Metaflumizone is a novel sodium channel blocker insecticide.

Metaflumizone is a novel semicarbazone insecticide, derived chemically from the pyrazoline sodium channel blocker insecticides (SCBIs) discovered at Philips-Duphar in the early 1970s, but with greatly improved mammalian safety. This paper describes studies confirming that the insecticidal action of metaflumizone is due to the state-dependent blockage of sodium channels. Larvae of the moth Spodoptera eridania injected with metaflumizone became paralyzed, concomitant with blockage of all nerve activity. Furthermore, tonic firing of abdominal stretch receptor organs from Spodoptera frugiperda was blocked by metaflumizone applied in the bath, consistent with the block of voltage-dependent sodium channels. Studies on native sodium channels, in primary-cultured neurons isolated from the CNS of the larvae of the moth Manduca sexta and on Para/TipE sodium channels heterologously expressed in Xenopus (African clawed frog) oocytes, confirmed that metaflumizone blocks sodium channels by binding selectively to the slow-inactivated state, which is characteristic of the SCBIs. The results confirm that metaflumizone is a novel sodium channel blocker insecticide.

Maintenance of GABA receptor function of small-diameter cockroach neurons by adenine nucleotides.

Small diameter (<20 microm) neurons from the sixth abdominal ganglion of the American cockroach, Periplaneta americana, were enzymatically isolated and responses to exogenously applied gamma-aminobutyric acid (GABA) were recorded using the whole-cell patch clamp technique. With a minimal intracellular medium, responses to repeated applications of GABA decreased to zero within a few minutes. The rate of rundown of GABA responses was decreased by the intracellular inclusion of the phosphatase inhibitors microcystin and okadaic acid, suggesting that phosphorylation is necessary for the maintenance of cockroach GABA receptor function. ATP (5 mM) prevented GABA response rundown. ADP (5 mM) also slowed GABA response rundown, but responses stabilized at a level about half that seen with ATP. In the presence of protein kinase A inhibitory peptide (PKI), ATP was only as efficacious as ADP in slowing rundown. PKI had no effect on the ability of ADP to slow rundown, suggesting that the beta-phosphate of ADP is not involved in PKA-dependent phosphorylation of the GABA receptor. These results suggest that in cockroach neurons, GABA receptor function is maintained intracellularly by adenine nucleotides, not only by phosphorylation, but also possibly by an interaction with a nucleotide recognition site unrelated to PKA-dependent phosphorylation.

Block of neuronal voltage-dependent K+ channels by diacylhydrazine insecticides.

RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), a novel insect growth regulator, also produces acute neurotoxic symptoms by selectively blocking the maintained voltage-dependent K+ current (IK) in nerve and muscle Salgado (1992). The effects of RH-5849 and an analog were examined on IK channels in internally-perfused crayfish giant axons. For bilaterally applied RH-5849, the concentration needed for 50% block (IC50) was 79 +/- 6 microM (mean +/- SEM, n = 3), with a Hill coefficient near 2. Block was independent of membrane potential, but dependent on time, with a speed proportional to concentration, suggesting an open channel block mechanism. In addition to their effects on IK, both diacylhydrazines were much weaker blockers of the voltage-dependent sodium current (INa). RH-5849 blocked IK from either side of the membrane, and was more potent when applied bilaterally. When RH-5849 was introduced inside the axon and internal perfusion was halted, IK increased within a few minutes to the control level, indicating that the compound diffused freely through the membrane and bound to a receptor within the plane of the membrane. The permeability coefficients measured in the stopped-flow experiments indicate that diacylhydrazines can diffuse readily throughout the body of a poisoned insect, consistent with the rapid onset of central nervous system symptoms following injection. The octanol:water partition coefficient of RH-5849 increased sharply from 145 to 258 at aqueous concentrations between 5 and 10 microM, suggesting that a new phase, possibly micellar, is formed in the octanol phase. This may be responsible for the anomalously high Hill coefficients for the channel blocking activity of the diacylhydrazines.

Immobilization of sodium channel gating charge in crayfish giant axons by the insecticide fenvalerate.

The type II pyrethroid fenvalerate is known to depolarize nerve membranes by keeping sodium channels in a very stable modified open state. We have performed experiments on crayfish giant axons to determine whether the asymmetric charge movement was affected in parallel with changes in sodium current. When all sodium channels were modified by repetitive stimulation in the presence of fenvalerate, on gating charge movement was reduced by 78%. When on gating currents were fractionated into three exponentially decaying components, it was found that fenvalerate selectively depressed the intermediate and slow components, leaving the fast component unchanged. Off gating currents could be fractionated into two exponentially decaying components. The fast component of off charge movement (tau = 50 microseconds at -160 mV) was abolished by fenvalerate, whereas the slow component was suppressed by 50%. These results are consistent with previous conclusions that a large fraction of the intermediate and slow on and slow off components and essentially all of the fast off components are related to sodium channel gating. We conclude that fenvalerate traps the gating charges of sodium channels in the open state.

Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels.

The membrane actions of three recently isolated polypeptide neurotoxins from the sea anemones Stichodactyla helianthus (toxin ShI), Condylactis gigantea (toxin CgII) and Calliactis parasitica (toxin CpI) were investigated on action potentials and voltage-clamp membrane currents of the giant axon of the crayfish Procambarus clarkii. The first two toxins were also tested on the cockroach (Periplaneta americana) giant axon. All three toxins were particularly lethal to crustaceans, moderately toxic to an insect (cockroach), and essentially non-toxic to a mammal (mouse). ShI and CgII were 50- to 100-fold more potent on crayfish than on cockroach axons; this difference in activity was correlated with the relative reversibility of their effects on these arthropod axons. The crustacean selectivity of these toxins is therefore due largely to their greater affinity for crustacean sodium channels. All three toxins prolonged crayfish giant axon action potentials by selectively slowing Na channel inactivation without greatly affecting activation. Before toxin treatment, inactivation was nearly exponential, with a time constant less than 1 msec. After treatment, the inactivation time course could be described as the sum of two exponentially decaying components, plus a large steady-state component. The major component possessed the slower (10-20 msec) time constant. The steady-state component increased with depolarization, causing the sodium channel steady-state inactivation curve to reach a minimum between -60 and -20 mV and then increase at more positive potentials. All three toxins shifted the peak sodium current-voltage relation to the left. This voltage shift was greater at 20 degrees C than at 10 degrees C. Maintained membrane depolarization during toxin wash-in delayed the appearance of modified Na channels. Also, prolonged depolarization of toxin-treated axons converted modified sodium channels back to normal ones. The toxins did not affect potassium and leakage currents. Our results indicate that the three crustacean-active sea anemone toxins share a common electrophysiological action on arthropod sodium channels, at least at the macroscopic level.

Slow voltage-dependent block of sodium channels in crayfish nerve by dihydropyrazole insecticides.

Previous current-clamp work has shown that dihydropyrazole insecticides block sodium channels in tonic sensory receptors and in axons depolarized by high K+ external solutions and that hyperpolarization removes the block [Pestic. Sci. 28:389-411 (1990)]. Voltage-clamp studies on internally perfused crayfish giant axons were done to confirm and extend these observations. At -100 mV dihydropyrazoles had little effect on the sodium current, but at more depolarized potentials they blocked it from either face of the membrane. The onset of block following a holding potential change or during wash-in of a dihydropyrazole was very slow, with a time constant of several minutes, and, although block could be removed with a similar time course by hyperpolarization, the effects of the insecticides could not be reversed by prolonged washing. Dihydropyrazoles did not affect delayed rectifier potassium currents in the axon. The voltage-dependent block could be described as a uniform shift of the steady state (slow) sodium inactivation (S infinity) curve in the direction of hyperpolarization, indicative of selective binding to inactivated states of the channel. Using hyperpolarizing prepulses to remove slow inactivation, block of sodium channels by dihydropyrazoles could be measured directly at holding potentials as positive as -50 mV, and it could be demonstrated that block saturated near -70 mV, consistent with a dependence on slow inactivation. The data were fit to a model tha assumes the dihydropyrazole binds to the slow-inactivated state of the channel on a one to one basis. Dissociation constants obtained from this analysis were similar to those obtained from analysis of inhibition of the binding of [benzoyl-2,5-3H]-batrachotoxinin A 20-alpha-benzoate by the same dihydropyrazoles. In axons whose fast or slow inactivation gates had been removed by N-bromoacetamide or trypsin, respectively, dihydropyrazoles still blocked sodium current, indicating that dihydropyrazoles can block the channel as well as enhance the normal slow inactivation process.

Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells.

(1) The kinetic properties of single sodium channels modified by the pyrethroid fenvalerate have been analyzed by patch clamp techniques using the cultured mouse neuroblastoma cells. (2) Fenvalerate drastically prolonged the open time of single sodium channels from the normal value of 5 ms to several hundred milliseconds during a depolarizing pulse. The channels remained open after termination of a depolarizing pulse for as long as several seconds. (3) The channel lifetime varied with the membrane potential, attained a maximum at -70 mV, and decreased with hyperpolarization and depolarization from -70 mV. (4) Prolonged openings of the modified channels allowed a current-voltage curve for a single channel to be plotted by sweeping a ramp pulse. The single channel conductance had a value of 11 pS and was linear over potentials ranging from 0 to -100 mV. (5) Power density spectral analysis of the open channel current noise indicated a single Lorentzian curve with a cut-off frequency at 90 Hz, indicating that the increase in noise during channel opening resulted from a relatively slow kinetic process. (6) The probability of the channel being modified by fenvalerate was independent of the length of time during which the channel was opened. This observation suggests that channel modification had taken place before the channel opened. This study of the prolonged opening at the single channel level provides a new insight into open channel properties and the kinetics of channel modification.

Interactions of the pyrethroid fenvalerate with nerve membrane sodium channels: temperature dependence and mechanism of depolarization.

Depolarization of nerve membranes is an important component of the mode of action of pyrethroids, and its negative temperature dependence parallels that of insecticidal activity. We studied the mechanism and temperature dependence of depolarization of crayfish giant axons by pyrethroids, using intracellular microelectrode and voltage clamp techniques. Membrane depolarization caused by tetramethrin and fenvalerate was greater at 10 degrees C than at 21 degrees C, and was reversible upon changing the temperature. Short-duration depolarizing pulses in voltage-clamped fenvalerate-treated axons induced prolonged sodium currents that are typical of other pyrethroids, but the decay of the tail current following repolarization was extremely slow, lasting several minutes at the large negative holding potential of -120 mV. At the normal resting potential, the tail current did not decay completely, and even without stimulation, a steady-state sodium current developed, which could account for the depolarization. The steady-state current induced by fenvalerate at the resting potential was much larger at 8 degrees C than at 21 degrees C, accounting for the negative temperature dependence of the depolarization. The negative temperature dependence of the steady-state current seems to be due ultimately to the great stabilizing effect of low temperature on the open-modified channel. When the steady-state current was induced at the resting potential, hyperpolarization to more negative potentials caused it to decay with exactly the same time course as tail currents induced by short-duration depolarizing pulses, indicating that both types of currents are carried by identically-modified channels. The modified channels were shown to be inactivated very slowly at potentials more positive than - 100 mV, accounting for the limited depolarization observed in micro-electrode experiments. Even when applied directly to the internal face of the membrane, the effect of fenvalerate on the sodium channel developed slowly, taking more than 90 min to reach its final level. Fenvalerate did not significantly affect potassium currents.

Voltage-dependent removal of sodium inactivation by N-bromoacetamide and pronase.

When perfused internally through crayfish giant axons, pronase removed sodium inactivation more than three times as fast at -100 mV as compared with -30 mV. N-bromoacetamide, applied internally, removed sodium inactivation twice as fast at -100 mV as at -30 mV, and the relative rate of removal declined with membrane depolarization in proportion to steady-state sodium inactivation. We conclude that in the closed conformation the sodium inactivation gate is partially protected from destruction by N-bromoacetamide and pronase.