodium current increased within 5 min to about 2&5-fold of the initial value. These two results show that ethanol can easily permeate through the membrane and could easily be diluted when it was applied from one side. So it

5 ALCOHOL DENATURATION OF Na+ CHANNELS is essential to apply the ethanol or longer hydrocarbon chain aliphatic alcohols (which are more lipophilic and more permeable than ethanol) from both sides of the membrane to observe the quantitative effect of alcohols on the sodium current. Otherwise the alcohol concentration in the membrane could not be controlled as e e e oo z CO 0) G) Time (min) Fig. 2. The decay of sodium conductance under intracellular perfusion and continuous voltage clamp. The sodium conductance (gna) obtained from a linear portion of the current-voltage curve was plotted as a function of time. On the ordinate the relative change of gtn. is expressed as a percentage. The arrows show the point when the internal and external solutions were exchanged between two same-constituent solutions neither of which contained alcohol. There was no large decrease in gn. during the experiment for 3 h. The decay time constant obtained by fitting with a single exponential was 1782 min. Data were obtained using protocol 1. required. These flow rate effects might partially explain the discrepancy between the dosage of alcohol which is needed to suppress the sodium current by 50% between the intact axon and the axon intracellularly perfused with solutions containing CsF (Haydon et al. 1984). Sodium currents remain for a long time in the absence of alcohol To estimate the run-down of the sodium current following rapid intracellular perfusion with solutions which did not contain alcohol, sodium currents were measured for 3 h during which the internal and external solutions were exchanged several times with solutions of the same constituents. The membrane currents were measured using protocol 1. As shown in Fig. 2, there was no notable decrease in the sodium conductance. The decline time constant was 1738 min, which means a 10 % decrease in the sodium conductance after 3 h. The mean decline time constant obtained from similar experiments was min (n = 6). The run-down in

6 528 F KUKITA AND S. MITAKU the absence of alcohol was sufficiently slow to measure the denaturation time course in sodium currents in the presence of alcohol. The run-down measured using protocol 2 was expected to be slower than that measured using protocol 1, because there was no harmful disturbance to the axon. A 0 B C o C -4-4 AI 6 _ L L- o 2 4 Time (ms) 6 o 2 4 Time (ms) J Time (ms) 6 D 100 z._ c 0 ac 0) 10 1 ci Time (min) Fig. 3. Changes of sodium currents and sodium conductances during and after the application of 82 mm 1-pentanol. Families of sodium current traces are shown in the upper panel. The traces in A and C were obtained before applying and after washing out 1-pentanol. The traces in B were obtained during 1-pentanol application. These traces were obtained at membrane potentials from -10 to 20 mv in 10 mv steps. The times when current traces were obtained are shown for A, B and C in D. D, the percentage change in 9Na plotted as a function of time. gna decreased at the time of 1-pentanol (Pent) application and recovered at the instance of washing out (Wash). Data were obtained using protocol 1. Denaturation effects by alcohols was accompanied by anaesthesia The change in the sodium current during and after the application of 82 mm 1-pentanol which is 5-fold larger than the concentration used for anaesthesia (Haydon et al. 1984) is shown in Fig. 3. To measure the sodium current continuously, the membrane potential was held throughout the experiment at -64 mv, because the resting membrane potential at the start of the experiment was -64 mv (protocol 1). This procedure could help to avoid the overestimation of the alcohol effect,

7 ALCOHOL DENATURATION OF Nae CHANNELS because this high concentration of alcohol depolarized the membrane potential at the most by 20 mv and produced slow inactivation of the sodium channel during the application of 1-pentanol. When 1-pentanol was applied, the sodium current almost disappeared within a few minutes and recovered after 1-pentanol was washed out. The time course of the sodium current before and after 1-pentanol application was unchanged (upper panel of Fig. 3). The change in the sodium conductance 9Na is plotted as a function of time (Fig. 3D). The sodium conductance, YNa' was obtained from the linear portion of the current-voltage relation curve as in Fig. 1. The sodium conductance decreased to about 1 % of control within a few minutes and remained in this state (which should be called anaesthesia). When 1-pentanol was completely washed out from both sides of the membrane, the sodium conductance recovered immediately but never reached the initial magnitude and stayed at this level for a long time. This procedure could be repeated several times. The three continuous trials are shown in Fig. 3. It is clear that a significant denaturation occurred during 1-pentanol application. The sodium conductance decreased 56, 34 and 18 % after 1-pentanol application for 15X5, 31 and 47 min, respectively. After washing 1- pentanol out, about 40 % of the remaining sodium channel did not recover after anaesthesia during the application of 1-pentanol at each trial. As shown later, this denaturation during the alcohol application occurred with a time constant of 28 min. There was also a slight decay in the sodium conductance even after the 1-pentanol was washed out. The decay time course in the first two trials was fitted with a single exponential function. The decay time constants obtained were 259 and 274 min. In the third trial the sodium conductance increased slightly but not significantly. The decay time course is shown by the dashed lines in Fig. 3D. This slow decay could be explained as having the same cause as the main fast decay and might be the cause of errors in estimating the denaturation kinetics during anaesthesia. During further analysis, we included all denaturation effects after alcohol application, by taking one data point in each recovery period. The error was at most around 10 % and was not significantly large to affect the final results. The sodium channel during the application of 82 mmi 1-pentanol is considered to be in a state of anaesthesia and after washing out the 1-pentanol seemed to be in a partly denatured state. The time course of the denaturation process is slower by a factor of 100 than that of anaesthesia. By repeating this procedure several times, the time course of the remaining sodium conductance could be obtained- as a function of the sum of the application time of 1-pentanol. In the usual experiment to examine the effect of anaesthesia, the concentration of alcohol used is less than one-fifth of that in our experiment (Haydon & Urban, 1983; Paternostre et al. 1983; Haydon et al. 1984). In this case the recovery is practically complete. We did not use this low concentration of alcohol to see the alcohol denaturation, because it was difficult to discriminate between the alcoholrelated denaturation and the natural denaturation shown in Fig. 2. The current-voltage relation of the sodium current before and after application of 90 mmi 1-pentanol is shown in Fig. 4A. The current-voltage relations were obtained after application of 1-pentanol for 15.5, 31 and 47 min. The current-voltage relation before and after the 1-pentanol application seemed 529

8 530 F KUKITA AND S. MITAKU ma cm r^f;v 100 B vm (mv) Fig. 4. A, current-voltage relations before and after the application of 90 mm 1-pentanol. The current-voltage relation was obtained just before the 1-pentanol application (0) and 155 (M), 31 (El), and 47 min (M) after application of 1-pentanol. B, sodium channel activation-voltage curves obtained just before the 1-pentanol application (0) and 15-5 (M), 31 (E1) and 47 min (U) after application of 1-pentanol. The sodium chord conductance, gna, defined by eqn (1), was normalized (to 1) to the maximum of the data before 1-pentanol application (0) and then plotted as a function of membrane potential. Each set of data was fitted using eqn (2). similar despite a large decrease in magnitude, to about one-fifth of the initial value, after the application. The sodium equilibrium potential, ENa, did not change significantly. The E values were 71, 71, 73 and 77 mv before, and after the 15X5, 31 and 47 min application of 1-pentanol, respectively. The chord conductance of the sodium channel is defined in eqn (1). c _ Na (1) N Vm -ENa' where gna' INa ENa and Vm are the sodium channel chord conductance, the sodium current, the sodium equilibrium potential and the membrane potential, respectively. qc is plotted as a function of the membrane potential to show the activation curve of the sodium channel (Fig. 4B). The sodium conductance decreased to 75, 44 and 20 % of the initial value after 15-5, 31 and 47 min application of 1-pentanol. The activation curve did not shift except for that in the last record which shifted in the depolarizing direction by about 9 mv and the sodium conductance was about 20 % of the initial value. In the last record, however, the ENa also shifted by 5 mv.

9 ALCOHOL DENATURATION OF Na+ CHANNELS There was no strong evidence that this could not be due to drift of the measuring system for the long-term continuous voltage clamp experiment. Usually there was no significant shift in ENa and the activation curve. The activation curves are fitted with eqn (2) N= 90Na + Na (2) I + e ' 2'' 2 where 9Na, 9 K and Vi are respectively, the sodium conductance at the most negative membrane potential, the sodium conductance at the most positive membrane potential, the steepness of the activation curve and the half-activation membrane potential. The values of gn,9 and K varied among data but were close to 0, 100 and 0.1 and are not mentioned here. Vi was -20, -16, -14 and -10 mv showing the activation curve shifted in the depolarizing direction, while ENa values were -71, -71, -72 and -77 mv. The mean values of Vi obtained from the data at 1-pentanol concentrations between 66 and 98 mm were (n = 9), (n = 9), (n = 9) and (n = 6) mv for the control and after the first, second and third application, respectively. The mean values of shift in Vi were (n = 9), (n = 9), (n = 6) mv after the first, second and third application. The values of ENa were (n= 9), (n= 9), (n= 9), (n = 6) mv for the control and after the first, second and third application, respectively. In the denatured state, there was not a large change in the activation curve and ENa. There was a tendency of a small shift of the membrane potential in the depolarizing direction due to the long-term voltage clamp and some effects of alcohol on the potential electrodes. Similar data were obtained after the application of ethanol at concentrations between 1P19 and 4 0 M. The mean values of Vi were (n = 10), (n= 10), (n=8) and (n=5)mv; the shifts of Vi were 0, 3+7 (n = 10), -1 ±4 (n= 8) and -5+4 (n= 5) mv; and the mean values of ENa were (n = 10), (n = 10), (n=8), (n = 5) mv; all results for the control and after the first, second and third application. Denaturation effects were observed when 1-propanol was applied at concentrations between 643 and 1286 mm. The mean values of Vi were (n = 8), (n = 8), (n= 7) and (n = 7) mv; the mean values of shift in Vi were 0, (n = 8), (n = 8), and (n = 7) mv; and the mean values of ENa were (n=8), (n= 8), (n= 8), and (n = 7) mv; all results for the control and after the first, second and third application. Denaturation effects were obtained when 1-butanol was applied at concentrations between 203 and 284 mm. The mean values of Vi were (n = 4), (n=4), (n = 3) and (n = 3) mv; the shift of Vi was 0, (n = 4), (n = 3) and (n =3) mv; and the mean values of ENa were (n = 4), (n = 4), (n = 3) and 74 ± 2 (n = 3) mv; all results for the control and after the first, the second and third application. 531

10 532 F KUKITA AND S. MITAKU Time dependence of the denaturation process as a function of time of anaesthesia In Fig. 5 the remaining Na+ conductance was plotted as a function of the sum of the 1-pentanol application time, i.e. the time during which the axon was anaesthetized. The data obtained using protocol 1 are plotted in Fig. 5A. The remaining sodium conductance seems to decrease as a single exponential, decaying A B 1 -Pentanol 1 -Pentanol c C: lo 0 o Time (min) Time (min) Fig. 5. A, the percentage of remaining gna, values obtained at 1-pentanol concentrations of 66 (O), 74 (0), 82 (O), 92 (-) and 98 mm (A) are plotted on a logarithmic scale as a function of the sum of the I1-pentanol application time. gn,a decays as a single exponential function. The straight lines through the data points are obtained by fitting data points with a single exponential function. The data were obtained using protocol 1. B, plot of 9Na. values obtained at 1-pentanol concentrations of 74 (O), 82 (0), 90 (O) and 98 mm (-) using protocol 2. with a time constant of 320 min at a I1-pentanol concentration of 82 mm. As the I1-pentanol concentration increased, the decay time constant decreased. The decay time constants at I1-pentonal concentrations of 66, 74, 82, 90 and 98 mm are 124, 88, 28, 12-5 and 4-6 min, respectively (Fig. 5A). The data obtained using protocol 2 are plotted in Fig. 5B. The decay time constants obtained using protocol 2 were 158, 94, 67 and 22 min at I1-pentanol concentrations of 74, 82, 90 and 98 mm, respectively and are larger than those obtained with protocol 1. In Fig. 6A, the remaining sodium conductance is plotted as a function of the sum of the application time of ethanol. The remaining sodium conductance seems to decrease as a single exponential, decaying with a time constant of 315 min at an ethanol concentration of 1 59 M. As the ethanol concentration increased, the decay time constant decreased. The decay time constants obtained with protocol I at

11 ALCOHOL DENATURATION OF Nae CHANNELS ethanol concentrations of 1P19, 1P59 and 3-17 M were 517, (n= 3) and 11P7 min, respectively (Fig. 6A). The decay time constants obtained with protocol 2 at ethanol concentrations of 2-0, 2-38, 3 0, 3-17 and 4 0 M were 111, 111, 23-2, 24-7 and 6-2 min, respectively. The family of data in Fig. 6A obtained with protocol 1 and that in Fig 6B obtained with protocol 2 were qualitatively similar, but the values of decay time constants obtained with protocol 2 are slightly larger. A B Ethanol - Ethanol C C O1 co 50 C C C ~~~~~~~~~~~~~~CD 0) ~ im (mn0im)mn L L Time (min) Time (min) Fig. 6. The decay of gx as the function of the sum of ethanol application time. A, the percentage of remaining ga values obtained at ethanol concentrations of 1P19 (0), 1P59 (0), 1P59 (E), 1P59 (U) and 3 17 M (A) plotted on a logarithmic scale as a function of the sum of the ethanol application time. gk. decays as a single exponential function. The straight lines through the data points are obtained by fitting data points with a single exponential function. The data were obtained using protocol 1. B, plot of gna values obtained at ethanol concentrations of 2-0 (0), 2-38 (0), 3 0 ((El), 3-17 (U) and 4 0 M (A) using protocol 2. Similar data obtained with protocol 2 at a different concentration of 1-propanol and 1-butanol are shown in Fig. 7. The remaining sodium conductance decreased in a single exponential manner. The decay time constants at the 1-propanol concentrations of 0 643, 0 804, 0 965, and M are (n= 2), 126 ± 12 (n= 2), 85 ± 9 (n = 2), 16 and 7 3 min, respectively (Fig. 7A). Those at 1-butanol concentrations of 203, 243 and 284 mm are (n = 2), 141 and 30 min, respectively (Fig. 7B). Difference between the denatured state and the anaesthetized state Records similar to those in Fig. 3 were obtained before, during and after the application of 159 M ethanol. When the ethanol was applied, the sodium current decreased to one-third within a few minutes and recovered after the ethanol was

12 534 5F KUKITA AND S. MITAKU washed out. The time course of the sodium current before and after the ethanol application was unchanged. During ethanol application, the time course seemed to be slightly slower (Fig. 8B) as reported by previous investigators (Haydon et al. 1984). The change in the sodium conductance (gna) is plotted as a function of time (Fig. 8D). As shown in Fig. 3, recovery from the anaesthesia was not complete A 100^ 1-Propanol Butanol B H50 C Y ' !~~~~~~ (a.co8 10 _, v10_* Time (min) Time (min) Fig. 7. The decay of gna as a function of the application time of I1-propanol or 1-butanol. A, gna values obtained at 1-propanol concentrations of 0-64 (O, *), 0-8 (El, I), 0 97 (E, A), 1-13 (V) and 1-29 M (V) are plotted as a function of the sum of the 1-propanol application time. The data were obtained using protocol 2. B, gna values obtained at 1-butanol concentrations of 203 (O, *), 243 (a) and 284 mm (M) are plotted as a function of the sum of the I1-butanol application time. The data were obtained using protocol 2. when the ethanol was completely washed out from both sides of the membrane. However, at each instance of 1-59 M ethanol application, the sodium conductance decreased to about 40 % of that before the ethanol application within a few minutes and then slowly decreased during the ethanol application. Fitting the data during the application of ethanol with a double exponential function, the time course of the anaesthetic effect could be roughly estimated. The fast phase of the anaesthesia has a time constant of around 1 min (perhaps smaller than this value, because the speed of the solution exchange was not so fast). The slow phase was a hundred times -slower than the fast phase, but the time constant was half of the denaturation time constant. These slow phases might show the mixture of some slow phase in the anaesthesia and the time course of the denaturation process. It is clear that anaesthesia occurs in the remaining intact sodium channels and has a- time course faster than the denaturation process by a factor of about 100. shown in Fig. 9A. The The current-voltage relation of the sodium current is current-voltage relations before and after ethanol application seemed similar despite a small decrease in magnitude after the ethanol application. However, the

13 ALCOHOL DENATURATION OF Nat CHANNELS 535 current-voltage relation during ethanol application was much different from these. The peak current was around 40 % of that in the absence of ethanol and shifted in the depolarizing direction but the sodium equilibrium potential EN. did not change. The values of ENa were 70, 71 and 70 mv before and after the first and the second application of ethanol. During ethanol application, ENa values were -70, -70 and A B C 'i Time (ms) Time (ms) Time (ms) D A 100 Ethanol C ed Z 4-c Time (min) Fig. 8. The change of sodium currents and sodium conductances during and after the application of 1-59 M ethanol. The traces of sodium currents were obtained before (A), during (B) and after (C) the application of ethanol. The sodium current is decreased to less than 40 % of the control at an ethanol concentration of 1t59 M. The times at which current traces were obtained are shown with A, B and C in D. D, percentage change of Yka is plotted as a function of time. When ethanol was applied, the sodium current decreased more slowly than in the case of 82 mm 1-pentanol shown in Fig. 3. When ethanol was washed out (Wash), gna recovered quickly. Data were obtained using protocol mv and were not different from those in the denatured state. Sodium conductance, g9, is plotted as a function of the membrane potential to show the activation curve of the sodium channel (Fig. 9B). The activation curve shifted in the depolarizing direction by about 22 mv during ethanol application. This is one of the typical features of the anaesthesia. Vi values were -16, -18 and -19 mv before and after the first and second applications of ethanol, whereas it was 3 mv during the application of ethanol. The mean values of Vi and ENa were 4X1+1X9 mv (n=8) and 69X4+ 1D1 mv (n=8) during 1X59 M ethanol application. The mean shift of Vi was mv (n= 8) during 1'59 M ethanol application.

14 536 F KUKITA AND S. MITAKU In the denaturation state, there was not an obvious change in ENa and VI, but in the anaesthetized state, there was a shift of Vi of about 22 mv. To distinguish the difference between the anaesthetized state and the denatured state, we compared the data of both states in which the suppression of the sodium conductance was A ma cm-2 B F Vm (mv) Fig. 9. The current-voltage relation and the sodium channel activation-voltage curve in the denatured state and the anaesthetized state caused by a 1P59 M ethanol application. A, current-voltage relations before (0) and after the first (O) and second (A) application of 1P59 M ethanol are shown together with those obtained during the application of ethanol (@, U). The data plotted are from the same experiment as in Fig. 8. B, sodium channel activation-voltage curves are plotted before (0) and after the first (a) and second (A) application of 1P59 M ethanol together with those obtained during the application of ethanol (@). The data were obtained from the data in A. The sodium chord conductance, gn., defined in eqn (1), was normalized (to 1) to the maximum of the control and then plotted as a function of membrane potential. Data were fitted with eqn (2). identical. Among the data obtained with four species of alcohol and at various concentrations, we collected the data in which the remaining sodium conductance was around 40 % of the initial value and was the same as in the anaesthetized state in the presence of 1-59 M ethanol. The denatured state in which % (n= 14) of the sodium conductance remained showed only a small shift in Vi of 1P0+5 5 mv (n= 14), while in the anaesthetized state there was a large shift of 23+4 mv (n= 8). It is not plausible that the main cause of the shift in Vi is an artifactual shift due to the uncompensated part of the series resistance. If this was the case, there would be a bigger shift in the less denatured state. However, the value of Vi did not change significantly during the course of denaturation. The large shift in the anaesthetized state should not be considered to be the series resistance-related artifact but a

15 ALCOHOL DENATURATION OF Na+ CHANNELS 537 typical characteristic of the anaesthesia which is quite different from the denatured state. Therefore the denatured state could not be thought to be the partly anaesthetized state caused by the unremovable alcohols Pentanol 1 -Propanol \ 1007 ~Ethanol 10 I -Butanol Aloohol concentration (M) Fig. 10. The relation between the decay time constant and the alcohol concentration of four alcohols. The logarithm of the decay time constant, td (in min), is plotted as a function of the logarithm of alcohol concentration in aqueous solutions (M). Data obtained using protocol 1 are plotted for ethanol (@) and 1-pentanol (U). Data obtained using protocol 2 are plotted for ethanol (0), 1-propanol (A), 1-butanol (V) and 1-pentanol (L). The straight line was drawn by fitting each group of data points with eqn (3). The alcohol concentration dependence of the denaturation time constants The logarithms of the decay time constants at the application of four species of alcohols are plotted as a function of the logarithm of alcohol concentration (Fig. 10). The time constant of the denaturation process for four types of alcohol decreased in the same manner as the concentration of each alcohol increased. The time constants obtained with each alcohol shifted in the lower concentration direction as the carbon number of the aliphatic chain increased. The decay time constant can be expressed by eqn (3): td TACAN, (3) where CA, td, TA amd Nare, respectively, the alcohol concentration in the aqueous solution, the denaturation time constant at CA, the proportional constant which has the value of the time constant when CA is 1 M and the number of alcohol molecules which bind to one sodium channel protein. The slopes of the line drawn with a curve fitting eqn (3) through the data points are almost identical. Hence the

16 538 F KUKITA AND S. MITAKU values of N (which is minus the slope of each line) are similar. This value for 1- pentanol was 8-6 in the data obtained with protocol 1 and 6-7 in the data obtained with protocol 2. The value of N in the data for 1-butanol was 5-8 and that for 1- propanol was 4-5. The value of N for ethanol was 4-1 in the data obtained with protocol 1 and was 4-4 in the data obtained with protocol 2. These values of Nwere distributed in the narrow range between 4-1 and 8-6. Equation (3) is a simple deduction from the following reaction formula: Pr + NCM Pr*C N (4) where Pr, CM and Pr*CMN are, respectively, the sodium channel concentration in the membrane, the concentration of alcohol in the membrane and the concentration of denatured sodium channel protein to which Nalcohols are binding. The time constant of this reaction can be described by eqn (5): td= TM CMN (5) where TM is the proportional constant whose value is the time constant at CM of 1 M. CM Pmemb CA, (6) where Pmemb is the partition coefficient of alcohol between the lipid membrane and the aqueous solution. Using eqns (5) and (6), we can derive eqn (3). These equations mean that several alcohol molecules inside the membrane bind to a restricted number of binding sites of the sodium channel protein at the rate limiting step of the denaturation. The two groups of data points are plotted for ethanol and 1-pentanol, which were obtained using the different protocols. The data obtained when the membrane potential was held throughout the experiment at the initial holding potential (protocol 1) seem to be shifted in the lower concentration direction, but in each group of data the concentration dependence is similar. The N values are plotted as a function of the carbon number of the alcohol (Fig. 11 A). The N values are linearly related to the carbon number and tend to increase as the chain length of the alcohol increases, but are in a narrow range of values. The logarithm of alcohol concentrations at which the denaturation time constant, td, was 20 min and those at which the half-denaturation time, 4, was 2 h are plotted as a function of the carbon number of the alcohol aliphatic chain. These concentrations are expressed as the exponential function of the carbon number (Fig. 1 lb). The alcohol concentrations at which the denaturation time constant is 20 min are 2-92 and 3-18 M for ethanol, 1-16 M for 1-propanol, 316 mm for 1-butanol and 84 and 102 mm for 1-pentanol. The alcohol concentrations at which t is 2 h are 1'73 and 1-95 M for ethanol, 0-72 M for 1-propanol, 218 mm for 1-butanol and 65 and 74 mm for 1-pentanol. The alcohol concentrations which cause 1 % denaturation in 24 h are (n = 2), 164, 68 and P5 (n = 2) mm for ethanol, 1-propanol, 1-butanol and 1-pentanol, respectively. The values of these concentrations are in the range of dosage for anaesthesia (Armstrong & Binstock, 1964; Haydon et al. 1984; Mitaku,

17 ALCOHOL DENATURATION OF Na+ CHANNELS 539 A td TAr C 10 -N 10 5 B 8 N 6 c I 0-05 I Carbon number Carbon number Fig. 11. The value of Nand the alcohol concentrations in the solution and in the membrane as a function of the carbon number of the alcohol. A, the values of N were obtained by fitting the data in Fig. 10 with eqn (3); Nis a linear function of the carbon number of the alcohol. B, the alcohol concentrations in the solution (CA) which caused denaturation with a time constant of 20 min (0) and those at which the sodium channel denatures by half in 2 h (O) are plotted on a logarithmic scale as the function of the carbon number. Those concentrations are roughly expressed as the exponential function of the carbon number. The alcohol concentrations in the membrane (CM) were obtained by multiplying CA by the partition coefficient of each alcohol. The partition coefficients we used are 0-15 for ethanol, 0-57 for 1-propanol, 2-0 for 1-butanol and 6-6 for 1-pentanol (Lyon et al. 1981). The concentrations at td = 20 min and ti = 2 h are about 0-8 (@) and 0 5 M (U). They did not strongly depend on the carbon number. Data obtained with two different protocols are averaged for ethanol and 1-pentanol. Kukita & Kasai, 1990). Therefore, the effects of these alcohols are reversible at the dosage for anaesthesia, which is usually difficult to demonstrate because of unknown damage in the experiment (Haydon et al. 1984). In other words, our experiment is well designed so as to remove this unknown damage, because we used a higher concentration of alcohol. The concentration of alcohol in the membrane (C) is obtained as in eqn (6) using the partition coefficient (Pmemb) of alcohol, which is defined by the ratio of alcohol concentration in the membrane and in the aqueous solution. The values of Pmemb we used are 0-15, 057, 2-0 and 6-6 for ethanol, 1- propanol, 1-butanol and 1-pentanol, respectively (Lyon, McComb, Schreurs & Goldstein, 1981). CM is plotted as a function of the carbon number of the alcohol. The value is around 0-8 M for the data at a td of 20 min and 0 5 M at a ti of 2 h and does not depend on the carbon number of the alcohol, especially when the carbon number is more than 3. Using other data of partition coefficients, the concentration of alcohol

18 540 5F KUKITA AND S. MITAKU in the membrane did not depend on the hydrocarbon chain length but their value depends on the data of the partition coefficients and the hydrophobic solvents which were used to obtain the data, because the logarithm of partition coefficients is generally a linear function of the hydrocarbon chain length (Leo, Hansch & Elkins, 1971). Therefore, we can conclude that the concentration in the membrane which is needed to denature the sodium channel does not depend on the species of alcohol. This means that the first step of the denaturation is the penetration of alcohols into the membrane as in anaesthesia (Seeman, 1972). DISCUSSION We have demonstrated that the denaturation of the sodium channel seemed to start by the binding of several alcohol molecules to a restricted number of binding sites of the sodium channel. This result is consistent with results obtained in the bacteriorhodopsin of the Halobacterium halobium membrane. In that preparation, the binding of alcohol causes the breaking of hydrogen bonds among segments of the protein to cause the partial denaturation keeping the secondary structure intact (Mitaku et al. 1988). We can suppose that the mechanism of stabilizing the sodium channel protein is similar to that for the bacteriorhodopsin, because the critical alcohol concentration to cause the denaturation is almost the same in both proteins (Mitaku et al. 1988, 1990). Considering the primary structure of the sodium channel protein, the four repeating structures consisting of six homologous hydrophobic segments are considered to pass through the membrane (Noda et al. 1984, 1986), and could be stabilized within the membrane by hydrogen bonds among these segments because in the hydrophobic environment the hydrogen bond is the major force stabilizing the global structure of membrane proteins (Engleman, 1982; Rosenbuch, 1985; Mitaku et al. 1988; Popot & Engelman, 1990). Assuming that alcohol molecules dissolve uniformly in the membrane, the critical alcohol concentration of 0 5 M is equivalent to 3 x 106 molecules um-2. Considering that the density of sodium channels is 300 molecules sm-2 (Conti, De Felice & Wanke, 1975), roughly alcohol molecules can be available to be accepted by one sodium channel. Among molecules, only a few molecules seemed to bind to one sodium channel. Therefore, the slow denaturation process was not attributed to the deficiency of alcohol molecules but to the slow process of the binding and/or the denaturation process itself. Denaturation effects appear to be independent of anaesthesia, because the denatured sodium channels were not available for anaesthesia. There was also a significant difference in the sodium channel activation-voltage curve between the denatured and the anaesthetized states. Therefore, the irreversible denaturation could not be explained by anaesthetic effects caused by the remaining alcohol in the membrane. It was not plausible that alcohol could remain in the membrane at a fixed ratio at each wash-out of alcohol, so as to increase the concentration of alcohol remaining in the membrane. Even if it might be possible, it is difficult to discriminate between irreversible storage of alcohol in the membrane and the irreversible binding to the sodium channel protein. Furthermore, alcohols irreversibly trapped into some storage site could not act as anaesthetics like the free alcohol molecules.

19 ALCOHOL DENATURATION OF Na+ CHANNELS The number of binding sites of alcohol in the sodium channel, which is related to the denaturation process, ranges from four to eight. The number of binding sites has the tendency to increase as the hydrocarbon chain of the alcohol increases. There are two possible explanations. The access of the alcohol to the binding sites might be sterically restricted slightly as the hydrocarbon chain length increases. As the hydrocarbon chain length decreases, the contribution of the effects of alcohol accessing the sodium channel from aqueous solutions cannot be neglected, which might mask the concentration dependence of the effects of alcohol in the membrane. The natural run-down has a time constant of more than 1000 min and is much slower than the denaturation process produced by alcohol application. However, in some data using a low concentration of alcohol, for example data from ethanol application, the denaturation process was so slow that its time constant might be underestimated. This could be one of the reasons that we obtained a slightly lower critical concentration of denaturation in ethanol application and the smaller value of Nfor ethanol and 1-propanol. However, the natural run-down is thought not to affect the general characteristics of alcohol-induced denaturation. The short-chain alcohol has a depolarizing effect at the dosage needed to anaesthetize the axon (Armstrong & Binstock, 1974; Haydon et al. 1984) and the longer-chain alcohols are reported to enhance the sodium inactivation process (Oxford & Swenson, 1979; Haydon & Urban, 1983; Haydon et al. 1984). In our experiment, the membrane is depolarized as much as 20 mv during the alcohol application but after washing out the alcohol completely, the resting potential returned to the value before the application but is larger by several millivolts than before. We usually used a longer preconditioning hyperpolarization of -100 mv for 0 5 to 1 s to reduce the after-effects of this depolarization of the resting potential. However, this preconditioning hyperpolarization is not enough to remove this effect. So we usually held this membrane potential at -60 mv for more than 5 min to remove this effect as far as possible (protocol 2). It is true that if we did not carefully remove the after-effect of the depolarization, we usually obtained the result that the alcohol concentration is lower than in the result shown here (Mitaku et al. 1990). We used another protocol (protocol 1), in which the membrane potential was held at -60 mv throughout the experiment. Using this protocol, unexpectedly we could not increase the denaturation concentration but obtained slightly lower values which suggests there are other damaging effects due to the longer hyperpolarization in the period of depolarization during the alcohol application (Haydon et al. 1984). In any case, protocol 2 is the best way to remove the after-effects at present. In any experiment, the values of Nare not so different from each other, suggesting that the denaturation process and the membrane potential-dependent sodium channel inactivation (Meves, 1978) are independent. We did not use alcohols whose side chains are longer than that of pentanol, because the long-chain alcohol is thought to be ineffective for protein denaturation (Eisenbach et al. 1979). It is also well known that there is a cut-off of the effects of alcohol (Lyon et al. 1981; Hunt, 1985) in anaesthesia and alcohol intoxication. It is difficult to say that the similar number of binding sites is related to anaesthesia, because of the poor time resolution in the experiment shown in Figs 2 and 5. The reported values obtained from steady-state analysis of anaesthesia of 541

20 542 F KUKITA AND S. MITAKU the sodium channel in the squid giant axon (Paternostre et al. 1983) and acetylcholine binding capacity of the acetylcholine receptor (Miller, Firestone & Forman, 1987) are at most two. This means that anaesthesia is a different process from the denaturation process at the viewpoint of the binding site. In anaesthesia, it is difficult to clear the specific binding site in the protein, although there is argument whether the target of anaesthetic agents is lipid or protein in the membrane (Haydon et al. 1984). We could demonstrate that in the denaturation process the sodium channel is the most probable target. We described how we can analyse the irreversible effects of alcohols which have frequently been observed in anaesthesia using short chain aliphatic alcohols, the mechanism of which is not yet clear (Haydon et al. 1984). Kinetic analysis is better able to discriminate between the reversible effect we call anaesthesia and additional irreversible effects. We have tried to attribute these effects to the denaturation of the sodium channel protein. It is true that there are more ambiguous effects in experiments using a nerve preparation than in those using the isolated sodium channel protein. However, the squid sodium channel is a good model preparation in which no contribution of other macromolecules including G-proteins is appreciable. Even if we use a reconstituted membrane system, it is difficult to discriminate the direct effect of alcohol on the sodium channel membrane and the indirect effects on the lipid layer and so on and also there is the more serious problem of fragility of the membrane. The squid sodium channel is the best preparation to examine the alcohol effects at present because of the easy and quick intracellular perfusion and is one of the most conservative ion channels in which there are not large differences among species (Haydon et al. 1984; Hille, 1984). We wish to thank the members of Ine Fishery Co-operative for collecting the squid, and Professor S. Yamagishi for supporting this work. We thank Dr M. Ichikawa and Dr G. Matsumoto at the Electrotechnical Laboratory, Tsukuba for showing us their voltage clamp circuit and giving valuable comments on the fast axial wire voltage clamp. We also thank Dr Q. Bone, FRS at Plymouth Marine Biological Association Laboratory for reading this manuscript and for correcting the English. This work was partially supported by a grant from Ministry of Education, Culture and Science of Japan (No , ) to F.K. REFERENCES ARMSTRONG, C. M. & BINSTOCK, L. (1964). The effects of several alcohols on the properties of the squid giant axon. Journal of General Physiology 48, CONTI, F., INOUE, I., KUKITA, F. & STtHMER, W. (1984). Pressure dependence of sodium gating currents in the squid giant axon. European Biophysics Journal 11, CONTI, F., DE FELICE, L. J. & WANKE, E. (1975). Potassium and sodium ion current noise in the membrane of the squid giant axon. Journal of Physiology 248, EISENBACH, M., CAPLAN, S. R. & TANNY, G. (1979). Interaction of purple membrane with solvents. I. Applicability of solubility parameter mapping. Biochimica et Biophysica Acta 554, ENGLEMAN, D. M. (1982). An implication of the structure of bacteriorhodopsin. Globular membrane proteins are stabilized by polar interactions. Biophysical Journal 37, HAYDON, D. A., ELLIOTT, J. R. & HENDRY, B. M. (1984). Effects of anaesthetics on the squid giant axon. In Current Topics in Membranes and Transport, vol. 22, The Squid Axon, ed. BAKER, P. F., pp Academic Press. Inc., Orlando, FL, USA. HAYDON, D. A. & KIMURA, J. E. (1981). Some effects of n-pentane on the sodium and potassium currents of the squid giant axon. Journal of Physiology 312, HAYDON, D. A. & URBAN, B. W. (1983). The action of alcohols and other non-ionic surface active substances on the sodium current of the squid giant axon. Journal of Physiology 341,

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