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Modification of single sodium channels by the insecticide tetramethrin
344
Brain Research, 274 (1983) 344-349 Elsevier
Modification of single sodium channels by the insecticide tetramethrin DAISUKE YAMAMOTO, FRED N. QUANDT and TOSHIO NARAHASHI* Department of Pharmacology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, 1L 60611 (U.S.A.) (Accepted May 10th, 1983) Key words: sodium channel - - patch clamp - - insecticide tetramethrin - - pyrethroid - - neuroblastoma
(+)-trans-Tetramethrin, a pyrethroid insecticide, markedly prolongs the open time of single sodium channels recorded by the gigaohm-seal voltage clamp technique in a membrane patch excised from the N1E-115 neuroblastoma cell. Single channel conductance is not altered by tetramethrin. The modification by tetramethrin occurs in an all-or-none manner in a population of sodium channels. The observed tetramethrin-induced modification of single sodium channels is compatible with previous sodium current data from axons. One of the most dramatic technical developments in the field of excitable membranes is recording of single channel activity using patch voltage clamp techniques. This method was first applied to the ionic channels associated with the extrajunctional acetylcholine receptors of denervated muscle fibers 16. The recently developed gigaohm seal1 as combined with the isolation of a m e m b r a n e patch 3 has opened up an avenue for the study of interactions of various chemicals with m e m b r a n e ionic channels at a level close to the molecular event occurring in the channel. We have found that the pyrethroid insecticide tetramethrin modifies a population of the voltage-dependent sodium channel causing a greatly prolonged open state as a result of the reduction of the rate of closing. From voltage clamp studies with squid and crayfish giant axons and frog nodes of Ranvier, it has been proposed that the primary site of action of pyrethroids is the sodium channels of nerve membranes 14,26,27. In the tetramethrin-poisoned nerve membrane, a remarkably slow sodium current is generated following the normal peak sodium current during a prolonged step depolarizationg,10. The slow sodium current attains a peak with a time course of 100 ms to 1 s, and inactivates with a time course of 1-5 s depending on the m e m b r a n e potential. This slow current increases the depolarizing (negative) af* To whom correspondence should be addressed. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
terpotential under current clamp conditions which in turn triggers repetitive afterdischarges. These results, using conventional voltage clamp methods, raise several important issues. The prolonged current could be caused primarily by an alteration in the probability of individual sodium channel opening by tetramethrin. Alternatively, it may be due to the prolonged opening of individual sodium channels. Further, a question may be asked whether channel kinetics could be changed independently of single channel conductance. Sodium current retains some of its normal properties after exposure to the pyrethroids. Modification of channels could occur either in an all-or-none manner for a certain population or in a graded manner for most channels. These questions can be answered by directly measuring single channel parameters using patch clamp techniques. Experiments were performed with the somatic membrane of mouse neuroblastoma cell N1E-115. The cells were grown in Dulbecco's modified Eagle Medium with 10% fetal calf serum under a humidified atmosphere containing 10% CO 2. The cells were differentiated in 2% dimethylsulfoxide (DMSO)8. Single channel currents were recorded, using the gigaohm seal patch clamp technique 1. For the inside-out membrane patch, the pipette containing the external solution was applied onto the cell surface to establish gigaohm sealing. The bathing so-
345 with the extracellular solution shortly before use. All experiments were conducted at temperatures of 9-10 °C. Fig. 1A shows a series of currents recorded from a patch of membrane in response to step depolarizations from a holding potential o f - - 9 0 mV to - - 5 0 mV in normal solutions. Although no currents could be observed at the membrane potential o f - - 9 0 mV, inward currents (downward deflections) were elicited with varying latencies after the onset of the depolarization. In other experiments using outside-out membrane patches, T T X Was added to the extracellular solution at a concentration of 3 pM. The inward currents associated with step depolarizations disappeared completely following the application of TTX. Since sodium channels are selectively blocked by q T X in this and other preparations 12,19, this experiment associated these currents in membrane patches with the opening of sodium channels. The amplitudes of single sodium channel currents are fairly constant, and show a normal distribution with the mean + S.D. of 1.29 + 0.18 p A (n = 56) in the experiment illustrated in Fig. lB. The number of open states showing a lifetime greater than the time on the abscissa are plotted in Fig. 1C. When plotted in this manner, the ordinate represents the probability of not observing a closing event within any time following channel
lution was then changed to an internal solution. A holding potential was applied to the patch, and the pipette was pulled away from the cell surface. Thus the interior of the m e m b r a n e was exposed to the bath which contained the internal solution. Some experiments were performed with outside-out patch preparations. For the outside-out m e m b r a n e patch, the pipette containing the internal solution was applied onto the cell surface to establish gigaohm sealing. Additional suction was applied to break the membrane attached to the pipette tip. The pipette was then pulled away from the cell surface in the external solution, and in some cases its tip was sealed over with a patch of the membrane isolated from the cell. The external surface of the m e m b r a n e patch faced the bath which could be changed easily. The extracellular solution contained (mM): NaC1, 125; KCI, 5.5; CaCIz'2H20, 1.8; MgC12"6H20, 0.8; H E P E S , 20; dextrose, 25; sucrose, 20; p H 7.3. The intracellular solution contained (mM); CsF, 150; H E P E S , 20; NaH E P E S , 1; p H 7.2. (+)-trans-Tetramethrin was first dissolved in D M S O and then suspended in the intracellular solution. The amount of D M S O in the final solution was below 0.3% (v/v), which had no effect on the sodium current. Tetrodotoxin (TTX) was dissolved in 10% (v/v) citric acid solution at a concentration of 300/~M, and this stock solution was diluted
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(pA) OPEN TIME (msec) Io msec Fig. 1. Behavior of single sodium channels in an inside-out membrane patch excised from the neuroblastoma cell N 1E-115. The membrane is exposed to normal external and internal solutions. A: sample records of inward sodium currents (downward deflections) associated with step depolarizations from the holding membrane potential of--90 mV to --50 mV. Depolarizing steps were applied at a frequency of 0.3 Hz, and the figure represents an example of a series of consecutive records beginning at the bottom. B: histogram showing the distribution of the amplitudes of single channel currents. The mean + S.D. of the amplitude is 1.29 + 0.18 pA. C: distribution of the open time of single sodium channels. The number of events having an open time longer than that indicated on the abscissa is plotted. The distribution can be fitted by a single exponential function with a decay rate contant of 0.59 ms-L
346 mV) which would result in opening of all channels in the patch. Experiments on macroscopic currents revealed that the sodium conductance reached a maximum at about 0 mV, indicating that all channels were activated at that membrane potential TM. The maximum current amplitude, which was attained by a superimposition of all available channels in the patch, was divided by the amplitude of unitary current, giving a maximum value of channels in the patch. Typically 3-5 channels were present in a membrane patch. The inside-out membrane patch utilized for the experiment illustrated in Fig. 1 was next exposed to tetramethrin in order to evaluate alterations in the properties of the open state. Fig. 2A shows a series of membrane currents obtained after the addition of 60 /uM (+)-trans-tetramethrin to the internal perfusate. Step depolarizations from the holding potential o f 90 mV to - - 5 0 mV were associated with inward currents of approximately the same amplitudes as those of the control in Fig. 1A. As shown in Fig. 2B, the amplitudes of the single sodium channel currents show a mean _+ S.D. of 1.31 + 0.16 pA. This mean is similar to that obtained for the control value (1.29 pA). However, the lifetime of the open state of the
opening. For a Poisson closing process, this probability decays as a single exponential function. The decay rate constant could be estimated from a least squares regression to the distribution and was found to be 0.59 ms-1. This constant should correspond to the mean rate of channel closing. The reciprocal of the mean closing rate is 1.7 ms. Channels can be closed by the closure of either the activation (m) gate or the inactivation (h) gate. Experiments on macroscopic sodium currents in neuroblastoma cells indicated that the rate of inactivation at membrane potentials more negative than - - 2 0 mV was extremely slow. For example, the time constant for sodium current inactivation at - - 2 0 mV was about 45 ms 18. Therefore, the closure of channels at - - 5 0 mV would be predominantly due to the m gate mechanism. In these conditions the inactivation proceeds very slowly, and repetitive openings of the same channel are possible. Indeed one of the records illustrated in Fig. 1A shows multiple openings without overlapping. Although it is difficult to measure the exact number of channels present in a membrane patch, a rough estimate can be obtained by applying a large voltage step (e.g. to 0 mV from the holding potential o f - - l O 0 A 40
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Fig. 2. Behavior of single sodium channels in the same inside-out membrane patch as that in Fig. 1, but after exposure to an internal solution containing 60/~M (+)-trans-tetramethrin. A: sample records of inward sodium currents as in Fig. 1. Note that the time scale is 4 times slower than that used for Fig. 1. B: histogram showing the distribution of the amplitudes of single channel currents. The mean + S.D. is 1.31 4- 0.16 pA. C: distribution of the open time of single sodium channels. The number of events showing the open time longer than that indicated on the abscissa is plotted. The distribution of the open time longer than 5 ms is fitted by a single exponential function with a decay rate constant of 0.06 ms -1. The distribution of the open time shorter than 5 ms can be divided into two components: the component obtained by subtraction of the long time constant component from the total population is plotted in the inset using an expanded time scale, and can be fitted by a single exponential function with a decay rate constant of 0.53 ms -1. This value is very close to the rate constant obtained before application of tetramethrin (see Fig. 1C), and this component likely represents the unmodified population of sodium channels in the presence of tetramethrin.
347 sodium channel was markedly prolonged by tetramethrin (Fig. 2A). Note that the time scale in the figure is 4 times slower than that used in Fig. 1A for the control experiment. The probability distribution of the lifetime of the open state after exposure to tetramethrin is illustrated in Fig. 2C. Two points are clear from the figure. First, the channel open time can be much longer in patches exposed to tetramethrin than in the control condition; openings longer than 10 ms, which were normally non-existent, were observed at a high frequency. The reduction of the rate of closing for the open state of the tetramethrin-modified sodium channels accounts for the slow decay of the macroscopic sodium current in the poisoned nerve fibers as described earlier9A0,12A4,24-27. Second, the population now appears to be heterogeneous for this is not fitted by a single characteristic. The distribution of lifetimes for the open state of the sodium channel is best described by two exponentials during exposure. The number of openings with durations greater than 5 ms can be fitted by an exponential function corresponding to a mean closing rate of 0.06 ms -a, and the number of openings having a lifetime of less than 5 ms can be fitted by another exponential function with a mean closing rate of 0.53 ms -a, a value close to the control of 0.59 ms -1. This latter observation is consistent with the hypothesis that in the tetramethrin-treated m e m b r a n e there are two populations of open states for sodium channels, one opening with the normal lifetime and the other opening with a prolonged lifetime. Large prolongations of open channel lifetime were uniformly observed in the tetramethrin-modified sodium channels. Experiments with 3 m e m b r a n e patches gave values of 9.93 ms, 10.31 ms and 12.82 ms at - - 5 0 mV. These open states likely represent those for separate unmodified and modified channels, respectively. Such an all-ornone modification of sodium channels by tetramethrin was also suggested by previous observations of 'average' sodium currents from large populations of channels in crayfish and squid giant axons9, a0. The result described here constitutes a definitive demonstration in support of the notion. No data are available on the effect of tetramethrin on the macroscopic sodium current of neuroblastoma cells with which the present measurements of the microscopic sodium current can be compared. The
closed rates of the sodium channels of crayfish giant axons 9,12, squid giant axons 10, and frog nodes of Ranvier 25, as measured from the tail currents associated with step repolarizations, have been found to be markedly slowed after application of a variety of pyrethroids. For example, in squid giant axons, the rate of closure of sodium channels at - - 8 0 mV was slowed from the control value of 5.9 ms-a to 0.1 ms -1 after application of tetramethrin at 10 °C, representing a slowing by a factor of 5910. The absence of the marked effect of tetramethrin on the mean amplitude of single sodium channel currents suggests that single sodium channel conductance is unaltered by tetramethrin. In order to determine single channel conductance, we measured the amplitude of single channel current at several different potentials in the absence and presence of 45/~M tetramethrin. The resulting current-voltage relationships are shown in Fig. 3. The single channel conductance, calculated from the slope of the line through the open circles in the presence of tetramethfin, is 10 pS. This value is close to the conductance obtained in normal conditions (filled circles), 13 pS. In another experiment, single channel conductance was 15 pS in normal solution and 18 pS in solution containing tetramethrin. Thus it may be concluded
MEMBRANE POTENTIAL (mY) Er=llSmV -50 -40 -30 -20 -I0 0 I00 I101120 o v /: I-// Z UJ cr -1.0 cr -I.2 J bJ ]" : ~ TCONTTRMOELTHRI N Z -1.4 ~ \f ~10PS 45~uM
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Fig. 3. The relationship between amplitude of single sodium channel current and the membrane potential before (O) and after (O) exposure to 45/~M (+)-trans-tetramethrin in an inside-out membrane patch excised from the neuroblastoma cell N1E-115. The data are given in the mean + S.D. The solid line is the least squares fit to the mean values in the presence of tetramethrin, and the slope corresponds to a single channel conductance of 10 pS. The single channel conductance before exposure to tetramethrin as calculated from the two mean current values is 13 pS. The line can be extrapolated to a reversal potential of + 115 mV which is very close to the sodium equilibrium potential calculated by the Nernst equation (+ 117 mV).
348 that tetramethrin does not affect the conductance of
the normal channels did not open. This indicates a
single sodium channels.
large shift of voltage dependence of channel opening
Previous studies with tetramethrin9.t0, and other sodium channel modulators such as DDT2, n , grayanotoxine2, batrachotoxin (BTX) 5-7 and veratridine 20~21~23suggest that they bind to the sodium chan-
as produced by BTX, but not by tetramethrin. This difference correlates well with the observation that a
nel in its closed and/or open configuration to yield the m o d u l a t o r - b o u n d open sodium channel which carries
for m e m b r a n e s exposed to tetramethrin9,10. This action of B T X apparently arises due to the shift of the
a slow sodium current, and that both the unmodified,
activation of sodium channels toward more negative
normal sodium channels and the modified sodium channels co-exist in the m e m b r a n e exposed to a mod-
potentials, as observed for the average sodium currents recorded from axons 5-7 as well as the lack of any
ulator. The observations of two distinct populations of sodium channels in the presence of tetramethrin
type of inactivation process for modified channels 4.
and of batrachotoxin ~7, one with the normal open
sizeable N a - d e p e n d e n t depolarization of the membrane occurs after exposure to BTX 15 which is absent
These effects have not been observed for tetramethrin9,10. The second difference is a marked reduction
time and the other with prolonged open time, provide direct evidence for the notion of all-or-none modifications of the sodium channel.
of single sodium channel conductance by BTX, an effect which cannot be observed with tetramethrin.
However, there are two distinct differences between tetramethrin and B T X in their actions on single sodium channels 17. Whereas no spontaneous
We thank Dr. H. Yoshioka of Sumitomo Chemical C o m p a n y for his generous supply of tetramethrin samples. Thanks are also due to Mary Taft and Randy Reid for technical assistance, and Janet H e n d e r -
opening of sodium channels was detected in the m e m b r a n e patch exposed to tetramethrin, the BTXmodified channels opened spontaneously at large negative m e m b r a n e potentials (e.g. - - 8 0 mV) where
1 Hamill, O. P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F. J., Improved patch-clamp techniques for highresolution current recording from cells and cell-free membrane patches, Pflagers Arch. ges. Physiol., 391 (1981) 85-100. 2 Hille, B., Pharmacological modifications of the sodium channels of frog nerve, J. gen. Physiol., 51 (1968) 199-219. 3 Horn. R. and Patlak, J. B., Single channel currents from excised patches of muscle membrane, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 6930-6934. 4 Huang, L.-Y. M., Moran, N. and Ehrenstein, G., Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 2082-2085. 5 Khodorov, B. I., Chemicals as tools to study nerve fiber sodium channels: effects of batrachotoxin and some local anesthetics. In D. C. Tosteson, A. O. Yu and R. Latorre (Eds.), Membrane Transport Processes, Vol. 2, Raven Press. New York, 1978, pp. 153-174. 6 Khodorov, B. I. and Revenko, S. V., Further analysis of the mechanisms of action of batrachotoxin on the membrane of myelinated nerve, Neuroscience, 4 (1979) 1315-1330. 7 Khodorov, B. I., Peganov, E. M., Ravenko, S. V. and Shishkova, L. D., Sodium currents in voltage clamped nerve fiber of frog under the combined action of batrachotoxin and procaine, Brain Research, 84 (1975) 541-546. 8 Kimhi, Y., Palfrey, C., Spector, I., Barak, Y. and Littauer, U. Z., Maturation of neuroblastoma cells in the presence of
son, Terri W a r r e n and Veronica L. Whatley for secretarial assistance. This work was supported by N I H G r a n t NS14143.
9 10 11 12
13 14
15
16 17
dimethylsulfoxide, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 462-466. Lund, A. E. and Narahashi, T., Modification of sodium channel kinetics by the insecticide tetramethrin in crayfish giant axons, Neurotoxicology, 2 (1981) 213-229. Lund, A. E. and Narahashi, T., Kinetics of sodium channel modification by the insecticide tetramethrin in squid axon membranes, J. Pharmacol. exp. Ther., 219 (1981) 464-473. Lund, A. E. and Narahashi, T., Interaction of DDT with sodium channels in squid giant axon membranes, Neuroscience, 6 (1981) 2253-2258. Lund, A. E. and Narahashi, T., Kinetics of sodium channel modification as the basis for the variation in the nerve membrane effects of pyrethroids and DDT analogs, Pesticide Biochem. Physiol., in press. Narahashi, T., Chemicals as tools in the study of excitable membranes, Physiol. Rev., 54 (1974) 813-889. Narahashi, T. and Anderson, N. C., Mechanism of excitation block by the insecticide allethrin applied externally and internally to squid giant axons, Toxicol. appl. Pharmacol., 10 (1967) 529-547. Narahashi, T., Albuquerque, E. X. and Deguchi, T., Effects of batrachotoxin on membrane potential and conductance of squid giant axons, J. gen. Physiol., 58 (1971) 54-70. Neher, E. and Sakmann, B., Single-channel currents recorded from membrane of denervated frog muscle fibres, Nature (Lond.), 260 (1976) 779--802. Quandt, F. N. and Narahashi, T., Modification of single
349
18
19
20
21
22
Na t channels by batrachotoxin, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 6732-6736. Quandt, F. and Narahashi, T., Ionic currents underlying the action potential of neuroblastoma cells, Soc. Neurosci. Abstr., 5 (1979) 294. Quandt, F. N., Yeh, J. Z. and Narahashi, T., Contrast between open and closed block of single Na channel currents, Biophys. J., 37 (1982) 319a. Scruggs, V. M., The Veratridine Induced Sodium Conductance in the Squid Giant Axon, Ph. D. Dissertation, University of Miami, 1979. Scruggs, V. M. and Narahashi, T., Veratridine modification of the nerve membrane sodium channel, Biophys. J., 37 (1982) 320a. Seyama, I. and Narahashi, T., Modulation of sodium channels of squid nerve membranes by grayanotoxin I, J. Pharmacol, exp. Ther. , 219 (1981) 614-624.
23 Ulbricht, W., The effect of veratridine on excitable membranes of nerve and muscle, Ergebn. Physiol. Biol. Chem. exp. Pharmakol., 61 (1969) 17-71. 24 Vijverberg, H. P. M., Van der Zalm, J. M. and Van der Bercken, J., Similar mode of action of pyrethroids and DDT on sodium channel gating in myelinated nerves, Nature (Lond.), 295 (1982) 601-603. 25 Vijverberg, H. P. M., Van der Zalm, J. M., Van Kleef, R. G. D. M. and Van den Bercken, J., Temperature- and structure-dependent ineraction of pyrethroids with the sodium channels in frog node of Ranvier, Biochim. biophys. Acta, 728 (1983) 73-82. 26 Wang, C. M., Narahashi, T. and Scuka, M., Mechanism of negative temperature coefficient of nerve blocking action of allethrin, J. Pharmacol. exp. Ther., 182 (1972) 442-453. 27 Wouters, W. and Van den Bercken, J., Action of pyrethroids, Gen. Pharmacol., 9 (1978) 387-398.
Brain Research, 274 (1983) 344-349 Elsevier
Modification of single sodium channels by the insecticide tetramethrin DAISUKE YAMAMOTO, FRED N. QUANDT and TOSHIO NARAHASHI* Department of Pharmacology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, 1L 60611 (U.S.A.) (Accepted May 10th, 1983) Key words: sodium channel - - patch clamp - - insecticide tetramethrin - - pyrethroid - - neuroblastoma
(+)-trans-Tetramethrin, a pyrethroid insecticide, markedly prolongs the open time of single sodium channels recorded by the gigaohm-seal voltage clamp technique in a membrane patch excised from the N1E-115 neuroblastoma cell. Single channel conductance is not altered by tetramethrin. The modification by tetramethrin occurs in an all-or-none manner in a population of sodium channels. The observed tetramethrin-induced modification of single sodium channels is compatible with previous sodium current data from axons. One of the most dramatic technical developments in the field of excitable membranes is recording of single channel activity using patch voltage clamp techniques. This method was first applied to the ionic channels associated with the extrajunctional acetylcholine receptors of denervated muscle fibers 16. The recently developed gigaohm seal1 as combined with the isolation of a m e m b r a n e patch 3 has opened up an avenue for the study of interactions of various chemicals with m e m b r a n e ionic channels at a level close to the molecular event occurring in the channel. We have found that the pyrethroid insecticide tetramethrin modifies a population of the voltage-dependent sodium channel causing a greatly prolonged open state as a result of the reduction of the rate of closing. From voltage clamp studies with squid and crayfish giant axons and frog nodes of Ranvier, it has been proposed that the primary site of action of pyrethroids is the sodium channels of nerve membranes 14,26,27. In the tetramethrin-poisoned nerve membrane, a remarkably slow sodium current is generated following the normal peak sodium current during a prolonged step depolarizationg,10. The slow sodium current attains a peak with a time course of 100 ms to 1 s, and inactivates with a time course of 1-5 s depending on the m e m b r a n e potential. This slow current increases the depolarizing (negative) af* To whom correspondence should be addressed. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
terpotential under current clamp conditions which in turn triggers repetitive afterdischarges. These results, using conventional voltage clamp methods, raise several important issues. The prolonged current could be caused primarily by an alteration in the probability of individual sodium channel opening by tetramethrin. Alternatively, it may be due to the prolonged opening of individual sodium channels. Further, a question may be asked whether channel kinetics could be changed independently of single channel conductance. Sodium current retains some of its normal properties after exposure to the pyrethroids. Modification of channels could occur either in an all-or-none manner for a certain population or in a graded manner for most channels. These questions can be answered by directly measuring single channel parameters using patch clamp techniques. Experiments were performed with the somatic membrane of mouse neuroblastoma cell N1E-115. The cells were grown in Dulbecco's modified Eagle Medium with 10% fetal calf serum under a humidified atmosphere containing 10% CO 2. The cells were differentiated in 2% dimethylsulfoxide (DMSO)8. Single channel currents were recorded, using the gigaohm seal patch clamp technique 1. For the inside-out membrane patch, the pipette containing the external solution was applied onto the cell surface to establish gigaohm sealing. The bathing so-
345 with the extracellular solution shortly before use. All experiments were conducted at temperatures of 9-10 °C. Fig. 1A shows a series of currents recorded from a patch of membrane in response to step depolarizations from a holding potential o f - - 9 0 mV to - - 5 0 mV in normal solutions. Although no currents could be observed at the membrane potential o f - - 9 0 mV, inward currents (downward deflections) were elicited with varying latencies after the onset of the depolarization. In other experiments using outside-out membrane patches, T T X Was added to the extracellular solution at a concentration of 3 pM. The inward currents associated with step depolarizations disappeared completely following the application of TTX. Since sodium channels are selectively blocked by q T X in this and other preparations 12,19, this experiment associated these currents in membrane patches with the opening of sodium channels. The amplitudes of single sodium channel currents are fairly constant, and show a normal distribution with the mean + S.D. of 1.29 + 0.18 p A (n = 56) in the experiment illustrated in Fig. lB. The number of open states showing a lifetime greater than the time on the abscissa are plotted in Fig. 1C. When plotted in this manner, the ordinate represents the probability of not observing a closing event within any time following channel
lution was then changed to an internal solution. A holding potential was applied to the patch, and the pipette was pulled away from the cell surface. Thus the interior of the m e m b r a n e was exposed to the bath which contained the internal solution. Some experiments were performed with outside-out patch preparations. For the outside-out m e m b r a n e patch, the pipette containing the internal solution was applied onto the cell surface to establish gigaohm sealing. Additional suction was applied to break the membrane attached to the pipette tip. The pipette was then pulled away from the cell surface in the external solution, and in some cases its tip was sealed over with a patch of the membrane isolated from the cell. The external surface of the m e m b r a n e patch faced the bath which could be changed easily. The extracellular solution contained (mM): NaC1, 125; KCI, 5.5; CaCIz'2H20, 1.8; MgC12"6H20, 0.8; H E P E S , 20; dextrose, 25; sucrose, 20; p H 7.3. The intracellular solution contained (mM); CsF, 150; H E P E S , 20; NaH E P E S , 1; p H 7.2. (+)-trans-Tetramethrin was first dissolved in D M S O and then suspended in the intracellular solution. The amount of D M S O in the final solution was below 0.3% (v/v), which had no effect on the sodium current. Tetrodotoxin (TTX) was dissolved in 10% (v/v) citric acid solution at a concentration of 300/~M, and this stock solution was diluted
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(pA) OPEN TIME (msec) Io msec Fig. 1. Behavior of single sodium channels in an inside-out membrane patch excised from the neuroblastoma cell N 1E-115. The membrane is exposed to normal external and internal solutions. A: sample records of inward sodium currents (downward deflections) associated with step depolarizations from the holding membrane potential of--90 mV to --50 mV. Depolarizing steps were applied at a frequency of 0.3 Hz, and the figure represents an example of a series of consecutive records beginning at the bottom. B: histogram showing the distribution of the amplitudes of single channel currents. The mean + S.D. of the amplitude is 1.29 + 0.18 pA. C: distribution of the open time of single sodium channels. The number of events having an open time longer than that indicated on the abscissa is plotted. The distribution can be fitted by a single exponential function with a decay rate contant of 0.59 ms-L
346 mV) which would result in opening of all channels in the patch. Experiments on macroscopic currents revealed that the sodium conductance reached a maximum at about 0 mV, indicating that all channels were activated at that membrane potential TM. The maximum current amplitude, which was attained by a superimposition of all available channels in the patch, was divided by the amplitude of unitary current, giving a maximum value of channels in the patch. Typically 3-5 channels were present in a membrane patch. The inside-out membrane patch utilized for the experiment illustrated in Fig. 1 was next exposed to tetramethrin in order to evaluate alterations in the properties of the open state. Fig. 2A shows a series of membrane currents obtained after the addition of 60 /uM (+)-trans-tetramethrin to the internal perfusate. Step depolarizations from the holding potential o f 90 mV to - - 5 0 mV were associated with inward currents of approximately the same amplitudes as those of the control in Fig. 1A. As shown in Fig. 2B, the amplitudes of the single sodium channel currents show a mean _+ S.D. of 1.31 + 0.16 pA. This mean is similar to that obtained for the control value (1.29 pA). However, the lifetime of the open state of the
opening. For a Poisson closing process, this probability decays as a single exponential function. The decay rate constant could be estimated from a least squares regression to the distribution and was found to be 0.59 ms-1. This constant should correspond to the mean rate of channel closing. The reciprocal of the mean closing rate is 1.7 ms. Channels can be closed by the closure of either the activation (m) gate or the inactivation (h) gate. Experiments on macroscopic sodium currents in neuroblastoma cells indicated that the rate of inactivation at membrane potentials more negative than - - 2 0 mV was extremely slow. For example, the time constant for sodium current inactivation at - - 2 0 mV was about 45 ms 18. Therefore, the closure of channels at - - 5 0 mV would be predominantly due to the m gate mechanism. In these conditions the inactivation proceeds very slowly, and repetitive openings of the same channel are possible. Indeed one of the records illustrated in Fig. 1A shows multiple openings without overlapping. Although it is difficult to measure the exact number of channels present in a membrane patch, a rough estimate can be obtained by applying a large voltage step (e.g. to 0 mV from the holding potential o f - - l O 0 A 40
B
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Fig. 2. Behavior of single sodium channels in the same inside-out membrane patch as that in Fig. 1, but after exposure to an internal solution containing 60/~M (+)-trans-tetramethrin. A: sample records of inward sodium currents as in Fig. 1. Note that the time scale is 4 times slower than that used for Fig. 1. B: histogram showing the distribution of the amplitudes of single channel currents. The mean + S.D. is 1.31 4- 0.16 pA. C: distribution of the open time of single sodium channels. The number of events showing the open time longer than that indicated on the abscissa is plotted. The distribution of the open time longer than 5 ms is fitted by a single exponential function with a decay rate constant of 0.06 ms -1. The distribution of the open time shorter than 5 ms can be divided into two components: the component obtained by subtraction of the long time constant component from the total population is plotted in the inset using an expanded time scale, and can be fitted by a single exponential function with a decay rate constant of 0.53 ms -1. This value is very close to the rate constant obtained before application of tetramethrin (see Fig. 1C), and this component likely represents the unmodified population of sodium channels in the presence of tetramethrin.
347 sodium channel was markedly prolonged by tetramethrin (Fig. 2A). Note that the time scale in the figure is 4 times slower than that used in Fig. 1A for the control experiment. The probability distribution of the lifetime of the open state after exposure to tetramethrin is illustrated in Fig. 2C. Two points are clear from the figure. First, the channel open time can be much longer in patches exposed to tetramethrin than in the control condition; openings longer than 10 ms, which were normally non-existent, were observed at a high frequency. The reduction of the rate of closing for the open state of the tetramethrin-modified sodium channels accounts for the slow decay of the macroscopic sodium current in the poisoned nerve fibers as described earlier9A0,12A4,24-27. Second, the population now appears to be heterogeneous for this is not fitted by a single characteristic. The distribution of lifetimes for the open state of the sodium channel is best described by two exponentials during exposure. The number of openings with durations greater than 5 ms can be fitted by an exponential function corresponding to a mean closing rate of 0.06 ms -a, and the number of openings having a lifetime of less than 5 ms can be fitted by another exponential function with a mean closing rate of 0.53 ms -a, a value close to the control of 0.59 ms -1. This latter observation is consistent with the hypothesis that in the tetramethrin-treated m e m b r a n e there are two populations of open states for sodium channels, one opening with the normal lifetime and the other opening with a prolonged lifetime. Large prolongations of open channel lifetime were uniformly observed in the tetramethrin-modified sodium channels. Experiments with 3 m e m b r a n e patches gave values of 9.93 ms, 10.31 ms and 12.82 ms at - - 5 0 mV. These open states likely represent those for separate unmodified and modified channels, respectively. Such an all-ornone modification of sodium channels by tetramethrin was also suggested by previous observations of 'average' sodium currents from large populations of channels in crayfish and squid giant axons9, a0. The result described here constitutes a definitive demonstration in support of the notion. No data are available on the effect of tetramethrin on the macroscopic sodium current of neuroblastoma cells with which the present measurements of the microscopic sodium current can be compared. The
closed rates of the sodium channels of crayfish giant axons 9,12, squid giant axons 10, and frog nodes of Ranvier 25, as measured from the tail currents associated with step repolarizations, have been found to be markedly slowed after application of a variety of pyrethroids. For example, in squid giant axons, the rate of closure of sodium channels at - - 8 0 mV was slowed from the control value of 5.9 ms-a to 0.1 ms -1 after application of tetramethrin at 10 °C, representing a slowing by a factor of 5910. The absence of the marked effect of tetramethrin on the mean amplitude of single sodium channel currents suggests that single sodium channel conductance is unaltered by tetramethrin. In order to determine single channel conductance, we measured the amplitude of single channel current at several different potentials in the absence and presence of 45/~M tetramethrin. The resulting current-voltage relationships are shown in Fig. 3. The single channel conductance, calculated from the slope of the line through the open circles in the presence of tetramethfin, is 10 pS. This value is close to the conductance obtained in normal conditions (filled circles), 13 pS. In another experiment, single channel conductance was 15 pS in normal solution and 18 pS in solution containing tetramethrin. Thus it may be concluded
MEMBRANE POTENTIAL (mY) Er=llSmV -50 -40 -30 -20 -I0 0 I00 I101120 o v /: I-// Z UJ cr -1.0 cr -I.2 J bJ ]" : ~ TCONTTRMOELTHRI N Z -1.4 ~ \f ~10PS 45~uM
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Fig. 3. The relationship between amplitude of single sodium channel current and the membrane potential before (O) and after (O) exposure to 45/~M (+)-trans-tetramethrin in an inside-out membrane patch excised from the neuroblastoma cell N1E-115. The data are given in the mean + S.D. The solid line is the least squares fit to the mean values in the presence of tetramethrin, and the slope corresponds to a single channel conductance of 10 pS. The single channel conductance before exposure to tetramethrin as calculated from the two mean current values is 13 pS. The line can be extrapolated to a reversal potential of + 115 mV which is very close to the sodium equilibrium potential calculated by the Nernst equation (+ 117 mV).
348 that tetramethrin does not affect the conductance of
the normal channels did not open. This indicates a
single sodium channels.
large shift of voltage dependence of channel opening
Previous studies with tetramethrin9.t0, and other sodium channel modulators such as DDT2, n , grayanotoxine2, batrachotoxin (BTX) 5-7 and veratridine 20~21~23suggest that they bind to the sodium chan-
as produced by BTX, but not by tetramethrin. This difference correlates well with the observation that a
nel in its closed and/or open configuration to yield the m o d u l a t o r - b o u n d open sodium channel which carries
for m e m b r a n e s exposed to tetramethrin9,10. This action of B T X apparently arises due to the shift of the
a slow sodium current, and that both the unmodified,
activation of sodium channels toward more negative
normal sodium channels and the modified sodium channels co-exist in the m e m b r a n e exposed to a mod-
potentials, as observed for the average sodium currents recorded from axons 5-7 as well as the lack of any
ulator. The observations of two distinct populations of sodium channels in the presence of tetramethrin
type of inactivation process for modified channels 4.
and of batrachotoxin ~7, one with the normal open
sizeable N a - d e p e n d e n t depolarization of the membrane occurs after exposure to BTX 15 which is absent
These effects have not been observed for tetramethrin9,10. The second difference is a marked reduction
time and the other with prolonged open time, provide direct evidence for the notion of all-or-none modifications of the sodium channel.
of single sodium channel conductance by BTX, an effect which cannot be observed with tetramethrin.
However, there are two distinct differences between tetramethrin and B T X in their actions on single sodium channels 17. Whereas no spontaneous
We thank Dr. H. Yoshioka of Sumitomo Chemical C o m p a n y for his generous supply of tetramethrin samples. Thanks are also due to Mary Taft and Randy Reid for technical assistance, and Janet H e n d e r -
opening of sodium channels was detected in the m e m b r a n e patch exposed to tetramethrin, the BTXmodified channels opened spontaneously at large negative m e m b r a n e potentials (e.g. - - 8 0 mV) where
1 Hamill, O. P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F. J., Improved patch-clamp techniques for highresolution current recording from cells and cell-free membrane patches, Pflagers Arch. ges. Physiol., 391 (1981) 85-100. 2 Hille, B., Pharmacological modifications of the sodium channels of frog nerve, J. gen. Physiol., 51 (1968) 199-219. 3 Horn. R. and Patlak, J. B., Single channel currents from excised patches of muscle membrane, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 6930-6934. 4 Huang, L.-Y. M., Moran, N. and Ehrenstein, G., Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 2082-2085. 5 Khodorov, B. I., Chemicals as tools to study nerve fiber sodium channels: effects of batrachotoxin and some local anesthetics. In D. C. Tosteson, A. O. Yu and R. Latorre (Eds.), Membrane Transport Processes, Vol. 2, Raven Press. New York, 1978, pp. 153-174. 6 Khodorov, B. I. and Revenko, S. V., Further analysis of the mechanisms of action of batrachotoxin on the membrane of myelinated nerve, Neuroscience, 4 (1979) 1315-1330. 7 Khodorov, B. I., Peganov, E. M., Ravenko, S. V. and Shishkova, L. D., Sodium currents in voltage clamped nerve fiber of frog under the combined action of batrachotoxin and procaine, Brain Research, 84 (1975) 541-546. 8 Kimhi, Y., Palfrey, C., Spector, I., Barak, Y. and Littauer, U. Z., Maturation of neuroblastoma cells in the presence of
son, Terri W a r r e n and Veronica L. Whatley for secretarial assistance. This work was supported by N I H G r a n t NS14143.
9 10 11 12
13 14
15
16 17
dimethylsulfoxide, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 462-466. Lund, A. E. and Narahashi, T., Modification of sodium channel kinetics by the insecticide tetramethrin in crayfish giant axons, Neurotoxicology, 2 (1981) 213-229. Lund, A. E. and Narahashi, T., Kinetics of sodium channel modification by the insecticide tetramethrin in squid axon membranes, J. Pharmacol. exp. Ther., 219 (1981) 464-473. Lund, A. E. and Narahashi, T., Interaction of DDT with sodium channels in squid giant axon membranes, Neuroscience, 6 (1981) 2253-2258. Lund, A. E. and Narahashi, T., Kinetics of sodium channel modification as the basis for the variation in the nerve membrane effects of pyrethroids and DDT analogs, Pesticide Biochem. Physiol., in press. Narahashi, T., Chemicals as tools in the study of excitable membranes, Physiol. Rev., 54 (1974) 813-889. Narahashi, T. and Anderson, N. C., Mechanism of excitation block by the insecticide allethrin applied externally and internally to squid giant axons, Toxicol. appl. Pharmacol., 10 (1967) 529-547. Narahashi, T., Albuquerque, E. X. and Deguchi, T., Effects of batrachotoxin on membrane potential and conductance of squid giant axons, J. gen. Physiol., 58 (1971) 54-70. Neher, E. and Sakmann, B., Single-channel currents recorded from membrane of denervated frog muscle fibres, Nature (Lond.), 260 (1976) 779--802. Quandt, F. N. and Narahashi, T., Modification of single
349
18
19
20
21
22
Na t channels by batrachotoxin, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 6732-6736. Quandt, F. and Narahashi, T., Ionic currents underlying the action potential of neuroblastoma cells, Soc. Neurosci. Abstr., 5 (1979) 294. Quandt, F. N., Yeh, J. Z. and Narahashi, T., Contrast between open and closed block of single Na channel currents, Biophys. J., 37 (1982) 319a. Scruggs, V. M., The Veratridine Induced Sodium Conductance in the Squid Giant Axon, Ph. D. Dissertation, University of Miami, 1979. Scruggs, V. M. and Narahashi, T., Veratridine modification of the nerve membrane sodium channel, Biophys. J., 37 (1982) 320a. Seyama, I. and Narahashi, T., Modulation of sodium channels of squid nerve membranes by grayanotoxin I, J. Pharmacol, exp. Ther. , 219 (1981) 614-624.
23 Ulbricht, W., The effect of veratridine on excitable membranes of nerve and muscle, Ergebn. Physiol. Biol. Chem. exp. Pharmakol., 61 (1969) 17-71. 24 Vijverberg, H. P. M., Van der Zalm, J. M. and Van der Bercken, J., Similar mode of action of pyrethroids and DDT on sodium channel gating in myelinated nerves, Nature (Lond.), 295 (1982) 601-603. 25 Vijverberg, H. P. M., Van der Zalm, J. M., Van Kleef, R. G. D. M. and Van den Bercken, J., Temperature- and structure-dependent ineraction of pyrethroids with the sodium channels in frog node of Ranvier, Biochim. biophys. Acta, 728 (1983) 73-82. 26 Wang, C. M., Narahashi, T. and Scuka, M., Mechanism of negative temperature coefficient of nerve blocking action of allethrin, J. Pharmacol. exp. Ther., 182 (1972) 442-453. 27 Wouters, W. and Van den Bercken, J., Action of pyrethroids, Gen. Pharmacol., 9 (1978) 387-398.