Modulation of nerve membrane sodium channel activation by deltamethrin

Brain Research, 584 (1992) 71-76 Elsevier Science Publishers B.V.

71

BRES 17886

Modulation of nerve membrane sodium channel activation by deltamethrin Leslee

D. Brown

* and Toshio

Narahashi

Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611 (USA) (Accepted 11 February 1992)

Key words: Deltamethrin; Sodium channel; Pyrethroid; Squid axon

Deltamethrin is a highly potent pyrethroid insecticide that causes hypersensitivity, choreoathetosis, tremors, and paralysis in mammals. It is known to modify the sodium channel in such a way as to prolong the tail current associated with step repolarization following a depolarizing pulse. Using the axial-wire voltage-clamp technique with the giant axon of the squid Loligo pealei, we have demonstrated that deltamethrin also greatly slows the opening of the sodium channel. This was first observed as a decrease, by as much as 80%, in the peak sodium current flowing during a short, 10 ms depolarization. Current flowing through these slowly opening deltamethrin modified sodium channels was observed during the first depolarizing pulse after deltamethrin exposure and developed with a time constant of 320 ms. This supports the idea that deltamethrin can modify sodium channels when they are in the closed or resting state. Further, evidence of this hypothesis was provided by experiments using 0.1 and 10 #M deltamethrin and measuring the tail current amplitude after depolarizing pulses of varying duration (1-1200 ms). The mean time constant for the increase in tail current amplitude was almost concentration independent; 253 ms at 0.1/zM and 193 ms at 10 tzM. We conclude that deltamethrin modifies the activation kinetics of sodium channels in such a way as to slow opening and that this modification occurs predominantly when channels are in the closed or resting state.

INTRODUCTION A v a r i e t y o f c h e m i c a l s a r e k n o w n to i n t e r a c t with t h e n e r v e m e m b r a n e s o d i u m c h a n n e l resulting in c h a n g e s in g a t i n g kinetics. T h e s e c h e m i c a l s can b e classified into two l a r g e c a t e g o r i e s , b l o c k e r s a n d m o d u l a t o r s 4'15. Blocking a g e n t s i n c l u d e t h e w a t e r - s o l u b l e h e t e r o c y c l i c g u a n i d i n e s , t e t r o d o t o x i n a n d saxitoxin, a n d local anesthetics. C h e m i c a l s t h a t m o d i f y t h e g a t i n g kinetics o f s o d i u m c h a n n e l s i n c l u d e lipid s o l u b l e polycyclic c o m p o u n d s , such as v e r a t r i d i n e , aconitine, grayanotoxin, and batrachotoxin, and the pyrethroid a n d D D T insecticides. T h e p r e s e n t study is c o n c e r n e d with t h e c h a r a c t e r i s t i c s o f the m o d i f i c a t i o n o f the gating kinetics o f squid axon s o d i u m c h a n n e l s by t h e highly p o t e n t p y r e t h r o i d insecticide, d e l t a m e t h r i n (Fig. 1). T h e p y r e t h r o i d insecticides can b e d i v i d e d into two classes b a s e d o n the c h e m i c a l structure. T h e t y p e I p y r e t h r o i d s such as a l l e t h r i n a n d t e t r a m e t h r i n lack a

c y a n o g r o u p at t h e a - p o s i t i o n on t h e carboxyl moiety, a n d have b e e n t h o r o u g h l y i n v e s t i g a t e d as to t h e m e c h a n i s m o f t h e i r n e u r o t o x i c i t y 3,12-14,16-2°,22-26,3°. T h e m o d i f i c a t i o n o f s o d i u m c h a n n e l g a t i n g kinetics c a u s e d by t h e type I p y r e t h r o i d s is well c h a r a c t e r i z e d at b o t h t h e w h o l e cell c u r r e n t 9,1°,27,28 a n d t h e single c h a n n e l level 32'33. H o w e v e r , t h e m e c h a n i s m o f action o f t h e t y p e II p y r e t h r o i d s a p p e a r s to b e m o r e c o m p l e x t h a n t h a t o f t h e t y p e I p y r e t h r o i d s . C h i n n a n d N a r a h a s h i 5,6 d e m o n s t r a t e d in single s o d i u m c h a n n e l studies with n e u r o b l a s t o m a cells t h a t d e l t a m e t h r i n , a type II p y r e t h r o i d , stabilizes a variety o f c h a n n e l states by r e d u c i n g t h e t r a n s i t i o n r a t e s b e t w e e n them, resulting

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Fig. 1. Structure of deltamethrin.

* Present address: Chemical Abstracts Service, 2540 Olentangy River Road, P.O. Box 3012, Columbus, OH 43210, U.S.A. Correspondence: Toshio Narahashi, Department of Pharmacology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, U.S.A. Fax: (1) (312) 5349.

72 in p r o l o n g e d openings. M o r e drastic p r o l o n g a t i o n of single sodium c h a n n e l o p e n i n g up to several seconds was observed after exposure of n e u r o b l a s t o m a cells to fenvalerate, a n o t h e r type II pyrethroid s. Also Brown

-20mV -lOOJ

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a n d N a r a h a s h i 2 have shown that d e l t a m e t h r i n greatly increases the time c o n s t a n t of s o d i u m c h a n n e l deacti-

i ~ Deltamethrin Peak

vation in squid axons; this also suggests slowing of the activation kinetics. A n o t h e r i m p o r t a n t q u e s t i o n is c o n c e r n e d with the c h a n n e l state in which pyrethroid modification takes place. E a r l i e r studies with squid a n d crayfish giant axons suggested that t e t r a m e t h r i n , a type I pyrethroid, modified the s o d i u m c h a n n e l in both closed a n d o p e n

1400uAlcmz

Fig. 2. Peak and steady-state currents as induced by 8 ms step depolarizing pulses to -20 mV from a holding potential of -100 mV before and after application of 10/~M deltamethrin.

in the closed or resting state.

cally. Exponential fits of the slowly developing sodium current flowing through the deltamethrin-modified sodium channels were computer-generated using a nonlinear least-squares fitting routine. Data are reported as mean + S.E.M. The deltamethrin sample was obtained from Roussel UCLAF (Marseille, France) and was composed of the active isomer, RU 22974, [(1R- l a( S * )-3a )]cyano(3-phenoxyphenyl)methyl-3-(2,2-dibromoethyenyl)-2,2-dimethylcyclopropane carboxylate (Fig. 1). A stock solution was prepared in dimethylsulfoxide (DMSO) at a concentration of 0.01 M and was stored below 0°C. The test solution was prepared by adding a small aliquot of the stock solution to the internal perfusate and shaking the mixture vigorously. This procedure resulted in a fine emulsion whose 'concentration' is given in molar units, although the actual amount of pyrethroid in solution was unknown. The test solution was kept in the dark until immediately prior to perfusion of the axon. The final DMSO concentration in the perfusate was less than 1.0% (v/v) and had no effect on the sodium currents. The other chemicals used were obtained from the followingsources: TTX, Sankyo Co. (Tokyo);TMA-CI and TMA-OH; Sigma Chemical Co. (St. Louis, MO); and CsOH, Aldrich Chemical Co. (Milwaukee, WI).

MATERIALS AND METHODS

RESULTS

states 9'1°. Studies with frog n o d e s of R a n v i e r suggested that b o t h type I a n d type II pyrethroids preferentially interfere with the s o d i u m c h a n n e l s in o p e n configuration 2s. However, our m o r e recent study with squid giant axons favors p r e d o m i n a n t l y closed c h a n n e l modification by p h e n o t h r i n a n d c y p h e n o t h r i n , although some c h a n n e l s are modified in their o p e n state zg. A n a l y s e s of single s o d i u m c h a n n e l s in the p r e s e n c e of f e n v a l e r a t e have also led to the c o n c l u s i o n of closed c h a n n e l m o d i f i c a t i o n s. T h e p u r p o s e of the p r e s e n t work was to characterize the slow activation kinetics of s o d i u m c h a n n e l s exposed to d e i t a m e t h r i n . T h e data p r e s e n t e d also suggest that d e l t a m e t h r i n m o d i f i c a t i o n of s o d i u m c h a n n e l s occurs

Experiments were performed with giant axons isolated from the squid Loligo pealei at the Marine Biological Laboratory, Woods Hole, MA. Axons were isolated, cleaned, and perfused internally using the roller method originally developed by Baker et al. 1 and modified by Narahashi and Anderson 21. Potassium-free artificial sea water was used as the external bathing medium and contained (mM): NaCI, 445; tetramethylammonium-Cl (TMA-CI), 10; CaCI2, 50; and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 5. pH was adjusted to 8.0 with 1 M NaOH. In order to avoid polarization of the axial wire and distortion of the membrane sodium current by accumulation or depletion of sodium ions in the periaxonal space during prolonged depolarizations, 70% to 80% of the peak sodium current was blocked by 4 nM tetrodotoxin (TTX) in the external solution. In experiments where TTX was not used, these problems were eliminated by using equimolar concentrations of sodium in both internal (50 mM) and external (50 mM) solutions and applying step depolarization to 0 inV. The internal perfusate contained (mM): CsOH, 250; glutamic acid, 250; NaF, 20; sucrose, 400; and sodiumphosphate buffer, 30. pH was adjusted to 7.3 with 1 M CsOH. The concentration of deltamethrin in the internal perfusate was 10/xM. The membrane potential and membrane current were recorded at 8-9°C using the axial-wire voltage-clamp method of Wu and Narahashi 31 with the modifications of Lund and Narahashi 1°. The holding potential for all experiments was - 100 mV. Command pulses, data acquisition and data analysis were controlled and performed by a PDP 11/23 computer (Cambridge Digital, Boston, MA). The membrane sodium current was fed into an analog/digital converter (14 bits resolution at a maximum sampling rate of 100 kHz), and the digitized data were stored on floppy diskettes. Leakage and capacitive currents were subtracted electroni-

N o r m a l s o d i u m c h a n n e l s were activated a n d inactivated d u r i n g a n 8 ms d e p o l a r i z a t i o n from a h o l d i n g p o t e n t i a l of - 1 0 0 m V (Fig. 2). T h e c u r r e n t - v o l t a g e (l-V) r e l a t i o n s h i p for n o r m a l ( t r i a n g l e s ) a n d d e l t a m e t h r i n - m o d i f i e d (squares) s o d i u m c h a n n e l s is shown in Fig. 3. P e a k s o d i u m c u r r e n t t h r o u g h n o r m a l s o d i u m c h a n n e l s r e a c h e d a m a x i m u m at a m e m b r a n e p o t e n t i a l of - 1 0 m V in this axon. T h e I - F relationship for s o d i u m c u r r e n t after the axon h a d b e e n perfused i n t e r n a l l y with 1 0 / x M d e l t a m e t h r i n is also shown. T h e decrease in p e a k s o d i u m c u r r e n t after d e l t a m e t h r i n t r e a t m e n t is p r e s u m a b l y d u e to m o d i f i c a t i o n of a p o p u lation of the s o d i u m channels. As will be shown later, the modified s o d i u m c h a n n e l s exhibit altered activation kinetics, a n d do n o t o p e n within the short 8 ms depolarization t h e r e b y failing to c o n t r i b u t e to the p e a k of s o d i u m current. T o avoid p r o b l e m s associated with the p r o l o n g e d d e p o l a r i z a t i o n s necessary to study s o d i u m c u r r e n t s flowing t h r o u g h the d e l t a m e t h r i n - m o d i f i e d sodium channels, e x p e r i m e n t s were c o n d u c t e d in the p r e s e n c e of 4 n M q-TX in the external solution (see Materials

73 and Methods for further explanation). Fig. 4A shows a representative family of sodium currents flowing through the normally activated and inactivated sodium channels after successive 10 ms depolarizations over the range from - 8 0 mV to + 4 0 mV. A family of sodium currents associated with prolonged 1 s depolarizing pulses after internal perfusion of the axon with 10 /zM deltamethrin is shown in Fig. 4B. Successive 1 s depolarizations over the range from - 8 0 mV to + 40 mV generated slowly developing sodium currents flowing through the deltamethrin-modified sodium channels. The I - V relationships for the sodium current from an axon before (triangles) or after (squares) deltamethrin treatment are shown in Fig. 5. A peak sodium current of 540 / z A / c m 2 at - 1 0 mV was observed during the short 10 ms depolarizing step before deltamethrin treatment. The steady-state currents showed much smaller amplitudes. Since the peak of sodium current was not clearly recorded during the 1 s depolarization in the presence of deltamethrin, the sodium current flowing at the end of the 1 s depolarization during deltamethrin perfusion was m e a s u r e d and plotted in Fig. 5. The steady-state current was markedly increased after application of deltamethrin. The slowly developing sodium current flowing through the deltamethrin-modified sodium channels is shown in Fig. 6A. A depolarizing step of at least 1 s in duration from the holding potential of - 1 0 0 mV was necessary to observe the full opening of the modified channels. The sodium current shown in Fig. 6A was elicited during the depolarizing pulse applied for the

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Fig. 4. Representative families of sodium currents flowing through normal and deltamethrin-modified sodium channels. Axons were depolarized (10 ms in control and 1 s after deltamethrin) successively over the potential range from -80 mV to + 40 mV from a holding potential of -100 mV. A: sodium current flowing through normal sodium channels. Almost complete inactivation occurs during the short 10 ms depolarization. B: family of sodium currents in an axon internally perfused with 10/zM deltamethrin for 20-30 min. Depolarizing pulses were 6 s apart to allow for complete decay of the prolonged sodium tail current. The inward sodium current is not inactivated during the long 1 s depolarization. External and internal sodium concentrations were 445 and 50 mM, respectively, and 4 nM TFX was added to the external solution.

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Fig. 3. The current-voltage relationships for sodium current before (triangles) and after (squares) exposure to deltamethrin. The axon was depolarized to various levels for 8 ms from a holding potential of 100 mV. Peak sodium current and steady-state sodium current are plotted as a function of the membrane potential during depolarized pulses. Normally activating sodium channels exhibited peak current of 2.5 mA/cm2 at - 10 mV. An 80% decrease in peak current was observed after internal perfusion of the axon with 10 /zM deltamethrin. External and internal sodium concentrations were 445 and 50 raM, respectively. -

first time after beginning internal perfusion of 10/~M deltamethrin. A holding potential of - 100 mV is sufficiently negative to ensure that the sodium channels are closed during the period of deltamethrin perfusion. The presence of this slowly developing sodium current during the first depolarizing step suggests that these channels were modified in the closed or resting state. A semi-logarithmic plot of this current (Fig. 6B) shows a time constant (~-) of opening equal to 320 ms. In further support of the premise that sodium channels are modified by deltamethrin largely in the closed or resting state, was the finding that the time constant for opening of deltamethrin-modified sodium channels was almost concentration independent. An experiment was conducted using 0.1 /zM and 1 0 / z M deltamethrin in the internal perfusate. Fig. 7A and B show the sodium tail currents flowing through deltamethrin-

74 modified channels that remain open upon repolarization to the holding potential ( - 100 mV) after depolarizations of varying durations (1 to 1200 ms) to 0 mV. The development of the initial amplitude of slow tail currents with lengthening of the pulse duration is fit by a single exponential function as shown in Fig. 7C. The time constants of tail current development were estimated to be 188 ms with 0.1 ~ M deltamethrin and 180 ms with 10/xM deltamethrin in the experiment shown in Fig. 7. The mean time constants were 253 _+ 43 ms (n = 4) and 193 _+ 37 ms (n = 4) for 0.1/xM and 10 ~ M deltamethrin, respectively.

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Fig. 6. Slowly developing sodium current flowing through deltamethrin-modified sodium channels. A: a depolarizing pulse of 1 s to 0 mV from a holding potential of -100 mV elicited a peak current followed by a slowly developing sodium current. The latter current flows through deltamethrin-modified channels. The sodium current shown here was generated during the first depolarizing pulse after 10/zM deltamethrin perfusion. B: semi-logarithmic plot of the slow current shown in A reveals a time constant of 320 ms, which represents the time for the modified channels to open.

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DISCUSSION In the axon perfused internally with deltamethrin, the peak sodium current is suppressed in amplitude and is followed by a slowly developing current. The slow current is elicited by the depolarizing pulse applied for the first time following internal perfusion of deltamethrin while holding the membrane potential at -100 mV at which no opening of sodium channels occurs. Furthermore, the peak sodium current in the presence of deltamethrin is activated and inactivated with the normal time courses although the amplitude is reduced. This reduced peak current is deemed to be due to opening of the sodium channels which remain unmodified during exposure to deltamethrin. Thus the slowly developing current represents opening of the sodium channels modified by deltamethrin while the membrane is held hyperpolarized without opening the

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-600 Fig. 5. The current-voltage relationships for sodium current before (triangles) and after (squares) internal perfusion of 10 /zM deltamethrin. This experiment was conducted with 4 nM TTX in the external solution to block 70-80% of the sodium current in order to prevent polarization of the axial-wire electrode. In the control experiment, peak and steady-state sodium currents were determined during 10 ms depolarizing pulses. After deltamethrin exposure the axon was depolarized for a period of 1 s to determine modified sodium current at steady-state level. There was a 72% decrease in current as compared to control peak current.

sodium channels. Additional evidence for closed channel modification is provided by the experiment in which the rate at which the deltamethrin-modified slow tail current develops is almost independent of the concentration. This experimental result confirms and extends the previously reported study in which a narrower concentration range of deltamethrin was used 29. The question as to whether the sodium channels are modified by pyrethroids in the open or closed state has been a matter of controversy. Hille 7 proposed open channel modification by DDT, whose action on the sodium channels is very similar to that of the type I pyrethroids u'27. Lund and Narahashi 1° proposed that the sodium channels can be modified by the type I pyrethroid tetramethrin in both open and closed states. Vijverberg et al. 28 suggested that both type I and type I1 pyrethroids preferentially interfere with the sodium channels in open configuration. A recent study with

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In conclusion, in the presence of deltamethrin the sodium channels of squid axons are modified largely in the closed state and the modified channels open slowly during a step depolarization. This results in a drastic decrease in the peak amplitude of the transient sodium current observed upon step depolarization. Acknowledgements. This study was supported by a grant from the

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National Institutes of Health (NS14143). Leslee D. Brown was supported by a National Research Service Award Postdoctoral Fellowship from the National Institutes of Health (ES05334). We thank Fukeun E. Chen for maintenance of the computer system and Vicky James-Houff for secretarial assistance.

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REFERENCES

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PULSE DURATION (AT)(msec) Fig. 7. Time constant for development of deltamethrin-modified sodium tail current is concentration independent as measured by the pulse protocol shown at the top. A and B show sodium tail currents flowing through sodium channels after perfusion of the axon with 0.1 /zM and 10 p.M deltamethrin, respectively. C: plot of sodium tail current amplitude after depolarizing pulses of varying duration (11200 ms). The data were fit by single exponential functions with time constants of 188 ms after 0.1 /xM and 180 ms after 10 # M deltamethrin, respectively.

the type I pyrethroid, phenothrin, and the type II pyrethroids, cyphenothrin and deltamethrin, concluded that the pyrethroid modification of the sodium channels occurs predominantly in the closed state 29. The rate at which the sodium channels were modified during a depolarization was largely independent of the pyrethroid concentration. More recently, a single channel study lends support to the notion of the closed channel modification caused by the type II pyrethroid fenvalerate s. The present study provides additional support for the closed channel modification caused by deltamethrin. Judging from a variety of experimental data, it appears that the sodium channels are modified by pyrethroids largely in the closed state and that the open channel modification could occur to some extent depending on the type of pyrethroid and the kind of nerve preparation. Thus, pyrethroids appear to have a higher affinity for the open sodium channel than the closed sodium channel.

1 Baker, P.F., Hodgkin, A.L. and Shaw, T.I., Replacement of the protoplasm of a giant nerve fiber with artificial solutions, Nature, 190 (1961) 885-887. 2 Brown, L.D. and Narahashi, T., Activity of tralomethrin to modify the nerve membrane sodium channel, Toxicol. Appl. Pharmacol., 89 (1987) 305-313. 3 Casida, J.E., Gammon, D.W., Glickman, A.H. and Lawrence, L.J., Mechanisms of selective action of pyrethroid insecticides, Annu. Rev. Pharmacol. Toxicol., 23 (1983) 413-438. 4 Catterall, W.A., Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes, Annu. Rev. Pharmacol. Toxicol., 20 (1980) 15-43. 5 Chinn, K. and Narahashi, T., Stabilization of sodium channel states by deltamethrin in mouse neuroblastoma cells, J. Physiol., 380 (1986) 191-207. 6 Chinn, K. and Narahashi, T., Temperature-dependent subconducting states and kinetics of deltamethrin-modified sodium channels of neuroblastoma cells, Pfliigers Arch., 413 (1989) 571579. 7 Hitle, B., Pharmacological modifications of the sodium channels of frog nerve, J. Gen. Physiol., 51 (1968) 199-219. 8 Holloway, S.F., Narahashi, T., Salgado, V.L and Wu, C.H., Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells, Pfliigers Arch., 414 (1989) 613-621. 9 Lund, A.E. and Narahashi, T., Modification of sodium channel kinetics by the insecticide tetramethrin in crayfish giant axons, Neurotoxicology, 2 (1981) 213-229. 10 Lund, A.E. and Narahashi, T., Kinetics of sodium channel modification by the insecticide tetramethrin in squid axon membranes, J. Pharmacol. Exp. Ther., 219 (1981) 464-473. 11 Lurid, A.E. and Narahashi, T., Kinetics of sodium channel modification as the basis for the variation in the nerve membrane effects of pyrethroids and DDT analogs, Pestic. Biochem. Physiol., 20 (1983) 203-216. 12 Narahashi, T., Effects of insecticides on excitable tissues. In J.W.L. Beament, J.E. T r e h e r n e and V.B. Wigglesworth (Eds.), Advances In Insect Physiology, Vol. 8, Academic Press, London and New York, 1971, pp. 1-93. 13 Narahashi, T., Effects of insecticides on nervous conduction and synaptic transmission. In C.F. Wilkinson (Ed.), Insecticide Biochemistry and Physiology, Plenum, New York, 1976, pp. 327-352. 14 Narahashi, T., Nerve membrane sodium channels as the target of pyrethroids. In T. Narahashi (Ed.), Cellular and Molecular Neurotoxicology, Raven, New York, 1984, pp. 85-108. 15 Narahashi, T., Pharmacology of nerve membrane sodium channels. In P.F. Baker (Ed.), Current Topics in Membranes and Transport, Vol. 22, The Squid Axon, Academic Press, New York, 1984, pp. 483-516. 16 Narahashi, T., Nerve membrane ionic channels as the primary target of pyrethroids, Neurotoxicology, 6 (1985) 3-22. 17 Narahashi, T., Mechanisms of action of pyrethroids on sodium

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