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ISSN : 1225-0171(Print)
ISSN : 2287-545X(Online)
Korean Journal of Applied Entomology Vol.61 No.1(Special Issue) pp.117-128
DOI : https://doi.org/10.5656/KSAE.2022.02.1.066

Modification of Insect Sodium Currents by a Pyrethroid Permethrin and Positive Cooperativity with Scorpion Toxins

Daewoo Lee1,2*, Michael E. Adams2
1Neuroscience Program, Department of Biological Sciences, Ohio University, Athens, OH 45701, USA
2Depts. of Entomol. and Cell Biol. & Neurosci., Univ. of California, Riverside, CA 92521, USA
*Corresponding author:Leed1@ohio.edu
December 30, 2021 February 21, 2022 February 22, 2022

Abstract


In this study, we have examined pyrethroid actions on sodium channels in the pest insect Heliothis virescens. The synthetic pyrethroid permethrin increased steady-state sodium current in H. virescens central neurons and prolonged tail currents (INa-tail) due to extreme slowing of sodium channel deactivation. Prolongation of INa-tail was evident at permethrin concentrations as low as 60 nM, which modified ~1.7% of sodium channels and 10 μM permethrin modified about 30% of channels. The average time constant (τ1) of tail current decay was ~335 ms for permethrin-modified channels. These modified channels activated at more negative potentials and showed slower activation kinetics, and failed to inactivate. Permethrin modification of sodium channels was dramatically potentiated by the α scorpion toxin LqhαIT, showing positive cooperativity between two binding sites. The amplitude of the tail current induced by 0.3 μM permethrin was enhanced ~8-fold by LqhαIT (200 pM). Positive cooperativity was also observed between permethrin and the insect-specific scorpion toxin AaIT as 10 nM permethrin potentiated the shift of voltage dependence caused by AaIT (~2-fold).



피레스로이드계 살충제 퍼메트린이 Heliothis virescens 중추신경세포에 있는 나트륨채널에 작용하는 기작을 전기생리학적으로 연구

이 대우1,2*, 마이클 아담스2
1오하이오 대학교, 생물학과, 신경과학프로그램
2캘리포니아 주립대학교, 곤충 및 세포신경생물학과

초록


본 연구는 피레스로이드계 살충제인 퍼메트린이 Heliothis virescens의 중추신경세포의 나트륨채널에 어떻게 작용하는 가를 전기생리학적으 로 관찰하였다. 퍼메트린은 나트륨채널의 꼬리전류(INa-tail)를 지속적으로 증가시켰으며 이러한 비정상적인 나트륨 전류증가가 나방류의 신경계에 과도한 흥분을 일으겨 살충작용을 하는 것으로 생각된다. 이러한 살충작용은 전갈독과 함께 사용했을때 약 8배의 증가가 있었음을 확인하였다. 전 갈독이 살충제의 독성을 강화하는 분자생리학적 기전연구가 계속되면 해충방제에 많은 기여를 할 것으로 생각된다.



    Pyrethroids, a class of insecticides widely used for pest control, are synthetic analogs of natural pyrethrins found in the flower heads of certain Chrysanthemum species. The main physiological consequence of pyrethroid poisoning is depolarization of the nerve membrane, which results in the repetitive firing and ultimate failure of impulse conduction (Narahashi, 1992, 1996;Field et al., 2017). Many studies over the past 40 years have demonstrated that sodium channels are the major target of pyrethroids in both mammals and invertebrates (Soderlund and Bloomquist, 1989;Narahashi, 1992, 1996, 2000;Trainer et al., 1997;Vais et al., 2001;Field et al., 2017;Kadala et al., 2019). The primary action of pyrethroids is to alter sodium channel gating kinetics.

    Sodium channels are composed of a large α-subunit and one or two smaller auxiliary subunits. The α-subunit of the channel forms a functional pore, contains molecular elements for the channel gating, and also renders at least 7 distinct binding sites for exogenous neurotoxins including a pyrethroid binding site (Catterall, 1992;Gordon et al., 1996;Trainer et al., 1997;Gilles et al., 2003;Stevens et al., 2011;Dong et al., 2014). Although those binding sites are topologically distinct, strong allosteric interactions have been observed. Trainer et al. (1993, 1996) showed that pyrethroids enhance the binding affinity of batrachotoxin to allosteric Site 2. Similar synergy has been shown using a combination of a pyrethroid (RU39568) and Ptychodiscus brevis toxin-1 (Site 5) which increases binding of batrachotoxin ~1,000- fold. Understanding the allosteric interactions of pharmacological binding sites could reveal structure-function relationship for sodium channels, and offers improved strategies for reduced use of pyrethroids when combined with the joint application of other sodium channel modifiers manifesting positive cooperativity. In support of this idea, McCutchen et al. (1997) have demonstrated that genetically engineered baculovirus expressing scorpion toxins showed synergistic enhancement of pyrethroid insecticidal activity to lepidopteran larvae although the molecular and functional basis for this enhancement remains to be further explored.

    Despite intensive studies in mammals, little information is available for pyrethroid actions on functional sodium channels in insect neurons, in particular economically important species targeted by these insecticides (Kadala et al., 2019). Therefore, we asked two questions regarding pyrethroid actions in this study. First, it is well known that pyrethroids have high selectivity to insects over mammals. But the basis of pyrethroid selectivity was not properly evaluated at their molecular target because there are few quantitative studies on pyrethroid actions in modulating ‘intact’ sodium channels expressed in insect central neurons although many studies were done using heterologous expression systems (Dong et al., 2014). It is interesting to know whether this high selectivity is due to higher binding affinity to insect sodium channels or due to differences in the channel modification by pyrethroids. Second, allosteric interactions between neurotoxins were not quantitatively examined at the level of functional sodium channels both in mammals and insects although some forms of interactions were biochemically characterized (Gordon et al., 1996;Trainer et al., 1997;Gilles et al., 2003). Especially, it is of interest in characterizing interactions between pyrethroids and insecticidal scorpion toxins for the practical application into the pest control.

    In the previous work (Lee et al., 1999;Lee and Adams, 2000), we have examined functional properties of sodium channels in cultured neurons prepared from the central nervous system of a moth, Heliothis virescens. Neuronal cultures had been used to examine effects of a pyrethroid permethrin on sodium channels. This study provided a valuable reference point to quantitatively explain preferential selectivity of pyrethroids to insects over mammals at the level of functional sodium channels. We also examined the joint actions of insecticidal scorpion toxins and permethrin. Our study demonstrated that (1) LqhαIT, an α scorpion toxin, potentiates permethrin action, and (2) sodium channel modulation by insect β scorpion toxin AaIT is enhanced by permethrin. The data presented here will help our effort to reveal differences between mammalian and insect sodium channels as well as to understand the relationship between structure and function of sodium channels. This information will be also useful to develop better strategies for pest control.

    Materials and Methods

    Neurotoxins

    The synthetic pyrethroid permethrin (mixture of 79% cis and 21% trans-isomers; Chem Service Inc., West Chester, PA, USA) was dissolved in dimethyl sulfoxide (DMSO) to make stock solutions at concentrations of 10 and 100 mM and kept frozen at -20°C. The stock solution was diluted in the external recording solution (see below) to give final permethrin concentrations of 0.06 to 10 μM. Residual DMSO in control and test solutions was < 0.1% (v/v). Control solutions had no measurable effects on sodium currents. Tetrodotoxin (TTX) was purchased from Sigma (St. Louis, MO, USA) and LqhαIT and AaIT were kindly supplied by Dr. Eliahu Zlotkin (Hebrew University of Jerusalem, Israel). The toxins were native peptides purified from venoms of Androctonus australis and Leiurus quinquestriatus hebraeus as previously described (Zlotkin et al., 1971, 1994;Eitan et al., 1990).

    Preparation of H. virescens central neurons

    Neurons were prepared from thoracic and abdominal ganglia of adult male moths 2-9 days post-emergence (Lee and Adams, 2000). Ganglia were dissected in ice-cold saline containing (in mM): 100 NaCl, 4 KCl, 6 CaCl2, 10 HEPES, 5 glucose, and 137 mannitol, pH 7.0. After desheathing, ganglia were treated for 5-7 min (~ 37°C) with 0.5 mg/ml collagenase (Type IA, Sigma) plus 2 mg/ml dispase (Boehringer Mannheim, Germany) in calcium-free insect saline. After transferring into sterile conditions, ganglia were gently triturated in L-15 Leibovitz culture medium supplemented with (in mg/l) 700 glucose, 400 fructose, 60 succinate, 3000 TC yeastolate, 2800 lactalbumin hydrolysate, 60 imidazole, 100 μg/ml streptomycin, 100 unit/ml penicillin, and 1 μg/ml 20 hydroxyecdysone. Dissociated neuronal cell bodies were plated onto poly-D-lysine coated dishes and incubated for 1-3 days at room temperature.

    Whole-cell recordings of neuronal sodium currents

    Whole-cell recordings were made using 1-2 MΩ Sylgardcoated patch pipettes (Boralex glass; Dynalab, Rochester, NY, USA). Sodium currents were recorded using pipettes filled with (in mM): 100 CsF, 40 CsCl, 3 MgCl2, 10 EGTA, and 5 HEPES (pH 7.0). The extracellular recording solution contained (in mM): 100 NaCl, 50 Choline-Cl, 4 KCl, 2 CaCl2, 30 TEA-Cl, 1 4-AP, 10 HEPES and 10 glucose (pH 7.0). Currents were recorded using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA, USA) and filtered at 2 kHz. Neurons were maintained at a holding potential (VH) of -108 mV. Currents were evoked by brief depolarizing steps to a test potential (VT). All data shown in this paper were compensated for a liquid junctional potential of approximately -8 mV on average. Leak currents were on-line subtracted by using a P/4 procedure (Bezanilla and Armstrong, 1977), and data were discarded if voltage errors due to series resistance remaining after partial compensation were greater than 5 mV. Cell stimulation and data acquisition were performed using pCLAMP 5.51 software (Axon Instruments) in Dell 466/ MX personal computer.

    Results

    Permethrin increases the steady-state current and slows deactivation of sodium channels. Whole cell sodium currents in H. virescens neurons show properties typical of tetrodotoxinsensitive channels (Lee and Adams, 2000). Activation kinetics are rapid, fast inactivation was almost complete during the test potential (10 ms long), and channels deactivate along a rapid time course. All of these kinetics are altered upon application of permethrin (1 μM). As shown in Fig. 1A, permethrin produced a significant increase in steady-state current, and a dramatic prolongation of the tail current (INa-tail), the latter resulting from an extreme slowing of deactivation. Permethrin-induced changes resulted from modification of sodium channels as 100 nM TTX abolished the inward current (Fig. 1B). Whole-cell recordings in an extended time scale (Fig. 1C) showed that the permethrin-induced INa-tail slowly decayed with a time constant (τ) ranging from 120 to 600 msec; the average τ was 335 +/- 110 ms (n = 5).

    The magnitude of these permethrin-induced tail currents increased in a concentration-dependent manner (Fig. 2). Following each increment in permethrin concentration (Fig. 2A & B), INa-tail increased rapidly to a new steady-state value, which was used to plot the curve in Fig. 2C. This application protocol yielded reasonable concentration-response curves by eliminating two variables, which normally confound quantification of pyrethroid effects: 1) the pronounced lipophilicity of these agents, which results in their continual partitioning of the agent into the membrane, and 2) their use-dependent action. The tail current amplitude, taken ~3 min after exposure to each permethrin concentration, was analyzed to obtain percent values of the modified channels (%M) using the following equation (Tatebayashi and Narahashi, 1994;Lee et al, 1999):

    M (%) = {[INa-tail / (VH-Erev)] / [peak INa / (VT-Erev)]} × 100
    (1)

    where peak INa is a peak sodium current before exposure to permethrin. VT, VH and Erev are the test potential, holding potential and the reversal potential of sodium current, respectively. The effective range of permethrin concentration for channel modification was 0.06 - 10 μM (Fig. 2C). Permethrin concentrations beyond 10 μM caused generation of unstable currents and, therefore, were not examined further.

    Permethrin shifts voltage-dependent activation to more negative potentials but slows sodium channel opening

    In molluscan and mammalian neurons, pyrethroids shift voltage-dependent activation and inactivation of sodium channels to more negative potentials (Lund and Narahashi, 1981;Tatebayashi and Narahashi, 1994;Song and Narahashi, 1996). We examined whether permethrin also alters these gating properties of sodium channels in H. virescens neurons. Sodium currents were recorded in response to brief depolarizing steps (VT for 10 ms) from a holding potential of -108 mV in order to plot a typical peak current-voltage curve (I/V curve; Fig. 3A). Activation of inward currents occurred at around -43 mV, peaked near -23 mV and decreased at more positive potentials in the absence and presence of 1 μM permethrin. While a reduction in peak current (17.5%, n = 4) was evident in the presence of 1 μM permethrin (Fig. 3A), the midpoint for voltage-dependent activation remained unchanged in the presence of 1 μM permethrin (Fig. 3B). Membrane potentials necessary to activate 50% of channels were -24.6 ± 1.0 (n = 5) and -25.9 ± 0.2 mV (n = 5) before and after application of permethrin, respectively. In contrast to the previous studies, these data suggest that the early phase of sodium channel activation was not altered by permethrin.

    It has been reported that single sodium channels show delayed opening in the presence of pyrethroids (Chinn and Narahashi, 1986;Narahashi, 1992, 1996). Therefore, we hypothesized that permethrin-modified sodium channels open more slowly than unmodified channels. While the early phase (peak INa) of sodium current (Figs. 1, 3B) is due to activation of unmodified channels, the late phase is likely due to slow activation of permethrin-modified channels. This idea is consistent with our result showing that only 13% of sodium channels contribute to the tail current (INa-tail) at 1 μM permethrin. Furthermore, peak INa at 1 and 10 μM permethrin were reduced 17.5 and 34.5% respectively (n = 4), of which values closely approximate the percent of channels modified at those concentrations (13% and 29.4%, see Fig. 2C). All of these results suggest that modified channels exhibit gating kinetics different from those for unmodified channels.

    To further examine this hypothesis, we compared fast rising and falling time of sodium currents in the early phase before and after permethrin exposure (Fig. 4). Although the peak current was decreased (Fig. 4A), normalized currents (Fig. 4B) showed that activation and fast decay of the peak current were not changed in the presence of permethrin. Time to peak was plotted as a function of test potential before and after permethrin exposure (Fig. 4C). Fast decay (τ1) of sodium current during depolarization was also compared using a double-exponential fit (Fig. 4D). These properties were not changed, indicating that the early phase of sodium current is due to opening of unmodified sodium channels.

    On the basis of this hypothesis, therefore, we re-examined voltage-dependent activation of sodium channels. The amplitude of sodium currents was measured at about 50 ms after onset of VT, which is long enough to completely inactivate unmodified channels. Fig. 5A and B show superimposed currents before and after 1 μM permethrin exposure. Note that although the current amplitude measured at ~50 ms (Late INa) is smaller, the curve is clearly shifted to more negative potentials (Fig. 5C). Indeed, the plot of normalized conductance (gNa) shown in Fig. 5D demonstrates that voltage-dependent activation of the modified channel was shifted more than 10 mV to the hyperpolarizing direction (10.1 ± 0.7 mV, n = 3).

    The reduction of peak current by permethrin can be explained by the fact that modified sodium channels open slowly and hence do not contribute to generation of this early phase of current. We further examined the opening of modified channels, taking advantage of the fact that they open at more negative potential (see Fig. 5D). Voltage steps to -48 mV induced a slowly activating current without activating unmodified channels (Figs. 5 & 6A). The rising phase of current through pyrethroid-modified channels recorded at more positive potential (i.e. -18 mV, Figs 6B) could not be measured directly, because activation of unmodified channels masked the relatively small, modified current in the early phase. Therefore, we subtracted control current at -18 mV from modified current measured after application of permethrin (Fig. 6C), which revealed the existence of a slowly developing current over time. The results demonstrate that the modified channels activate slowly.

    The insect selective alpha-scorpion toxin LqhαIT potentiates permethrin action

    The α-scorpion toxin LqhαIT, slows inactivation of sodium channels in both mammals and insects (Eitan et al., 1990;Zilberberg et al., 1996;Lee and Adams, 2000;Lee et al., 2000;Gurevitz et al., 2015). Although this toxin modifies both mammalian and insect sodium channels, it has a higher selectivity (>10 fold) for insect channels. It has been reported that α scorpion toxins enhance the binding of pyrethroids to sodium channels (Trainer et al., 1997). In the present study, we examined whether their positive cooperativity exists at the level of functional sodium channels.

    The dose-dependent elevation of tail current by permethrin was measured in the absence and presence of LqhαIT. Application of 200 pM LqhαIT alone caused a slight elevation (<2%) of steady-state sodium current (Fig. 7A). Modification by low concentrations of permethrin was greatly enhanced in the presence of 200 pM LqhαIT. The percentage (%M) of the sodium channels modified was calculated according to Equation 1 and plotted as a function of permethrin concentration in the presence or absence of LqhαIT (Fig. 7B). As an example, the action of 300 nM permethrin was enhanced ~8-fold by LqhαIT. Since the slope of the curve was dramatically shifted in the presence of LqhαIT, it is likely that this toxin allosterically potentiates actions of permethrin, possibly by enhancing binding of permethrin to sodium channels.

    Permethrin enhances the action of the scorpion toxin AaIT

    Pyrethroids also increase the binding affinity of other pharmacological agents including batrachotoxin and brevetoxins, which bind to allosteric Sites 2 and 5, respectively (Trainer et al., 1993). It has been reported that pyrethroids enhance the toxicity of a recombinant baculovirus carrying a synthetic gene encoding the insect-specific scorpion toxin AaIT (McCutchen, 1997). We tested the hypothesis that permethrin potentiates the action of AaIT at the level of molecular target, H. virescens sodium channels.

    The major effect of AaIT on H. virescens sodium channels is to shift their voltage-dependent activation to more negative potentials (Fig. 8), resulting in repetitive firing of action potentials in insects (Lee and Adams, 2000;Zlotkin et al., 2000;Gurevitz et al., 2015). In the presence of 10 nM permethrin, which alone did not show any significant effect (refer to Fig. 2B), the action of AaIT (100 nM) was potentiated (Fig. 8A). Normalized currents before and after permethrin were superimposable (Fig. 8B), suggesting enhancement of AaIT binding by permethrin. This potentiation was observed only at test potentials more negative than -30 mV which was the effective range for AaIT as well (Fig. 8C & D). AaIT (100 nM) shifted the midpoint of sodium channel activation 6.5 ± 1.3 mV (n = 4) to negative direction and a further shift of 6.8 ± 2.3 mV (n = 3) was observed upon addition of 10 nM permethrin.

    Discussion

    Modification of H. virescens sodium channels by permethrin

    In this study, we have examined pyrethroid actions on sodium channels in the pest insect H. virescens. Permethrin induced prolonged sodium tail currents and increased steady-state current. Furthermore permethrin shifts voltage-dependent activation to the hyperpolarizing direction. Thus permethrin-induced changes in sodium channels account for depolarization and repetitive firing in the nervous system, resulting in hyperexcitation and paralysis of insects.

    The most evident alteration by permethrin was the prolonged sodium tail current. Normal (unmodified) channel opening and closing is extremely fast (sub-millisecond) due to rapid movement of the voltage sensor (S4) in response to membrane potential change (Hille, 2001;Catterall, 2017). Modification by permethrin drastically slows channel deactivation, evident as a large tail current upon returning to the holding potential. A similar slowing of voltage sensor movement was observed when a pyrethroid fenvalerate modified deactivation of crayfish and mammalian sodium channels (Salgado and Narahashi, 1993;Tatebayashi and Narahashi, 1994). The time constant of deactivation in permethrin-modified channels in H. virescens was 335.4 ms while the time constant of unmodified channel deactivation was not experimentally measured due to the technical limitations. Therefore, we deduced the time constant (~0.06 ms) on the basis of an assumption that activation is 5 times faster than inactivation (Tatebayashi and Narahashi, 1994;Hille, 2001), of which time constant was 0.3 ms for H. virescens sodium channels. Deactivation of the channel in the presence of permethrin was ~5,500 fold slower than that for unmodified channels. This magnitude is about 3 times greater than that (1,300-fold) reported in Tatebayashi and Narahashi (1994) whose work examined actions of a pyrethroid tetramethrin on TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons.

    Permethrin-modified channels also activate at more negative potentials and the time course of activation was extremely slow (Figs. 5 & 6). These observations provide further evidence that permethrin modifies gating charge movement both during opening and closing of the channel. As gating current and mutagenesis studies have demonstrated that the S4 segment is responsible for voltage-dependent activation (Kontis et al., 1997;Marban et al., 1998), permethrin binding is expected to alter voltage sensitivity and movement of S4 in the sodium channel.

    In the presence of permethrin, the whole-cell currents in H. virescens neurons showed an elevated level of steady-state sodium current. Several reports indicate that this is due to slowing of the fast inactivation process (Tatebayashi and Narahashi, 1994;Narahashi, 1996, 2000;Song and Narahashi, 1996). Our data also showed that steady-state currents were elevated by permethrin (Figs. 1 & 2), while unmodified sodium channels were completely inactivated within 10 ms after the same depolarization step. However, this change could be explained with slow activation of permethrin-modified sodium channels as seen in Fig. 6. If this is the case, we suspect that elevated steady-state currents are generated by opening of the same number of permethrin-modified sodium channels (%M) which cause the tail currents. Therefore, we measured steady-state currents at 10 ms after onset of depolarization step in the presence of 1 μM permethrin and normalized to the control peak sodium currents. Interestingly, 12.8+/-2.6% (n = 4) of channels appears to contribute to steady-state currents induced by 1 μM permethrin and is highly comparable with %M value (13%) at the same concentration of permethrin (Fig. 2B). These results strongly support the idea that permethrin-modified sodium channels are slowly activated, but not inactivated, to induce steady-state currents while unmodified sodium channels are completely inactivated within 10 ms after the same depolarization step. The idea is further supported by the shape of the tail current in the presence of permethrin. As proposed by Tatebayashi and Narahashi (1994), if inactivation is moderately slowed by permethrin, then a “hooked” tail current is expected due to slow relocation of inactivation particle to its open position in response to repolarization. Therefore, we concluded that elevated steady-state currents by permethrin are due to slow activation of modified sodium channels, not by slow inactivation. Taken together, our findings are consistent with the fact that permethrin binding on H. virescens sodium channels affects mobilization and voltage sensitivity of S4 sensor, manifesting slow opening of sodium channel, the shift of voltage-dependent activation, removal of channel inactivation, and prolonged tail current.

    High selectivity of pyrethroids on insect sodium channels

    Selective toxicity to insects over mammals is an important feature of improved insecticides. Although other mechanisms such as degradation and excretion of pyrethroids in animals may contribute to the selectivity of these agents, the primary factor should be at the site of action. Numerous literature accounts have focused on the qualitative and quantitative aspects of pyrethroid actions on mammalian sodium channels (Tatebayashi and Narahashi, 1994), while little quantitative analysis was done to show pyrethroid actions on ‘intact’ insect sodium channels. Therefore this study provides a valuable reference point to quantify selective toxicity of pyrethroids on sodium channels in pest insects.

    In this study, we have quantified permethrin effects by calculating %M values (= percent of the modified channels). Given that the magnitude of single channel conductance is not altered by pyrethroids (Yamamoto et al., 1983;Holloway et al., 1989; Chin and Narahashi, 1993), the amplitude of tail currents most likely results from opening of permethrin-modified channels. Therefore, %M can be quantified from the amplitude of the tail current if driving force of sodium influx is compensated (refer to Equation 1). It seems very reasonable to use %M for quantification of the modified channel by pyrethroids. In addition, %M analysis was fruitful to prove that alteration of a few channels (0.6%) is enough to induce repetitive firing of action potentials (Song and Narahashi, 1996).

    Song and Narahashi (1996) estimated that the overall difference in tetramethrin toxicity is 2,250-fold between mammals and invertebrates (i.e. squid and crayfish) which is in the same order of magnitude as the differences in measured LD50 values of 500- to 4,500-fold for a pyrethroid tetramethrin. The factors they considered are sodium channel sensitivity, temperaturedependent potency of pyrethroids and detoxification rate. The primary source for the difference came from the sensitivity of sodium channels to pyrethroids, which is ~50-fold higher in invertebrates. As shown in the Table 1, the selectivity we calculated in H. virescens neurons is 20 - 80-fold, similar to their estimation. However, we found that major source for this selectivity was an extreme slowing of the channel deactivation in H. virescens sodium channels by permethrin, not sensitivity of permethrin on insect sodium channels.

    Two features of permethrin-induced tail current are potentially important to impair nerve function. One is the amplitude of the tail current, which is proportional to the number of channels modified (%M) and thus provides a physiological way to estimate relative binding affinity (=sensitivity) of pyrethroids. The other is the time constant of deactivation in the presence of permethrin. Slow deactivation is expected to induce more severe alterations in neuronal functions and thus is related to higher toxicity. Therefore those two features determine the number of sodium ions passing through the channels. As seen in Table 1, charge transfer due to prolonged tail currents was used to determine pyrethroid selectivity on insect versus mammalian sodium channels. For this comparison, we assumed that mammalian and H. virescens neurons have comparable cell size (ø15-40 μm: Song and Narahashi, 1996;Li and Zhao, 1998;Lee and Adams, 2000) and peak INa (5-10 nA: Tatebayashi and Narahashi, 1994;Song and Narahashi, 1996;Lee and Adams, 2000). Song and Narahashi (1996) cited 10-fold lower %M value for mammalian sodium channels to pyrethroids. On the basis of our results, percent of channels modified by a pyrethoid (29.35% vs 12.03-22.77%, Table 1) was not different in TTX-S sodium channels in both animal groups. Even TTX-R sodium channels in DRG neurons show 3-fold higher %M. However, a marked difference was the time constant of deactivation. At 10 μM pyrethroid (permethrin or tetramethrin), the decay of H. virescens tail current is 20 - 80-fold slower than those of rat CNS and peripheral DRG sodium channels. This comparison shows that selective toxicity of pyrethroids primarily comes from distinct molecular determinants (e.g. S4 sensor) of channel gating in insect sodium channels, which are differently influenced by permethrin, not from those of pyrethroid binding site and affinity.

    Cooperative actions of pyrethroids and scorpion toxins

    We have shown that the permethrin-induced tail current was markedly increased in the presence of the α scorpion toxin Lqh αIT. Permethrin itself also enhances the shift of voltagedependent activation initially induced by an insect-specific scorpion toxin AaIT.

    LqhαIT binds to Site 3 on insect and mammalian sodium channels (Gordon et al., 1996;Stevens et al., 2011) which is distinct from the pyrethroid binding site (Trainer et al., 1997;Gilles et al., 2003;Field et al., 2017). The permethrin-induced tail current was increased about 3~8 fold in the presence of 200 pM LqhαIT, which itself causes marginal elevation of the steadystate current (Fig. 7). Our previous study (Lee and Adams, 2000) showed that LqhαIT modifies exclusively inactivation of sodium channels and does not alter deactivation of H. virescens sodium channels. Therefore, this potentiation is related to enhanced binding of permethrin which can be achieved by two possible mechanisms. The first possibility is related to preferential binding of permethrin to sodium channels in the open state induced by LqhαIT through removal of inactivation as pyrethroids are known to prefer open channels (Narahashi, 1992;1996). Second, allosteric interaction between two binding sites results in enhanced binding of permethrin to H. virescens sodium channels. The latter is more likely the mechanism accounting for increased tail current by permethrin as 40 pM LqhαIT did not modify sodium current at all (data not shown), but enhanced permethrin action. Indeed, binding of pyrethroids was enhanced by ~40% when an α scorpion toxin (LqTx) was co-incubated (Trainer et al., 1997). Therefore our finding confirms, for the first time, allosteric interaction between pyrethroids and α scorpion toxins at the level of functional sodium channels. This finding is also consistent with a report (Gilles et al., 2003) biochemically demonstrating the interaction between sites 3 and 7 (putative pyrethroid binding site) as permethrin increases binding of 120 pM LqhαIT to locust neuronal sodium channels.

    Votage-dependent activation was shifted to the direction of hyperpolarization by AaIT, an insect specific scorpion toxin which belongs to β scorpion toxin family (Gurevitz et al., 2015). It has been shown that the S3-S4 extracellular loop in domain II is essential for β scorpion toxin binding (Marcotte et al., 1997;Cestele et al., 1998;Stevens et al., 2011). The binding of β scorpion toxins trap the voltage sensor (IIS4) in activated (open) position resulting in the negative shift of voltagedependent activation. In this study, we showed that the shift of voltage-dependent activation by AaIT was further enhanced by 10 nM permethrin which alone caused no changes in activation of H. virescens sodium channels. Since normalized current recordings before and after 10 nM permethrin treatment showed no changes in the rise phase, the time constant and the tail current (Fig. 8), our result favors a hypothesis that permethrin enhances binding of AaIT on H. virescens sodium channel which traps the voltage-sensor (S4) in open position and thus, shifts voltage-dependent activation.

    Acknowledgments

    저의 학문적 스승이셨던 고 부경생 교수님의 추모논문집에 이 논문을 발표하게 됨을 개인적으로 매우 의미있게 생각합니 다. 논문을 준비하면서 참 스승이셨던 교수님을 다시한번 생각 할 수 있는 시간이었음을 감사드립니다. University of California- Riverside박사과정 대학원생으로 이논문실험을 하던 1995년 여름에 교수님과 사모님이 Riverside를 약1주일가량 방문하셨 을 때 , 이 실험결과와 곤충생리학 전반에 대해 귀한 조언을 해 주시고 지도해 주시던 일들이 생각납니다. 박사과정 후에는 다 른 분야를 연구하게 되어 이연구결과를 논문으로 투고하기가 쉽지 않았는데, 25년이 넘어 교수님으로 인해 출판하게 되니 교 수님의 생전동안의 제자사랑과 또 고인이 되신 후에도 스승님 의 사랑을 알게 하시니 참 감사하기도 하고 또 많이 그립고 아 쉽습니다. 다시 한 번 교수님의 명복을 빕니다.

    KSAE-61-1-117_F1.gif

    Modification of sodium channels in Heliothis virescens central neurons by permethrin. (A) 1 μM permethrin increased steady-state current during the test pulse and dramatically slowed deactivation, producing a prolonged tail current. (B) All current except for capacitive transients was completely abolished following application of 100 nM tetrodotoxin (TTX). (C) Extreme prolongation of sodium tail current (INa-tail) in the presence of 1 μM permethrin. Double-exponential fit yielded a time constant (τ1) of ~162 ms. The holding potential (VH) and test potential (VT) were -18 mV and -108 mV respectively. Each recording shown here was from a different neuron.

    KSAE-61-1-117_F2.gif

    Concentration-dependent elevation of sodium tail current by permethrin. (A) Example of prolonged tail currents caused by sequential applications of 0.6, 1, and 3 μM permethrin. Depolarizing pulses (-18 mV) were applied with 10 second intervals and the holding potential (VH) was -108 mV. Each application of progressively higher permethrin concentrations produced rapid increase of tail currents. (B) A plot showing relationship between tail current amplitude and time lapse after application of three successive concentrations. (C) Percentage of modified channels plotted as a function of permethrin concentration. Closed circle represents the mean of four individual experiments. Each open symbol indicates %M (see text for details) from one experiment. (Inset) Superimposed sodium currents recorded 3 min after application of various permethrin concentrations as indicated in the right.

    KSAE-61-1-117_F3.gif

    Voltage-dependent activation of sodium channels before and after exposure to 1 μM permethrin. (A) Typical I/V curves before and after exposure to permethrin. (B) Sodium conductancepotential (gNa-V) curves plotted using an equation gNa = INa / (VT - Erev), where INa is a peak sodium current evoked by the test potential (VT), and Erev is a reversal potential of sodium channels. Voltage-dependent activation of channels was expressed by normalized sodium conductance and plotted as a function of test potentials. Each point is an average of 5 experiments and Bars are SEMs in (B).

    KSAE-61-1-117_F4.gif

    Permethrin (1 μM) effects on fast rise and decay of H. virescens sodium currents. (A) Peak sodium current was reduced in the presence of permethrin, while steady-state current was increased. (B) Normalized sodium currents before and after 1 μM permethrin exposure showing that fast rise and decay were not modified by permethrin. Fast decay was fitted with doubleexponentials. (C) Time to peak was measured as a latency between peak and onset of depolarizing step, and plotted as a function of test potentials. (D) Fast decay was measured as a tau (τ1) determined from a double-exponential fit shown in (B). τ1 was plotted as a function of test potentials. Bars are SEMs.

    KSAE-61-1-117_F5.gif

    Voltage-dependent activation of permethrin-modified sodium channels. Superimposed sodium currents evoked by VT from -63 to -28 mV with 5 mV increment before (A) and after (B) 1 μM permethrin exposure. Sodium currents in the late phase were taken at the point indicated by upward arrows (Late INa at 50 ms from the onset of test potential) before (A) and after (B) application of permethrin and then, plotted as a function of test potential (C). Peak sodium currents (INa) were also plotted in (C) for comparison. Voltage dependence of the peak INa was not modified by permethrin (see Fig. 3) while peak INa was decreased. However, Late INa-voltage curve shifted to more negative potentials. There was little or no Late INa in control. Conductance-voltage curves (D) clearly show that voltage-dependent activation of permethrinmodified sodium channels was shifted to more negative potentials.

    KSAE-61-1-117_F6.gif

    Delayed activation of permethrin-modified sodium channels. (A) At a low membrane potential (-48 mV), permethrin modified sodium channels activated very slowly, while unmodified channels were not opened (control). (B) At a more depolarized potential (-18 mV), activation of modified channels is much faster compared to that at -48 mV, but it is still much delayed compared to that of unmodified channels. Because early part of current is masked by large unmodified currents, a sodium current recorded after permethrin exposure was subtracted from control current, yielding slowly increasing sodium current (Subtracted INa). 1 μM permethrin was applied in this experiment.

    KSAE-61-1-117_F7.gif

    Potentiation of permethrin action by 200 pM LqhαIT. (A) In the presence of 200 pM LqhαIT, permethrin action was dramatically potentiated. Numbers in the right are concentrations of permethrin applied. INa-tail was measured at the time point indicated by asterisk. (B) Concentration-response curve in the absence and presence of 200 pM LqhαIT. INa-tail was translated into % modified channels (%M). % modified channels were plotted as a function of permethrin concentration. LqhαIT dramatically enhanced permethrin action in terms of increased %M. Each point in the presence of 200 pM LqhαIT is an average of 3 experiments.

    KSAE-61-1-117_F8.gif

    Potentiation of AaIT action by permethrin. (A) Superimposed sodium currents evoked by VT of -43 mV (refer to a downarrow in D) before and after exposure to the toxins (AaIT or AaIT + permethrin). Applied at 100 nM AaIT, peak and steady-state sodium currents were increased. Additional application of 10 nM permethrin doubled the peak current. (B) Normalized currents in the presence of AaIT before and after permethrin exposure. There were no changes in rising and decaying phases of sodium currents, indicating permethrin does not directly alter actions of AaIT on the channels. I/V (C) and sodium conductance (D) curves in the absence and presence of AaIT, which caused the shift of I/V curve to negative potentials. 10 nM permethrin shifts the curve further to negative direction.

    Comparison of pyrethroid selectivity on insect versus mammalian sodium channels

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