44 Brain Research, 459 (198~) 44 53 Elsevier BRE 13882 Interaction of insecticides of the pyrethroid family with specific binding sites on the voltage-dependent sodium channel from mammalian brain Alain Lombet, Christiane Mourre and Michel Lazdunski Centre de Biochimie du CNRS, Parc Valrose, Nice (France) (Accepted 22 March 1988) Key words: Pyrethroid; Insecticide; Sodium channel; Batrachotoxin; Batrachotoxinin A 20-a-benzoate Measurement of neurotoxin binding in rat brain membranes and neurotoxin-activated 22Na+ influx in neuroblastoma cells were used to define the site and mechanism of action of pyrethroids and DDT on sodium channels. A highly potent pyrethroid, RU 39568, alone enhanced the binding of [3H]batrachotoxinin A 20-a-benzoate up to 30 times. This effect was amplified by the action of neurotoxins such as sea anemone toxins and brevetoxin acting at different sites of the sodium channel protein in brain membranes. The ability of various pyrethroids and DDT to enhance batrachotoxin binding was related to their capacity to activate tetrodotoxin sensitive 22Na+ uptake. These results point to an allosteric mechanism of pyrethroids and DDT action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels. Pyrethroids do not block [3H]tetrodotoxin binding, 125I-Anemoniasulcata toxin 2 binding, 125I-Tityusserrulatus toxin ~ binding at neurotoxin receptor sites 1, 3 and 4 respectively. Pyrethroids appear to act at a new neurotoxin receptor site on the sodium channel. The distribution of pyrethroid binding sites in rat brain was determined by quantitative autoradiographic procedures using the property of pyrethroids to reveal binding sites for [3H]batrachotoxinin A 20-a-benzoate. INTRODUCTION in culture 1°'12'29. This approach as well as binding studies t7 with tritiated and iodinated toxins specific A n u m b e r of naturally occurring neurotoxins acting at distinct binding sites have been used as tools to characterize various structural and functional aspects of Na + channels 2'11't337"33. Pyrethroids are synthetic derivatives of the natural toxins pyrethrins contained in the flowers of the Chrysanthemum species. They constitute nowadays the most interesting class of insecticides5 but they are also toxic in mammals. These compounds have been found to drastically prolong Na ÷ current in both vertebrate and invertebrate nerve m e m b r a n e s 16"24'35"38. Their mechanism of action on the Na ÷ channel has been studied in considerable detail with the patchclamp technique both at the single channel 9'3°'38 and at the whole cell 9'18'25'34 levels. Pyrethroids have also been found to induce 22Na+ influx through tetrodotoxin (TTX)-sensitive Na ÷ channels in n e u r o n a l cells for the Na + channel has shown that pyrethroids are not acting at the tetrodotoxin/saxitoxin binding site or at the batrachotoxin/veratridine site or else at specific sites involved in the recognition of polypeptide scorpion and sea a n e m o n e toxins. These data have led to the conclusion that pyrethroids have a specific binding site on the Na + channel protein which is distinct from binding sites previously identified for other toxins acting on the same channel. Receptors for several different classes of toxins acting on the Na + channel have now been biochemically identified using 3H-labeled or 125I-labeled toxins. This type of biochemical identification has not yet been possible with [3H]pyrethroids. The purpose of this paper is to describe the properties of binding of pyrethroids to the Na + channel protein using the allosteric interaction between the pyre- Correspondence: M. Lazdunski, Centre de Biochimie du Centre National de la Recherche Scientifique, Parc Valrose, 06034 Nice Cedex, France. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division) 45 throid binding site and the batrachotoxin binding site that has been identified with [3H]batrachotoxinin A 20-a-benzoate ([3H]BTX-B). Allosteric interactions between the pyrethroid binding site, the sea anemone toxin binding site and the brevetoxin binding site are also described. Autoradiographic procedures have been used to determine the distribution of pyrethroid binding sites in rat brains. A A ~'o 6 N10 x - l [3H]BTX-B m _u D. C All RU-$ AS2 m MATERIALS AND METHODS Materials Chemicals were obtained from the following sources: [3H]BTX-B (1.7 TBq/mmol, 45.2 Ci/mmol) and NaB3H4 (1.85 TBq/mmol, 50 Ci/mmol) from NEN, veratridine from Sigma. Anemonia sulcata toxin II (As2) was purified as previously described 31. Batrachotoxin was kindly provided by Dr. J. Daly (NIH, Bethesda, U.S.A.) and Ptychodiscus brevis toxin (brevetoxin-B, PbTx-2) by Dr. K. Nakanishi (Columbia University, New York, U.S.A.). Pyrethroids analogs were obtained from Dr. M. Roche (Procida-Roussel-Uclaf). 2,2-bis(p-Chlorophenyl)trichloroethane (p,p'-DDT) was a generous gift from Dr. J. Berg6 (INRA, Antibes, France). These insecticides were dissolved in Me2SO to prepare a 10 mM stock solution. Concentrations of Me2SO in binding experiments up to 1% had no effect on [3H]BTX-B binding to brain membranes. Binding experiments Microsomal fractions from rat brain homogenates were obtained according to Krueger et a1.15. Specific binding of [3H]BTX-B was measured using a previously described procedure 4 with minor modifications. The binding reaction was initiated by addition of 50/A of the membrane preparation containing 300-400 ag protein to a standard incubation buffer, 40 ~M As 2, 0.3/~M PbTx-2 and various unlabeled ellectors as indicated. The concentration of [3H]BTXB was generally 1.5-2.0 nM in a total assay volume of 0.5 ml. The standard incubation medium contained 130 mM N-methylglucamine, 5.4 mM KCI, 0.8 mM MgSO4, 1 mg/ml BSA and 50 mM HEPESTris buffer pH 7.5. After incubation at 22 °C for 1 h, two aliquots of 200 ~1 were filtered through Whatman GF/C glass fiber filters pretreated with 0.1% poly- x 0 .,. C AS= PbTx A u - i I R u - 5 As2As: A | 2 PbTiPbTx PbTx flu.l; Ru-S Ru-$ Ru-5 AI2 As2 PbTx PbTx RU-S Ru-S Fig. 1. Binding of [ 3 H ] B ~ - B to rat brain microsomes in the presence of various effectors. Binding of 2 nM [3H]BTX-Bwas measured at 22 °C with 0.35 mg/ml of protein in the absence (controls) or in the presence of 40 aM As2, 0.3 aM PbTx-2, 1 aM RU 39568 (RU-6), 10aM RU 39568 (RU-5) or a combination of these toxins at the same concentrations. In all cases, non-specific binding was measured in the presence of 100 aM veratridine and represented 500 dpm. Inset: binding of [3H]PbTx-3 to rat brain microsomesin the presence of various effectors. Binding of [3H]PbTx-3 was measured at 4 °C with 0.35 mg/ml of protein in the absence (control) or in the presence of 40 aM As2, 10 aM RU 39568 (RU-5) or a combination of these toxins. Non-specificbinding was measured in the presence of 10aM unlabeled PbTx-2. In all cases data are given as mean + S.E.M. (n = 3). ethyleneimine and immediately washed 3 times with 5 ml of an ice-cold 150 mM Tris-C1 buffer at pH 7.4. The radioactivity remaining on the filter was extracted with Biofluor (NEN) as scintillator and counted in a Packard Tricarb 3350 spectrophotometer. The non-specific binding component was measured in the presence of 100 a M veratridine since veratridine is known to bind to the same site as BTXB 8. Tetrodotoxin was not included in the incubations because it could interfere with [3H]BTX-B binding at 22 °C (ref. 20). Binding of [3H]PbTx-3 was measured by rapid filtration in the same conditions as described for [3H]BTX-B binding. Preparation of [3H]PbTx-3 was carfled out as described 27 by reduction of PbTx-2 with NaB3H4. [3H]PbTx-3 had a specific radioactivity of 0.65 Ci/mmol. 22Na+ influx measurements using neuroblastoma cells N I E 115 mouse neuroblastoma cells were grown 46 and plated as p r e v i o u s l y d e s c r i b e d ~5. 22Na~ influx 20 A measurements w e r e carried out as p r e v i o u s l y re- p o r t e d mA2'29 except for N - m e t h y l g l u c a m i n e replac- O ing choline chloride. T h e activating effect of the different p y r e t h r o i d s tested was m e a s u r e d in the presence of 100BM v e r a t r i d i n e . A utoradiographic procedures -9 W h o l e rat brains w e r e quickly r e m o v e d , f r o z e n -8 -7 tog [s'rx] (S) and the brain sections (15 tim thickness) w e r e prep a r e d using a p r e v i o u s l y d e s c r i b e d p r o c e d u r e 2°. T h e sections w e r e i n c u b a t e d for 2 h at 4 °C in the s a m e standard i n c u b a t i o n m e d i u m as d e s c r i b e d for binding e x p e r i m e n t s d e s c r i b e d a b o v e with [ 3 H ] B T X - B (1.5 nM) in p r e s e n c e or a b s e n c e of A s 2 (40 t i M ) plus R u 39568 (10/~M) as indicated. T h e non-specific binding c o m p o n e n t was d e t e r m i n e d by a d d i n g v e r a t r i d i n e (0.1 raM) 15 min prior to the a d d i t i o n of [3H]BTX-B. A t the end of the i n c u b a t i o n , the sections w e r e w a s h e d twice for 5 s in Tris 100 m M in the p r e s e n c e of | , m i 0 . 1 % B S A and twice for 5 s in w a t e r . A part of the 100 200 300 BTX bound(f tool/assay) 100 B ! 80 60 slices was used to p r e p a r e and to q u a n t i f y a u t o r a d i o grams as p r e v i o u s l y d e s c r i b e d 21. T h e o t h e r labeled brain sections w e r e r e m o v e d and c o u n t e d . RESULTS A number of Na + channel effectors have been pre- \ U. 1_ 201 -9 -8 -7 tog [re'x] (M) -6 ~ . 100 200 3OO B'I'X bound (tmol/assaY) Fig. 2. A, B: inhibition of [3H]BTX-B binding in the presence of various effectors. A: Scatchard analysis of [3H]BTX-B binding in the presence of positive effectors. Inset: binding of 2 nM [3H]BTX-B was measured in standard binding medium without (O) or with 40/~M As2 (©), with 40 pM As 2 + 0.3 ~uM PbTx-2 (11), with l pM RU 39568 (C3) with 10/lM RU 39568 (A) and increasing concentrations of unlabeled BTX. Non-specific binding in the presence of 100 ~M veratridine is illustrated by the dotted line. Main panel: specific binding calculated from the data presented in inset is illustrated as a Scatchard plot. At each concentration of BTX from the data presented in inset, the dpm of specifically bound [3H]BTX-B were determined and converted to total specifically bound BTX (labeled plus unlabeled) using the specific radioactivity appropriate for the concentration of unlabeled BTX added. Free BTX (labeled plus unlabeled) was calculated as the difference between total added BTX and bound BTX. Bound/free was then plotted vs bound in the form of a Scatchard representation. The regression lines were drawn for values of Bmax = 300 fmol and Kj = 460 nM (0), K d = 200 nM (O), K d = 70 nM (11), K d = 40 nM (D) and Kd = 15 nM (&). B: Scatchard analysis of the effect of pyrethroids on [3H]BTX-B binding in the presence of 40 pM As 2 and 0.3 pM PbTx-2. BTX displacement curves (inset) and BTX saturation curves (main panel) were obtained as in Fig. 1 in absence of pyrethroid (11) or in the presence of 1 ktM deltamethrin (O), 10 pM deltamethrin (O), 1 pM RU 39568 (A) and 10 ~M RU 39568 (&). The regression lines are drawn for values of Bmax = 300 fmol and K d = 70 nM (11), Ko = 29 nM (©), K d = 12.5 nM (O), Kj = 4.8 n M ( & ) and K d = 2.9 nM (A). 47 viously shown to increase specific [3H]BTX-B binding to Na ÷ channels. They include polypeptide toxins such as sea anemone and scorpion toxins 8 and nonpeptidic toxins such as brevetoxins 7. Fig. 1 confirms that the sea anemone toxin As 2 and PbTx-2 alone increase [3H]BTX-B binding. Enhancement of [3H]BTX-B binding was also observed with the pyrethroid molecule R U 39568 at concentrations of 1 nM (RU-6) and 10/~M (RU-5) (Fig. 1, I). The specific binding of [3H]BTX-B in all these conditions was inhibited by unlabeled batrachotoxin as it will be seen later in more detail. Fig. 1 also presents the interesting observation that there is an additive activation not only between As2 and PbTx-2 but also between As 2 and pyrethroids and between PbTx-2 and pyrethroids (Fig. 1, II). A very important increase of specific [3H]BTX-B binding is observed with a mixture of the 3 different toxins As2, PbTx-2 and the pyrethroid which all appear to work additively (Fig. 1, III). The [3H]PbTx-3 binding site has been recently identified 27. The sea anemone toxin As 2 enhances [3H]PbTx-3 binding. Pyrethroids (RU 39568) also increase [3H]PbTx-3 binding and the mixture of As2 and the pyrethroid produces a larger enhancement than the one observed with As 2 or the pyrethroid alone (Inset, Fig. 1). Properties of inhibition of [3H]BTX-B binding by unlabeled BTX-B are presented in the two insets of Fig. 2. The data have been converted into Scatchard type representations that are linear and indicate a single class of receptor sites for [3H]BTX-B in the presence of the different combinations of binding eflectors. K d values were 460 nM for BTX-B when [3H]BTX-B binding experiments were carried out in the absence of any other toxin than batrochotoxin. K o was 200 nM in the presence of As2 (40/~M), 40 nM in the presence of 1/tM R U 39568 and 15 nM in the presence of 10/zM RU 39568 (Fig. 2A). The maximum binding capacity was unchanged by either As2 or the pyrethroid (Br, ax = 2.2 pmol of BTX-B bound per mg of protein). The K d value for BTX-B was found to be 70 nM in the presence of the mixture of As2 and PbTx-2. It decreased to 29 and 12 nM upon addition of 1 and 10/~M of the pyrethroid deltamethrin to this mixture. Addition of 1 and 10/~M of R U 39568 to the mixture instead of deltamethrin shifted the K d value to 4.8 and 2.9 nM respectively (Fig. 2B). With all toxin mixtures the Bmax value remained at -8, --ry/ -7, -6, -5 -4 A - - - m, 5 ,._yj 0 -t,z/F x +~ d) -J - r - - T - - -,- Ru3956 2 * - -,- --T- ff -// Z log [Pyr] (M) Fig. 3. A, B: effects of pyrethroids on [3H]BTX-Bbinding and 22Na+ influx. A: effect of different pyrethroids on the binding of [3H]BTX-Bin the presence of 40/xM As2 and 0.3/~M PbTx2. The binding of 2 nM [3H]BTX-Bwas measured in the presence of increasing concentrations of RU 39568 (0) and RU 39130 (rq). Levels of total binding in absence of pyrethroid (~t) and of the non-specific binding (+) in the presence of 100/~M veratridine are represented by the solid line and the dotted line respectively. B: effect of pyrethroids on 22Na+ influx in N1E 115 neuroblastoma cells in the presence of 100/~M veratridine. 22Na+ influx was measured in the presence of increasing concentration of RU 39568 (0) and RU 39130 (D). Levels of 22Na+ uptake measured in the presence of 100/~M veratridine ('k) and in the presence of 100/~M veratridine and 1/~M "I"FX (+) are represented by the solid line and the dotted line respectively. 2.2 pmol/mg of microsomal protein. Enhancement of [3H]BTX-B binding to Na ÷ channels in synaptosomal membranes by pyrethroids is concentration-dependent as we have already seen in Fig. 2. Dose-response curves for two pyrethroids, RU 39568 and R U 39130 are given in Fig. 3A. Halfmaximal activations were seen at Ko. 5 values of 0.2 and 2.5/~M respectively. The same type of difference in efficacy between the two pyrethroids (K0 5 values of 0.1 and 5 ~tM) is shown in Fig. 3B using the 22Na+ influx technique to follow the functional activity of this class of toxins on the Na + channel in neuroblastoma cells. A classical compound belonging to another class of 48 _4 ¸ ae Iog[})DT](M) '0 0 .,-7 - 6 -5 -4 ~-" . . . . J2 ~ / 2 [ 2 OT 9221 RU39130~)eltamethrin / an . . . . -8 ~ .~ 6-" 4 -s log [DDT] (M) 4 4 -g log Kol~rethroid on BTX ~ n a n g (M) C IX~7 Ru - 4 - s_ -,i Fig. 4. Effect of DDT on [3H]BTX-B binding and Na + influx. Main panel: the effect of DDT was measured on the binding of 2 nM [3H]BTX-B, in the presence of 40/aM As2 and 0.3/aM PbTx-2 (100%). The stimulation obtained with increasing concentrations of DDT (0) was expressed as % of the control (100%). The non-specific binding was obtained in the presence of 100/aM veratridine. A K0.5 value of 15/aM was observed for DDT. Inset A: the effect of DDT on 22Na+ influx in N1E 115 was measured in the presence of 100/aM veratridine. The halfmaximum increase in 22Na+ uptake occurred at 40/aM DDT. Basal 22Na+ uptake was measured at each concentration of DDT in the presence of 1/aM TTX (dotted line). Inset B: a comparison of the stimulation produced by DDT and RU 39568 on [3H]BTX-Bbinding in the presence of As2 and PbTx-2. insecticide, D D T , also enhances [3H]BTX-B binding to rat brain membranes in the presence of both Ase and PbTx-2. The enhancement of the binding is not as large as for pyrethroids (see Fig. 4, inset B). A 1.8fold increase was found for D D T as compared to a 9fold increase for RU 39568 in the same type of experiment. The dose-response curve shown in the main panel of Fig. 4 indicates a K0. 5 value of 15/zM for D D T . D D T stimulates [3H]BTX-B binding to synaptosomal membranes in the same range of concentration in which it activates the tetrodotoxin-sensitive 22Na+ influx through the Na ÷ channel in neuroblastoma cells (K0. 5 = 40/~M) (Fig. 4, inset A). Fig. 5 compares the efficacy of pyrethroids to bind to the pyrethroid receptor and the efficacy of pyrethroids to activate the Na ÷ channel. This correlation includes the data obtained with D D T . A good correlation has been observed (r = 0.990) between K0.5 values corresponding to half-maximum enhancement of [3H]BTX-B binding to brain membranes and Ko.5 values for activation of the Na + channel in neuroblastoma cells by the same compounds. -4 Fig. 5. Correlation between efficacies of various pyrethroids (and DDT) in enhancing specific [3H]BTX-B binding to rat brain membranes and activating TTX-sensitive 22Na÷ uptake in neuroblastoma N1E 115 cells. Kd (half-stimulation of [3H]BTX-B binding obtained in the presence of As2 and PbTx2) was plotted vs K0.5 (half-stimulation of 22Na÷ uptake obtained in the presence of 100/~M veratridine). Correlation was described for RU 39568 (D), RU 24501, cis-cypermethrin (©), RU 39130 (A), deltamethrin (0), RU 29221 (11) and DDT (A). Quantitative autoradiographic measurements were used to analyze the distribution of pyrethroid binding sites in the CNS. Since direct experiments with [3H]pyrethroids were not possible, we analyzed pyrethroid-induced [3H]BTX-B binding. Other authors have recently analyzed scorpion venom-induced [3H]BTX-B binding 37. Incubations for brain slices with [3H]BTX-B (1.5 nM) either alone or in the presence of As2 failed to provide a significant specific labeling of the [3H]BTX-B binding site. However things become different in the presence of pyrethroids. [3H]BTX-B (1.5 nM) binding to brain slices in the presence of the pyrethroid R U 39568 (10/~M) alone or associated to the sea anemone toxin As2 (40 btM) lead to a clear identification of a large enough component of [3H]BTX-B binding which could be prevented in the presence of veratridine (100/aM). These results obtained on brain slices are in agreement with results previously described for brain membranes (Fig. 2). The CNS distribution of [3H]BTX-B binding revealed in the presence of R U 39568 is heterogeneous (Fig. 61,3,5,7 and Table 1) while the non-specific binding component was uniformly distributed throughout the CNS (47 + 7% of the total binding). Fig. 62,4,6,8 shows the total binding of [3H]BTX-B alone in the binding medium. Under these conditions, [3H]BTX- 49 B binding is uniformly distributed and is identical to the non-specific binding component of [3H]BTX-B measured in the presence of veratridine (not shown). The same result was observed for [3H]BTX-B binding in the sole presence of As2. A specific binding component only appeared in the presence of the toxin mixture containing the pyrethroid. Therefore [3H]BTX-B binding sites are only revealed in brain areas where pyrethroid binding sites are present. Structures that are particularly rich in [3H]BTX-B receptors in the presence of the pyrethroid, are the different areas of the neocortex, the central gray and to a lesser extent the anterior thalamic nuclei, the superior colliculus and substantia nigra (Table I). Regions with the lowest densities of pyrethroid-induced [3H]BTX-B binding include the caudate putamen and medulla oblongata. DISCUSSION Numerous toxins are active on the voltage-sensitive Na ~ channel and some of them have been essential in revealing new aspects of Na ÷ channel structure and function 1. These toxins bind at separate receptor sites t7'19'25'33. Sites that have been well identified by binding studies using labeled toxins are those for: (i) tetrodotoxin and saxitoxin that inhibit Na + flux through the Na ÷ channel (site 1); (ii) batrachotoxin and other lipid soluble toxins like veratridine, aconitine or grayanotoxin that cause a persistent activation of Na ÷ channels at the resting potential by shifting the voltage-dependence of the activation and by blocking inactivation (site 2); (iii) scorpion toxins from North Africa and North America and sea anemone toxins that slow down Na ÷ channel inactivation (site 3); (iv) another class of scorpion toxins that shifts the voltage-dependence of Na ÷ channel activation to more negative membrane potentials (site 4); (v) brevetoxins and ciguatoxin that cause repetitive firing and depolarization of excitable membranes by activating the Na ÷ channel (site 5). A good number of [3H]pyrethroids have been assayed in this laboratory to try to characterize pyrethroid binding sites. Unfortunately, it turned out that even with the most active of these insecticides, it was impossible to identify a specific binding component on rat brain synaptosomes or on insect neuronal membranes (D. Pauron and J. Barhanin, unpub- I CPu Sp 2 CeCx D( G MC MI v 3 4 Fig. 6. Autoradiographic distribution of [3H]BTX-B binding sites in rat brain. 1,3,5,7: sections were incubated with [3H]BTX-B (1.5 nM) in presence of As2 (40/tM) and Ru 39568 (10/tM). 2,4,6,8: sections were only incubated with [3H]BTX-B (1.5 nM). Abbreviations: AH, Ammon's Horn; CeCx, cerebellar cortex; CG, central gray; CPu, caudate putamen; DG, dentate gyrus; FrCx, frontoparietal cortex; G, geniculate nuclei; MM, mammillary bodies; MO, medulla oblongata; SN, substantia nigra; Sp, septum; StCx, striate cortex. 50 lished results), T h e non-specific binding c o m p o n e n t vetoxin, do n o t c h a n g e the m a x i m a l binding capacity of [3H]pyrethroids was always t o o high as c o m p a r e d of B T X - B to brain m e m b r a n e s , to the specific b i n d i n g c o m p o n e n t . F u r t h e r m o r e , it crease the affinity of B T X - B for its specific r e c e p t o r h o w e v e r t h e y in- has already b e e n s h o w n p r e v i o u s l y that p y r e t h r o i d s site. This increase in the affinity for [ 3 H ] B T X - B ex- are w i t h o u t effect on t h e b i n d i n g of 3 H - l a b e l e d t e t r o - plains the e n h a n c e m e n t of [ 3 H ] B T X - B binding at dotoxin (A. L o m b e t , u n p u b l i s h e d results) and 1251- n o n - s a t u r a t i n g c o n c e n t r a t i o n s (2 n M ) of [3H]BTX-B. labeled sea a n e m o n e toxins 36 and s c o r p i o n toxins 3 to A n e w synthetic p y r e t h r o i d , R U 39568, i n c r e a s e d the their respective r e c e p t o r s . affinity of [ 3 H ] B T X - B for its b i n d i n g site by a factor This p a p e r d e m o n s t r a t e s for the first t i m e that of 30 while A s 2 and P b T x - 2 only i n c r e a s e d the affinity pyrethroids, similarly to sea a n e m o n e toxins and to by factors of 2.3 and 4.3 r e s p e c t i v e l y 7,s. T h e mixtur/e b r e v e t o x i n PbTx-2, binding. of As 2 and P b T x - 2 at c o n c e n t r a t i o n s at which t h e y Pyrethroids, similarly to sea a n e m o n e toxin and bre- saturate their binding site p r o d u c e d a 7.5-fold in- enhance [3H]BTX-B TABLE I Distribution of [3H] B TX-B binding sites revealed in the presence of R U 39568 and As 2 in rat brain Results, expressed in fmol/mg protein, are the mean + S.E.M. of 6-8 separate autoradiographic measurements. Each value is the difference between the total binding and the non-specific binding and is determined with 1.5 nM [3H]BTX-B associated with RU 39568 (10/~M) and As 2 (40/~M). Density variations between corresponding right and left nuclei were always similar or smaller than S.E.M. given in Table I. Brain structures Neocortex Frontoparietal cortex, motor area Frontoparietal cortex, sensory area Cingulate cortex Temporal cortex auditory area Striate cortex Entorhinal cortex Primary olfactory cortex Specific binding 97.5 105.5 104.0 120.5 92.0 72.5 52.5 + + + + + + + Brain structures 17.4 8.9 11.6 3.1 1.7 18.4 2.7 Hippocampalformation Ammon's Horn, strati oriens and radiatum Ammon's Horn, stratum lacunosum moleculare Dentate gyrus Subiculum 41.5 ± 1.7 63.5 + 1.6 74.0 ± 2.2 Cerebellar cortex Molecular layer Granular layer White matter 34.5 + 1.3 30.0 + 4.5 4.5 + 0.2 Forebrain Accumbens nucleus Caudate putamen Lateral septal nucleus Medial septal nucleus Nuclei of the diagonal band of broca Basolateral amygdaloid nucleus 28.5 41.5 25.0 34.0 27.5 65.5 49.0 ± 1.1 + + ± ± + + 0.2 3.6 0.5 5.5 0.9 3.6 Specific binding Thalamus Anterior thalamic nuclei Laterodorsal thalamic nucleus Ventroposterior thalamic nucleus Centromedian thalamic nucleus Geniculate nuclei Habenula 92.0 77.5 53.0 76.0 80.0 34.5 Hypothalamus Anterior hypothalamic area Ventromedian hypothalamic nucleus Posterior hypothalamic nucleus 70.0 ± 10.2 66.0 + 0.8 73.0 ± 7.2 Midbrain Mammillary nuclei Ventral tegmental nucleus Substantia nigra Interpeduncular nuclei Central gray Superior colliculus Inferior colliculus Pontine nuclei Median raphe nucleus Deep mesencephalic nuclei 53.0 40.5 81.0 68.0 104.0 90.5 77.5 54.0 53.0 30.0 + + + + + ± 9.8 1.1 3.7 4.3 3.2 4.2 _+0.7 ± 2.0 ± 0.8 + 3.6 ± 3.7 ± 3.7 ± 9.4 ± 7.1 +_ 3.5 ___2.0 Medulla oblongata Cochlear nuclei Nucleus of the spinal tract of the trigeminal nerve Vestibular nuclei Reticular pontine nuclei 25.5 + 4.3 28.5 +_ 1.2 20.5 + 2.9 Myelinated fiber tracts Corpus callosum Internal capsule 36.0 +__0.6 21.5 + 0.6 Brain average 59.5 50.0 + 7.5 51 crease in affinity for BTX-B at its binding site. The addition of RU 39568 to these two toxins further increased the affinity of BTX-B which then became 100-fold higher than in the absence of any toxin. The functional channel protein is known to be constituted by a single polypeptide of about 270 kD that bears all the identified binding sites for the different toxins 1,17,26. Depending on the membrane potential and on the time after a step depolarization, the channel can exist in the form of a non-conducting or of a conducting form. Batrachotoxin is known to bind preferentially to the conducting form 14'28. Therefore, toxins like sea anemone toxins or brevetoxins or pyrethroids or mixtures of these toxins that increase the probability of finding the Na ÷ channel in the open form 6a7'25 will of course favor the binding of batrachotoxin. Cumulative effects of sea anemone toxins, brevetoxins and pyrethroids on [3H]BTX-B binding imply of course that these 3 categories of Na ÷ channel effectors have different types of receptors and also that these receptors are distinct from the batrachotoxin binding sites. The stimulation of [3H]PbTx-3 binding by both As 2 and pyrethroids and by the mixture of the two (Fig. 1, inset) confirms that the 3 types of toxins bind to 3 different types of receptors. Conclusions regarding the different types of binding sites and allosteric interactions between the different receptor sites for different toxins coming from [3H]BTX-B binding experiments are perfectly consistent with 22Na÷ flux studies through the voltagesensitive Na ÷ channel in neuroblastoma cells. Previous studies from this laboratory have shown that batrachotoxin, veratridine, grayanotoxin and sea anemone toxin and also a-scorpion toxins act synergistically with pyrethroids 12. For all these reasons, it is now clear that pyrethroids bind to a special category of receptor sites (site 6). The good correlation between binding results on brain membranes and 22Na+ flux data (Fig. 5) in neuroblastoma cells clearly indicates that receptor sites for pyrethroids that have been biochemically identified in Fig. 3 are those that are responsible for Na ÷ channel activation under the influence of this class of insecticides. Electrophysiological experiments TM have suggested that DDT, in spite of its difference in structure with pyrethroids, act similarly and at the same binding site as pyrethroids. Moreover, acquisition of pyrethroid resistance in insects is often accompanied by resistance to DDT 23. Observations made in this paper also show the analogy between D D T and pyrethroids. Data for DDT are well integrated into the correlation presented in Fig. 5. The direct autoradiographic localization of pyrethroid binding sites with labeled pyrethroids is not possible at present. Therefore, pyrethroid binding sites have been indirectly localized by their capacity to reveal [3H]BTX-B binding to the CNS. The distribution of [3H]BTX-B binding sites which then reveal the distribution of pyrethroid binding sites is similar but not identical to the previously identified distribution of tritiated tetrodotoxin or saxitoxin binding sites 22 that are also known to reside on voltage-dependent Na + channels. Large amounts of tetrodotoxin and saxitoxin binding sites were previously found 22 in hippocampus (region 3 of the Ammon's Horn), in substantia nigra and in the molecular layer of the cerebellar cortex. No such property was found for pyrethroid binding sites revealed from [3H]BTX-B binding. The probable interpretation of these results is linked to the existence of sub-types of voltage-sensitive Na + channels. Among these sub-types some are sensitive to tetrodotoxin and saxitoxin, others are resistant to the two toxins and require much higher toxin concentrations to be blocked. Tritiated tetrodotoxin and saxitoxin probably label selectively the sub-type of Na + channels with a high affinity for the toxins. Conversely pyrethroid-induced [3H]BTX-B binding will reveal both types of Na + channels since pyrethroids have previously been shown to act with the same efficacy on tetrodotoxin-sensitive and tetrodotoxin-resistant Na + channels 3. The assay developed in this work to analyze pyrethroid binding properties to the Na + channel in mammalian neuronal membranes can probably be used with insect neuronal membranes (in preparation) and serve to analyse the structure-function relationships of these important compounds in relation with their insecticide properties and to analyse the mechanism of acquired resistance of insects to insecticides of the pyrethroid family32. ACKNOWLEDGEMENTS We are very grateful to Dr. Daly for his generous 52 gift of b a t r a c h o t o x i n , to Dr. K. N a k a n i s h i for b r e v e - to C. W i d m a n n for e x p e r t technical assistance and to toxin-B, to Dr. H. Schweitz for purification of A s 2 M. Valetti and C. R o u l i n a t - B e t t e l h e i m for skillful and to Drs. R o c h e and C o v e n t ( P r o c i d a ) for a gen- secretarial help d u r i n g the p r e p a r a t i o n of this m a n u - erous gift of the d i f f e r e n t p y r e t h r o i d m o l e c u l e s . W e script. This w o r k was s u p p o r t e d by the C e n t r e N a t i o - are grateful to Drs. J . R . de W e i l l e , G. R o m e y and nal de la R e c h e r c h e S c i e n t i f i q u e ( A T P 1217). J.-P. V i n c e n t and J. B a r h a n i n for fruitful discussion, REFERENCES 1 Agnew, W.S., Tomiko, S.A., Rosenberg, R.L., Emerick, M.C. and Cooper, E.C., The structure and function of the voltage-sensitive Na channel. In C.Y. 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