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How do chemically defended animals resist their own toxins? This intriguing question on the concept of autotoxicity is at the heart of how species interactions evolve. In this issue of Molecular Ecology (Molecular Ecology, 2024, 33), Bodawatta and colleagues report on how Papua New Guinean birds coopted deadly neurotoxins to create lethal mantles that protect against predators and parasites. Combining chemical screening of the plumage of a diverse collection of passerine birds with genome sequencing, the researchers unlocked a deeper understanding of how some birds sequester deadly batrachotoxin (BTX) from their food without poisoning themselves. They identified that birds impervious to BTX bear amino acid substitutions in the toxin-binding site of the voltage-gated sodium channel Nav1.4, whose function is essential for proper contraction and relaxation of vertebrate muscles. Comparative genetic and molecular docking analyses show that several of the substitutions associated with insensitivity to BTX may have become prevalent among toxic birds through positive selection. Intriguingly, poison dart frogs that also co-opted BTX in their lethal mantles were found to harbour similar toxin insensitivity substitutions in their Nav1.4 channels. Taken together, this sets up a powerful model system for studying the mechanisms behind convergent molecular evolution and how it may drive biological diversity.

Biological toxins are entrenched within ecosystems. Thus, animals are often exposed to such toxins, and how they adapt can be a key determinant of their evolutionary trajectories. In this review, we provide an overview of the diversity of toxin resistance mechanisms, with a focus on animals that sequester toxins from their diet and their natural predators and parasites. We propose a structured framework in which to study toxin resistance by recategorizing and reorganizing known mechanisms into avoidance, metabolism, and target categories. Then, using this framework, we review evidence regarding how animals resist four widely studied compounds: tetrodotoxin, batrachotoxin, cardiotonic steroids, and pyrrolizidine alkaloids. Based on the available data, we conclude that toxin resistance and sequestration are interrelated from both ecological and evolutionary perspectives. To conclude, we highlight open questions regarding toxin resistance and review its importance as a field. En los ecosistemas las toxinas de origen biológico son componentes intrínsecos. Por esta razón, los animales se ven expuestos frecuenciamente a dichas toxinas y la forma en que se adaptan puede ser un factor que determina su trayectoria evolutiva. Esta revisión ofrece una visión general de la diversidad de mecanismos de resistencia a toxinas, centrándose en animales que secuestran toxinas de su dieta y en sus depredadores y parásitos naturales. En este texto se propone un marco estructural para estudiar la resistencia a toxinas mediante la recategorización y reorganización de mecanismos conocidos en categorías de: evación, metabolismo y moléculas diana. A continuación, utilizando este marco, revisamos la literatura científica en busca de evidencia sobre cómo los animales resisten a cuatro compuestos ampliamente estudiados: tetrodotoxina, batracotoxina, esteroides cardiotónicos y alcaloides de pirrolizidina. A partir de los datos disponibles, llegamos a la conclusión de que la resistencia y la retención de toxinas están interrelacionadas tanto desde el punto de vista ecológico como evolutivo. Por último, destacamos algunas preguntas abiertas en torno a la resistencia a las toxinas y resaltamos su importancia como campo de estudio en el futuro. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (NaVs) are targets for a number of acute poisons. Many of these agents act as allosteric modulators of channel activity and serve as powerful chemical tools for understanding channel function. Herein, we detail studies with batrachotoxin (BTX), a potent steroidal amine, and three ester derivatives prepared through de novo synthesis against recombinant NaV subtypes (rNaV1.4 and hNaV1.5). Two of these compounds, BTX-B and BTX-cHx, are functionally equivalent to BTX, hyperpolarizing channel activation and blocking both fast and slow inactivation. BTX-yne-a C20-n-heptynoate ester-is a conspicuous outlier, eliminating fast but not slow inactivation. This property differentiates BTX-yne among other NaV modulators as a unique reagent that separates inactivation processes. These findings are supported by functional studies with bacterial NaVs (BacNaVs) that lack a fast inactivation gate. The availability of BTX-yne should advance future efforts aimed at understanding NaV gating mechanisms and designing allosteric regulators of NaV activity.

Frogs in the genus Phyllobates are known for the presence of batrachotoxin, a highly toxic alkaloid, in their skin. Nevertheless, Phyllobates frogs from Costa Rica and Panama (P. lugubris and P. vittatus) are considered non-toxic, as they have been reported to harbor low concentrations of this alkaloid. However, the potential toxicity of Central American Phyllobates has not been assessed experimentally. Our goal was to determine the toxicity of the whole skin of P. vittatus, an endemic species from the Southeastern Pacific region of Costa Rica. We performed median lethal dose (LD50) tests in mice to determine general toxicity, and an irritant assay based on the behavioral responses of mice to subcutaneous injection, to determine differences in irritability, as a measure of toxicity, among three study localities. Using UPLC-ESI-QTOF, we obtained chemical profiles of the methanolic extract of frog skins. Due to the absence of mortality at the studied doses, we were unable to estimate LD50. However, we recorded a list of toxicity symptoms in mice that are consistent with cardiotoxic effects, and found that mice presented more symptoms at higher concentrations of skin extracts during the first hour of the LD50 assays, recovering completely at all doses by the end of the assay. On the other hand, we did not detect differences in irritability among studied localities. Additionally, we putatively identified three toxic alkaloids (Batrachotoxinin A, DHQ 251A and Lehm 275A). This study provides the first experimental data on the toxicity and associated symptoms in mice, as well as the chemical profile of the skin of P. vittatus. We suggest that the skin alkaloids of P. vitattus may confer a chemical defense towards predators. [ABSTRACT FROM AUTHOR]

Toxicity is widespread among living organisms, and evolves as a multimodal phenotype. Part of this phenotype is the ability to avoid self‐intoxication (autoresistance). Evolving toxin resistance can involve fitness tradeoffs, so autoresistance is often expected to evolve gradually and in tandem with toxicity, resulting in a correlation between the degrees of toxicity and autoresistance among toxic populations. We investigate this correlation in Phyllobates poison frogs, notorious for secreting batrachotoxin (BTX), a potent neurotoxin that targets sodium channels, using ancestral sequence reconstructions of BTX‐sensing areas of the muscular voltage‐gated sodium channel. Reconstructions suggest that BTX resistance arose at the root of Phyllobates, coinciding with the evolution of BTX secretion. After this event, little or no further evolution of autoresistance seems to have occurred, despite large increases in toxicity throughout the history of these frogs. Our results, therefore, provide no evidence in favor of an evolutionary correlation between toxicity and autoresistance, which conflicts with previous work. Future research on the functional costs and benefits of mutations putatively involved in BTX resistance, as well as their prevalence in natural populations, should shed light on the evolutionary mechanisms driving the relationship between toxicity and autoresistance in Phyllobates frogs. [ABSTRACT FROM AUTHOR]

The steroidal ABCD-ring system of the potent neurotoxin batrachotoxin was efficiently assembled in a convergent fashion. Bridgehead radical coupling between the simple AB-ring and D-ring fragments (3 and 4) formed the sterically congested linkage at the C9-oxygen-attached tetrasubstituted carbon. The C-ring was then cyclized by the Pd/Ni-promoted Ullmann reaction of the vinyl triflate and vinyl bromide of 19, giving rise to tetracyclic structure 1. [ABSTRACT FROM AUTHOR]

Poison-dart Phyllobates terribilis frogs sequester lethal amounts of steroidal alkaloid batrachotoxin (BTX) in their skin as a defense mechanism against predators. BTX targets voltage-gated Na+ channels and enables them to open persistently. How BTX autoresistance arises in such frogs remains a mystery. The BTX receptor has been delineated along the Na+ channel inner cavity, which is formed jointly by four S6 transmembrane segments from domains D1 to D4. Within the P. terribilis muscle Na+ channel, five amino acid (AA) substitutions have been identified at D1/S6 and D4/S6. We therefore investigated the role of these naturally occurring substitutions in BTX autoresistance by introducing them into rat Nav1.4 muscle Na+ channel, both individually and in combination. Our results showed that combination mutants containing an N1584T substitution all conferred a complete BTX-resistant phenotype when expressed in mammalian HEK293t cells. The single N1584T mutant also retained its functional integrity and became exceptionally resistant to 5 μM BTX, aside from a small residual BTX effect. Single and combination mutants with the other four S6 residues (S429A, I433V, A445D, and V1583I) all remained highly BTX sensitive. These findings, along with diverse BTX phenotypes of N1584K/A/D/T mutant channels, led us to conclude that the conserved N1584 residue is indispensable for BTX actions, probably functioning as an integral part of the BTX receptor. Thus, complete BTX autoresistance found in P. terribilis muscle Na+ channels could emerge primarily from a single AA substitution (asparagine→threonine) via a single nucleotide mutation (AAC→ACC). [ABSTRACT FROM AUTHOR]

An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous quantification of batrachotoxin and epibatidine in plasma. Plasma samples were pretreated by liquid-liquid extraction with acetonitrile and methanol. The toxins were separated on a reversed phase C18-column (2.1 mm × 50 mm, 1.7 μm) using a formic acid/acetonitrile gradient elution. Quantification was carried out by mass chromatography with each product ion referenced against midazolam-d 4 as an internal standard (IS). The two toxins and the IS were separated within 2 min. The calibration curves for the two toxins spiked into human plasma showed good linearities in the range from 2.5 to 250 ng/mL. The detection limits were estimated to be 0.5 ng/mL for batrachotoxin and 1 ng/mL for epibatidine with a signal-to-noise ratio of 3:1. Overall recoveries ranged from 69.6% to 98.2%, and no significant matrix effects were observed. The intra- and interday accuracies were 94.7–102.3%, and the precisions were 1.0–10.3%. This method was successfully applied for the quantification of batrachotoxin and epibatidine in rat plasma samples taken after intraperitoneal administration of the toxins. This is the first report to use UPLC-MS/MS to simultaneously quantify batrachotoxin and epibatidine in plasma samples. [ABSTRACT FROM AUTHOR]

The steroidal neurotoxin (−)-batrachotoxin functions as a potent agonist of voltage-gated sodium ion channels (NaVs). Here we report concise asymmetric syntheses of the natural (−) and non-natural (+) antipodes of batrachotoxin, as well both enantiomers of a C-20 benzoate–modified derivative. Electrophysiological characterization of these molecules against NaV subtypes establishes the non-natural toxin enantiomer as a reversible antagonist of channel function, markedly different in activity from (−)-batrachotoxin. Protein mutagenesis experiments implicate a shared binding side for the enantiomers in the inner pore cavity of NaV. These findings motivate and enable subsequent studies aimed at revealing how small molecules that target the channel inner pore modulate NaV dynamics. [ABSTRACT FROM AUTHOR]

The in vivo antidote activity of 72 diterpenoid alkaloids isolated from plants of the genera Aconitum, Delphinium, and Consolida and their derivatives was tested against poisoning of mice by a lethal dose of aconitine. The structure-activity relationship was analyzed. The use of diterpenoid alkaloids as versatile antidotes against poisoning of animals and humans by aconitine-, veratridine-, and batrachotoxin-type neurotoxins was justified. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium and calcium channels play key roles in the physiology of excitable cells. The alpha-1 subunit of these channels folds from a polypeptide chain of four homologous repeats. In each repeat, the cytoplasmic halves of the pore-lining helices contain exceptionally conserved asparagines. Such conservation implies important roles, which are unknown. Mutations of the asparagines affect activation and inactivation gating as well as the action of pore-targeting ligands, including local anesthetics and steroidal agonists batrachotoxin and veratridine. In the absence of the open-channel structures, underlying mechanisms are unclear. Here, we modeled the pore module of Cav1.2 and Nav1.4 channels and their mutants in the open and closed states using the X-ray structures of potassium and sodium channels as templates. The energy of each model was Monte Carlo-minimized. The asparagines do not face the pore in the modeled states. In the open-channel models, the asparagine residue in a given repeat forms an inter-repeat H-bond with a polar residue, which is typically nine positions downstream from the conserved asparagine in the preceding repeat. The H-bonds, which are strengthened by surrounding hydrophobic residues, would stabilize the open channel and shape the open-pore geometry. According to our calculation, the latter is much more sensitive to mutations of the asparagines than the closed-pore geometry. Rearrangement of inter-repeat contacts may explain effects of these mutations on the voltage dependence of activation and inactivation and action of pore-targeting ligands. [ABSTRACT FROM AUTHOR]

Abstract: Frogs of the genus Phyllobates from Colombia are known to contain the highly toxic alkaloid batrachotoxin, but species from Central America exhibit only very low levels or are entirely free of this toxin. In the present study alcohol extracts from 101 specimens of Phyllobates lugubris and Phyllobates vittatus and 21 of three sympatric species (Dendrobates pumilio, Dendrobates auratus, Dendrobates granuliferus) from Costa Rica were analyzed by gas chromatography-mass spectrometry. Whereas the extracts of the Dendrobates species exhibited typical profiles of toxic alkaloids, those of the two Phyllobates species contained low levels of few alkaloids only, batrachotoxin was not detected. Although the feeding pattern of the Dendrobates and Phyllobates species are similar as revealed by examination of their stomach content (mainly ants and mites), the Phyllobates species are poorly sequestering alkaloids from their food source in contrast to the Dendrobates frogs. [Copyright &y& Elsevier]

Ion channels are targets for many naturally occurring toxins and small-molecule drugs. Despite great progress in the X-ray crystallography of ion channels, we still do not have a complete understanding of the atomistic mechanisms of channel modulation by ligands. In particular, the importance of the simultaneous interaction of permeant ions with the ligand and the channel protein has not been the focus of much attention. Considering these interactions often allows one to rationalize the highly diverse experimental data within the framework of relatively simple structural models. This has been illustrated in earlier studies on the action of local anesthetics, sodium channel activators, as well as blockers of potassium and calcium channels. Here, we discuss the available data with a view to understanding the use-, voltage-, and current carrying cation-dependence of the ligand action, paradoxes in structure–activity relationships, and effects of mutations in these ion channels. [Copyright &y& Elsevier]

Ion channels in cell membranes are targets for a multitude of ligands including naturally occurring toxins, illicit drugs, and medications used to manage pain and treat cardiovascular, neurological, autoimmune, and other health disorders. In the past decade, the x-ray crystallography revealed 3D structures of several ion channels in their open, closed, and inactivated states, shedding light on mechanisms of channel gating, ion permeation and selectivity. However, atomistic mechanisms of the channel modulation by ligands are poorly understood. Increasing evidence suggest that cationophilic groups in ion channels and some ligands may simultaneously coordinate permeant cations, which form indispensible (but underappreciated) components of respective receptors. This review describes ternary ligand-metal-channel complexes predicted by means of computer-based molecular modeling. The models rationalize a large body of experimental data including paradoxes in structure-activity relationships, effects of mutations on the ligand action, sensitivity of the ligand action to the nature of current-carrying cations, and action of ligands that bind in the ion-permeation pathway but increase rather than decrease the current. Recent mutational and ligand-binding experiments designed to test the models have confirmed the ternary-complex concept providing new knowledge on physiological roles of metal ions and atomistic mechanisms of action of ion channel ligands. [ABSTRACT FROM AUTHOR]

Abstract: Here we report on a novel fluorescent analog of phenytoin as a potential inhibitor of neuropathic pain with potential use as an imaging agent. Compound 2 incorporated a heptyl side chain and dansyl moiety onto the parent compound phenytoin and produced greater displacement of BTX from sodium channels and greater functional blockade with greatly reduced toxicity. Compound 2 reduced mechano-allodynia in a rat model of neuropathic pain and was visualized ex vivo in sensory neuron axons with two-photon microscopy. These results suggest a promising strategy for developing novel sodium channel inhibitors with imaging capabilities. [Copyright &y& Elsevier]

Abstract: Deltamethrin, a pyrethroid insecticide, and BTG 502, an alkylamide insecticide, target voltage-gated sodium channels. Deltamethrin binds to a unique receptor site and causes prolonged opening of sodium channels by inhibiting deactivation and inactivation. Previous 22Na+ influx and receptor binding assays using mouse brain synaptoneurosomes showed that BTG 502 antagonized the binding and action of batrachotoxin (BTX), a site 2 sodium channel neurotoxin. However, the effect of BTG 502 has not been examined directly on sodium channels expressed in Xenopus oocytes. In this study, we examined the effect of BTG 502 on wild-type and mutant cockroach sodium channels expressed in Xenopus oocytes. Toxin competition experiments confirmed that BTG 502 antagonizes the action of BTX and possibly shares a common receptor site with BTX. However, unlike BTX which causes persistent activation of sodium channels, BTG 502 reduces the amplitude of peak sodium current. A previous study showed that BTG 502 was more toxic to pyrethroid-resistant house flies possessing a super-kdr (knockdown resistance) mechanism than to pyrethroid-susceptible house flies. However, we found that the cockroach sodium channels carrying the equivalent super-kdr mutations (M918T and L1014F) were not more sensitive to BTG 502 than the wild-type channel. Instead, a kdr mutation, F1519I, which reduces pyrethroid binding, abolished the action of BTG 502. These results provide evidence the actions of alkylamide and pyrethroid insecticides require a common sodium channel residue. [Copyright &y& Elsevier]

The article surveys the concentrations of defensive toxins of the batrachotoxin family of steroidal alkaloid neurotoxins in various tissues of the New Guinean genus Hooded Pitohui dichrous using radioligand binding assays. The areas of the bird's body detected with the highest toxin are the skin and feathers and toxins were also found in skeletal muscle, heart and liver. Also examined are the morphological or histological adaptations of the skin and feathers for storing and secreting toxins using scanning and transmission electron microscopy.

Investigation on the total synthesis of batrachotoxin 1 revealed that the cyclic ketals could be cleaved to ketone with IBX in DMSO at 70-81 °C under very mild conditions. A variety of functional groups showed no interference under these conditions except vinyl bromides, which, in fact, inhibits the single electron transfer to IBX. [ABSTRACT FROM AUTHOR]

Abstract: Bird species in the avian genus Pitohui contain potent neurotoxic alkaloids that may be used for defense. The genus comprises multiple species that are endemic to New Guinea and were presumed to belong to the family Pachycephalidae or Colluricinclidae, within the core corvoidea, an ancient Australasian radiation of crow-like birds. In order to understand the evolution of toxicity within the genus Pitohui, we sequenced three mitochondrial and two nuclear gene segments and reconstructed a phylogeny of the genus Pitohui and its putative relatives. We show that the genus Pitohui is polyphyletic, and consists of five different lineages. Using Bayesian ancestral state reconstruction, we estimate that toxicity likely evolved multiple times within this group. Furthermore, because the morphological and behavioral similarity among these poisonous birds appears to have evolved convergently, we hypothesize that this may be a possible example of Müllerian mimicry in birds. The Morningbird of Palau, Micronesia, that has often been included in the genus Pitohui, actually belongs in the genus Pachycephala and offers an intriguing case of pronounced evolution on a remote oceanic island. [Copyright &y& Elsevier]

Abstract: Voltage-gated sodium, calcium, and potassium channels generate electrical signals required for action potential generation and conduction and are the molecular targets for a broad range of potent neurotoxins. These channels are built on a common structural motif containing six transmembrane segments and a pore loop. Their pores are formed by the S5/S6 segments and the pore loop between them, and they are gated by bending of the S6 segments at a hinge glycine or proline residue. The voltage sensor domain consists of the S1–S4 segments, with positively charged residues in the S4 segment serving as gating charges. The diversity of toxin action on these channels is illustrated by sodium channels, which are the molecular targets for toxins that act at six or more distinct receptor sites on the channel protein. Both hydrophilic low molecular weight toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Hydrophobic alkaloid toxins and related lipid-soluble toxins act at intramembrane sites and alter voltage-dependent gating of sodium channels via an allosteric mechanism. In contrast, polypeptide toxins alter channel gating by voltage-sensor trapping through binding to extracellular receptor sites, and this toxin interaction has now been modeled at the atomic level for a β-scorpion toxin. The voltage-sensor trapping mechanism may be a common mode of action for polypeptide gating modifier toxins acting on all of the voltage-gated ion channels. [Copyright &y& Elsevier]

Abstract: Sodium channel activators, batrachotoxin and veratridine, cause sodium channels to activate easier and stay open longer than normal channels. Traditionally, this was explained by an allosteric mechanism. However, increasing evidence suggests that activators can bind inside the pore. Here, we model the open sodium channel with activators and propose a novel mechanism of their action. The activator-bound channel retains a hydrophilic pathway for ions between the ligand and conserved asparagine in segment S6 of repeat II. One end of the activator approaches the selectivity filter, decreasing the channel conductance and selectivity. The opposite end reaches the gate stabilizing it in the open state. [Copyright &y& Elsevier]

Abstract: A 96-well-microplate-based ion flux method utilizing readily available autoradiographic phosphorimaging detection is described. Nicotinic acetylcholine receptor-mediated 22Na influx in four cultured cell lines provided satisfactory concentration-response data for epibatidine and several other nicotinic agonists. The data were consistent with data obtained using standard 6-well assays. Assays for nicotinic-receptor-mediated 86Rb efflux produced data similar to data obtained with the 22Na influx assay. However, assays for 45Ca influx were not successful, although 45Ca was readily detected and quantified. Voltage-gated sodium channel-mediated 22Na influx in a neuroblastoma cell line allowed assay of the effects of such sodium channel activators as batrachotoxin and a pumiliotoxin B/scorpion venom combination. Phosphorimaging detection allows for reliable beta counting of up to 1200 simultaneous samples with excellent sensitivity and is amenable for application to high-throughput screening. [Copyright &y& Elsevier]

The presence and properties of voltage-gated Na+ channels in mesenteric artery smooth muscle cells (SMCs) were studied using whole cell patch-clamp recording. SMCs from mouse and rat mesenteric arteries were enzymatically dissociated using two dissociation protocols with different enzyme combinations. Na+ and Ca2+ channel currents were present in myocytes isolated with collagenase and elastase. In contrast, Na+ currents were not detected, but Ca2+ currents were present in cells isolated with papain and collagenase. Ca2+ currents were blocked by nifedipine. The Na+ current was insensitive to nifedipine, sensitive to changes in the extracellular Na+ concentration, and blocked by tetrodotoxin with an IC50 at 4.3 nM. The Na+ conductance was half maximally activated at -16 mV, and steady-state inactivation was half-maximal at -53 mV. These values are similar to those reported in various SMC types. In the presence of 1 μM batrachotoxin, the Na+ conductance voltage relationship was shifted by 27 mV in the hyperpolarizing direction, inactivation was almost completely eliminated, and the deactivation rate was decreased. The present study indicates that TTX-sensitive, voltage-gated Na+ channels are present in SMCs from the rat and mouse mesenteric artery. The presence of these channels in freshly isolated SMC depends critically on the enzymatic dissociation conditions. This could resolve controversy about the presence of Na+ channels in arterial smooth muscle. [ABSTRACT FROM AUTHOR]

Batrachotoxin is a potent modulator of voltage-gated sodium channels, leading to irreversible depolarisation of nerves and muscles, fibrillation, arrhythmias and eventually cardiac failure. Since its discovery, field researchers also reported numbness after their skin came into contact with this toxin. Intrigued by this phenomenon, we determined the effect of batrachotoxin on the voltage-gated sodium channel Nav1.8, which is considered to be a key player in nociception. As a result, we discovered that batrachotoxin profoundly modulates this channel: the inactivation process is severely altered, the voltage-dependence of activation is shifted towards more hyperpolarised potentials resulting in the opening of Nav1.8 at more negative membrane potentials and the ion selectivity is modified. [Copyright &y& Elsevier]

Voltage-gated Na+ channels are heteromeric membrane glycoproteins responsible for the generation of action potentials. A number of diverse lipid-soluble neurotoxins, such as batrachotoxin, veratridine, aconitine, grayanotoxins, pyrethroid insecticides, brevetoxins and ciguatoxin, target voltage-gated Na+ channels for their primary actions. These toxins promote Na+ channel opening, induce depolarization of the resting membrane potential, and thus drastically affect the excitability of nerve, muscle and cardiac tissues. Poisoning by these lipid-soluble neurotoxins causes hyperexcitability of excitable tissues, followed by convulsions, paralysis and death in animals. How these lipid-soluble neurotoxins alter Na+ channel gating mechanistically remains unknown. Recent mapping of receptor sites within the Na+ channel protein for these neurotoxins using site-directed mutagenesis has provided important clues on this subject. Paradoxically, the receptor site for batrachotoxin and veratridine on the voltage-gated Na+ channel α-subunit appears to be adjacent to or overlap with that for therapeutic drugs such as local anaesthetics (LAs), antidepressants and anticonvulsants. This article reviews the physiological actions of lipid-soluble neurotoxins on voltage-gated Na+ channels, their receptor sites on the S6 segments of the Na+ channel α-subunit and a possible linkage between their receptors and the gating function of Na+ channels. [Copyright &y& Elsevier]

Reserpine inhibited batrachotoxin-elicited sodium influx in guinea pig brain synaptoneurosomes with an IC50 of about 1 μM. In the presence of brevetoxin the IC50 increased to about 80 μM. Reserpine inhibited binding of batrachotoxinin-A [3H]benzoate ([3H]BTX-B) binding in a complex manner causing a partial inhibition from 0.001 to 0.08 μM, then a rebound stimulation from 0.1 to 0.8 μM, followed by complete inhibition by 80 μM. The stimulation was prevented by the presence of brevetoxin; reserpine then smoothly inhibited binding with an IC50 of about 1 μM. Reserpine at 1 μM slightly reduced the off-rate of [3H]BTX-B binding measured in the presence of veratridine, while at a concentration of 50 μM it enhanced the off-rate, presumably by an allosteric mechanism. Reserpine at 0.3–10 μM elicited a partial inhibition of the binding of [3H]brevetoxin-3. The local anesthetic dibucaine had effects similar to reserpine: It partially inhibited binding of [3H]brevetoxin. The presence of brevetoxin reduced the potency of dibucaine as an inhibitor of batrachotoxin-elicited sodium influx from an IC50 of about 2 μM to an IC50 of about 50 μM. The results suggest that reserpine binds at both a local anesthetic site to cause allosteric inhibition of batrachotoxin-binding and action, but that it also binds to another site causing, like brevetoxin, an enhancement of batrachotoxin-binding and action. Local anesthetics also may bind to the brevetoxin site. [ABSTRACT FROM AUTHOR]

Biochemical evidence indicates that veratridine (VTD) and batrachotoxin (BTX) share a common binding site in Na+ channels. Under whole-cell voltage-clamp conditions, we examined this single receptor hypothesis by studying the VTD phenotype in BTX-resistant muscle Na+ channels, µ1-I433K, N434K, L437K, F1579K, and N1584K. Derived from point mutations at segments D1–S6 and D4–S6, these mutant Na+ channels are resistant to 5 µM BTX when expressed in human embryonic kidney cells. In contrast to the wild-type phenotype, VTD at 200 µM elicits little or no maintained current during a test pulse at +50 mV, and little or no "tail" current after the test pulse in all BTX-resistant mutant channels. Paradoxically, VTD retains its ability to inhibit the peak Na+ current in BTX-resistant mutant Na+ channels. To explain these mutant phenotypes, we propose a two-step binding reaction scheme. An initial VTD-binding interaction with the Na+ channel results in the inhibition of peak current amplitude, and a second binding reaction results in the trapping of VTD within the D1–S6 and D4–S6 domain interface. The failure of BTX-resistant mutant Na+ channels to trap VTD suggests that segments of D1–S6 and D4–S6 form a common receptor for VTD and BTX. [ABSTRACT FROM AUTHOR]

The effects of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7), a novel neuroprotective compound, on the voltage-sensitive sodium channels (VSSC) were examined in the rat brain and cardiac myocytes. NS-7 inhibited [3H]batrachotoxinin A 20α-benzoate (BTX) binding (neurotoxin receptor site 2) in brain membranes with a Ki value of 1 μM , while the compound was less effective in the cardiac myocytes (Ki = 13 μM). Aconitine, on the other hand, inhibited [3H]BTX binding to brain membranes and cardiac myocytes with the same potency. In contrast, NS-7 had no affinity for [3H]saxitoxin binding in brain (neurotoxin receptor site 1). In superfused slices of the rat cerebral cortex, NS-7 inhibited the veratridine (5 μM)-evoked glutamate release in a concentration-dependent manner, the IC50 value of which was 7.7 μM, whereas the compound showed a weak and not significant suppression of KCl-evoked glutamate release. The tissue concentrations of NS-7 in the rat cerebral cortex and heart were 89 and 28 nmole/g tissue, respectively, 5 min after its intravenous injection (8 mg/kg). Furthermore, in the cerebral cortex, NS-7 distributed preferentially to the membrane-enriched synaptosomal fraction. Since neurotoxin receptor site 2 is located in the transmembrane region of the VSSC moiety, the channel function may be substantially inhibited by a peripheral administration of NS-7. These results suggest that the blockade of neurotoxin receptor site 2 of VSSC in the brain contributes to the neuroprotective action of NS-7. [ABSTRACT FROM AUTHOR]

The effects of aconitine and batrachotoxin (BTX) on Na currents and gating currents were studied on voltage clamped frog nodes of Ranvier. The relation between steady-state values of charge movement and test pulse potential ( Q-E) was compared with the fraction of open channels at a given potential ( F-E). In aconitine-treated nodes the half potential, E, of the F-E curve was −87 mV and the slope factor, k, was 6.6 mV. Compared to normal nodes aconitine shifted the F-E curve by −53 mV along the voltage axis without significantly altering the slope. The parameters of the Q- E curve of aconitine-treated nodes were E=−63 mV and k=18.8 mV compared with E=−30 mV and k=17 mV in a normal node. For the Q- E curve E was −76 mV, fairly close to E of the F-E curve, but the slope remained much smaller than that of the F-E curve. Thus, in aconitine-treated nodes, the normalized Q- E and Q- E curves stay below the F-E curve over almost the whole potential range, a situation different from that in untreated or chloramine-T-treated nodes (Drews 1987). Similar results were obtained with BTX. The F-E curve of BTX-treated fibres had a half potential E=−85.1 mV and a slope factor k=4.7 mV. In contrast to aconitine-treated nodes, the F-E curve showed a secondary rise at E>−50 mV. Compared to the F-E curve the half potential of the Q- E curve was more positive ( E=−49.2 mV) and the slope was smaller ( k=18.7 mV, similar to k=17 mV measured in normal nodes). Even when Q was used instead of Q the slope of the two curves remained very different. By contrast, Dubois et al. (1983) found that both curves coincide after BTX treatment. I could reproduce this result by using pulses of relatively long duration (4 or 15 ms). However, my experiments suggest that such long pulses are likely to produce artefacts in both the on- and off-response. Q-E and F-E curves similar to those observed on aconitine- or BTX-treated nodes can be calculated from the models of Neumcke et al. (1978) and Edmonds (1987 a, b). [ABSTRACT FROM AUTHOR]