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Interaction of Tetramethrin and Deltamethrin at the Single Sodium Channel in Rat Hippocampal Neurons
NeuroToxicology 22 (2001) 329±339
Interaction of Tetramethrin and Deltamethrin at the Single Sodium Channel in Rat Hippocampal Neurons Haruhiko Motomura, Toshio Narahashi* Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611-3008, USA Received 2 October 2000; accepted 6 February 2001
Abstract Type I and type II pyrethroids are known to modulate the sodium channel to cause persistent openings during depolarization and upon repolarization. Although there are some similarities between the two types of pyrethroids in their actions on sodium channels, the pattern of modi®cation of sodium currents is different between the two types of pyrethroids. In the present study, interactions of the type I pyrethroid tetramethrin and the type II pyrethroid deltamethrin at rat hippocampal neuron sodium channels were investigated using the inside-out single-channel patch clamp technique. Deltamethrin-modi®ed sodium channels opened much longer than tetramethrin-modi®ed sodium channels. When 10 mM tetramethrin was applied to membrane patches that had been exposed to 10 mM deltamethrin, deltamethrin-modi®ed prolonged single sodium currents disappeared and were replaced by shorter openings which were characteristic of tetramethrin-modi®ed channel openings. These single-channel data are compatible with previous whole-cell competition study between type I and type II pyrethroids. These results are interpreted as being due to the displacement of the type II pyrethroid molecule by the type I pyrethroid molecule from the same binding site or to the allosteric interaction of the two pyrethroid molecules at separate sodium channel sites. # 2001 Elsevier Science Inc. All rights reserved.
Keywords: Pyrethroid; Tetramethrin; Deltamethrin; Sodium channel; Hippocampal neuron; Singlechannel
INTRODUCTION Pyrethroids are synthetic derivatives of pyrethrins, which are toxic components contained in the ¯owers of Chrysanthemum cinerariaefolium. The pyrethroids are used widely as near-ideal insecticides due to their high insecticidal potencies, low mammalian toxicities and biodegradability. Sodium channels are known to be one of the most important target sites of pyrethroids (Narahashi, 1985, 1992, 1996; Ruigt, 1984; Soderlund and Bloomquist, 1989; Vijverberg and van den Bercken, 1990; Bloomquist, 1996). * Corresponding author. Tel.: 1-312-503-8284; fax: 1-312-503-1700. E-mail address: [email protected] (T. Narahashi).
Pyrethroids are divided into two groups by their chemical structures: type I pyrethroids are devoid of a cyano moiety at the a-position, and type II pyrethroids have an a-cyano moiety. Type I pyrethroids cause hyperexcitation, ataxia, convulsions and eventual paralysis in mammals, whereas type II pyrethroids produce hypersensitivity, choreoathetosis, tremors, and paralysis (Narahashi, 1985; Verschoyle and Aldridge, 1980; Vijverberg and van den Bercken, 1990). Pyrethroids seem to stabilize gating particles of the sodium channel (Salgado and Narahashi, 1993), resulting in slowing of the movements of both the activation and inactivation gates (Chinn and Narahashi, 1986), and shift the voltage dependence of the gates in the hyperpolarizing direction (Tabarean and Narahashi, 1998; Tatebayashi and Narahashi, 1994). These changes cause
0161-813X/01/$ ± see front matter # 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 1 ) 0 0 0 2 3 - 7
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a prolonged ¯ow of sodium current into the cell, leading to a sustained membrane depolarization. Although the basic actions of pyrethroids on sodium channels are similar between the two types of pyrethroids, the degree of modi®cation of sodium currents is different; single sodium channel currents are prolonged to a greater extent with type II than type I pyrethroids (Chinn and Narahashi, 1986; Holloway et al., 1989; Yamamoto et al., 1983; Motomura and Narahashi, 2000). Biochemical and molecular biological studies demonstrated that pyrethroids seem to have a speci®c binding site (Trainer et al., 1997) and that point mutations rendered the sodium channels resistant to pyrethroids (Pittendrigh et al., 1997; Park et al., 1997, 2000; Schuler et al., 1998; Lee et al., 1999a,b), suggesting the putative binding sites of pyrethroids in the sodium channels. However, no report has been published concerning the relationship between binding sites of type I and II pyrethroids using molecular biological or binding experiments. We previously reported interactions of type I and II pyrethroids at sodium channels using whole-cell patch clamp techniques (Song et al., 1996). In both tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels of rat dorsal root ganglion neurons, tetramethrin (type I) and fenvalerate (type II) prolonged the sodium channel tail current that was generated upon repolarization following a depolarizing pulse (Song et al., 1996). Fenvalerate-induced tail current decayed very slowly and was irreversible after washing with drug-free solution, whereas tetramethrininduced tail current decayed faster and was reversible after washout. When tetramethrin was applied to the fenvalerate-treated cell, the prolonged tail current was replaced by a much shorter tail current characteristic of the tetramethrin-treated cell. Furthermore, washing out of both tetramethrin and fenvalerate resulted in an appearance of the prolonged tail current characteristic of fenvalerate-treated cell. It was suggested that the type I pyrethroid molecule displaces the type II pyrethroid molecule from the binding site in the sodium channel protein, or that type I and II pyrethroids bind to separate sodium channel sites which interact allosterically with each other. However, since the whole-cell tail current induced by pyrethroids results form changes in a variety of single-channel parameters, the aforementioned wholecell data do not allow us to conclude all-or-none changes from fenvalerate-type to tetramethrin-type modi®cation of channel kinetics. It is imperative to obtain data at the single-channel level. We now address this issue in the present study using single-channel
techniques, which are expected to provide more straightforward evidence. We compared channel gating behaviors of tetramethrin (type I)- and deltamethrin (type II)-modi®ed single-channel currents, and examined interactions of the two pyrethroids at the individual channel level. MATERIALS AND METHODS Dissociation and Culture of Neurons Although our previous study (Song et al., 1996) was performed using rat dorsal root ganglion neurons which contain both TTX-S and TTX-R sodium channels, in the present study, hippocampal neurons were chosen for three reasons. (1) The single-channel current amplitude of TTX-S channels were larger than that of TTX-R channels (Song and Narahashi, 1996b). Therefore, the measurement of channel openings were relatively easy. (2) Since the openings of unmodi®ed TTX-S channels were very brief, the distinction between the unmodi®ed and pyrethroid-modi®ed channel openings was easier that that in TTX-R channels. (3) Pyrethroid-modi®ed channels sometimes opened at subconductance levels (Chinn and Narahashi, 1986). The current amplitude of subconductance level openings of pyrethroid-modi®ed TTX-S channels could be very similar to that of full-level openings of TTX-R channels if dorsal root ganglion cells were used. This might cause dif®culties in analysis. Hippocampal neurons were prepared from 17-dayold embryos of a Sprague±Dawley pregnant rat under methoxy¯urane anaesthesia. After an adequate level of anesthesia was reached as indicated by lack of responses to pinching the hind legs, a transverse incision was made in the lower abdomen of the pregnant rat and the fetuses were withdrawn from the abdomen. The heads of the fetuses were immediately cut-off, and the hippocampi were dissected and collected in a calciumand magnesium-free phosphate-buffered saline solution (PBS). The mother rat was euthanized immediately thereafter by decapitation. Neurons were dissociated by repeated passage through a ¯ame-narrowed Pasteur pipette and diluted with Neurobasal Medium supplemented with B-27 and 2 mM L-glutamic acid (GIBCO/Life Technologies, Grand Island, NY). The ®nal suspension was plated onto 12 mm poly-L-lysine-coated coverslips at a density of 200,000 cells/well. Cells were maintained in a humidi®ed atmosphere of 90% air and 10% CO2 at 378C, and were used for experiments within 3 weeks of
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culture. Neurons having triangle cell bodies or pyramidal morphology were selected for experiments. Electrical Recording Single-channel currents were recorded using the inside-out con®guration of the patch clamp technique (Hamill et al., 1981). Patch pipettes fabricated from borosilicate glass capillary tubes (1.5 mm inner diameter) had a resistance of 5±10 MO when ®lled with pipette solution. The pipette solution contained (mM): NaCl, 250; CsCl, 5; CaCl2, 1.8; MgCl2, 1; and 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES) 5. It should be noted that the pipette sodium concentration was doubled to 250 mM to obtain larger single sodium channel currents (Motomura et al., 1995). The pH was adjusted to 7.4 with NaOH. Normal Krebs solution contained (mM): NaCl, 120; KCl, 5; CaCl2, 1.8; MgCl2, 1; HEPES, 5; and glucose, 25. The pH was adjusted to 7.4 with NaOH. After making a gigaohm seal in the Krebs solution, it was replaced by the test bath solution which contained (mM): NaF, 1.4; CsF 145; sucrose, 200; HEPES, 5; and EGTA, 5. The pH was adjusted to 7.2 with CsOH. After the solution exchange, the membrane patch was excised. Single-channel currents passing through the pipette were recorded by a patch clamp ampli®er (Axopatch 200A, Axon Instruments, Foster City, CA). The currents were ®ltered at 2±10 kHz with a four-pole Bessel ®lter, digitalized at a rate of 10±50 kHz (at least ®ve times larger than the ®ltering frequency) through an analog-to-digital converter (Digidata 1200, Axon Instruments), and stored on hard disk for later analysis. The baseline of current at the holding potential was continuously recorded with a pen recorder. Programmed sequences of voltage pulses (by the software pClamp6, Axon Instruments) were applied to the preparation from the computer using a digital-to-analog converter (Digidata 1200). Voltage steps were applied after an initial stabilization period of about 15 min. All experiments were performed at 22 18C. Results are expressed as means S:D:, and n represents the number of patches unless otherwise stated. Analysis of Single-Channel Currents pClamp6 and TAC 3.0 (Bruxton Corporation, Seattle, WA) softwares were used for single-channel current analysis. A Gaussian ®ltration was applied if necessary. Capacitive and leakage currents were eliminated by subtraction of an averaged null trace from a
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trace with channel openings. Each corrected trace was carefully checked by eye and used for further analysis. Openings and closings of the channel were detected using the half-amplitude threshold analysis (Colquhoun and Sigworth, 1995). When the effective cutoff frequency was 2 and 5 kHz, the rise times of the signal (Tr) were approximately 180 and 70 ms, respectively. Openings or closings having a duration longer than Tr were selected for dwell histogram analysis. Chemicals The insecticidally active type I pyrethroid ()-transisomer of tetramethrin (97.8% purity) was provided by Sumitomo Chemical Co. (Takarazuka, Japan). The type II pyrethroid deltamethrin was provided by Roussel UCLAF (Marseille, France). Unlike our previous whole-cell study (Song et al., 1996), deltamethrin instead of fenvalerate was used. Deltamethrin-modi®ed single sodium channel properties are better established than fenvalerate-modi®ed channels in our previous studies (Chinn and Narahashi, 1986). Also, we con®rmed that deltamethrin had similar actions to fenvalerate on both whole-cell TTX-S and TTX-R sodium currents in respect to interactions of two types of pyrethroids (unpublished). Stock solutions of tetramethrin and deltamethrin were made in dimethylsulfoxide (DMSO) at a concentration of 10 mM. Final concentrations of pyrethroids and DMSO were 1±10 mM and 0.1% v/v, respectively. All control experiments were performed in the presence of DMSO (0.1% v/v) and compared with drug effect. All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). RESULTS Normal Single Sodium Channel Openings During Depolarization Normal single sodium channel currents were recorded using the membrane patches with insideout con®guration. Fig. 1A shows representative traces recorded at the ®nal cut-off ®ltration of 5 kHz. The membrane was depolarized to 60 mV from a holding potential of 100 mV for a duration of 45 ms. Very brief openings were observed in response to depolarizations. In the presence of 200 nM TTX in the pipette, these channel openings were not observed (n 4). In order to mimic whole-cell sodium currents, consecutive single-channel traces were summed and
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Fig. 1. Single sodium channel currents recorded from cultured rat hippocampal neurons. (A) Single-channel currents were evoked by a test pulse to 60 mV from a holding potential of 100 mV. The duration of test pulses was 45 ms, and the interpulse interval was 5 s. The final cut-off frequency of filtering was 5 kHz. In this and subsequent figures, downward and upward deflections represent inward and outward currents, respectively. (B) Averaged currents of normal channels at 60 mV (a) and 50 mV (b). Fifty consecutive single-channel traces recorded were averaged. (C) The open time histogram ( 60 mV) for normal sodium channels.
divided by the number of summed traces to calculate averaged currents. Fig. 1B represents the averaged control currents at 60 mV (a) and 50 mV (b). At 60 mV, the averaged current did not show apparent rising and decay phases, but at 50 mV their phases were obvious. The channel opening pattern and TTX sensitivity indicated that this single-channel activity was derived from TTX-S sodium channels. The open time histogram for normal sodium channels is shown in Fig. 1C. Events having longer durations than Tr (i.e. 0.07 ms at 5 kHz) were selected. All opening events were <1 ms. The time constant calculated by ®tting with a single exponential function was
0.089 ms, although the reliability of their values were somewhat limited because of the limitation of ®ltering. Tetramethrin-Modified Single-Channel Currents During Depolarization Tetramethrin was included in the internal solution at a concentration of 10 mM. The patch membranes were depolarized for 100 ms to 60 mV from a holding potential of 120 mV. As shown in Fig. 2A, two types of openings were observed: one was a very brief opening which may have represented the activity of normal channels, the other was a much longer opening
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Fig. 2. Single sodium channel currents during 10 mM tetramethrin application. (A) Single-channel currents were evoked by a test pulse to 60 mV from a holding potential of 100 mV during 10 mM tetramethrin application. The duration of test pulses was 100 ms. The final cut-off frequency of filtering was 2 kHz. (B) The burst length histogram ( 60 mV) for channel openings during tetramethrin application. Curves superimposed on the histograms are double exponential fits to the data points.
which may have represented the activity of tetramethrin-modi®ed channels. According to previous whole-cell experiments with rat dorsal root ganglion neurons (Tatebayashi and Narahashi, 1994) and rat cerebellar Purkinje neurons (Song and Narahashi, 1996a), and single-channel experiments with rat dorsal root ganglion neurons (Song and Narahashi, 1996b), 10 mM tetramethrin modi®es only a fraction of sodium channels. The situation was similar in the present experiment with rat hippocampal neurons. To assemble appropriate burst length histograms from tetramethrin-modi®ed single-channel traces was dif®cult. All closed time histograms should be compiled at ®rst from a patch containing only one channel in order to determine the upper limit of brief
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close duration in a burst (Colquhoun and Sigworth, 1995). However, the patch membrane having only one channel rarely showed long openings after tetramethrin application and the collection of tetramethrin-modi®ed channel opening data was dif®cult in the present study. This could be due to the relatively low sensitivity of TTX-S sodium channels to 10 mM tetramethrin (Song and Narahashi, 1996a; Tatebayashi and Narahashi, 1994). Therefore, most patches having a tetramethrin-modi®ed channel contained at least another unmodi®ed channel, causing dif®culties in compiling reliable closed time histograms. We de®ned here that a closure or de¯ection to baseline shorter than 1 ms which is located between two opening events should be regarded as an intraburst closure or transition to subconductance levels. The burst length histogram shown in Fig. 2B was analyzed by using this criterion. The histogram had two peaks which were well ®tted by two exponential functions. The time constants for two components were 0.20 and 7.69 ms. The fast component may correspond to unmodi®ed channel openings, and the slow component may correspond to tetramethrin-modi®ed channel openings. Although we observed the two patterns of openings in tetramethrin-treated membrane patches during depolarization, the objective distinction between unmodi®ed and modi®ed channel openings is not easy. There are atleast two possible interpretations of these two patterns of openings. First, brief and longer openings are deemed to represent unmodi®ed and tetramethrinmodi®ed channel openings, respectively (Song and Narahashi, 1996b). Alternatively, both opening patterns are due to tetramethrin-modi®ed channels but different modes of openings. Since the time constant of fast component consisting of brief openings was similar to that of normal channel openings, we assumed the ®rst interpretation that brief and longer openings represented unmodi®ed and tetramethrin-modi®ed channel openings, respectively. Single-Channel Currents upon Repolarization During Tetramethrin Application In order to record tetramethrin-modi®ed singlechannel currents upon repolarization effectively, various protocols with different voltage levels and lengths of depolarizing pulses were tested. Among them, the protocol shown in Fig. 3A could effectively evoke single-channel currents upon repolarization; membrane patches were depolarized to 30 mV from a holding potential of 100 mV for 5 ms, and subsequently repolarized to 100 mV. In response to this
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(but, see the bottom trace of Fig. 2A). Our data show that the percentage of traces having openings upon repolarization following depolarizations to 60 mV for 100 ms was <2% of the total number of traces. This phenomenon was compatible with previous whole-cell experiments showing that longer depolarizations decreased the amplitude of tail currents (Tatebayashi and Narahashi, 1994). Thus, it can be concluded that tetramethrin-modi®ed channels rarely opened during repolarization after long depolarizations. Single-Channel Currents Recorded from Neurons with the Pretreatment of Type II Pyrethroid Deltamethrin
Fig. 3. Tetramethrin induces single-channel current openings upon repolarization. (A) Membrane patches were depolarized to 30 mV for 5 ms, and subsequently repolarized to 100 mV during 10 mM tetramethrin application. The final cut-off frequency of filtering was 2 kHz. (B) The burst length histogram during repolarization during tetramethrin application. The histogram was derived from traces upon repolarization to 100 mV from 30 mV.
protocol, most openings upon repolarization were continuations of openings during depolarizaion. Channel openings upon repolarization were observed for a period of tens of milliseconds. The burst length histogram upon repolarization showed one peak which was ®tted by a single exponential function with a time constant of 8.26 ms (Fig. 3B). On the contrary, prolonged depolarizations with durations of 100 ms or more could rarely evoke tetramethrin-modi®ed channel openings during repolarization. For instance, openings upon repolarization to 100 mV preceded by a 100 ms-depolarizations to 60 mV were very rare
Since actions of type II pyrethroids including deltamethrin on sodium channels were irreversible (Chinn and Narahashi, 1986; Song et al., 1996), the modi®cation of sodium channels by deltamethrin in the present study was done by the pretreatment; hippocampal neurons were treated with 10 mM deltamethrin in the incubator for 5 20 min and used for experiments. Fig. 4A shows representative traces during depolarization after deltamethrin treatment. Two patterns of openings could be seen: one was very brief openings which are similar to normal channel openings (the ®rst and second traces in Fig. 4A), while the other type was markedly prolonged openings (the third and ®fth traces in Fig. 4A). This observation was common with singlechannel currents during tetramethrin application although the open durations of deltamethrin-modi®ed currents were much longer than those of tetramethrinmodi®ed currents. Fig. 4B illustrates typical traces showing extremely prolonged openings during depolarization and subsequent repolarization. Channels opened during depolarization remained open for hundreds of milliseconds regardless of the length of depolarization and the subsequent repolarized level. This characteristic prolonged opening was regarded as being derived from deltamethrin-modi®ed channels and is compatible with the previous study using neuroblastoma cells (Chinn and Narahashi, 1986). The observation that most openings of deltamethrin-modi®ed channels during repolarization were continuations of openings during depolarization regardless of various voltage protocols is in good agreement with the previous whole-cell data showing that the tail current of deltamethrin-modi®ed channels did not exhibit an obvious rising phase which is characteristic of tetramethrin-modi®ed channels (Tabarean and Narahashi, 1998; Tatebayashi and Narahashi, 1994). Thus, the opening behavior of deltamethrin-modi®ed channels
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Fig. 4. Single sodium currents after the treatment with 10 mM deltamethrin. Representative traces after deltamethrin treatment in response to different protocols. (A) Membrane patches were depolarized to 60 mV for 80 ms, and subsequently repolarized to 100 mV. (B) Membrane patches were depolarized to 0 mV for 30 ms, and subsequently repolarized to 100 mV. The final cut-off frequencies of filtering were 1 and 2 kHz in A and B, respectively. (C) The open time histogram ( 60 mV) for events having shorter durations than 5 ms after deltamethrin treatment.
were different from that of tetramethrin-modi®ed channels which rarely opened upon repolarization after prolonged depolarizations. Since these prolonged openings were often terminated by the end of depolarizing pulse or by the end of recording protocol, it was dif®cult to compile accurate open time histograms during depolarization or repolarization. Instead, in order to evaluate openings of unmodi®ed channels, openings with shorter than 5 ms in response to depolarizations to 60 mV after deltamethrin treatment were complied as the open time histogram (Fig. 4C). All opening events were shorter than 2 ms and the time constant of histogram was 0.27 ms, which is similar to that of the fast component
during tetramethrin application (Fig. 2B). Thus, it was concluded that the openings with shorter durations than 2 ms were derived from unmodi®ed channels after the treatment with deltamethrin. Definition of Tetramethrin- and DeltamethrinModified Channel Openings at the Single-Channel Level Based on the present single-channel data, we de®ned the criteria for distinction between unmodi®ed and modi®ed channels. Openings longer than 1 ms (longer than normal channel openings, see Fig. 1C) during depolarization are de®ned as being due to
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tetramethrin-modi®ed channels. Similarly, in the presence of deltamethrin, channel openings longer than 2 ms (see Fig. 4C) during depolarization are de®ned as being due to deltamethrin-modi®ed channels. With long depolarizations, tetramethrin-modi®ed channels opened for tens of milliseconds during depolarization but rarely open upon repolarization. By contrast, deltamethrin-modi®ed channels opened for hundreds of milliseconds during depolarization and during subsequent repolarization. Comparison of Open Frequencies Between Tetramethrin- and Deltamethrin-Modified Channels Another important difference between tetramethrinand deltamethrin-modi®ed channels were found with
the open frequency. In order to compare the open frequency between tetramethrin- and deltamethrinmodi®ed channels, the percentages of traces having modi®ed channel openings were estimated according to the de®nitions above. We selected the patches having no overlapping modi®ed channels. The open frequency at 10 mM was 17:96 4:97% (n 5) and 3:40 2:46% (n 5) for tetramethrin- and deltamethrin-modi®ed channels, respectively. Chinn and Narahashi (1986) reported that deltamethrin-modi®ed sodium channels of neuroblastoma cells showed low open frequency. Thus, the difference in open frequencies between tetramethrin- and deltamethrinmodi®ed channels were large (approximately ®ve times), suggesting that not only openings but also closed periods of deltamethrin-modi®ed channels were prolonged.
Fig. 5. Interactions of tetramethrin and deltamethrin on sodium channels. Representative single-channel traces after the treatment with deltamethrin at 10 mM (A), and during the subsequent application of tetramethrin at 10 mM (B). Single-channel currents were obtained by applying the protocols indicated in the figure. The final cut-off frequency of filtering was 1 kHz. See text for further explanation.
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Interaction of Tetramethrin and Deltamethrin at the Single-Channel Level Interactions of tetramethrin and deltamethrin were investigated at the single sodium channel level. Single sodium channel currents during the treatment with deltamethrin at 10 mM, and after washout of deltamethrin, those during subsequent application of tetramethrin at 10 mM were recorded and compared. Since the actions of deltamethrin on sodium channels are irreversible after washout (Chinn and Narahashi, 1986; Song et al., 1996), channel openings during the subsequent exposure to tetramethrin represented the results of interactions between tetramethirn and deltamethrin. Sodium channels opened for hundreds of milliseconds after the treatment with deltamethrin (Fig. 5A) in response to prolonged depolarizations. However, during additional tetramethrin application, sodium channels opened for tens of milliseconds in response to depolarizations to 60 mV for 100 ms (Fig. 5B), which were very similar to openings in the presence of tetramethrin alone. The time constant of slow component of openings during additional tetramethrin application was 7.51 ms (the histogram not shown), which was very close to the time constant of slow component (7.69 ms) in Fig. 2B. The extremely prolonged openings which can be regarded as deltamethrin-modi®ed channel openings were not observed during the tetramethrin application. Thus, it can be concluded that during tetramethrin application, the extremely prolonged openings caused by deltamethrin disappeared and shorter openings which were similar to tetramethrin-modi®ed channel openings occurred on depolarizations (n 3). It was suggested that the tetramethrin molecule displaced the deltamethrin molecule from the sodium channel binding site. DISCUSSION In order to investigate interactions of tetramethrin (type I) and deltamethrin (type II) at the single sodium channel, tetramethrin- and deltamethrin-modi®ed sodium channel openings were compared. Tetramethrin-modi®ed channels opened at most for tens of milliseconds during depolarization (Fig. 2) and upon repolarization (Fig. 3). Although deltamethrin-modi®ed channels opened only at a very low frequency, if they opened, they were kept open for hundreds of milliseconds during depolarization and upon subsequent repolarization (Fig. 4A and B). This difference in
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behavior between tetramethrin- and deltamethrin-modi®ed channels is useful to distinguish between two types of channels during simultaneous exposure to these pyrethroids. The opening frequency of channels is a critical factor to estimate the percentage of modi®ed channels at various concentrations of pyrethroids. The estimate of opening frequency in previous whole-cell experiments was performed with an assumption that opening frequencies for normal and modi®ed channels are the same (Song and Narahashi, 1996a,b; Tabarean and Narahashi, 1998; Tatebayashi and Narahashi, 1994). However, the large difference in the opening frequencies between tetramethrin- and deltamethrin-modi®ed channels at the single-channel level in the present study suggested that the estimate of the percentage of deltamethrin-modi®ed channels in previous whole-cell experiments could be underestimated. For instance, the percentages of tetramethrin- and deltamethrinmodi®ed TTX-S sodium channels in rat dorsal root ganglion were 3.5 and 4.5%, respectively, at 1 mM of each pyrethroid (Tatebayashi and Narahashi, 1994; Tabarean and Narahashi, 1998). However, the actual percentages of modi®ed channels could be 3.5 and 22.5% (i.e. 4.5% as multiplied by 5, the difference in opening frequency) with an assumption that the opening frequencies for normal and tetramethrin-modi®ed channels are the same. Song et al. (1996) reported interactions of type I (tetramethrin) and type II (fenvalerate) pyrethroids using whole-cell patch clamp technique. When fenvalerate application was followed by tetramethrin application, the markedly prolonged tail currents induced by fenvalerate disappeared and the less prolonged tail currents appeared which were very similar to tail currents observed during treatment with tetramethrin alone. The most straightforward interpretation is, as Song et al. (1996) suggested, that the type I pyrethroid molecule displaces the type II pyrethroid molecule from the binding site in the sodium channel protein, or that type I and II pyrethroids bind to separate sodium channel sites which interact allosterically with each other. Our single-channel data showed that characteristic deltamethrin-modi®ed channel openings disappeared during tetramethrin application and that the opening patterns during a combined application of tetramethrin and deltamethrin seem to be similar to those during application of tetramethrin alone. It should be noted that our single-channel data showed possible interactions of the two types of pyrethroids during depolarization, while the previous study (Song et al., 1996) suggested such interactions based only on
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whole-cell tail currents upon repolarization. Thus, our single-channel data are in good agreement with the previous whole-cell data. Interestingly, we could not observe tetramethrinand deltamethrin-modi®ed channel openings at the same time in a single membrane patch, although it was possible that deltamethrin-modi®ed channel openings occurred even after tetramethrin application. Either type I or type II pyrethroid modi®ed only a fraction of TTX-S sodium channels at a concentration of 1±10 mM, suggesting that some normal unmodi®ed channels may have been contained in the patch. Although pyrethroids modify the sodium channel at its resting state, opening increases the af®nity for pyrethroids (see Narahashi, 1996). The deltamethrin-modi®ed channels are kept open much longer than the normal sodium channels, providing the tetramethrin molecules with more chances to cause open channel modi®cation. Therefore, it is suggested that the type I pyrethroid tetramethrin molecules might get access more easily to type II pyrethroid deltamethrinmodi®ed channels than to the normal unmodi®ed sodium channels, resulting in displacement of deltamethrin-modi®ed channel openings by tetramethrinmodi®ed channels openings. Molecular biological studies reported putative binding sites of pyrethroids on sodium channels. For instance, Pittendrigh et al. (1997) demonstrated that several point mutations in Drosophila sodium channel rendered insects resistant to pyrethroids. These point mutations were located in the Segment 6 (S6) of Domain III (DIII), the intracellular loops between the S4 and S5 of DI and DIII, and the extracellular loop between S5 and S6 of DIII. Lee et al. (1999a) also reported the altered properties of insect neuron sodium channels that are resistant to pyrethroids; the single mutation of valine to methionine in the S6 (DI) transmembrane segment rendered sodium channels less sensitive to pyrethroids. However, none of these studies can account for the mechanism of action of pyrethroids on sodium channels or the de®nite binding sites of pyrethroids. The present single-channel and previous whole-cell studies (Song et al., 1996) showed possible interactions of two types of pyrethroids at the same or adjacent binding sites of sodium channels. One possible interpretation about this phenomenon is the following: the deltamethrin molecule bound to the site on sodium channels is kicked out by the tetramethrin molecule due to its lower binding af®nity to the site, but remained in the membrane phase because of its high lipophilicity. After washout of tetramethrin, the deltamethrin molecule located still in the membrane can
get access to the binding sites again, resulting in extremely prolonged openings. Interactions of pyrethroids demonstrated by the electrophysiological studies may provide insights into the molecular mechanisms of pyrethroid actions. ACKNOWLEDGEMENTS This work was supported by a grant from the National Institutes of Health NS14143. We thank Nayla Hasan for technical assistance and Julia Irizarry for secretarial assistance.
REFERENCES Bloomquist JR. Ion channels as targets for insecticides. Annu Rev Entomol 1996;41:163±90. Chinn K, Narahashi T. Stabilization of sodium channel states by deltamethrin in mouse neuroblastoma cells. J Physiol (London) 1986;380:191±207. Colquhoun D, Sigworth FJ. Fitting and statistical analysis of single-channel records. In: Sakmann B, Neher E, editors. Single-channel recording. New York: Plenum Press, 1995. p. 483±587. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. PfluÈg Arch 1981;391:85±100. Holloway SF, Narahashi T, Salgado VL, Wu CH. Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells. PfluÈg Arch 1989;414: 613±21. Lee D, Park Y, Brown TM, Adams ME. Altered properties of neuronal sodium channels associated with genetic resistance to pyrethroids. Mol Pharmacol 1999;55:584±93. Lee SH, Smith TJ, Knipple DC, Soderlund DM. Mutations in the house fly Vsscl sodium channel gene associated with super-kdr resistance abolish the pyrethroid sensitivity of Vsscl/tipE sodium channels expressed in Xenopus oocytes. Insect Biochem Mol Biol 1999;29:185±94. Motomura H, Narahashi T. Temperature dependence of pyrethroid modificaiton of single sodium channels in rat hippocampal neurons. J Membr Biol 2000;177:23±9. Motomura H, Fujikawa S, Tashiro N, Ito Y, Ogata N. Singlechannel analysis of two types of Na currents in rat dorsal root ganglia. PfluÈg Arch 1995;431:221±9. Narahashi T. Nerve membrane ionic channels as the primary target of pyrethroids. NeuroToxicology 1985;6:3±22. Narahashi T. Nerve membrane Na channels as targets of insecticides. Trends Pharmacol Sci 1992;13:236±41. Narahashi T. Neuronal ion channels as the target sites of insecticides. Pharmacol Toxicol 1996;78:1±14. Park Y, Taylor MF, Feyereisen R. A valine421 to methionine mutation in IS6 of the hscp voltage-gated sodium channel associated with pyrethroid resistance in Heliothis virescens F. Biochem Biophys Res Commun 1997;239:688±91.
H. Motomura, T. Narahashi / NeuroToxicology 22 (2001) 329±339 Park Y, Lee D, Taylor MFJ, Holloway JW, Ottea JA, Adams ME, Feyereisen R. A mutation Leu1029 to His in Heliothis virescens F-hscp sodium channel gene associated with a nerve-insensitivity mechanism of resistance to pyrethroid insecticides. Pestic Biochem Physiol 2000;66:1±8. Pittendrigh B, Reenan R, ffrench-Constant RH, Ganetzky B. Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides. Mol Gen Genet 1997;256:602±10. Ruigt GSF. Pyrethroids. In: Kerkut GA, Gilbert LI, editors. Comprehensive insect physiology, biochemistry and pharmacology, Vol. 12. Oxford: Pergamon Press, 1984 (Chapter 7) p. 183±263. Salgado VL, Narahashi T. Immobilization of sodium channel gating charge in crayfish giant axons by the insecticide fenvalerate. Mol Pharmacol 1993;43:626±34. Schuler TH, Martinez-Torres D, Tompson AJ, Denholm I, Devonshire AL, Duce IR, Williamson MS. Toxicological, electrophysiological and molecular characterization of knockdown resistance to pyrethroid insecticides in the diamondback moth. Pestic Biochem Physiol 1998;59:169±82. Soderlund DM, Bloomquist JR. Neurotoxic actions of pyrethroid insecticides. Annu Rev Entomol 1989;34:77±96. Song JH, Narahashi T. Modulation of sodium channels of rat cerebellar Purkinje neurons by the pyrethroid tetramethrin. J Pharmacol Exp Ther 1996;277:445±53.
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Song JH, Narahashi T. Differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channels. Brain Res 1996;712:258±64. Song JH, Nagata K, Tatebayashi H, Narahashi T. Interactions of tetramethrin, fenvalerate and DDT at the sodium channel in rat dorsal root ganglion neurons. Brain Res 1996;708:29±37. Tabarean IV, Narahashi T. Potent modulation of tetrodotoxinsensitive and tetrodotoxin-resistant sodium channels by the type II pyrethroid deltamethrin. J Pharmacol Exp Ther 1998;284:958±65. Tatebayashi H, Narahashi T. Differential mechanism of action of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels. J Pharmacol Exp Ther 1994;270:595±603. Trainer VL, McPhee JC, Boutelet-Bochan H, Baker C, Scheuer T, Babin D, Demoute JP, Guedin D, Catterall WA. High affinity binding of pyrethroids to the alpha subunit of brain sodium channels. Mol Pharmacol 1997;51:651±7. Verschoyle RD, Aldridge WN. Structure-activity relationships of some pyrethroids in rats. Arch Toxicol 1980;45:325±9. Vijverberg HPM, van den Bercken J. Neurotoxicological effects and the mode of action of pyrethroid insecticides. Crit Rev Toxicol 1990;21:105±26. Yamamoto D, Quandt FN, Narahashi T. Modification of single sodium channels by the insecticide tetramethrin. Brain Res 1983;274:344±9.
Interaction of Tetramethrin and Deltamethrin at the Single Sodium Channel in Rat Hippocampal Neurons Haruhiko Motomura, Toshio Narahashi* Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611-3008, USA Received 2 October 2000; accepted 6 February 2001
Abstract Type I and type II pyrethroids are known to modulate the sodium channel to cause persistent openings during depolarization and upon repolarization. Although there are some similarities between the two types of pyrethroids in their actions on sodium channels, the pattern of modi®cation of sodium currents is different between the two types of pyrethroids. In the present study, interactions of the type I pyrethroid tetramethrin and the type II pyrethroid deltamethrin at rat hippocampal neuron sodium channels were investigated using the inside-out single-channel patch clamp technique. Deltamethrin-modi®ed sodium channels opened much longer than tetramethrin-modi®ed sodium channels. When 10 mM tetramethrin was applied to membrane patches that had been exposed to 10 mM deltamethrin, deltamethrin-modi®ed prolonged single sodium currents disappeared and were replaced by shorter openings which were characteristic of tetramethrin-modi®ed channel openings. These single-channel data are compatible with previous whole-cell competition study between type I and type II pyrethroids. These results are interpreted as being due to the displacement of the type II pyrethroid molecule by the type I pyrethroid molecule from the same binding site or to the allosteric interaction of the two pyrethroid molecules at separate sodium channel sites. # 2001 Elsevier Science Inc. All rights reserved.
Keywords: Pyrethroid; Tetramethrin; Deltamethrin; Sodium channel; Hippocampal neuron; Singlechannel
INTRODUCTION Pyrethroids are synthetic derivatives of pyrethrins, which are toxic components contained in the ¯owers of Chrysanthemum cinerariaefolium. The pyrethroids are used widely as near-ideal insecticides due to their high insecticidal potencies, low mammalian toxicities and biodegradability. Sodium channels are known to be one of the most important target sites of pyrethroids (Narahashi, 1985, 1992, 1996; Ruigt, 1984; Soderlund and Bloomquist, 1989; Vijverberg and van den Bercken, 1990; Bloomquist, 1996). * Corresponding author. Tel.: 1-312-503-8284; fax: 1-312-503-1700. E-mail address: [email protected] (T. Narahashi).
Pyrethroids are divided into two groups by their chemical structures: type I pyrethroids are devoid of a cyano moiety at the a-position, and type II pyrethroids have an a-cyano moiety. Type I pyrethroids cause hyperexcitation, ataxia, convulsions and eventual paralysis in mammals, whereas type II pyrethroids produce hypersensitivity, choreoathetosis, tremors, and paralysis (Narahashi, 1985; Verschoyle and Aldridge, 1980; Vijverberg and van den Bercken, 1990). Pyrethroids seem to stabilize gating particles of the sodium channel (Salgado and Narahashi, 1993), resulting in slowing of the movements of both the activation and inactivation gates (Chinn and Narahashi, 1986), and shift the voltage dependence of the gates in the hyperpolarizing direction (Tabarean and Narahashi, 1998; Tatebayashi and Narahashi, 1994). These changes cause
0161-813X/01/$ ± see front matter # 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 6 1 - 8 1 3 X ( 0 1 ) 0 0 0 2 3 - 7
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a prolonged ¯ow of sodium current into the cell, leading to a sustained membrane depolarization. Although the basic actions of pyrethroids on sodium channels are similar between the two types of pyrethroids, the degree of modi®cation of sodium currents is different; single sodium channel currents are prolonged to a greater extent with type II than type I pyrethroids (Chinn and Narahashi, 1986; Holloway et al., 1989; Yamamoto et al., 1983; Motomura and Narahashi, 2000). Biochemical and molecular biological studies demonstrated that pyrethroids seem to have a speci®c binding site (Trainer et al., 1997) and that point mutations rendered the sodium channels resistant to pyrethroids (Pittendrigh et al., 1997; Park et al., 1997, 2000; Schuler et al., 1998; Lee et al., 1999a,b), suggesting the putative binding sites of pyrethroids in the sodium channels. However, no report has been published concerning the relationship between binding sites of type I and II pyrethroids using molecular biological or binding experiments. We previously reported interactions of type I and II pyrethroids at sodium channels using whole-cell patch clamp techniques (Song et al., 1996). In both tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels of rat dorsal root ganglion neurons, tetramethrin (type I) and fenvalerate (type II) prolonged the sodium channel tail current that was generated upon repolarization following a depolarizing pulse (Song et al., 1996). Fenvalerate-induced tail current decayed very slowly and was irreversible after washing with drug-free solution, whereas tetramethrininduced tail current decayed faster and was reversible after washout. When tetramethrin was applied to the fenvalerate-treated cell, the prolonged tail current was replaced by a much shorter tail current characteristic of the tetramethrin-treated cell. Furthermore, washing out of both tetramethrin and fenvalerate resulted in an appearance of the prolonged tail current characteristic of fenvalerate-treated cell. It was suggested that the type I pyrethroid molecule displaces the type II pyrethroid molecule from the binding site in the sodium channel protein, or that type I and II pyrethroids bind to separate sodium channel sites which interact allosterically with each other. However, since the whole-cell tail current induced by pyrethroids results form changes in a variety of single-channel parameters, the aforementioned wholecell data do not allow us to conclude all-or-none changes from fenvalerate-type to tetramethrin-type modi®cation of channel kinetics. It is imperative to obtain data at the single-channel level. We now address this issue in the present study using single-channel
techniques, which are expected to provide more straightforward evidence. We compared channel gating behaviors of tetramethrin (type I)- and deltamethrin (type II)-modi®ed single-channel currents, and examined interactions of the two pyrethroids at the individual channel level. MATERIALS AND METHODS Dissociation and Culture of Neurons Although our previous study (Song et al., 1996) was performed using rat dorsal root ganglion neurons which contain both TTX-S and TTX-R sodium channels, in the present study, hippocampal neurons were chosen for three reasons. (1) The single-channel current amplitude of TTX-S channels were larger than that of TTX-R channels (Song and Narahashi, 1996b). Therefore, the measurement of channel openings were relatively easy. (2) Since the openings of unmodi®ed TTX-S channels were very brief, the distinction between the unmodi®ed and pyrethroid-modi®ed channel openings was easier that that in TTX-R channels. (3) Pyrethroid-modi®ed channels sometimes opened at subconductance levels (Chinn and Narahashi, 1986). The current amplitude of subconductance level openings of pyrethroid-modi®ed TTX-S channels could be very similar to that of full-level openings of TTX-R channels if dorsal root ganglion cells were used. This might cause dif®culties in analysis. Hippocampal neurons were prepared from 17-dayold embryos of a Sprague±Dawley pregnant rat under methoxy¯urane anaesthesia. After an adequate level of anesthesia was reached as indicated by lack of responses to pinching the hind legs, a transverse incision was made in the lower abdomen of the pregnant rat and the fetuses were withdrawn from the abdomen. The heads of the fetuses were immediately cut-off, and the hippocampi were dissected and collected in a calciumand magnesium-free phosphate-buffered saline solution (PBS). The mother rat was euthanized immediately thereafter by decapitation. Neurons were dissociated by repeated passage through a ¯ame-narrowed Pasteur pipette and diluted with Neurobasal Medium supplemented with B-27 and 2 mM L-glutamic acid (GIBCO/Life Technologies, Grand Island, NY). The ®nal suspension was plated onto 12 mm poly-L-lysine-coated coverslips at a density of 200,000 cells/well. Cells were maintained in a humidi®ed atmosphere of 90% air and 10% CO2 at 378C, and were used for experiments within 3 weeks of
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culture. Neurons having triangle cell bodies or pyramidal morphology were selected for experiments. Electrical Recording Single-channel currents were recorded using the inside-out con®guration of the patch clamp technique (Hamill et al., 1981). Patch pipettes fabricated from borosilicate glass capillary tubes (1.5 mm inner diameter) had a resistance of 5±10 MO when ®lled with pipette solution. The pipette solution contained (mM): NaCl, 250; CsCl, 5; CaCl2, 1.8; MgCl2, 1; and 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES) 5. It should be noted that the pipette sodium concentration was doubled to 250 mM to obtain larger single sodium channel currents (Motomura et al., 1995). The pH was adjusted to 7.4 with NaOH. Normal Krebs solution contained (mM): NaCl, 120; KCl, 5; CaCl2, 1.8; MgCl2, 1; HEPES, 5; and glucose, 25. The pH was adjusted to 7.4 with NaOH. After making a gigaohm seal in the Krebs solution, it was replaced by the test bath solution which contained (mM): NaF, 1.4; CsF 145; sucrose, 200; HEPES, 5; and EGTA, 5. The pH was adjusted to 7.2 with CsOH. After the solution exchange, the membrane patch was excised. Single-channel currents passing through the pipette were recorded by a patch clamp ampli®er (Axopatch 200A, Axon Instruments, Foster City, CA). The currents were ®ltered at 2±10 kHz with a four-pole Bessel ®lter, digitalized at a rate of 10±50 kHz (at least ®ve times larger than the ®ltering frequency) through an analog-to-digital converter (Digidata 1200, Axon Instruments), and stored on hard disk for later analysis. The baseline of current at the holding potential was continuously recorded with a pen recorder. Programmed sequences of voltage pulses (by the software pClamp6, Axon Instruments) were applied to the preparation from the computer using a digital-to-analog converter (Digidata 1200). Voltage steps were applied after an initial stabilization period of about 15 min. All experiments were performed at 22 18C. Results are expressed as means S:D:, and n represents the number of patches unless otherwise stated. Analysis of Single-Channel Currents pClamp6 and TAC 3.0 (Bruxton Corporation, Seattle, WA) softwares were used for single-channel current analysis. A Gaussian ®ltration was applied if necessary. Capacitive and leakage currents were eliminated by subtraction of an averaged null trace from a
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trace with channel openings. Each corrected trace was carefully checked by eye and used for further analysis. Openings and closings of the channel were detected using the half-amplitude threshold analysis (Colquhoun and Sigworth, 1995). When the effective cutoff frequency was 2 and 5 kHz, the rise times of the signal (Tr) were approximately 180 and 70 ms, respectively. Openings or closings having a duration longer than Tr were selected for dwell histogram analysis. Chemicals The insecticidally active type I pyrethroid ()-transisomer of tetramethrin (97.8% purity) was provided by Sumitomo Chemical Co. (Takarazuka, Japan). The type II pyrethroid deltamethrin was provided by Roussel UCLAF (Marseille, France). Unlike our previous whole-cell study (Song et al., 1996), deltamethrin instead of fenvalerate was used. Deltamethrin-modi®ed single sodium channel properties are better established than fenvalerate-modi®ed channels in our previous studies (Chinn and Narahashi, 1986). Also, we con®rmed that deltamethrin had similar actions to fenvalerate on both whole-cell TTX-S and TTX-R sodium currents in respect to interactions of two types of pyrethroids (unpublished). Stock solutions of tetramethrin and deltamethrin were made in dimethylsulfoxide (DMSO) at a concentration of 10 mM. Final concentrations of pyrethroids and DMSO were 1±10 mM and 0.1% v/v, respectively. All control experiments were performed in the presence of DMSO (0.1% v/v) and compared with drug effect. All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). RESULTS Normal Single Sodium Channel Openings During Depolarization Normal single sodium channel currents were recorded using the membrane patches with insideout con®guration. Fig. 1A shows representative traces recorded at the ®nal cut-off ®ltration of 5 kHz. The membrane was depolarized to 60 mV from a holding potential of 100 mV for a duration of 45 ms. Very brief openings were observed in response to depolarizations. In the presence of 200 nM TTX in the pipette, these channel openings were not observed (n 4). In order to mimic whole-cell sodium currents, consecutive single-channel traces were summed and
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Fig. 1. Single sodium channel currents recorded from cultured rat hippocampal neurons. (A) Single-channel currents were evoked by a test pulse to 60 mV from a holding potential of 100 mV. The duration of test pulses was 45 ms, and the interpulse interval was 5 s. The final cut-off frequency of filtering was 5 kHz. In this and subsequent figures, downward and upward deflections represent inward and outward currents, respectively. (B) Averaged currents of normal channels at 60 mV (a) and 50 mV (b). Fifty consecutive single-channel traces recorded were averaged. (C) The open time histogram ( 60 mV) for normal sodium channels.
divided by the number of summed traces to calculate averaged currents. Fig. 1B represents the averaged control currents at 60 mV (a) and 50 mV (b). At 60 mV, the averaged current did not show apparent rising and decay phases, but at 50 mV their phases were obvious. The channel opening pattern and TTX sensitivity indicated that this single-channel activity was derived from TTX-S sodium channels. The open time histogram for normal sodium channels is shown in Fig. 1C. Events having longer durations than Tr (i.e. 0.07 ms at 5 kHz) were selected. All opening events were <1 ms. The time constant calculated by ®tting with a single exponential function was
0.089 ms, although the reliability of their values were somewhat limited because of the limitation of ®ltering. Tetramethrin-Modified Single-Channel Currents During Depolarization Tetramethrin was included in the internal solution at a concentration of 10 mM. The patch membranes were depolarized for 100 ms to 60 mV from a holding potential of 120 mV. As shown in Fig. 2A, two types of openings were observed: one was a very brief opening which may have represented the activity of normal channels, the other was a much longer opening
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Fig. 2. Single sodium channel currents during 10 mM tetramethrin application. (A) Single-channel currents were evoked by a test pulse to 60 mV from a holding potential of 100 mV during 10 mM tetramethrin application. The duration of test pulses was 100 ms. The final cut-off frequency of filtering was 2 kHz. (B) The burst length histogram ( 60 mV) for channel openings during tetramethrin application. Curves superimposed on the histograms are double exponential fits to the data points.
which may have represented the activity of tetramethrin-modi®ed channels. According to previous whole-cell experiments with rat dorsal root ganglion neurons (Tatebayashi and Narahashi, 1994) and rat cerebellar Purkinje neurons (Song and Narahashi, 1996a), and single-channel experiments with rat dorsal root ganglion neurons (Song and Narahashi, 1996b), 10 mM tetramethrin modi®es only a fraction of sodium channels. The situation was similar in the present experiment with rat hippocampal neurons. To assemble appropriate burst length histograms from tetramethrin-modi®ed single-channel traces was dif®cult. All closed time histograms should be compiled at ®rst from a patch containing only one channel in order to determine the upper limit of brief
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close duration in a burst (Colquhoun and Sigworth, 1995). However, the patch membrane having only one channel rarely showed long openings after tetramethrin application and the collection of tetramethrin-modi®ed channel opening data was dif®cult in the present study. This could be due to the relatively low sensitivity of TTX-S sodium channels to 10 mM tetramethrin (Song and Narahashi, 1996a; Tatebayashi and Narahashi, 1994). Therefore, most patches having a tetramethrin-modi®ed channel contained at least another unmodi®ed channel, causing dif®culties in compiling reliable closed time histograms. We de®ned here that a closure or de¯ection to baseline shorter than 1 ms which is located between two opening events should be regarded as an intraburst closure or transition to subconductance levels. The burst length histogram shown in Fig. 2B was analyzed by using this criterion. The histogram had two peaks which were well ®tted by two exponential functions. The time constants for two components were 0.20 and 7.69 ms. The fast component may correspond to unmodi®ed channel openings, and the slow component may correspond to tetramethrin-modi®ed channel openings. Although we observed the two patterns of openings in tetramethrin-treated membrane patches during depolarization, the objective distinction between unmodi®ed and modi®ed channel openings is not easy. There are atleast two possible interpretations of these two patterns of openings. First, brief and longer openings are deemed to represent unmodi®ed and tetramethrinmodi®ed channel openings, respectively (Song and Narahashi, 1996b). Alternatively, both opening patterns are due to tetramethrin-modi®ed channels but different modes of openings. Since the time constant of fast component consisting of brief openings was similar to that of normal channel openings, we assumed the ®rst interpretation that brief and longer openings represented unmodi®ed and tetramethrin-modi®ed channel openings, respectively. Single-Channel Currents upon Repolarization During Tetramethrin Application In order to record tetramethrin-modi®ed singlechannel currents upon repolarization effectively, various protocols with different voltage levels and lengths of depolarizing pulses were tested. Among them, the protocol shown in Fig. 3A could effectively evoke single-channel currents upon repolarization; membrane patches were depolarized to 30 mV from a holding potential of 100 mV for 5 ms, and subsequently repolarized to 100 mV. In response to this
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(but, see the bottom trace of Fig. 2A). Our data show that the percentage of traces having openings upon repolarization following depolarizations to 60 mV for 100 ms was <2% of the total number of traces. This phenomenon was compatible with previous whole-cell experiments showing that longer depolarizations decreased the amplitude of tail currents (Tatebayashi and Narahashi, 1994). Thus, it can be concluded that tetramethrin-modi®ed channels rarely opened during repolarization after long depolarizations. Single-Channel Currents Recorded from Neurons with the Pretreatment of Type II Pyrethroid Deltamethrin
Fig. 3. Tetramethrin induces single-channel current openings upon repolarization. (A) Membrane patches were depolarized to 30 mV for 5 ms, and subsequently repolarized to 100 mV during 10 mM tetramethrin application. The final cut-off frequency of filtering was 2 kHz. (B) The burst length histogram during repolarization during tetramethrin application. The histogram was derived from traces upon repolarization to 100 mV from 30 mV.
protocol, most openings upon repolarization were continuations of openings during depolarizaion. Channel openings upon repolarization were observed for a period of tens of milliseconds. The burst length histogram upon repolarization showed one peak which was ®tted by a single exponential function with a time constant of 8.26 ms (Fig. 3B). On the contrary, prolonged depolarizations with durations of 100 ms or more could rarely evoke tetramethrin-modi®ed channel openings during repolarization. For instance, openings upon repolarization to 100 mV preceded by a 100 ms-depolarizations to 60 mV were very rare
Since actions of type II pyrethroids including deltamethrin on sodium channels were irreversible (Chinn and Narahashi, 1986; Song et al., 1996), the modi®cation of sodium channels by deltamethrin in the present study was done by the pretreatment; hippocampal neurons were treated with 10 mM deltamethrin in the incubator for 5 20 min and used for experiments. Fig. 4A shows representative traces during depolarization after deltamethrin treatment. Two patterns of openings could be seen: one was very brief openings which are similar to normal channel openings (the ®rst and second traces in Fig. 4A), while the other type was markedly prolonged openings (the third and ®fth traces in Fig. 4A). This observation was common with singlechannel currents during tetramethrin application although the open durations of deltamethrin-modi®ed currents were much longer than those of tetramethrinmodi®ed currents. Fig. 4B illustrates typical traces showing extremely prolonged openings during depolarization and subsequent repolarization. Channels opened during depolarization remained open for hundreds of milliseconds regardless of the length of depolarization and the subsequent repolarized level. This characteristic prolonged opening was regarded as being derived from deltamethrin-modi®ed channels and is compatible with the previous study using neuroblastoma cells (Chinn and Narahashi, 1986). The observation that most openings of deltamethrin-modi®ed channels during repolarization were continuations of openings during depolarization regardless of various voltage protocols is in good agreement with the previous whole-cell data showing that the tail current of deltamethrin-modi®ed channels did not exhibit an obvious rising phase which is characteristic of tetramethrin-modi®ed channels (Tabarean and Narahashi, 1998; Tatebayashi and Narahashi, 1994). Thus, the opening behavior of deltamethrin-modi®ed channels
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Fig. 4. Single sodium currents after the treatment with 10 mM deltamethrin. Representative traces after deltamethrin treatment in response to different protocols. (A) Membrane patches were depolarized to 60 mV for 80 ms, and subsequently repolarized to 100 mV. (B) Membrane patches were depolarized to 0 mV for 30 ms, and subsequently repolarized to 100 mV. The final cut-off frequencies of filtering were 1 and 2 kHz in A and B, respectively. (C) The open time histogram ( 60 mV) for events having shorter durations than 5 ms after deltamethrin treatment.
were different from that of tetramethrin-modi®ed channels which rarely opened upon repolarization after prolonged depolarizations. Since these prolonged openings were often terminated by the end of depolarizing pulse or by the end of recording protocol, it was dif®cult to compile accurate open time histograms during depolarization or repolarization. Instead, in order to evaluate openings of unmodi®ed channels, openings with shorter than 5 ms in response to depolarizations to 60 mV after deltamethrin treatment were complied as the open time histogram (Fig. 4C). All opening events were shorter than 2 ms and the time constant of histogram was 0.27 ms, which is similar to that of the fast component
during tetramethrin application (Fig. 2B). Thus, it was concluded that the openings with shorter durations than 2 ms were derived from unmodi®ed channels after the treatment with deltamethrin. Definition of Tetramethrin- and DeltamethrinModified Channel Openings at the Single-Channel Level Based on the present single-channel data, we de®ned the criteria for distinction between unmodi®ed and modi®ed channels. Openings longer than 1 ms (longer than normal channel openings, see Fig. 1C) during depolarization are de®ned as being due to
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tetramethrin-modi®ed channels. Similarly, in the presence of deltamethrin, channel openings longer than 2 ms (see Fig. 4C) during depolarization are de®ned as being due to deltamethrin-modi®ed channels. With long depolarizations, tetramethrin-modi®ed channels opened for tens of milliseconds during depolarization but rarely open upon repolarization. By contrast, deltamethrin-modi®ed channels opened for hundreds of milliseconds during depolarization and during subsequent repolarization. Comparison of Open Frequencies Between Tetramethrin- and Deltamethrin-Modified Channels Another important difference between tetramethrinand deltamethrin-modi®ed channels were found with
the open frequency. In order to compare the open frequency between tetramethrin- and deltamethrinmodi®ed channels, the percentages of traces having modi®ed channel openings were estimated according to the de®nitions above. We selected the patches having no overlapping modi®ed channels. The open frequency at 10 mM was 17:96 4:97% (n 5) and 3:40 2:46% (n 5) for tetramethrin- and deltamethrin-modi®ed channels, respectively. Chinn and Narahashi (1986) reported that deltamethrin-modi®ed sodium channels of neuroblastoma cells showed low open frequency. Thus, the difference in open frequencies between tetramethrin- and deltamethrinmodi®ed channels were large (approximately ®ve times), suggesting that not only openings but also closed periods of deltamethrin-modi®ed channels were prolonged.
Fig. 5. Interactions of tetramethrin and deltamethrin on sodium channels. Representative single-channel traces after the treatment with deltamethrin at 10 mM (A), and during the subsequent application of tetramethrin at 10 mM (B). Single-channel currents were obtained by applying the protocols indicated in the figure. The final cut-off frequency of filtering was 1 kHz. See text for further explanation.
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Interaction of Tetramethrin and Deltamethrin at the Single-Channel Level Interactions of tetramethrin and deltamethrin were investigated at the single sodium channel level. Single sodium channel currents during the treatment with deltamethrin at 10 mM, and after washout of deltamethrin, those during subsequent application of tetramethrin at 10 mM were recorded and compared. Since the actions of deltamethrin on sodium channels are irreversible after washout (Chinn and Narahashi, 1986; Song et al., 1996), channel openings during the subsequent exposure to tetramethrin represented the results of interactions between tetramethirn and deltamethrin. Sodium channels opened for hundreds of milliseconds after the treatment with deltamethrin (Fig. 5A) in response to prolonged depolarizations. However, during additional tetramethrin application, sodium channels opened for tens of milliseconds in response to depolarizations to 60 mV for 100 ms (Fig. 5B), which were very similar to openings in the presence of tetramethrin alone. The time constant of slow component of openings during additional tetramethrin application was 7.51 ms (the histogram not shown), which was very close to the time constant of slow component (7.69 ms) in Fig. 2B. The extremely prolonged openings which can be regarded as deltamethrin-modi®ed channel openings were not observed during the tetramethrin application. Thus, it can be concluded that during tetramethrin application, the extremely prolonged openings caused by deltamethrin disappeared and shorter openings which were similar to tetramethrin-modi®ed channel openings occurred on depolarizations (n 3). It was suggested that the tetramethrin molecule displaced the deltamethrin molecule from the sodium channel binding site. DISCUSSION In order to investigate interactions of tetramethrin (type I) and deltamethrin (type II) at the single sodium channel, tetramethrin- and deltamethrin-modi®ed sodium channel openings were compared. Tetramethrin-modi®ed channels opened at most for tens of milliseconds during depolarization (Fig. 2) and upon repolarization (Fig. 3). Although deltamethrin-modi®ed channels opened only at a very low frequency, if they opened, they were kept open for hundreds of milliseconds during depolarization and upon subsequent repolarization (Fig. 4A and B). This difference in
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behavior between tetramethrin- and deltamethrin-modi®ed channels is useful to distinguish between two types of channels during simultaneous exposure to these pyrethroids. The opening frequency of channels is a critical factor to estimate the percentage of modi®ed channels at various concentrations of pyrethroids. The estimate of opening frequency in previous whole-cell experiments was performed with an assumption that opening frequencies for normal and modi®ed channels are the same (Song and Narahashi, 1996a,b; Tabarean and Narahashi, 1998; Tatebayashi and Narahashi, 1994). However, the large difference in the opening frequencies between tetramethrin- and deltamethrin-modi®ed channels at the single-channel level in the present study suggested that the estimate of the percentage of deltamethrin-modi®ed channels in previous whole-cell experiments could be underestimated. For instance, the percentages of tetramethrin- and deltamethrinmodi®ed TTX-S sodium channels in rat dorsal root ganglion were 3.5 and 4.5%, respectively, at 1 mM of each pyrethroid (Tatebayashi and Narahashi, 1994; Tabarean and Narahashi, 1998). However, the actual percentages of modi®ed channels could be 3.5 and 22.5% (i.e. 4.5% as multiplied by 5, the difference in opening frequency) with an assumption that the opening frequencies for normal and tetramethrin-modi®ed channels are the same. Song et al. (1996) reported interactions of type I (tetramethrin) and type II (fenvalerate) pyrethroids using whole-cell patch clamp technique. When fenvalerate application was followed by tetramethrin application, the markedly prolonged tail currents induced by fenvalerate disappeared and the less prolonged tail currents appeared which were very similar to tail currents observed during treatment with tetramethrin alone. The most straightforward interpretation is, as Song et al. (1996) suggested, that the type I pyrethroid molecule displaces the type II pyrethroid molecule from the binding site in the sodium channel protein, or that type I and II pyrethroids bind to separate sodium channel sites which interact allosterically with each other. Our single-channel data showed that characteristic deltamethrin-modi®ed channel openings disappeared during tetramethrin application and that the opening patterns during a combined application of tetramethrin and deltamethrin seem to be similar to those during application of tetramethrin alone. It should be noted that our single-channel data showed possible interactions of the two types of pyrethroids during depolarization, while the previous study (Song et al., 1996) suggested such interactions based only on
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whole-cell tail currents upon repolarization. Thus, our single-channel data are in good agreement with the previous whole-cell data. Interestingly, we could not observe tetramethrinand deltamethrin-modi®ed channel openings at the same time in a single membrane patch, although it was possible that deltamethrin-modi®ed channel openings occurred even after tetramethrin application. Either type I or type II pyrethroid modi®ed only a fraction of TTX-S sodium channels at a concentration of 1±10 mM, suggesting that some normal unmodi®ed channels may have been contained in the patch. Although pyrethroids modify the sodium channel at its resting state, opening increases the af®nity for pyrethroids (see Narahashi, 1996). The deltamethrin-modi®ed channels are kept open much longer than the normal sodium channels, providing the tetramethrin molecules with more chances to cause open channel modi®cation. Therefore, it is suggested that the type I pyrethroid tetramethrin molecules might get access more easily to type II pyrethroid deltamethrinmodi®ed channels than to the normal unmodi®ed sodium channels, resulting in displacement of deltamethrin-modi®ed channel openings by tetramethrinmodi®ed channels openings. Molecular biological studies reported putative binding sites of pyrethroids on sodium channels. For instance, Pittendrigh et al. (1997) demonstrated that several point mutations in Drosophila sodium channel rendered insects resistant to pyrethroids. These point mutations were located in the Segment 6 (S6) of Domain III (DIII), the intracellular loops between the S4 and S5 of DI and DIII, and the extracellular loop between S5 and S6 of DIII. Lee et al. (1999a) also reported the altered properties of insect neuron sodium channels that are resistant to pyrethroids; the single mutation of valine to methionine in the S6 (DI) transmembrane segment rendered sodium channels less sensitive to pyrethroids. However, none of these studies can account for the mechanism of action of pyrethroids on sodium channels or the de®nite binding sites of pyrethroids. The present single-channel and previous whole-cell studies (Song et al., 1996) showed possible interactions of two types of pyrethroids at the same or adjacent binding sites of sodium channels. One possible interpretation about this phenomenon is the following: the deltamethrin molecule bound to the site on sodium channels is kicked out by the tetramethrin molecule due to its lower binding af®nity to the site, but remained in the membrane phase because of its high lipophilicity. After washout of tetramethrin, the deltamethrin molecule located still in the membrane can
get access to the binding sites again, resulting in extremely prolonged openings. Interactions of pyrethroids demonstrated by the electrophysiological studies may provide insights into the molecular mechanisms of pyrethroid actions. ACKNOWLEDGEMENTS This work was supported by a grant from the National Institutes of Health NS14143. We thank Nayla Hasan for technical assistance and Julia Irizarry for secretarial assistance.
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