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Block of Two Subtypes of Sodium Channels in Cockroach Neurons by Indoxacarb Insecticides
NeuroToxicology 26 (2005) 455–465
Block of Two Subtypes of Sodium Channels in Cockroach Neurons by Indoxacarb Insecticides Xilong Zhao 1, Tomoko Ikeda 1, Vincent L. Salgado 2, Jay Z. Yeh 1, Toshio Narahashi 1,* 1
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA 2 Bayer CropScience, Global Biology Insecticides, Monheim, Germany Received 21 October 2004; accepted 18 March 2005 Available online 26 April 2005
Abstract Indoxacarb, a novel insecticide, and its decarbomethoxyllated metabolite, DCJW, are known to block voltage-gated Na+ channels in insects and mammals, but the mechanism of block is not yet well understood. The present study was undertaken to characterize the action of indoxacarb and DCJW on cockroach Na+ channels. Na+ currents were recorded using the whole-cell patch clamp technique from neurons acutely dissociated from thoracic ganglia of the American cockroach Periplaneta americana L. Two types of tetrodotoxin-sensitive Na+ currents were observed, with different voltage dependencies of channel inactivation. Type-I Na+ currents were inactivated at more negative potentials than typeII Na+ currents. As a result, these two types of Na+ channels responded to indoxacarb compounds differentially. At a holding potential of 100 mV, type-I Na+ currents were inhibited reversibly by 1 mM indoxacarb and irreversibly by 1 mM DCJW in a voltage-dependent manner, whereas type-II Na+ currents were not affected by either of the compound. However, type-II Na+ currents were inhibited by indoxacarb or DCJW at more depolarizing membrane potentials, ranging from 60 to 40 mV. The slow inactivation curves of type-I and type-II Na+ channels were significantly shifted in the hyperpolarizing direction by indoxacarb and DCJW, suggesting that these compounds have high affinities for the inactivated state of the Na+ channels. It was concluded that the differential blocking actions of indoxacarb insecticides on type-I and type-II Na+ currents resulted from their different voltage dependence of Na+ channel inactivation. The irreversible nature of DCJW block may be partially responsible for its potent action in insects.
# 2005 Elsevier Inc. All rights reserved. Keywords: Sodium channel; Pyrazoline; Insecticide; Indoxacarb; Cockroach neuron
INTRODUCTION Indoxacarb is a new broad-spectrum insecticide effective against insects belonging to at least 10 orders and well over 30 families. It exhibits low mammalian toxicity and shows no cross-resistance to carbamates, pyrethroids, spinosad, cyclodienes, benzoylureas or organophosphates (Zhao et al., 2002; Wing et al., 2004). Indoxacarb was the first commercialized Na+ channel blocking insecticide of the oxadiazine class. Indoxacarb’s selective toxicity against insects is due in part to the fact that it is a * Corresponding author. Tel.: +1 312 503 8284; fax: +1 312 503 1700. E-mail address: [email protected] (T. Narahashi).
proinsecticide, bioactivated in insects by esterase and amylase-like enzyme to the decarbomethoxyllated derivative DCJW, which is considered to be the active form for the insecticidal activity (Wing et al., 1998; McCann et al., 2001; Sugiyama et al., 2001). Whereas indoxacarb has been shown to modulate the nicotinic acetylcholine receptors in mammalian neurons (Zhao et al., 1999; Narahashi, 2001), its biological activity is primarily due to the inhibition of voltage-gated Na+ channels in a way similar to the pyrazolines. The blocking action of pyrazolines on Na+ channels was described in detail by a voltage clamp study using the crayfish giant axons (Salgado, 1992). Pyrazolines selectively bind to the slow-inactivated state of the Na+ channel leading to inhibition of Na+
0161-813X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.03.007
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currents, a mechanism totally different from pyrethroids, which prolongs the openings of Na+ channels (Narahashi, 1996). A recent in vitro study showed that DCJW blocked the action potential, and Na+ channels in cockroach dorsal unpaired median neurons (Lapied et al., 2001), but voltage dependence could not be demonstrated, leading to the conclusion that DCJW did not share a common mode of action with pyrazolines. However, in our previous study (Zhao et al., 2003), DCJW was more potent than indoxacarb in blocking mammalian Na+ channels, and both acted in a manner similar to pyrazolines in crayfish axons (Salgado, 1992). The present study was undertaken to better characterize the mechanism of the blocking action of indoxacarb insecticides on cockroach Na+ channels.
MATERIALS AND METHODS Isolation of Cockroach Neurons The American cockroaches, Periplaneta americana L., were purchased from Carolina Biological Supply Company (Burlington, NC), and were reared in the dark at 29 8C on rat chow (Ralston Purina Company, St. Louis, MO) and water. Neurons from three thoracic ganglia of an adult male cockroach were prepared according to a modified version of the technique of Pinnock and Sattelle (1987). Briefly, three thoracic ganglia were isolated, desheathed and incubated for 20 min in cockroach saline solution containing collagenase (type-IA, 0.5 mg/ml, Sigma–Aldrich, St. Louis, MO) and hyaluronidase (type-IS, 1 mg/ml, Sigma–Aldrich) at room temperature (22 8C). The ganglia were then rinsed three times with cockroach saline solution, and mechanically dissociated by repeated gentle suctions using a series of Pasteur pipettes of decreasing tip diameter. Cells were allowed to settle on glass coverslips pre-coated with poly-Llysine hydrobromide (MW 70,000–150,000, Sigma) for about 1 h. The coverslip was then transferred into the recording chamber and perfused with the external solution. The neurons were prepared on the day of experiments. Solutions The cockroach saline solution for cell isolation contained (in mM): NaCl 200, KCl 3.1, CaCl2 5, MgCl2 4, Sucrose 50 and HEPES-acid 10. The pH was adjusted to 7.2 with NaOH and the osmolarity was
400 mOsm. For the patch clamp current recording experiments, the external solution superfusing the cells contained (in mM): NaCl 100, tetraethylammonium (TEA)-Cl 100, KCl 3.1, CaCl2 2, MgCl2 7,4-aminopyridine (4-AP) 3 and HEPES-acid 10. The pH was adjusted to 7.3 with TEA-OH and the osmolarity was 400 mOsm. The internal solution in the recording pipette contained (in mM): NaCl 15, CsCl 80, CsF 80, EGTA 5, HEPES-acid 10, ATP-Mg2+ 2 and MgCl2 1. The pH was adjusted to 7.3 with CsOH and the osmolarity was 400 mOsm. Electrophysiological Recordings and Data Analysis Currents were recorded using the whole-cell patch clamp technique at room temperature (22–24 8C). Large neurons, 50–100 mm in diameter with pyriform bodies, were selected. Recording electrodes were made of borosilicate glass capillary tubes (1.5–1.8 mm inner diameter, Kimble, Vineland, NJ) using a two-step vertical puller (Narishige, Tokyo, Japan), and coated with dental wax to reduce the capacitive currents. The resistance of pipette was 1.0–1.5 MV, when filled with the internal solution. An Ag–AgCl pellet/3 M KCl– agar bridge was used for the reference electrode. Membrane currents were recorded using an Axo-Patch 200A amplifier (Axon Instruments, Union City, CA). Signals were digitized by a 14-bit analog-to-digital converter, filtered with a Bessel filter at 5 kHz and stored on a PC computer. Series resistance was compensated 70–75%. Capacitive and leakage currents were digitally subtracted using the P-P/4 procedure. The liquid junction potential between internal and external solutions was 4 mV on an average, which was used to compensate for the membrane potential. All data are expressed as mean S.E.M. and n represents the number of the cells examined. All figures represent typical examples from at least three independent experiments. The statistical significance between two samples was tested by Student’s t-test. Chemicals Indoxacarb [indeno(1,2-e)(1,3,4)oxadiazine-4a (3H)-carboxylic acid, 7-chloro-2,5-dihydro-2-(((methoxycarbonyl)-((4-trifluoromethoxy) phenyl)amino)carbonyl)-methyl ester] (DPX-MP062, 75:25 mixture of active and inactive enantiomers) and its metabolite DCJW were provided by E.I. DuPont de Nemours (Newark, DE), and were first dissolved in dimethylsulfoxide to prepare the stock solutions. The stock solutions (1 or 10 mM) were then diluted to desired
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concentrations with the external solution shortly before experiments. The final concentrations of dimethylsulfoxide in test solutions were 0.1% (v/v) or less, which had no effect on the Na+ currents when applied externally. Because both indoxacarb and DCJW are highly hydrophobic compounds, our drug application system was built with Teflon tubing and glass syringes, and was cleaned with alcohol after each experiment to prevent the contamination.
RESULTS Two Types of Na+ Currents The cockroach thoracic ganglia contain neurons of different sizes (10–100 mm) and different shapes. Most of them are with mainly round or pyriform somata with axon remnants. Na+ currents were rarely observed in small round-shaped neurons or large neurons without axon remnants, but were reliably recorded in large pyriform neurons. This is consistent with the fact that voltage-gated Na+ channels are mainly located on the axon or the initial segment of the soma, instead of the apical pole of cockroach DUM neurons (Amat et al., 1998; French et al., 1993). Therefore, in the present study the pyriform neurons with axon remnants were selected for experiments. Three types of inward currents were induced by step depolarization to 10 mV from a holding potential of 100 mV in cockroach neurons. One type of current
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was characterized by a fast onset and fast decay (Fig. 1A). This current disappeared reversibly by eliminating Na+ ions from the external solution (Fig. 1B and C). It was completely blocked by the Na+ channel blocker tetrodotoxin (TTX) at 400 nM (Fig. 1E), but not by the calcium channel blocker CdCl2 at 50 mM (Fig. 1D). Therefore, this type of current was carried by Na+ ions, and was designated the type-I Na+ current. In most neurons, a second type of Na+ current was found to co-exist with a current which had slow onset and slow decay and was blocked by 50 mM CdCl2 or elimination of external Ca2+ but not by 400 nM TTX (Fig. 2Aa). The fast transient component, but not the slow component, disappeared either by eliminating Na+ ions from the external solution (Fig. 2Ab) or by applying 400 nM TTX (Fig. 2Cb). The slow component, but not the fast component, of the current was reversibly eliminated by the removal of external Ca2+ or by 50 mM CdCl2 (Fig. 2Cc). Therefore, the fast component is the Na+ current through the voltagegated Na+ channel, whereas the slow component is the calcium current through the voltage-gated calcium channel (Fig. 2B and D). The Na+ current obtained in the presence of 50 mM CdCl2 was designated as the type-II Na+ current. It differed from type-I Na+ current. Inactivation of Type-I and Type-II Na+ Currents Na+ channels undergo two kinetically distinct types of inactivation: fast inactivation and slow inactivation, which form the basis for differentiating type-I and
Fig. 1. Characteristics of type-I Na+ currents recorded from a cockroach thoracic ganglion neuron. (A) The fast inactivating transient current evoked by a step depolarization to 10 mV from a holding potential of 100 mV, in the presence of 100 mM Na+ in external solution. (B) Current recorded following superfusion of Na+-free solution. (C) Recovery of the current after re-superfusion of 100 mM Na+ solution. (D) The current was not changed by bath application of 50 mM CdCl2. (E) The current was quickly blocked by 400 nM TTX. (F) Current recovered after washing with a TTX-free 100 mM Na+ solution.
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Fig. 2. Mixed type currents comprising Na+ and Ca2+ currents recorded from cockroach thoracic ganglion neurons. The currents were evoked by 30-ms test pulses to 10 mV, at a holding potential of 100 mV. (A) With normal external solution containing 100 mM NaCl, the current showed a fast transient component and a slow component (trace Aa). The fast component was eliminated when Na+ was replaced by TMA-Cl (trace Ab). The remaining slow component was eliminated by 50 mM CdCl2 (trace Ac). (B) Subtraction of Ab from Aa yields Na+ current (type-II). Subtraction of Ac from Ab reveals Ca2+ current. (C) Current (Ca) recorded from another neuron with normal external solution also exhibited fast and slow components. The fast component was removed by 400 nM TTX (trace Cb), whereas the slow component was removed by 50 mM CdCl2 (trace Cc). The entire current was completely blocked by TTX and CdCl2 (trace Cd). (D) Na+ and Ca2+ currents were obtained by subtraction of Cd from Cc and Cd from Cb, respectively.
type-II Na+ currents. In the present study, the fast inactivation was studied by applying a series of 150ms conditioning pulses ranging from 120 to +20 mV (in steps of 10 mV) that were long enough to allow the inactivation to reach the steady state level. Each conditioning pulse was followed by a repolarization to 100 mV for 1 ms, and then a 30 ms test pulse to 10 mV. The fast steady state inactivation curve, obtained by plotting the normalized peak Na+ current
against the conditioning potential, is shown in Fig. 3A. The slow steady state inactivation curve was obtained by applying longer conditioning pulses (1 min), as shown in Fig. 3B. Type-I and type-II Na+ currents differed in both fast steady state inactivation and slow steady state inactivation. The mid-point potentials for inactivation (V0.5 s) were more negative for type-I than for type-II Na+ currents, for both fast and slow inactivation (Fig. 3).
Fig. 3. Fast and slow steady state inactivation of type-I and type-II Na+ channels in cockroach thoracic ganglion neurons. The steady state fast inactivation data (A) were obtained from currents evoked by 30 ms test pulses to 10 mV following 150-ms conditioning pulses ( 120 to +20 mV) from a holding potential of 100 mV. The steady state slow inactivation data (B) were obtained from currents evoked by 30-ms test pulses to 10 mV, following a longer (1 min) conditioning pulses ( 130 to 60 mV for type-I and 100 to 30 mV for type-II) from a holding potential of 100 mV. The peak amplitudes of Na+ currents associated with test pulses were normalized to its respective maximum value, and are plotted as a function of the conditioning potential. The curves are drawn according to the equation I/Imax = 1/{1 + exp[(V V0.5)/k]}, where V is conditioning potential, V0.5 is the potential at which the current reduces to half maximum (Imax), and k is the slope factor (potential required for an e-fold change). The voltage dependence of inactivation occurs at more negative potentials for type-I Na+ channels than for type-II Na+ channels.
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The V0.5 values for fast inactivation were 64.3 and 39.8 mV for type-I and type-II Na+ currents, respectively. The V0.5 values for slow inactivation were 86.8 and 52.7 mV for type-I and type-II Na+ currents, respectively. Indoxacarb Insecticides on Type-I Na+ Currents DCJW, a metabolite of indoxacarb, was recognized as an active component for the insecticidal activity, and blocked the compound action potentials and Na+ channels in both insect and mammalian neurons (Wing et al., 1998; Lapied et al., 2001; Sugiyama et al., 2001; Zhao et al., 2003). Fig. 4 shows a sharp contrast between indoxacarb and DCJW with respect to the reversibility of type-I Na+ current block. Na+ currents were evoked by step depolarizations to 10 mV from a holding potential of 100 mV, at an interval of 1 min. After several stable control recordings had been established, indoxacarb or DCJW at a concentration of 1 mM was applied in the bath. The amplitude of Na+ currents was quickly suppressed by 77.1 5.3% (n = 4) and 78.3 11.6% (n = 4) by indoxacarb and DCJW, respectively. However, the Na+ current suppression caused by indoxacarb was completely reversible after washout, whereas the current suppression caused by DCJW was irreversible 10 min after washout with insecticide-free media. Similar results were obtained in all the four cells tested. The irreversible nature of DCJW block of type-I Na+ currents was further corroborated by experiments in which the holding potential was changed (Fig. 5). Before application of DCJW, a change in holding
Fig. 5. Time course and irreversibility of blocking action of 100 nM DCJW on type-I Na+ channels. Currents were evoked by test pulses to 10 from 100 or 80 mV holding potentials, at an interval of 1 min. The Na+ currents were smaller at a holding potential of 80 mV than at 100 mV due to Na+ channel inactivation. DCJW at a concentration of 100 nM was applied at 80 mV for 10 min to produce a nearly complete block. No recovery was seen by a 10-min washout with a normal external solution or by hyperpolarization to 100 mV.
potential from 100 to 80 mV decreased the current amplitude as expected from the steady state inactivation curve (Fig. 3). The current amplitude at a holding potential of 80 mV was further decreased but the time to peak of the current was not changed during the bath perfusion of 100 nM DCJW. Washout with DCJW-free solutions caused no recovery. Changing the holding potential from 80 to 100 mV still failed to resurrect the Na+ current. Indoxacarb Insecticides on Type-II Na+ Currents
Fig. 4. The differential actions of indoxacarb and DCJW on type-I Na+ channels of cockroach thoracic ganglion neurons. Currents were evoked by a 10-ms step depolarization to 10 mV from a holding potential of 100 mV at an interval of 1 min. The peak current was inhibited by 77.1 5.3% (n = 4) by 1 mM indoxacarb and 84.1 5.3% (n = 3) by 1 mM DCJW. Upon washing with insecticide-free solution, the block by indoxacarb was reversed, while the block by DCJW was not reversed.
Type-II Na+ currents were isolated from the mixed type of currents by application of 50 mM CdCl2 to block the Ca2+ currents, as shown in the insets of Fig. 6. In contrast to type-I Na+ currents, type-II Na+ currents were unaffected by either 1 mM indoxacarb or 1 mM DCJW applied in the bath for 10 min (Fig. 6), when the Na+ current was evoked from the membrane holding potential of 100 mV. Previous studies demonstrated that the blocking action of the pyrazoline-type insecticides, indoxacarb and DCJW, on Na+ channels was voltage-dependent in
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Fig. 6. Indoxacarb (A) and DCJW (B) had no effect on type-II Na+ channels at a concentration of 1 mM at 100 mV membrane potential. Control Na+ currents were recorded with 10 mV test pulses from a holding potential of 100 mV in the presence of 50 mM CdCl2 at an interval of 5 s. Indoxacarb or DCJW was applied through bath perfusion for 10 min. During drug application, the Na+ currents were monitored at an interval of 30 s. The insets show the typical Na+ current traces before (dotted lines) and after (solid lines) bath perfusion of indoxacarb (A) or DCJW (B).
crayfish giant axons and rat dorsal root ganglion neurons (Salgado, 1992; Zhao et al., 2003). However, a recent study of DCJW using cockroach ganglion neurons did not support the above findings (Lapied et al., 2001). To further resolve the discrepancy of voltagedependent block by indoxacarb insecticides on cockroach Na+ channels, type-II Na+ currents were recorded at different holding potentials ( 100, 80, 60 and 40 mV) at an interval of 5 s. The sample traces of type-II Na+ currents recorded at different holding potentials in the absence and presence of insecticides are shown in Fig. 7A. In the absence of the insecticides, the peak Na+ current began to decrease progressively after changing the holding potentials from 100 to 80, 60 and 40 mV (open circles in Fig. 7B and C). However, in the presence of indoxacarb or DCJW at a concentration of 1 mM, the decline in Na+ currents was accentuated, especially at holding potentials of 60 and 40 mV (filled circles in Fig. 7B and C). Thus, the degree of block of Na+ current by a given concentration of indoxacarb or DCJW was modulated by changing the holding potential. The results clearly showed that the blocking action of indoxacarb insecticides on type-II Na+ currents was voltage-dependent. Indoxacarb Insecticides on Na+ Channel Inactivation To further characterize the voltage-dependent blocking action of indoxacarb, type-II Na+ currents were evoked by step depolarizations to 10 mV at an interval of 5 s from sequential changes in holding potentials
of 100, 50, and back to 100, in the absence of test drug. The Na+ current gradually decreased in amplitude as the holding potential changed from 100 to 50 mV (Fig. 8A, opened circles). The peak amplitude of Na+ currents was not changed when the neurons were treated with indoxacarb at 100 nM for 10 min at a holding potential of 100 mV. However, indoxacarb treatment exacerbated the decrease in Na+ currents when the holding potential was changed from 100 to 50 mV (Fig. 8A, filled circles). The Na+ current in the presence of indoxacarb was completely restored by switching the holding potential from 50 to 100 mV (Fig. 8A). However, simple washout of indoxacarb at a holding potential of 50 mV did not cause any recovery of currents (Fig. 8B). Complete recovery was observed only when the holding potential was brought back to 100 mV (Fig. 8B). Similar results were obtained with DCJW (Fig. 9). Following bath perfusion of 1 mM DCJW for 10 min, the peak amplitude of Na+ currents was not changed at a holding potential of 100 mV, but was decreased at 50 mV. The decreased amplitude of Na+ currents at 50 mV recovered upon hyperpolarization from 50 to 100 mV, but was not restored by washout of DCJW at 50 mV. The above results of voltage dependency of block clearly suggest that indoxacarb insecticides enhance the inactivation of type-II Na+ channel in cockroach neurons. In addition, the different inhibitory effects of indoxacarb insecticides on type-I and type-II Na+ currents at 100 mV holding potential could be due to a high affinity binding of insecticides to the inactivated states of the channel, because the type-I Na+
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inactivation were analyzed using the protocol shown in Fig. 3. Indoxacarb or DCJW was applied in the bath for 15 min at a concentration of 100 nM at a holding potential of 100 mV. Fig. 10A illustrates the effects of indoxacarb on the voltage dependence of fast and slow inactivation of type-II Na+ currents. Indoxacarb significantly shifted the mid-point of the slow inactivation from 55.1 2.2 to 61.1 1.9 mV (n = 5, p < 0.05) while its effect on the fast inactivation was not significant with the mid-point of inactivation changing from 43.4 1.3 to 46.6 1.9 mV (n = 4, p > 0.05). Similarly, DCJW shifted the slow inactivation curve significantly from 56.1 3.4 to 72.3 3.3 mV (n = 4, p < 0.05), and its shift of the fast inactivation curve from 41.6 2.9 to 45.0 3.4 mV was not significant (n = 5, p > 0.05). The effects of DCJW on Na+ inactivation of type-I Na+ currents were also studied. The slow inactivation curve was significantly shifted by DCJW changing from 88.8 1.5 to 97.1 1.6 mV (n = 3, p < 0.05), whereas the shift of the fast inactivation curve from 67.3 1.5 to 71.5 0.7 mV was not significant (n = 5, p > 0.05) (Fig. 10B). The slope factors of fast and slow inactivation curves in both types of Na+ currents were not changed by indoxacarb insecticides (Fig. 10). These results suggested that indoxacarb insecticides induced the voltage-dependent inhibition of type-I and type-II Na+ currents by selective binding to the slow-inactivated state of the channel.
DISCUSSION Two Kinds of Na+ Currents Fig. 7. Inhibition of type-II Na+ channels by 1 mM indoxacarb and DCJW is dependent on holding potentials. Na+ currents were evoked by test pulses to 10 mV from various holding potentials ( 100, 80, 60 and 40 mV) in the presence of 50 mM CdCl2. Currents were recorded at an interval of 5 s in the absence (opened circles) or presence (filled circles) of insecticides. Indoxacarb or DCJW at 1 mM was applied in bath for 10 min before recording the currents. Representative Na+ currents are shown for control (Aa) and in the presence of 1 mM DCJW (Ab). The results shown here (B and C) were compiled from different neurons and the currents in the presence of insecticides were normalized to the one at 100 mV. Data are expressed as mean S.E.M. Current suppression in the presence of 1 mM indoxacarb (B) or DCJW (C) were accentuated by holding potentials, especially at 60 and 40 mV.
channel undergoes more inactivation at 100 mV than does the type-II Na+ channel, as shown in Fig. 3. In order to verify the validity of the above notion of the role of Na+ channel inactivation in the action of indoxacarb insecticides, the steady state fast and slow
Voltage-gated Na+ channels in dorsal unpaired median neurons of the cockroach are located mainly on the axon and the initial segment of the soma, as shown by immunocytochemical staining (French et al., 1993; Amat et al., 1998). The physiological and pharmacological properties of the fast voltage-gated Na+ currents in both giant axons and dorsal unpaired median neurons of the cockroach were studied using voltage clamp techniques (Sattelle et al., 1979; Pelhate et al., 1979; Lapied et al., 1990; Grolleau and Lapied, 2000). In the present study, two kinds of Na+ currents (type-I and type-II) were recorded in cockroach thoracic ganglion neurons. Type-I and type-II Na+ currents completely disappeared by elimination of Na+ ions from the external solution and were sensitive to the blocking action of TTX. Type-II but not type-I Na+ currents were always accompanied by a slow component of current
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Fig. 8. Indoxacarb inhibition of type-II Na+ channels can be reversed by hyperpolarization. Na+ currents were evoked by step depolarizations to 10 from 100 or 50 mV holding potential in the presence of 50 mM CdCl2. (A) Control Na+ currents were first recorded at 100 mV, then at 50 mV holding potential for 2.5 min at an interval of 5 s (open circles). Following complete recovery from channel inactivation after switching the holding potential back to 100 mV, neurons were treated with 100 nM indoxacarb for 10 min at a holding potential of 100 mV. Na+ currents were then recorded using the same protocol as above (solid circles). Indoxacarb had no effect on type-II Na+ current when the neuron was held at 100 mV and exerted a potent inhibition when it was held at 50 mV. (B) Time-course of changes in Na+ currents during and after application of indoxacarb at holding potentials of 100 and 50 mV. Recovery from indoxacarb block was not seen after washout at a holding potential of 50 mV, but occurred at a holding potential of 100 mV during the application of indoxacarb and after its washout.
that could be eliminated by application of Cd2+ or Ca2+-free external solutions. It was concluded that the slow component of current was generated by Ca2+ channels. Type-I and type-II Na+ currents exhibited differential inactivation properties. The steady state fast and slow inactivation of type-I Na+ channels occurred at more negative membrane potentials than did in type-II Na+ channels. The existence of two types of Na+ channels was reported in the cockroach giant axon (Yawo et al., 1985) and dorsal unpaired median TAG
Fig. 9. DCJW inhibition of type-II Na+ channels can be reversed by hyperpolarization. Na+ currents were recorded with the protocol as described in Fig. 8. Following recovery from channel inactivation after switching the holding potential from 50 back to 100 mV, neurons were treated with 1 mM DCJW for 10 min at 100 mV holding potential without channel activation. Na+ currents were then recorded using the same protocol as above. The peak amplitude of Na+ currents was not changed by DCJW at 100 mV holding potential, but was quickly reduced at 50 mV holding potential. Na+ currents largely recovered from inactivation and DCJW inhibition by changing the holding potential from 50 to 100 mV, but no recovery from DCJW occurred after washout at a holding potential of 50 mV.
neurons (Lapied et al., 1990). Under voltage clamp conditions (Lapied et al., 1990), in about 80% of the cells tested, the transient Na+ currents were reported with half-inactivation at 41.1 mV and a saxitoxinsensitive maintained Na+ current was observed in the identical remaining cells. Our type-II Na+ currents might be similar to the transient Na+ current reported by Lapied et al. (1990) because the mid-point for the fast inactivation were nearly identical. Functional diversity and tissue-specific distributions of the Na+ channels in mammals are achieved by selective expression of at least nine different Na+ channel genes (Goldin, 2002). In insects, however, only one functional Na+ channel gene was reported, i.e. para in Drosophila melanogaster (Warmke et al., 1997; Zhou et al., 2004). Recent studies have demonstrated that alternative splicing and RNA-editing of a cockroach Na+ channel gene generate a wide array of functionally and pharmacologically distinct Na+ channels (Tan et al., 2002; Song et al., 2004; Liu et al., 2004). It is possible that the two types of Na+ currents identified in our study and those from other laboratories are generated by two splicing and/or RNA-editing variants of a Na+ channel gene in Periplaneta americana. Sensitivity of Two Types of Na+ Currents to Indoxacarb Insecticides Both type-I and type-II Na+ currents were TTXsensitive, but had differential sensitivity to the blocking action of indoxacarb insecticides. Type-I and type-II
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Fig. 10. Modification of steady state inactivation of type-II (A) and type-I (B) Na+ channels by indoxacarb and DCJW. The voltage dependencies of steady state fast inactivation and slow inactivation of both type-II and type-I Na+ channels were measured and analyzed by the method, as illustrated in Fig. 3. Following exposure to 100 nM indoxacarb (A, filled squares) or 100 nM DCJW (B, filled circles) for 15 min at a holding potential of 100 mV, the fast and slow inactivation curves were compared to the corresponding control (A, opened squares; B, opened circles). DCJW and indoxacarb caused a significant hyperpolarizing shift of slow steady state Na+ channel inactivation curve.
Na+ currents in cockroach neurons responded differently to the blocking action of indoxacarb and its metabolite DCJW. The blocking effect of indoxacarb on Na+ currents depended largely on the membrane potential, as observed in crayfish axons for pyrazoline block (Salgado, 1992). However, the sensitivity of type-I Na+ currents to indoxacarb and DCJW was different from that of type-II Na+ currents. At a membrane potential of 100 mV, where type-II Na+ channels were not inactivated while type-I Na+ channels underwent substantial inactivation, type-I Na+ currents were blocked by indoxacarb or DCJW at a concentration as low as 100 nM. In contrast, type-II Na+ currents were not blocked by indoxacarb or DCJW at 100 mV, and were blocked only at less negative potentials ranging from 60 to 40 mV, where some inactivation occurred. The voltage-dependent suppression of Na+ currents by indoxacarb insecticides is very similar to that in rat dorsal root ganglion neurons (Zhao et al., 2003). The hyperpolarization shift in voltage dependence of slow inactivation suggests that insecticides bind to the inactivated state with a higher affinity than to the resting state of the Na+ channel. Therefore, the differential sensitivity of two types of Na+ currents to indoxacarb and DCJW is largely due to the different voltage dependence of slow inactivation.
Different Insecticidal Potency of Indoxacarb and DCJW Indoxacarb was much weaker than its metabolite DCJW as an insecticide (Tsurubuchi et al., 2001a). Studies using extracellular recording electrodes showed that DCJW, but not indoxacarb, appeared to be the active form in lepidopteran (Manduca sexta) larval motor nerve preparations (Wing et al., 1998). Previous studies using mammalian neurons also showed that indoxacarb was about 10 times less potent than DCJW in blocking TTX-sensitive and TTX-resistant Na+ channels (Zhao et al., 2003; Tsurubuchi et al., 2001b; Nagata et al., 1998). In the present study, both indoxacarb and DCJW strongly blocked type-I and type-II Na+ channels. Indoxacarb block of type-I Na+ channels at 100 mV membrane potential was reversible, while DCJW block was irreversible. The difference in reversibility between indoxacarb and DCJW may be partially responsible for the higher insecticidal activity of DCJW as compared to indoxacarb. In conclusion, the present study demonstrated that indoxacarb and its metabolite DCJW blocked the two types of Na+ channels of cockroach neurons in a manner dependent on the membrane potential. The steady state inactivation curve of type-I Na+ channels
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has its voltage dependence resided at the membrane potential more hyperpolarized than the type-II Na+ channels. The voltage dependence of block caused by indoxacarb and DCJW was due to their higher affinity for the inactivated state than for the resting state of the channel. The observation that indoxacarb block was reversible after washout while DCJW block of type-I Na+ channels was irreversible might provide an explanation for the more potent insecticidal activity of DCJW.
ACKNOWLEDGEMENTS This study was supported by National Institutes of Health grant NS14143. Samples of indoxacarb (DPXMP062) and its metabolite, DCJW, were provided by E.I. DuPont de Nemours and Company, Newark, Delaware. The authors wish to thank Dr. Ke Dong (Michigan State University, MI) for the helpful discussion on insect sodium channels. The authors thank Nayla Hasan and Sandra Guy for technical assistances and Julia Irizarry for secretarial assistance.
REFERENCES Amat C, Lapied B, French AS, Hue B. Na+-dependent neuritic spikes initiate Ca2+-dependnet somatic plateau action potentials in insect dorsal paired median neurons. J Neurophysiol 1998;80:2718–26. French AS, Sanders EJ, Duszyk. Prasad S, Torkkeli PH, Haskins J, et al. Immunocytochemical localization of sodium channels in an insect central nervous system using a site-directed antibody. J Neurobiol 1993;24:939–48. Goldin AL. Evolution of voltage-gated Na+ channels. J Exp Biol 2002;205(Pt. 5):575–84. Grolleau F, Lapied B. Dorsal unpaired median neurones in the insect central nervous system: towards a better understanding of the ionic mechanisms underlying spontaneous electrical activity. J Exp Biol 2000;203:1633–48. Lapied B, Male´ cot CO, Pelhate M. Patch-clamp study of the properties of the sodium current in cockroach single isolated adult aminergic neurones. J Exp Biol 1990;151:387– 403. Lapied B, Grolleau F, Sattelle DB. Indoxacarb, an oxadiazine insecticide, blocks insect neuronal sodium channels. Br J Pharmacol 2001;132:87–595. Liu Z, Song W, Dong K. Persistent tetradotoxin-sensitive sodium current resulting from U-to-C RNA-editing of an insect sodium channel. Proc Natl Acad Sci 2004;101: 11862–7. McCann SF, Annis GD, Shapiro R, Piotrowski DW, Lahm GP, Long JK, et al. The discovery of indoxacarb: oxadiazines as a new class of pyrozoline-type-Insecticides. Pest Management Sci 2001;57:153–64.
Nagata K, Ikeda T, Honda H, Shono T. Suppression of voltagegated sodium currents by the dihydropyrazole insecticide, DPXJW062 in rat dorsal root ganglion neurons. J Pesticide Sci 1998;23:62–4. Narahashi T. Neuronal ion channels as the target sites of insecticides. Pharmacol Toxicol 1996;78:1–14. Narahashi T. Recent progress in the mechanism of action of insecticides: pyrethroids, fipronil and indoxacarb. J Pesticide Sci 2001;26:277–85. Pelhate M, Hue B, Sattelle DB. Pharmacological properties of axonal sodium channels in the cockroach Periplaneta Americana L. II. Slowing of sodium current turn-off by Condylactis toxin. J Exp Biol 1979;83:49–58. Pinnock RD, Sattelle DB. Dissociation and maintenance in vitro of neurones from adult cockroach (Periplaneta americana) and housefly (Musca domestica). J Neurosci Methods 1987;20:195– 202. Salgado VL. Slow voltage-dependent block of sodium channels in crayfish nerve by dihydropyrazole insecticides. Mol Pharmacol 1992;41:120–6. Sattelle DB, Pelhate M, Hue B. Pharmacological properties of axonal sodium channels in the cockroach Periplaneta americana L. I. Selective block by synthetic saxitoxin. J Exp Biol 1979;83:41–8. Song W, Liu Z, Tan J, Nomura Y, Dong K. RNA-editing generates tissue-specific sodium channels with distinct gating properties. J Biol Chem 2004;279:32554–61. Sugiyama S, Tsurubuchi Y, Karasawa A, Nagata K, Kono Y, Shono T. Insecticidal activity and cuticular penetration of indoxacarb and its N-decarbomethoxylated metabolite in organophosphorus insecticide-resistant and susceptible strains of the housefly, Musca domestica (L.). J Pesticide Sci 2001;26: 117–20. Tan J, Liu Z, Nomura Y, Goldin AL, Dong K. Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels. J Neurosci 2002;22: 5300–9. Tsurubuchi Y, Karasawa A, Nagata K, Shono T, Kono Y. Insecticidal activity of oxadiazine insecticide indoxacarb and its Ndecarbomethoxylated metabolite and their modulations of voltage-gated sodium channels. Appl Entomol Zool 2001;36:381– 5. Tsurubuchi Y, Zhao X, Nagata K, Kono Y, Nishimura K, Yeh JZ, et al. Modulation of tetrodotoxin-resistant sodium channels by dihydropyrazol insecticide RH-3421 in rat dorsal root ganglion neurons. Neurotoxicology 2001;22:743–53. Warmke JW, Reenan RA, Wang P, Qian S, Arena JP, Wang J, et al. Functional expression of Drosophila para sodium channels. Modulation by the membrane protein tipE and toxin pharmacology. J Gen Physiol 1997;110:119–33. Wing KD, Schnee ME, Sacher M, Connair M. A novel oxadiazine insecticide is bioactivated in lepidopteran larvae. Arch Insect Biochem Physiol 1998;37:91–103. Wing KD, Andaloro JT, McCann SF, Salgado VL. Indoxacarb and the sodium channel blocker insecticides: chemistry, physiology and biology in insects. In: Gilbert LI, Iatrou K, Gill SS, editors. Comprehensive molecular insect science. Oxford: Elsevier; 2004. 31–53. Yawo H, Kojima H, Kuno M. Low-threshold, slow-inactivating Na+ potentials in the cockroach giant axon. J Neurophysiol 1985;54:1087–100.
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Block of Two Subtypes of Sodium Channels in Cockroach Neurons by Indoxacarb Insecticides Xilong Zhao 1, Tomoko Ikeda 1, Vincent L. Salgado 2, Jay Z. Yeh 1, Toshio Narahashi 1,* 1
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA 2 Bayer CropScience, Global Biology Insecticides, Monheim, Germany Received 21 October 2004; accepted 18 March 2005 Available online 26 April 2005
Abstract Indoxacarb, a novel insecticide, and its decarbomethoxyllated metabolite, DCJW, are known to block voltage-gated Na+ channels in insects and mammals, but the mechanism of block is not yet well understood. The present study was undertaken to characterize the action of indoxacarb and DCJW on cockroach Na+ channels. Na+ currents were recorded using the whole-cell patch clamp technique from neurons acutely dissociated from thoracic ganglia of the American cockroach Periplaneta americana L. Two types of tetrodotoxin-sensitive Na+ currents were observed, with different voltage dependencies of channel inactivation. Type-I Na+ currents were inactivated at more negative potentials than typeII Na+ currents. As a result, these two types of Na+ channels responded to indoxacarb compounds differentially. At a holding potential of 100 mV, type-I Na+ currents were inhibited reversibly by 1 mM indoxacarb and irreversibly by 1 mM DCJW in a voltage-dependent manner, whereas type-II Na+ currents were not affected by either of the compound. However, type-II Na+ currents were inhibited by indoxacarb or DCJW at more depolarizing membrane potentials, ranging from 60 to 40 mV. The slow inactivation curves of type-I and type-II Na+ channels were significantly shifted in the hyperpolarizing direction by indoxacarb and DCJW, suggesting that these compounds have high affinities for the inactivated state of the Na+ channels. It was concluded that the differential blocking actions of indoxacarb insecticides on type-I and type-II Na+ currents resulted from their different voltage dependence of Na+ channel inactivation. The irreversible nature of DCJW block may be partially responsible for its potent action in insects.
# 2005 Elsevier Inc. All rights reserved. Keywords: Sodium channel; Pyrazoline; Insecticide; Indoxacarb; Cockroach neuron
INTRODUCTION Indoxacarb is a new broad-spectrum insecticide effective against insects belonging to at least 10 orders and well over 30 families. It exhibits low mammalian toxicity and shows no cross-resistance to carbamates, pyrethroids, spinosad, cyclodienes, benzoylureas or organophosphates (Zhao et al., 2002; Wing et al., 2004). Indoxacarb was the first commercialized Na+ channel blocking insecticide of the oxadiazine class. Indoxacarb’s selective toxicity against insects is due in part to the fact that it is a * Corresponding author. Tel.: +1 312 503 8284; fax: +1 312 503 1700. E-mail address: [email protected] (T. Narahashi).
proinsecticide, bioactivated in insects by esterase and amylase-like enzyme to the decarbomethoxyllated derivative DCJW, which is considered to be the active form for the insecticidal activity (Wing et al., 1998; McCann et al., 2001; Sugiyama et al., 2001). Whereas indoxacarb has been shown to modulate the nicotinic acetylcholine receptors in mammalian neurons (Zhao et al., 1999; Narahashi, 2001), its biological activity is primarily due to the inhibition of voltage-gated Na+ channels in a way similar to the pyrazolines. The blocking action of pyrazolines on Na+ channels was described in detail by a voltage clamp study using the crayfish giant axons (Salgado, 1992). Pyrazolines selectively bind to the slow-inactivated state of the Na+ channel leading to inhibition of Na+
0161-813X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.03.007
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currents, a mechanism totally different from pyrethroids, which prolongs the openings of Na+ channels (Narahashi, 1996). A recent in vitro study showed that DCJW blocked the action potential, and Na+ channels in cockroach dorsal unpaired median neurons (Lapied et al., 2001), but voltage dependence could not be demonstrated, leading to the conclusion that DCJW did not share a common mode of action with pyrazolines. However, in our previous study (Zhao et al., 2003), DCJW was more potent than indoxacarb in blocking mammalian Na+ channels, and both acted in a manner similar to pyrazolines in crayfish axons (Salgado, 1992). The present study was undertaken to better characterize the mechanism of the blocking action of indoxacarb insecticides on cockroach Na+ channels.
MATERIALS AND METHODS Isolation of Cockroach Neurons The American cockroaches, Periplaneta americana L., were purchased from Carolina Biological Supply Company (Burlington, NC), and were reared in the dark at 29 8C on rat chow (Ralston Purina Company, St. Louis, MO) and water. Neurons from three thoracic ganglia of an adult male cockroach were prepared according to a modified version of the technique of Pinnock and Sattelle (1987). Briefly, three thoracic ganglia were isolated, desheathed and incubated for 20 min in cockroach saline solution containing collagenase (type-IA, 0.5 mg/ml, Sigma–Aldrich, St. Louis, MO) and hyaluronidase (type-IS, 1 mg/ml, Sigma–Aldrich) at room temperature (22 8C). The ganglia were then rinsed three times with cockroach saline solution, and mechanically dissociated by repeated gentle suctions using a series of Pasteur pipettes of decreasing tip diameter. Cells were allowed to settle on glass coverslips pre-coated with poly-Llysine hydrobromide (MW 70,000–150,000, Sigma) for about 1 h. The coverslip was then transferred into the recording chamber and perfused with the external solution. The neurons were prepared on the day of experiments. Solutions The cockroach saline solution for cell isolation contained (in mM): NaCl 200, KCl 3.1, CaCl2 5, MgCl2 4, Sucrose 50 and HEPES-acid 10. The pH was adjusted to 7.2 with NaOH and the osmolarity was
400 mOsm. For the patch clamp current recording experiments, the external solution superfusing the cells contained (in mM): NaCl 100, tetraethylammonium (TEA)-Cl 100, KCl 3.1, CaCl2 2, MgCl2 7,4-aminopyridine (4-AP) 3 and HEPES-acid 10. The pH was adjusted to 7.3 with TEA-OH and the osmolarity was 400 mOsm. The internal solution in the recording pipette contained (in mM): NaCl 15, CsCl 80, CsF 80, EGTA 5, HEPES-acid 10, ATP-Mg2+ 2 and MgCl2 1. The pH was adjusted to 7.3 with CsOH and the osmolarity was 400 mOsm. Electrophysiological Recordings and Data Analysis Currents were recorded using the whole-cell patch clamp technique at room temperature (22–24 8C). Large neurons, 50–100 mm in diameter with pyriform bodies, were selected. Recording electrodes were made of borosilicate glass capillary tubes (1.5–1.8 mm inner diameter, Kimble, Vineland, NJ) using a two-step vertical puller (Narishige, Tokyo, Japan), and coated with dental wax to reduce the capacitive currents. The resistance of pipette was 1.0–1.5 MV, when filled with the internal solution. An Ag–AgCl pellet/3 M KCl– agar bridge was used for the reference electrode. Membrane currents were recorded using an Axo-Patch 200A amplifier (Axon Instruments, Union City, CA). Signals were digitized by a 14-bit analog-to-digital converter, filtered with a Bessel filter at 5 kHz and stored on a PC computer. Series resistance was compensated 70–75%. Capacitive and leakage currents were digitally subtracted using the P-P/4 procedure. The liquid junction potential between internal and external solutions was 4 mV on an average, which was used to compensate for the membrane potential. All data are expressed as mean S.E.M. and n represents the number of the cells examined. All figures represent typical examples from at least three independent experiments. The statistical significance between two samples was tested by Student’s t-test. Chemicals Indoxacarb [indeno(1,2-e)(1,3,4)oxadiazine-4a (3H)-carboxylic acid, 7-chloro-2,5-dihydro-2-(((methoxycarbonyl)-((4-trifluoromethoxy) phenyl)amino)carbonyl)-methyl ester] (DPX-MP062, 75:25 mixture of active and inactive enantiomers) and its metabolite DCJW were provided by E.I. DuPont de Nemours (Newark, DE), and were first dissolved in dimethylsulfoxide to prepare the stock solutions. The stock solutions (1 or 10 mM) were then diluted to desired
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concentrations with the external solution shortly before experiments. The final concentrations of dimethylsulfoxide in test solutions were 0.1% (v/v) or less, which had no effect on the Na+ currents when applied externally. Because both indoxacarb and DCJW are highly hydrophobic compounds, our drug application system was built with Teflon tubing and glass syringes, and was cleaned with alcohol after each experiment to prevent the contamination.
RESULTS Two Types of Na+ Currents The cockroach thoracic ganglia contain neurons of different sizes (10–100 mm) and different shapes. Most of them are with mainly round or pyriform somata with axon remnants. Na+ currents were rarely observed in small round-shaped neurons or large neurons without axon remnants, but were reliably recorded in large pyriform neurons. This is consistent with the fact that voltage-gated Na+ channels are mainly located on the axon or the initial segment of the soma, instead of the apical pole of cockroach DUM neurons (Amat et al., 1998; French et al., 1993). Therefore, in the present study the pyriform neurons with axon remnants were selected for experiments. Three types of inward currents were induced by step depolarization to 10 mV from a holding potential of 100 mV in cockroach neurons. One type of current
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was characterized by a fast onset and fast decay (Fig. 1A). This current disappeared reversibly by eliminating Na+ ions from the external solution (Fig. 1B and C). It was completely blocked by the Na+ channel blocker tetrodotoxin (TTX) at 400 nM (Fig. 1E), but not by the calcium channel blocker CdCl2 at 50 mM (Fig. 1D). Therefore, this type of current was carried by Na+ ions, and was designated the type-I Na+ current. In most neurons, a second type of Na+ current was found to co-exist with a current which had slow onset and slow decay and was blocked by 50 mM CdCl2 or elimination of external Ca2+ but not by 400 nM TTX (Fig. 2Aa). The fast transient component, but not the slow component, disappeared either by eliminating Na+ ions from the external solution (Fig. 2Ab) or by applying 400 nM TTX (Fig. 2Cb). The slow component, but not the fast component, of the current was reversibly eliminated by the removal of external Ca2+ or by 50 mM CdCl2 (Fig. 2Cc). Therefore, the fast component is the Na+ current through the voltagegated Na+ channel, whereas the slow component is the calcium current through the voltage-gated calcium channel (Fig. 2B and D). The Na+ current obtained in the presence of 50 mM CdCl2 was designated as the type-II Na+ current. It differed from type-I Na+ current. Inactivation of Type-I and Type-II Na+ Currents Na+ channels undergo two kinetically distinct types of inactivation: fast inactivation and slow inactivation, which form the basis for differentiating type-I and
Fig. 1. Characteristics of type-I Na+ currents recorded from a cockroach thoracic ganglion neuron. (A) The fast inactivating transient current evoked by a step depolarization to 10 mV from a holding potential of 100 mV, in the presence of 100 mM Na+ in external solution. (B) Current recorded following superfusion of Na+-free solution. (C) Recovery of the current after re-superfusion of 100 mM Na+ solution. (D) The current was not changed by bath application of 50 mM CdCl2. (E) The current was quickly blocked by 400 nM TTX. (F) Current recovered after washing with a TTX-free 100 mM Na+ solution.
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Fig. 2. Mixed type currents comprising Na+ and Ca2+ currents recorded from cockroach thoracic ganglion neurons. The currents were evoked by 30-ms test pulses to 10 mV, at a holding potential of 100 mV. (A) With normal external solution containing 100 mM NaCl, the current showed a fast transient component and a slow component (trace Aa). The fast component was eliminated when Na+ was replaced by TMA-Cl (trace Ab). The remaining slow component was eliminated by 50 mM CdCl2 (trace Ac). (B) Subtraction of Ab from Aa yields Na+ current (type-II). Subtraction of Ac from Ab reveals Ca2+ current. (C) Current (Ca) recorded from another neuron with normal external solution also exhibited fast and slow components. The fast component was removed by 400 nM TTX (trace Cb), whereas the slow component was removed by 50 mM CdCl2 (trace Cc). The entire current was completely blocked by TTX and CdCl2 (trace Cd). (D) Na+ and Ca2+ currents were obtained by subtraction of Cd from Cc and Cd from Cb, respectively.
type-II Na+ currents. In the present study, the fast inactivation was studied by applying a series of 150ms conditioning pulses ranging from 120 to +20 mV (in steps of 10 mV) that were long enough to allow the inactivation to reach the steady state level. Each conditioning pulse was followed by a repolarization to 100 mV for 1 ms, and then a 30 ms test pulse to 10 mV. The fast steady state inactivation curve, obtained by plotting the normalized peak Na+ current
against the conditioning potential, is shown in Fig. 3A. The slow steady state inactivation curve was obtained by applying longer conditioning pulses (1 min), as shown in Fig. 3B. Type-I and type-II Na+ currents differed in both fast steady state inactivation and slow steady state inactivation. The mid-point potentials for inactivation (V0.5 s) were more negative for type-I than for type-II Na+ currents, for both fast and slow inactivation (Fig. 3).
Fig. 3. Fast and slow steady state inactivation of type-I and type-II Na+ channels in cockroach thoracic ganglion neurons. The steady state fast inactivation data (A) were obtained from currents evoked by 30 ms test pulses to 10 mV following 150-ms conditioning pulses ( 120 to +20 mV) from a holding potential of 100 mV. The steady state slow inactivation data (B) were obtained from currents evoked by 30-ms test pulses to 10 mV, following a longer (1 min) conditioning pulses ( 130 to 60 mV for type-I and 100 to 30 mV for type-II) from a holding potential of 100 mV. The peak amplitudes of Na+ currents associated with test pulses were normalized to its respective maximum value, and are plotted as a function of the conditioning potential. The curves are drawn according to the equation I/Imax = 1/{1 + exp[(V V0.5)/k]}, where V is conditioning potential, V0.5 is the potential at which the current reduces to half maximum (Imax), and k is the slope factor (potential required for an e-fold change). The voltage dependence of inactivation occurs at more negative potentials for type-I Na+ channels than for type-II Na+ channels.
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The V0.5 values for fast inactivation were 64.3 and 39.8 mV for type-I and type-II Na+ currents, respectively. The V0.5 values for slow inactivation were 86.8 and 52.7 mV for type-I and type-II Na+ currents, respectively. Indoxacarb Insecticides on Type-I Na+ Currents DCJW, a metabolite of indoxacarb, was recognized as an active component for the insecticidal activity, and blocked the compound action potentials and Na+ channels in both insect and mammalian neurons (Wing et al., 1998; Lapied et al., 2001; Sugiyama et al., 2001; Zhao et al., 2003). Fig. 4 shows a sharp contrast between indoxacarb and DCJW with respect to the reversibility of type-I Na+ current block. Na+ currents were evoked by step depolarizations to 10 mV from a holding potential of 100 mV, at an interval of 1 min. After several stable control recordings had been established, indoxacarb or DCJW at a concentration of 1 mM was applied in the bath. The amplitude of Na+ currents was quickly suppressed by 77.1 5.3% (n = 4) and 78.3 11.6% (n = 4) by indoxacarb and DCJW, respectively. However, the Na+ current suppression caused by indoxacarb was completely reversible after washout, whereas the current suppression caused by DCJW was irreversible 10 min after washout with insecticide-free media. Similar results were obtained in all the four cells tested. The irreversible nature of DCJW block of type-I Na+ currents was further corroborated by experiments in which the holding potential was changed (Fig. 5). Before application of DCJW, a change in holding
Fig. 5. Time course and irreversibility of blocking action of 100 nM DCJW on type-I Na+ channels. Currents were evoked by test pulses to 10 from 100 or 80 mV holding potentials, at an interval of 1 min. The Na+ currents were smaller at a holding potential of 80 mV than at 100 mV due to Na+ channel inactivation. DCJW at a concentration of 100 nM was applied at 80 mV for 10 min to produce a nearly complete block. No recovery was seen by a 10-min washout with a normal external solution or by hyperpolarization to 100 mV.
potential from 100 to 80 mV decreased the current amplitude as expected from the steady state inactivation curve (Fig. 3). The current amplitude at a holding potential of 80 mV was further decreased but the time to peak of the current was not changed during the bath perfusion of 100 nM DCJW. Washout with DCJW-free solutions caused no recovery. Changing the holding potential from 80 to 100 mV still failed to resurrect the Na+ current. Indoxacarb Insecticides on Type-II Na+ Currents
Fig. 4. The differential actions of indoxacarb and DCJW on type-I Na+ channels of cockroach thoracic ganglion neurons. Currents were evoked by a 10-ms step depolarization to 10 mV from a holding potential of 100 mV at an interval of 1 min. The peak current was inhibited by 77.1 5.3% (n = 4) by 1 mM indoxacarb and 84.1 5.3% (n = 3) by 1 mM DCJW. Upon washing with insecticide-free solution, the block by indoxacarb was reversed, while the block by DCJW was not reversed.
Type-II Na+ currents were isolated from the mixed type of currents by application of 50 mM CdCl2 to block the Ca2+ currents, as shown in the insets of Fig. 6. In contrast to type-I Na+ currents, type-II Na+ currents were unaffected by either 1 mM indoxacarb or 1 mM DCJW applied in the bath for 10 min (Fig. 6), when the Na+ current was evoked from the membrane holding potential of 100 mV. Previous studies demonstrated that the blocking action of the pyrazoline-type insecticides, indoxacarb and DCJW, on Na+ channels was voltage-dependent in
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Fig. 6. Indoxacarb (A) and DCJW (B) had no effect on type-II Na+ channels at a concentration of 1 mM at 100 mV membrane potential. Control Na+ currents were recorded with 10 mV test pulses from a holding potential of 100 mV in the presence of 50 mM CdCl2 at an interval of 5 s. Indoxacarb or DCJW was applied through bath perfusion for 10 min. During drug application, the Na+ currents were monitored at an interval of 30 s. The insets show the typical Na+ current traces before (dotted lines) and after (solid lines) bath perfusion of indoxacarb (A) or DCJW (B).
crayfish giant axons and rat dorsal root ganglion neurons (Salgado, 1992; Zhao et al., 2003). However, a recent study of DCJW using cockroach ganglion neurons did not support the above findings (Lapied et al., 2001). To further resolve the discrepancy of voltagedependent block by indoxacarb insecticides on cockroach Na+ channels, type-II Na+ currents were recorded at different holding potentials ( 100, 80, 60 and 40 mV) at an interval of 5 s. The sample traces of type-II Na+ currents recorded at different holding potentials in the absence and presence of insecticides are shown in Fig. 7A. In the absence of the insecticides, the peak Na+ current began to decrease progressively after changing the holding potentials from 100 to 80, 60 and 40 mV (open circles in Fig. 7B and C). However, in the presence of indoxacarb or DCJW at a concentration of 1 mM, the decline in Na+ currents was accentuated, especially at holding potentials of 60 and 40 mV (filled circles in Fig. 7B and C). Thus, the degree of block of Na+ current by a given concentration of indoxacarb or DCJW was modulated by changing the holding potential. The results clearly showed that the blocking action of indoxacarb insecticides on type-II Na+ currents was voltage-dependent. Indoxacarb Insecticides on Na+ Channel Inactivation To further characterize the voltage-dependent blocking action of indoxacarb, type-II Na+ currents were evoked by step depolarizations to 10 mV at an interval of 5 s from sequential changes in holding potentials
of 100, 50, and back to 100, in the absence of test drug. The Na+ current gradually decreased in amplitude as the holding potential changed from 100 to 50 mV (Fig. 8A, opened circles). The peak amplitude of Na+ currents was not changed when the neurons were treated with indoxacarb at 100 nM for 10 min at a holding potential of 100 mV. However, indoxacarb treatment exacerbated the decrease in Na+ currents when the holding potential was changed from 100 to 50 mV (Fig. 8A, filled circles). The Na+ current in the presence of indoxacarb was completely restored by switching the holding potential from 50 to 100 mV (Fig. 8A). However, simple washout of indoxacarb at a holding potential of 50 mV did not cause any recovery of currents (Fig. 8B). Complete recovery was observed only when the holding potential was brought back to 100 mV (Fig. 8B). Similar results were obtained with DCJW (Fig. 9). Following bath perfusion of 1 mM DCJW for 10 min, the peak amplitude of Na+ currents was not changed at a holding potential of 100 mV, but was decreased at 50 mV. The decreased amplitude of Na+ currents at 50 mV recovered upon hyperpolarization from 50 to 100 mV, but was not restored by washout of DCJW at 50 mV. The above results of voltage dependency of block clearly suggest that indoxacarb insecticides enhance the inactivation of type-II Na+ channel in cockroach neurons. In addition, the different inhibitory effects of indoxacarb insecticides on type-I and type-II Na+ currents at 100 mV holding potential could be due to a high affinity binding of insecticides to the inactivated states of the channel, because the type-I Na+
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inactivation were analyzed using the protocol shown in Fig. 3. Indoxacarb or DCJW was applied in the bath for 15 min at a concentration of 100 nM at a holding potential of 100 mV. Fig. 10A illustrates the effects of indoxacarb on the voltage dependence of fast and slow inactivation of type-II Na+ currents. Indoxacarb significantly shifted the mid-point of the slow inactivation from 55.1 2.2 to 61.1 1.9 mV (n = 5, p < 0.05) while its effect on the fast inactivation was not significant with the mid-point of inactivation changing from 43.4 1.3 to 46.6 1.9 mV (n = 4, p > 0.05). Similarly, DCJW shifted the slow inactivation curve significantly from 56.1 3.4 to 72.3 3.3 mV (n = 4, p < 0.05), and its shift of the fast inactivation curve from 41.6 2.9 to 45.0 3.4 mV was not significant (n = 5, p > 0.05). The effects of DCJW on Na+ inactivation of type-I Na+ currents were also studied. The slow inactivation curve was significantly shifted by DCJW changing from 88.8 1.5 to 97.1 1.6 mV (n = 3, p < 0.05), whereas the shift of the fast inactivation curve from 67.3 1.5 to 71.5 0.7 mV was not significant (n = 5, p > 0.05) (Fig. 10B). The slope factors of fast and slow inactivation curves in both types of Na+ currents were not changed by indoxacarb insecticides (Fig. 10). These results suggested that indoxacarb insecticides induced the voltage-dependent inhibition of type-I and type-II Na+ currents by selective binding to the slow-inactivated state of the channel.
DISCUSSION Two Kinds of Na+ Currents Fig. 7. Inhibition of type-II Na+ channels by 1 mM indoxacarb and DCJW is dependent on holding potentials. Na+ currents were evoked by test pulses to 10 mV from various holding potentials ( 100, 80, 60 and 40 mV) in the presence of 50 mM CdCl2. Currents were recorded at an interval of 5 s in the absence (opened circles) or presence (filled circles) of insecticides. Indoxacarb or DCJW at 1 mM was applied in bath for 10 min before recording the currents. Representative Na+ currents are shown for control (Aa) and in the presence of 1 mM DCJW (Ab). The results shown here (B and C) were compiled from different neurons and the currents in the presence of insecticides were normalized to the one at 100 mV. Data are expressed as mean S.E.M. Current suppression in the presence of 1 mM indoxacarb (B) or DCJW (C) were accentuated by holding potentials, especially at 60 and 40 mV.
channel undergoes more inactivation at 100 mV than does the type-II Na+ channel, as shown in Fig. 3. In order to verify the validity of the above notion of the role of Na+ channel inactivation in the action of indoxacarb insecticides, the steady state fast and slow
Voltage-gated Na+ channels in dorsal unpaired median neurons of the cockroach are located mainly on the axon and the initial segment of the soma, as shown by immunocytochemical staining (French et al., 1993; Amat et al., 1998). The physiological and pharmacological properties of the fast voltage-gated Na+ currents in both giant axons and dorsal unpaired median neurons of the cockroach were studied using voltage clamp techniques (Sattelle et al., 1979; Pelhate et al., 1979; Lapied et al., 1990; Grolleau and Lapied, 2000). In the present study, two kinds of Na+ currents (type-I and type-II) were recorded in cockroach thoracic ganglion neurons. Type-I and type-II Na+ currents completely disappeared by elimination of Na+ ions from the external solution and were sensitive to the blocking action of TTX. Type-II but not type-I Na+ currents were always accompanied by a slow component of current
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Fig. 8. Indoxacarb inhibition of type-II Na+ channels can be reversed by hyperpolarization. Na+ currents were evoked by step depolarizations to 10 from 100 or 50 mV holding potential in the presence of 50 mM CdCl2. (A) Control Na+ currents were first recorded at 100 mV, then at 50 mV holding potential for 2.5 min at an interval of 5 s (open circles). Following complete recovery from channel inactivation after switching the holding potential back to 100 mV, neurons were treated with 100 nM indoxacarb for 10 min at a holding potential of 100 mV. Na+ currents were then recorded using the same protocol as above (solid circles). Indoxacarb had no effect on type-II Na+ current when the neuron was held at 100 mV and exerted a potent inhibition when it was held at 50 mV. (B) Time-course of changes in Na+ currents during and after application of indoxacarb at holding potentials of 100 and 50 mV. Recovery from indoxacarb block was not seen after washout at a holding potential of 50 mV, but occurred at a holding potential of 100 mV during the application of indoxacarb and after its washout.
that could be eliminated by application of Cd2+ or Ca2+-free external solutions. It was concluded that the slow component of current was generated by Ca2+ channels. Type-I and type-II Na+ currents exhibited differential inactivation properties. The steady state fast and slow inactivation of type-I Na+ channels occurred at more negative membrane potentials than did in type-II Na+ channels. The existence of two types of Na+ channels was reported in the cockroach giant axon (Yawo et al., 1985) and dorsal unpaired median TAG
Fig. 9. DCJW inhibition of type-II Na+ channels can be reversed by hyperpolarization. Na+ currents were recorded with the protocol as described in Fig. 8. Following recovery from channel inactivation after switching the holding potential from 50 back to 100 mV, neurons were treated with 1 mM DCJW for 10 min at 100 mV holding potential without channel activation. Na+ currents were then recorded using the same protocol as above. The peak amplitude of Na+ currents was not changed by DCJW at 100 mV holding potential, but was quickly reduced at 50 mV holding potential. Na+ currents largely recovered from inactivation and DCJW inhibition by changing the holding potential from 50 to 100 mV, but no recovery from DCJW occurred after washout at a holding potential of 50 mV.
neurons (Lapied et al., 1990). Under voltage clamp conditions (Lapied et al., 1990), in about 80% of the cells tested, the transient Na+ currents were reported with half-inactivation at 41.1 mV and a saxitoxinsensitive maintained Na+ current was observed in the identical remaining cells. Our type-II Na+ currents might be similar to the transient Na+ current reported by Lapied et al. (1990) because the mid-point for the fast inactivation were nearly identical. Functional diversity and tissue-specific distributions of the Na+ channels in mammals are achieved by selective expression of at least nine different Na+ channel genes (Goldin, 2002). In insects, however, only one functional Na+ channel gene was reported, i.e. para in Drosophila melanogaster (Warmke et al., 1997; Zhou et al., 2004). Recent studies have demonstrated that alternative splicing and RNA-editing of a cockroach Na+ channel gene generate a wide array of functionally and pharmacologically distinct Na+ channels (Tan et al., 2002; Song et al., 2004; Liu et al., 2004). It is possible that the two types of Na+ currents identified in our study and those from other laboratories are generated by two splicing and/or RNA-editing variants of a Na+ channel gene in Periplaneta americana. Sensitivity of Two Types of Na+ Currents to Indoxacarb Insecticides Both type-I and type-II Na+ currents were TTXsensitive, but had differential sensitivity to the blocking action of indoxacarb insecticides. Type-I and type-II
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Fig. 10. Modification of steady state inactivation of type-II (A) and type-I (B) Na+ channels by indoxacarb and DCJW. The voltage dependencies of steady state fast inactivation and slow inactivation of both type-II and type-I Na+ channels were measured and analyzed by the method, as illustrated in Fig. 3. Following exposure to 100 nM indoxacarb (A, filled squares) or 100 nM DCJW (B, filled circles) for 15 min at a holding potential of 100 mV, the fast and slow inactivation curves were compared to the corresponding control (A, opened squares; B, opened circles). DCJW and indoxacarb caused a significant hyperpolarizing shift of slow steady state Na+ channel inactivation curve.
Na+ currents in cockroach neurons responded differently to the blocking action of indoxacarb and its metabolite DCJW. The blocking effect of indoxacarb on Na+ currents depended largely on the membrane potential, as observed in crayfish axons for pyrazoline block (Salgado, 1992). However, the sensitivity of type-I Na+ currents to indoxacarb and DCJW was different from that of type-II Na+ currents. At a membrane potential of 100 mV, where type-II Na+ channels were not inactivated while type-I Na+ channels underwent substantial inactivation, type-I Na+ currents were blocked by indoxacarb or DCJW at a concentration as low as 100 nM. In contrast, type-II Na+ currents were not blocked by indoxacarb or DCJW at 100 mV, and were blocked only at less negative potentials ranging from 60 to 40 mV, where some inactivation occurred. The voltage-dependent suppression of Na+ currents by indoxacarb insecticides is very similar to that in rat dorsal root ganglion neurons (Zhao et al., 2003). The hyperpolarization shift in voltage dependence of slow inactivation suggests that insecticides bind to the inactivated state with a higher affinity than to the resting state of the Na+ channel. Therefore, the differential sensitivity of two types of Na+ currents to indoxacarb and DCJW is largely due to the different voltage dependence of slow inactivation.
Different Insecticidal Potency of Indoxacarb and DCJW Indoxacarb was much weaker than its metabolite DCJW as an insecticide (Tsurubuchi et al., 2001a). Studies using extracellular recording electrodes showed that DCJW, but not indoxacarb, appeared to be the active form in lepidopteran (Manduca sexta) larval motor nerve preparations (Wing et al., 1998). Previous studies using mammalian neurons also showed that indoxacarb was about 10 times less potent than DCJW in blocking TTX-sensitive and TTX-resistant Na+ channels (Zhao et al., 2003; Tsurubuchi et al., 2001b; Nagata et al., 1998). In the present study, both indoxacarb and DCJW strongly blocked type-I and type-II Na+ channels. Indoxacarb block of type-I Na+ channels at 100 mV membrane potential was reversible, while DCJW block was irreversible. The difference in reversibility between indoxacarb and DCJW may be partially responsible for the higher insecticidal activity of DCJW as compared to indoxacarb. In conclusion, the present study demonstrated that indoxacarb and its metabolite DCJW blocked the two types of Na+ channels of cockroach neurons in a manner dependent on the membrane potential. The steady state inactivation curve of type-I Na+ channels
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has its voltage dependence resided at the membrane potential more hyperpolarized than the type-II Na+ channels. The voltage dependence of block caused by indoxacarb and DCJW was due to their higher affinity for the inactivated state than for the resting state of the channel. The observation that indoxacarb block was reversible after washout while DCJW block of type-I Na+ channels was irreversible might provide an explanation for the more potent insecticidal activity of DCJW.
ACKNOWLEDGEMENTS This study was supported by National Institutes of Health grant NS14143. Samples of indoxacarb (DPXMP062) and its metabolite, DCJW, were provided by E.I. DuPont de Nemours and Company, Newark, Delaware. The authors wish to thank Dr. Ke Dong (Michigan State University, MI) for the helpful discussion on insect sodium channels. The authors thank Nayla Hasan and Sandra Guy for technical assistances and Julia Irizarry for secretarial assistance.
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