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All or none block of single Na+ channels by tetrodotoxin
Neuroscience Letters, 54 (1985) 77-83 Elsevier Scientific Publishers Ireland Ltd.
77
NSL 03142
ALL OR NONE BLOCK OF SINGLE Na + CHANNELS BY TETRODOTOXIN
FRED N. QUANDT*, 1.2. YEH and T. NARAHASHI
Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611 (U.S.A.) (Received August 28th, 1984; Revised version received and accepted November 14th, 1984)
Key words: tetrodotoxin - sodium channel - neuroblastoma - patch clamp - channel blocker - singlechannel analysis
The effects of tetrodotoxin on single Na + -channel currents recorded from excised patches of neuroblastoma cells were examined. Tetrodotoxin was found to cause a dose-dependent reduction in the frequency at which Na + channels conduct during a series of depolarizations. Surviving conducting states had normal open times and current amplitudes. These effects could be explained by a model which includes initial binding of tetrodotoxin to a closed state of the channel with stable, complete block during the time the channel would normally be gated open.
One approach to a molecular description of the nature of the voltage-gated Na + channel in neuronal membrane has been to characterize the actions of toxins and drugs [7, 11]. Tetrodotoxin causes a specific and reversible block of Na + current across neuronal membranes with high affinity [12]. Block of Na+ channels by tetrodotoxin (TTX) or saxitoxin appears to be due to an interaction with structures within the channel involved with ion permeation [5]. For neuronal membranes, block by these compounds does not seem to vary in a time- and voltage-dependent manner [4, 15, 16]. However, TTX block of Na + current in heart cells is 'use dependent' [2]. The object of the study reported here was to examine the mechanism of block of Na + channels by TTX using single-channel analysis. Single-channel analysis was applied to study this question, since this method can give direct information concerning the conductance, lifetime and probability of occurrence of states of the channel. The action of TTX was best described by a model which does not require the Na + channel to be open for block to proceed. Preliminary reports have been presented [8, 10]. Current was recorded from patches of membrane isolated from NIE-II5 neuroblastoma cells. Details of this technique as adapted to this preparation have *Author for correspondence at present address: Department of Medical Physiology, University of Calgary, Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alta. T2N 4NI. Canada.
78
been described elsewhere (9)- For experiments investigating the effects of TTX an 'outside-out' patch configuration was used . This configuration is described in the paper by Hamill et al. [3]. The solution on the extracellular side of the membrane had the following composition (rrrM): Na + 125, K + 5.5, Ca 2 + 1.8, Mg 2 + 0.8, CI135.7, HEPES 20, glucose 25. The pH was adjusted to 7.3 with NaOH, and sucrose was added to bring the osmolarity to 330 mOsm. TTX was added by dilution from a 10 -4 M stock solution containing 5 mM citric acid. For the experiments employing outside-out patches, the solution on the intracellular side of the membrane was composed of the following (mM): K + 100, Na + I, eth yleneglycoltetraacetic acid (EGTA) 20, glutamic acid 50, HEPES 21. The osmolarity was adjusted to 300 mOsm with 100 mM sucrose and the pH was adjusted to 7.2 with KOH. Fig . I shows an example of an experiment to investigate the actions of TTX on Control
3nM TTX
Fig. I. Effects of TTX on single Na + -channel currents. Examples of current records obt ained from an outside-out patch of membrane in response to a series of depolarizations. The bottom-most traces show the time course of the depolarization used. Left panel : representative traces recorded in normal external saline. The patch was depolarized repet itively for 40 ms from a holding potent ial of -90 mV to a potential of - 30 mV at 3 s intervals (temperature: IOOe). Downward steps in each trace are inward currents due to the openin g of individual channel s. Right panel: after exposure to 3 nM TTX. the appearance of individual op enings is normal. Many traces showed no opening of Na + channels. as represented b)' the top-most trac e.
79
the frequency and properties of Na + -channel conducting states. The figure shows selected records of membrane current in response to depolarizations to - 30 mV from a holding potential of - 90 mV. Inward currents observed under these conditions have been shown to be associated with the voltage-gated Na + channel by pharmacological and physiological criteria [9). The selected records on the left, obtained in normal saline, are very similar in appearance to the surviving conducting states in the presence of 3 nM TTX, which are shown in the right panel. Histograms of the amplitude and histograms of the duration of these conducting states are shown in Fig. 2A, B; respectively. The mean amplitude of single-channel 8
A
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/'
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Fig. 2. Characteristics of the conducting state of the Na + channel are not altered by TIX. A: histogram analysis of the distribution of the current amplitudes of conducting states, under control conditions (top) and during exposure to TIX (bottom). The solid lines plot normal distributions with mean amplitude and standard deviation, as cited in the text, for either the control condition or during exposure to TIX. B: comparison of open times of Na + channels before (top) and during TIX application (bottom). The number of channels having open times greater than the time indicated are plotted. Three cases of overlapping openings were excluded from the analysis in both the control and TIX conditions. The mean open time was 4.6 ms for control conditions and 5.4 ms during the application of TIX. The solid lines plot exponential distributions of open times for each population, given the number of observations and mean. Data was pooled from different runs for this patch.
80
current was 1.23 pA in the control condition (S.D. = 0.20 pA) with a mean open time of 4.6 rns. During the application ofTTX. the mean current amplitude was I.12 pA (S.D. = 0.16 pA) and the mean open time was 5.4 ms. This result is true for higher TTX concentrations, as well. In one typical experiment following exposure to 20 nM TTX, surviving channels had a mean current amplitude of 1.24 ± .24 pA and a mean open time of 2.1 ms. Control records gave a mean amplitude of 1.47 ± .37 pA and mean open time of 2.2 ms (-40 mY, 9°C). Thus, TTX does not have an apparent effect on either of these parameters. Sigworth [14] analyzed the variance of currents recorded from node of Ranvier before and during exposure to TTX. The results of this study showed that the conductance of the blocked channel is similar to the conductance of the closed channel. During exposure to TTX, many records did not contain Na + channel conducting states, as illustrated in the upper-most record of the right panel in Fig. I. For this patch, 57 openings were observed over a series of 60 depolarizations in the absence of TTX, while only 42 openings were observed for 95 depolarizatio~s with 3 nM TTX. The inhibition of the entry of Na + channels into the conducting state by TTX increased as the concentration of the toxin was increased. The average frequency of opening was measured over a series of depolarizations under control conditions and during exposure to varying concentrations of TTX for 6 patches of membrane. Inhibition of opening was calculated for each case. The average inhibition for each concentration is plotted in Fig. 3. The data could be reasonably fit by a 100
•
•
•
• 20 Ol...01
• -'--
-L..
K d=2 nM
10
~.."._-
100
TTX Concentration (nM) Fig. 3. Dose-dependent inhibition of the frequency of Na + -channel opening by TTX. The frequency of Na + -channel entry into a conducting state was determined by measuring the number of openings (N) in response to a series of pulses. The number of openings in response to the same number of pulses was then determined in the presence of TTX. The percent inhibition was calculated according to Eqn. 3 in the text. The graph represents pooled data from 6 membrane patches. In some cases, one patch was exposed to more than one concentration of TTX. The points give the mean effect for each concentration. Recording conditions varied between patches. Temperature ranged from 9 to 13°C, and depolarizations varied from -56 to -30 mV, from a holding potential of -90 mY. Under control conditions, one or two openings were observed per depolarization in each experiment. The solid line is plotted according to Eqn. 4, given in the text, with Kd=2 nM.
81
dose-response curve (solid line) having a Hill coefficient of 1.0 and a dissociation constant (Kd) of 2 nM. The actions described for the effect ofTTX on single Na + channels are most consistent with a model which do es not require channels to open before they can be blocked: K, TTx
C1t7° K
b
CB-->L.- O B
~
In the above model, TTX can be bind to a closed or inactivated state of the channel (C) with forward rate constant, Kj, to produce a closed and blocked state (CB). Kb is the dissociation rate. Kj and Kb are assumed to be independent of voltage. 0 and OB represent states which have been gated open, but are conducting and nonconducting, respectively. (X is the average opening rate constant and {3 is the average closing rate constant. In this simplified model, (X is actually the weighted opening rate for transitions from either resting or inactivated states, at either the holding potential or potential during a depolarization. Since TTX docs not normally alter Na + -channel 'gating' currents, it is likely that the channel is free to gate open in the presence of TTX [I]. No evidence for TTX binding to the open state, with formation of the OB state. was obtained in our experiments, since the open time and single-channel current in the presence of TTX was comparable to control values. If the total time a channel spends in its open state is small compared to the total time during which activity is observed, the following equations can be developed. The number of openings for a series of depolarizations (N) in the control case is then given by: Nconlrol = 1] • Tobs . (X
(I)
where 1] is the number of channels in any patch and is assumed to be constant. T obs is the total observation time. Since the opening rate at the holding potential is 0, only the openings during each depolarization need be determined. In the presence of TTX, (2)
The percentage inhibition of the number of observed open states by TTX is then: 070 Inhibition = (Nconlrol - Nnx)/Nconlrol
(3)
This last equation can be simplified to a dose-response relationship:
% Inhibition = [TTX]/([TTX] + Kd)
(4)
where Kd is the dissociation constant for TTX. K; is given by : Kd=Kb/Kj
(5)
82
As shown in Fig. 3, our data could be modeled well by Eqn. 4 with KcI equal to 2 nM, a value close to that observed for the block of voltage-dependent Na + currents in various nerve and muscle cells [12]. Thus, the experimental data can be described by assuming that TTX binds to a state of the channel normally leading to the conducting state and that the occupancy of the channel by TTX is stable during the period the channel is normally gated open. Although block is explained by TTX interactions with the closed channel, it should be noted that our experiment docs not exclude an additional open-channel blocking component for TTX if reactions with the open state are slow compared to the time the channel is gated open. TTX kinetics have been estimated to be quite slow. Schwarz et al. [13] estimated the kinetics of association and dissociation of TTX by measuring the rate of onset and recovery of TTX block of Na + currents at the frog node of Ranvier. Using their values, for 3 nM TTX, the average waiting time for a blocking event is about 0.3 x 106 ms. Therefore, the probability of seeing a blocking event during the 5 ms the channel is open in our experiments would be only one out of approximately 100,000 trials. Dissociation of TTX froni the channel is likewise slow and would only be observed in one out of every 10,000 openings. Recently, Krueger et al. [6] have examined the actions of saxitoxin (STX) on batrachotoxin-activated Na + channels. The fraction of channels that remained conducting in the presence of STX was not markedly variable over the range of potentials where modulation of channel opening was found. The result suggests that STX may not interact selectively with either open or closed states of the channel. We would like to thank Mary Taft for maintaining the cells in culture and Rae Barolet for secretarial assistance. These studies were supported by NIH Grants NSl4144 and ES02330. I Armstrong. C.M . and Bezanilla, F.• Charge movem ent associated with the opening and closing of activation gates of the Na channels. J. Gen . Physiol ., 63 (1974) 533-552. 2 Cohen, C.J ., Dean, B.P ., Colastsky, T .J. and Tsien, R. W., Tetrodotoxin block of sodium channels in rabb it Purkinje fibers. Interactions between toxin binding and channel gating, J. Gen . Physiol., 48 (1981) 383-411. 3 Hamill, O .P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high resolution current record ing from cells and cell free membrane patches, Pfliigers Arch. Ges. Physiol., 391 (1981) 85-100. 4 Hille, D., Pharmacological modifications of the sodium channels of frog nerve, J. Gen. Physiol., 51 (1968) 199-219. 5 Hille, B., The receptor for tetrodotoxin and saxitoxin: a structural hypothesis, Biophys. J., 15 (1975) 615-619. 6 Krueger, B.K., Worley, J.F., 11\ and French, R.J., Single sodium channels from rat brain incorporated into planar lipid bilayer membranes, Nature (Lond .), 303 (1983) 172-175 . 7 Narahashi, T., Chemicals as tools in the study of excitable membranes, Physiol. Rev., 54 (1974) 813-866 . 8 Quandt, F. and Narahashi, T., Contrast between open and closed block of single Na channel currents, Biophys. J., 37 (1982) 319a (abstract).
83 9 Quandt, F.N. and Narahashi, T., 1\lodification of single Na channels by batrachotoxin, Proc. Natl. Acad. Sci. USA, 79 (1982) 6732-6736. 10 Quandt, F.N. and Narahashi, T., Pharmacology of single sodium channels, Biophys, J.; 41 (1983) 279a (abstract). 11 Ritchie, l.1\I., A pharmacological approach to the structure of sodium channels in myelinated axons, Ann. Rev. Neurosci., 2 (1979) 341-362. 12 Ritchie, l.M. and Rogart, R.B., The binding of saxitoxin and tetrodotoxin to excitable tissue, Rev. Physiol. Biochem. Pharmacol., 79 (1977) 2-50. 13 Schwarz, l.R., Ulbricht, W. and Wagner, H.H., The rate of action of tetrodotoxin on myelinated nerve fibres of Xenopus laevis and Rana esculenta, J, Physiol. (Lond.), 233 (1973) 161-194. 14 Sigworth, F.l., The conductance of sodium channels under conditions of reduced current at the node of Ranvier, r. Physiol. (Lond.), 307 (1980) 131-142. 15 Takata, 1\1.,Moore, l.W., Kao, C.Y. and Fuhrman, F.A., Blockage of sodium conductance increase in lobster giant axon by tarichatoxin (tetrodotoxin), 1. Gen, Physiol., 49 (1966) 977-988. 16 Ulbricht, W. and Wagner, H.H., The influence of pH on equilibrium effects of tetrodotoxin on myelinated nerve fibers of Rana esculenta, J. Physiol. (Lond.) 252 (1975) 159-184.
77
NSL 03142
ALL OR NONE BLOCK OF SINGLE Na + CHANNELS BY TETRODOTOXIN
FRED N. QUANDT*, 1.2. YEH and T. NARAHASHI
Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611 (U.S.A.) (Received August 28th, 1984; Revised version received and accepted November 14th, 1984)
Key words: tetrodotoxin - sodium channel - neuroblastoma - patch clamp - channel blocker - singlechannel analysis
The effects of tetrodotoxin on single Na + -channel currents recorded from excised patches of neuroblastoma cells were examined. Tetrodotoxin was found to cause a dose-dependent reduction in the frequency at which Na + channels conduct during a series of depolarizations. Surviving conducting states had normal open times and current amplitudes. These effects could be explained by a model which includes initial binding of tetrodotoxin to a closed state of the channel with stable, complete block during the time the channel would normally be gated open.
One approach to a molecular description of the nature of the voltage-gated Na + channel in neuronal membrane has been to characterize the actions of toxins and drugs [7, 11]. Tetrodotoxin causes a specific and reversible block of Na + current across neuronal membranes with high affinity [12]. Block of Na+ channels by tetrodotoxin (TTX) or saxitoxin appears to be due to an interaction with structures within the channel involved with ion permeation [5]. For neuronal membranes, block by these compounds does not seem to vary in a time- and voltage-dependent manner [4, 15, 16]. However, TTX block of Na + current in heart cells is 'use dependent' [2]. The object of the study reported here was to examine the mechanism of block of Na + channels by TTX using single-channel analysis. Single-channel analysis was applied to study this question, since this method can give direct information concerning the conductance, lifetime and probability of occurrence of states of the channel. The action of TTX was best described by a model which does not require the Na + channel to be open for block to proceed. Preliminary reports have been presented [8, 10]. Current was recorded from patches of membrane isolated from NIE-II5 neuroblastoma cells. Details of this technique as adapted to this preparation have *Author for correspondence at present address: Department of Medical Physiology, University of Calgary, Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, Alta. T2N 4NI. Canada.
78
been described elsewhere (9)- For experiments investigating the effects of TTX an 'outside-out' patch configuration was used . This configuration is described in the paper by Hamill et al. [3]. The solution on the extracellular side of the membrane had the following composition (rrrM): Na + 125, K + 5.5, Ca 2 + 1.8, Mg 2 + 0.8, CI135.7, HEPES 20, glucose 25. The pH was adjusted to 7.3 with NaOH, and sucrose was added to bring the osmolarity to 330 mOsm. TTX was added by dilution from a 10 -4 M stock solution containing 5 mM citric acid. For the experiments employing outside-out patches, the solution on the intracellular side of the membrane was composed of the following (mM): K + 100, Na + I, eth yleneglycoltetraacetic acid (EGTA) 20, glutamic acid 50, HEPES 21. The osmolarity was adjusted to 300 mOsm with 100 mM sucrose and the pH was adjusted to 7.2 with KOH. Fig . I shows an example of an experiment to investigate the actions of TTX on Control
3nM TTX
Fig. I. Effects of TTX on single Na + -channel currents. Examples of current records obt ained from an outside-out patch of membrane in response to a series of depolarizations. The bottom-most traces show the time course of the depolarization used. Left panel : representative traces recorded in normal external saline. The patch was depolarized repet itively for 40 ms from a holding potent ial of -90 mV to a potential of - 30 mV at 3 s intervals (temperature: IOOe). Downward steps in each trace are inward currents due to the openin g of individual channel s. Right panel: after exposure to 3 nM TTX. the appearance of individual op enings is normal. Many traces showed no opening of Na + channels. as represented b)' the top-most trac e.
79
the frequency and properties of Na + -channel conducting states. The figure shows selected records of membrane current in response to depolarizations to - 30 mV from a holding potential of - 90 mV. Inward currents observed under these conditions have been shown to be associated with the voltage-gated Na + channel by pharmacological and physiological criteria [9). The selected records on the left, obtained in normal saline, are very similar in appearance to the surviving conducting states in the presence of 3 nM TTX, which are shown in the right panel. Histograms of the amplitude and histograms of the duration of these conducting states are shown in Fig. 2A, B; respectively. The mean amplitude of single-channel 8
A
I~
CONTROL
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/'
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Fig. 2. Characteristics of the conducting state of the Na + channel are not altered by TIX. A: histogram analysis of the distribution of the current amplitudes of conducting states, under control conditions (top) and during exposure to TIX (bottom). The solid lines plot normal distributions with mean amplitude and standard deviation, as cited in the text, for either the control condition or during exposure to TIX. B: comparison of open times of Na + channels before (top) and during TIX application (bottom). The number of channels having open times greater than the time indicated are plotted. Three cases of overlapping openings were excluded from the analysis in both the control and TIX conditions. The mean open time was 4.6 ms for control conditions and 5.4 ms during the application of TIX. The solid lines plot exponential distributions of open times for each population, given the number of observations and mean. Data was pooled from different runs for this patch.
80
current was 1.23 pA in the control condition (S.D. = 0.20 pA) with a mean open time of 4.6 rns. During the application ofTTX. the mean current amplitude was I.12 pA (S.D. = 0.16 pA) and the mean open time was 5.4 ms. This result is true for higher TTX concentrations, as well. In one typical experiment following exposure to 20 nM TTX, surviving channels had a mean current amplitude of 1.24 ± .24 pA and a mean open time of 2.1 ms. Control records gave a mean amplitude of 1.47 ± .37 pA and mean open time of 2.2 ms (-40 mY, 9°C). Thus, TTX does not have an apparent effect on either of these parameters. Sigworth [14] analyzed the variance of currents recorded from node of Ranvier before and during exposure to TTX. The results of this study showed that the conductance of the blocked channel is similar to the conductance of the closed channel. During exposure to TTX, many records did not contain Na + channel conducting states, as illustrated in the upper-most record of the right panel in Fig. I. For this patch, 57 openings were observed over a series of 60 depolarizations in the absence of TTX, while only 42 openings were observed for 95 depolarizatio~s with 3 nM TTX. The inhibition of the entry of Na + channels into the conducting state by TTX increased as the concentration of the toxin was increased. The average frequency of opening was measured over a series of depolarizations under control conditions and during exposure to varying concentrations of TTX for 6 patches of membrane. Inhibition of opening was calculated for each case. The average inhibition for each concentration is plotted in Fig. 3. The data could be reasonably fit by a 100
•
•
•
• 20 Ol...01
• -'--
-L..
K d=2 nM
10
~.."._-
100
TTX Concentration (nM) Fig. 3. Dose-dependent inhibition of the frequency of Na + -channel opening by TTX. The frequency of Na + -channel entry into a conducting state was determined by measuring the number of openings (N) in response to a series of pulses. The number of openings in response to the same number of pulses was then determined in the presence of TTX. The percent inhibition was calculated according to Eqn. 3 in the text. The graph represents pooled data from 6 membrane patches. In some cases, one patch was exposed to more than one concentration of TTX. The points give the mean effect for each concentration. Recording conditions varied between patches. Temperature ranged from 9 to 13°C, and depolarizations varied from -56 to -30 mV, from a holding potential of -90 mY. Under control conditions, one or two openings were observed per depolarization in each experiment. The solid line is plotted according to Eqn. 4, given in the text, with Kd=2 nM.
81
dose-response curve (solid line) having a Hill coefficient of 1.0 and a dissociation constant (Kd) of 2 nM. The actions described for the effect ofTTX on single Na + channels are most consistent with a model which do es not require channels to open before they can be blocked: K, TTx
C1t7° K
b
CB-->L.- O B
~
In the above model, TTX can be bind to a closed or inactivated state of the channel (C) with forward rate constant, Kj, to produce a closed and blocked state (CB). Kb is the dissociation rate. Kj and Kb are assumed to be independent of voltage. 0 and OB represent states which have been gated open, but are conducting and nonconducting, respectively. (X is the average opening rate constant and {3 is the average closing rate constant. In this simplified model, (X is actually the weighted opening rate for transitions from either resting or inactivated states, at either the holding potential or potential during a depolarization. Since TTX docs not normally alter Na + -channel 'gating' currents, it is likely that the channel is free to gate open in the presence of TTX [I]. No evidence for TTX binding to the open state, with formation of the OB state. was obtained in our experiments, since the open time and single-channel current in the presence of TTX was comparable to control values. If the total time a channel spends in its open state is small compared to the total time during which activity is observed, the following equations can be developed. The number of openings for a series of depolarizations (N) in the control case is then given by: Nconlrol = 1] • Tobs . (X
(I)
where 1] is the number of channels in any patch and is assumed to be constant. T obs is the total observation time. Since the opening rate at the holding potential is 0, only the openings during each depolarization need be determined. In the presence of TTX, (2)
The percentage inhibition of the number of observed open states by TTX is then: 070 Inhibition = (Nconlrol - Nnx)/Nconlrol
(3)
This last equation can be simplified to a dose-response relationship:
% Inhibition = [TTX]/([TTX] + Kd)
(4)
where Kd is the dissociation constant for TTX. K; is given by : Kd=Kb/Kj
(5)
82
As shown in Fig. 3, our data could be modeled well by Eqn. 4 with KcI equal to 2 nM, a value close to that observed for the block of voltage-dependent Na + currents in various nerve and muscle cells [12]. Thus, the experimental data can be described by assuming that TTX binds to a state of the channel normally leading to the conducting state and that the occupancy of the channel by TTX is stable during the period the channel is normally gated open. Although block is explained by TTX interactions with the closed channel, it should be noted that our experiment docs not exclude an additional open-channel blocking component for TTX if reactions with the open state are slow compared to the time the channel is gated open. TTX kinetics have been estimated to be quite slow. Schwarz et al. [13] estimated the kinetics of association and dissociation of TTX by measuring the rate of onset and recovery of TTX block of Na + currents at the frog node of Ranvier. Using their values, for 3 nM TTX, the average waiting time for a blocking event is about 0.3 x 106 ms. Therefore, the probability of seeing a blocking event during the 5 ms the channel is open in our experiments would be only one out of approximately 100,000 trials. Dissociation of TTX froni the channel is likewise slow and would only be observed in one out of every 10,000 openings. Recently, Krueger et al. [6] have examined the actions of saxitoxin (STX) on batrachotoxin-activated Na + channels. The fraction of channels that remained conducting in the presence of STX was not markedly variable over the range of potentials where modulation of channel opening was found. The result suggests that STX may not interact selectively with either open or closed states of the channel. We would like to thank Mary Taft for maintaining the cells in culture and Rae Barolet for secretarial assistance. These studies were supported by NIH Grants NSl4144 and ES02330. I Armstrong. C.M . and Bezanilla, F.• Charge movem ent associated with the opening and closing of activation gates of the Na channels. J. Gen . Physiol ., 63 (1974) 533-552. 2 Cohen, C.J ., Dean, B.P ., Colastsky, T .J. and Tsien, R. W., Tetrodotoxin block of sodium channels in rabb it Purkinje fibers. Interactions between toxin binding and channel gating, J. Gen . Physiol., 48 (1981) 383-411. 3 Hamill, O .P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high resolution current record ing from cells and cell free membrane patches, Pfliigers Arch. Ges. Physiol., 391 (1981) 85-100. 4 Hille, D., Pharmacological modifications of the sodium channels of frog nerve, J. Gen. Physiol., 51 (1968) 199-219. 5 Hille, B., The receptor for tetrodotoxin and saxitoxin: a structural hypothesis, Biophys. J., 15 (1975) 615-619. 6 Krueger, B.K., Worley, J.F., 11\ and French, R.J., Single sodium channels from rat brain incorporated into planar lipid bilayer membranes, Nature (Lond .), 303 (1983) 172-175 . 7 Narahashi, T., Chemicals as tools in the study of excitable membranes, Physiol. Rev., 54 (1974) 813-866 . 8 Quandt, F. and Narahashi, T., Contrast between open and closed block of single Na channel currents, Biophys. J., 37 (1982) 319a (abstract).
83 9 Quandt, F.N. and Narahashi, T., 1\lodification of single Na channels by batrachotoxin, Proc. Natl. Acad. Sci. USA, 79 (1982) 6732-6736. 10 Quandt, F.N. and Narahashi, T., Pharmacology of single sodium channels, Biophys, J.; 41 (1983) 279a (abstract). 11 Ritchie, l.1\I., A pharmacological approach to the structure of sodium channels in myelinated axons, Ann. Rev. Neurosci., 2 (1979) 341-362. 12 Ritchie, l.M. and Rogart, R.B., The binding of saxitoxin and tetrodotoxin to excitable tissue, Rev. Physiol. Biochem. Pharmacol., 79 (1977) 2-50. 13 Schwarz, l.R., Ulbricht, W. and Wagner, H.H., The rate of action of tetrodotoxin on myelinated nerve fibres of Xenopus laevis and Rana esculenta, J, Physiol. (Lond.), 233 (1973) 161-194. 14 Sigworth, F.l., The conductance of sodium channels under conditions of reduced current at the node of Ranvier, r. Physiol. (Lond.), 307 (1980) 131-142. 15 Takata, 1\1.,Moore, l.W., Kao, C.Y. and Fuhrman, F.A., Blockage of sodium conductance increase in lobster giant axon by tarichatoxin (tetrodotoxin), 1. Gen, Physiol., 49 (1966) 977-988. 16 Ulbricht, W. and Wagner, H.H., The influence of pH on equilibrium effects of tetrodotoxin on myelinated nerve fibers of Rana esculenta, J. Physiol. (Lond.) 252 (1975) 159-184.