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The activation of the action potential Na+ ionophore by veratridine and batrachotoxin is time- and concentration-dependent and completely reversible. Batrachotoxin acts more slowly than veratridine. The concentration dependence of activation at equilibrium suggests reversible interaction of each toxin with a single class of independent sites having dissociation constants at physiologic ion concentrations of 80 plus or minus 13 muM for veratridine and 0.4 plus or minus muM for batrachotoxin. The maximum velocity of Na+ uptake at 50 mM Na+ is 128 plus or minus 12 nmol/min/mg in the presence of batrachotoxin compared to 48 plus or minus 4 nmol/min/mg in the presence of veratridine. Treatment of cells with excess veratridine in addition to batrachotoxin inhibits batrachotoxin-dependent 22-Na+ uptake. The concentration dependence of this inhibition suggests that it reflects competitive displacement of batrachotoxin from its binding site by veratridine. The activation by veratridine and batrachotoxin is inhibited in a competitive manner by divalent cations. The inhibition by divalent cations exhibits significant ion specificity with Mn-2+ greater than Co-2+ greater than Ni-2+ greater than Ca-2+ greater than Mg-2+ greater than Sr-2+. The inhibition constants (KI) for Ca-2+ are 0.84 mM for veratridine-dependent 22-Na+ uptake and 1.2 mM for batrachotoxin-dependent 22-Na+ uptake. The activation by veratridine and batrachotoxin is inhibited in a noncompetitive manner by tetrodotoxin. The apparent KD for tetrodotoxin as 11 plus or minus 1 nM in the presence of 150 mM Na+ and approximately 8.5 nM in 50 mM Na+. Divalent cations do not affect the apparent KD for tetrodotoxin. A hypothesis is presented which suggests that batrachotoxin, veratridine, and divalent cations interact with an activation site associated with the action potential Na+ ionophore, whereas tetrodotoxin interacts with a physically and functionally independent site involved in the transport of monovalent cations by the ionophore.
Batrachotoxin has been reported to inhibit fast axonal transport. We have examined the effect of batrachotoxin on saltatory organelle movements in N115 neuroblastoma cells (a model of fast axonal transport) using time-lapse video intensification microscopy. Batrachotoxin (0.1-1.0 microM) inhibits saltatory organelle movement. Contrary to previously published hypotheses, this inhibition of intra-axonal movement depends upon the action of batrachotoxin on action potential Na+ channels. Evidence for this conclusion is: (1) the range of concentrations of batrachotoxin which inhibit saltatory organelle movement is consistent with the dose-response curve for the activation of action potential Na+ channels by batrachotoxin in N18 neuroblastoma cells; (2) tetrodotoxin, which blocks action potential Na+ channels, prevents the inhibition of organelle movements by batrachotoxin; (3) batrachotoxin has no effect on saltatory movement in cells, including some neuroblastoma cell lines, which lack action potential Na+ channels; and (4) in Na+-free or low Na+ media, batrachotoxin does not block organelle movement. We suggest that changes in internal ion concentrations which follow the influx of Na+ are responsible for the inhibition of fast axonal transport by batrachotoxin.
Batrachotoxin under certain conditions has a strong depolarizing effect on the innervated membrane of the monocellular electroplax preparation from the electric eel, El-ectrophorus electricus. No effect is observed when the toxin (50-200 nM) is applied to the resting membrane for periods up to 1 hr. However, if the membrane is exposed to batrachotoxin and the cell is subjected to stimulation at a stimulus voltage slightly above the threshold for action potential firing, a progressive prolongation of the action potential and concomitant progressive depolarization of the innervated membrane is observed. When the membrane is depolarized by 15-20 mV, a further abrupt all-or-none depolarization occurs, and the potential attains a steady-state value between 0 and -10 mV. Brief stimulation of a cell in the presence of batrachotoxin is sufficient to define a batrachotoxin-treated cell, even though negligible depolarization occurs. If depolarizing agents such as carbamoylcholine or potassium chloride are introduced to such a cell in concentrations that normally produce a 20-30 mV depolarization, the abrupt all-or-none depolarization immediately occurs. All-or-none depolarizations arising from either electrical stimulation or depolarizing agents are unaffected by d-tubocurarine but are completely reversed by tetrodotoxin. Batrachotoxin thus appears to activate only the action potential sodium channels. In the batrachotoxin-treated membrane, these channels can attain stable steady states in either a closed configuration at the normal resting potential or in an open configuration after complete depolarization. A striking hysteresis cycle thus can be generated, which is strongly indicative of a voltage-dependent interaction of the toxin with the action potential sodium channels.
The binding site for batrachotoxin, a lipid-soluble neurotoxin acting at Na+ channel receptor site 2, was localized using a photoreactive radiolabeled batrachotoxin derivative to covalently label purified and reconstituted rat brain Na+ channels. In the presence of the brevetoxin 1 from Ptychodiscus brevis and the pyrethroid RU51049, positive allosteric enhancers of batrachotoxin binding, a protein with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subunit was specifically covalently labeled. The region of the alpha subunit specifically photolabeled by the photoreactive batrachotoxin derivative was identified by antibody mapping of proteolytic fragments. Even after extensive trypsinization, and anti-peptide antibody recognizing an amino acid sequence adjacent to Na+ channel transmembrane segment IS6 was able to immunoprecipitate up to 70% of the labeled peptides. Analysis of a more complete digestion with trypsin or V8 protease indicated that the batrachotoxin receptor site is formed in part by a portion of domain I. The identification of a specifically immunoprecipitated photolabeled 7.3-kDa peptide containing transmembrane segment S6 from domain I restricted the site of labeling to residues Asn-388 to Glu-429 if V8 protease digestion was complete or Leu-380 to Glu-429 if digestion was incomplete. These results implicate the S6 transmembrane region of domain I of the Na+ channel alpha subunit as an important component of the batrachotoxin receptor site.
Batrachotoxins, including many congeners not previously described, were detected, and relative amounts were measured by using HPLC-mass spectrometry, in five species of New Guinean birds of the genus Pitohui as well as a species of a second toxic bird genus, Ifrita kowaldi. The alkaloids, identified in feathers and skin, were batrachotoxinin-A cis-crotonate (1), an allylically rearranged 16-acetate (2), which can form from 1 by sigmatropic rearrangement under basic conditions, batrachotoxinin-A and an isomer (3 and 3a, respectively), batrachotoxin (4), batrachotoxinin-A 3'-hydroxypentanoate (5), homobatrachotoxin (6), and mono- and dihydroxylated derivatives of homobatrachotoxin. The highest levels of batrachotoxins were generally present in the contour feathers of belly, breast, or legs in Pitohui dichrous, Pitohui kirhocephalus, and Ifrita kowaldi. Lesser amounts are found in head, back, tail, and wing feathers. Batrachotoxin (4) and homobatrachotoxin (6) were found only in feathers and not in skin. The levels of batrachotoxins varied widely for different populations of Pitohui and Ifrita, a result compatible with the hypothesis that these birds are sequestering toxins from a dietary source.
Batrachotoxin is present in remarkably high amounts in the skin of Phyllobates terribilis. Levels of batrachotoxin tend to be reduced when P. terribilis is maintained in captivity, but even after being confined for up to 6 years, these frogs were still at least five times more toxic than other Phyllobates species used by natives for poisoning blowgun darts. Batrachotoxin was not detectable in F1 progeny reared to maturity in captivity. Nerve and muscle preparations from wild-caught frogs and from the nontoxic F1 frogs were both insensitive to batrachotoxin. The regulatory site controlling sodium-channel activation and permeability appears to have been minimally altered to prevent interaction with batrachotoxin, but is still sensitive to other sodium conductance activators (veratridine, grayanotoxin) to which the frogs arenot exposed naturally.
BACKGROUND AND OBJECTIVE: To investigate the effects of barbiturates on batrachotoxin-modified sodium channels from different regions of the human heart. Single sodium channels from human atria were studied and compared with existing data from the human ventricle and from the central nervous system. METHODS: Sodium channels from preparations of human atrial muscle were incorporated into planar lipid bilayers in the presence of batrachotoxin, a sodium channel activator. The steady-state behaviour of single sodium channels was recorded in symmetrical 500 mmol NaCl before and after the addition of pentobarbital 0.34-1.34 mmol. RESULTS: The batrachotoxin-treated human atrial sodium channel had an average single-channel conductance of 23.8 +/- 1.6 pS in symmetrical 500 mmol NaCl and a channel fractional open time of 0.83 +/- 0.06. The activation mid-point potential was -98.0 +/- 2.3 mV. Extracellular tetrodotoxin (a specific sodium channel blocking agent) blocked these channels with a k(1/2) = 0.53 micromol at 0 mV. Pentobarbital reduced the time average conductance of single atrial sodium channels in a concentration-dependent manner (ID50 = 0.71 mmol). In the same way, the steady-state activation was shifted to more hyperpolarized potentials (-10.6 mV at 0.67 mmol pentobarbital). CONCLUSIONS: The properties of batrachotoxin-modified sodium channels from human atrial tissue did not differ greatly from those described for ventricular sodium channels in the literature. Our data yielded no explanation for the observed functional diversity. However, cardiac sodium channels differ from those found in the central nervous system.
Reserpine inhibited batrachotoxin-elicited sodium influx in guinea pig brain synaptoneurosomes with an IC50 of about 1 microM. In the presence of brevetoxin the IC50 increased to about 80 microM. 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 microM, then a rebound stimulation from 0.1 to 0.8 microM, followed by complete inhibition by 80 microM. The stimulation was prevented by the presence of brevetoxin; reserpine then smoothly inhibited binding with an IC50 of about 1 microM. Reserpine at 1 microM slightly reduced the off-rate of [3H]BTX-B binding measured in the presence of veratridine, while at a concentration of 50 microM it enhanced the off-rate, presumably by an allosteric mechanism. Reserpine at 0.3-10 microM 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 microM to an IC50 of about 50 microM. 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.
We have studied the effect of local anesthetics QX 572, which is permanently charged, and benzocaine, which is neutral, on batrachotoxin-activated sodium channels in mouse neuroblastoma N18 cells. The dose-response curves for each drug suggest that QX 752 and benzocaine each act on a single class of binding sites. The dissociation constants are 3.15 X 10(-5) M for QX 572 and 2.65 X 10(-4) M for benzocaine. Equilibrium and kinetic experiments indicate that both drugs are competitive inhibitors of batrachotoxin. When benzocaine and QX 572 are present with batrachotoxin, they are much more effective at inhibiting Na+ flux than would be predicted by a one-site model. Our results indicate that QX 572 and benzocaine bind to separate sites, each of which interacts competitively with batrachotoxin.
A fluorescent N- methylanthraniloyl derivative of the potent depolarizing agent batrachotoxin has been used to probe the structural and conformational properties of the neurotoxin receptor site on the voltage-dependent sodium channel. Batrachotoxin A 20-alpha-N- methylanthranilate (BTX-NMA) retains high affinity for its receptor site on the synaptosomal sodium channel with a Kd between 78 and 91 nM and an average site capacity of 2 pmol/mg of synaptosomal protein in the presence of Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin. The fluorescence emission of BTX-NMA upon binding to synaptosomes indicates a hydrophobic environment. Toxin V from L. quinquestriatus, an allosteric activator, effects a 20-nm red shift in the spectrum of bound BTX-NMA and a 4-fold enhancement in the fluorescence quantum yield disclosing a conformational change into a hydrophilic environment. Fluorescence resonance energy transfer measurements show that the distance separating the receptor sites is 37 +/- 10 A. Thus, the binding of alpha-scorpion toxin must involve conformational changes that extend over large distances from the batrachotoxin-binding locus. This information together with the distance measurements between the tetrodotoxin and alpha-scorpion toxin receptors and the conformational transition associated with this distance upon batrachotoxin addition indicate a conformationally flexible channel with coupling of sites through the polyatomic framework of individual subunits or through extensive alterations in subunit/subunit interactions.
(1) Single myelinated nerve fibers of Rana esculenta were treated with the steroidal alkaloid batrachotoxin, and Na+ currents and Na+-current fluctuations were measured near the resting potential under voltage-clamp conditions. Between test pulses the fibres were held at hyperpolarizing membrane potentials. (2) The spectral density of Na+-current fluctuations was fitted by the sum of a 1/f component and a Lorentzian function. The time constant tau c = 1/(2 pi fc) obtained from the corner frequency fc of the Lorentzian function approximately agreed with the activation time constant tau m of the macroscopic currents. (3) The conductance gamma of a single Na+ channel modified by batrachotoxin was calculated from the integral of the Lorentzian function and the steady-state Na+ current. At the resting potential V = 0 we obtained gamma - 1.6 pS, higher gamma-values of 3.2 and 3.45 pS were found at V = --8 and --16 mV, respectively. (4) The conductance of a modified Na+ channel is significantly lower than the values 6.4 to 8.85 pS reported in the literature for normal Na+ channels. Hence, our experiments are in agreement with the view that batrachotoxin acts in an 'all-or-none' manner on Na+ channels and creates a distinct population of modified channels.
A combined structural and electrophysiologic analysis on the effects of subarachnoid injections of batrachotoxin was conducted in rats. Swelling of neuronal perikarya, accompanied by areas of clearing, satellitosis, fatty metamorphosis and central chromatolysis were demonstrated in the spinal anterior horns. Studies on isolated extensor digitorum longus (fast) and soleus (slow) muscles showed membrane depolarization in the extensor 24 h after injection, with progressive augmentation of this phenomenon in animals given multiple injections of the toxin. Differential sensitivity of fast and slow muscles was evident: soleus was only slightly affected. Subarachnoid injections of batrachotoxin, which induced swelling of neuronal perikarya and axonal processes, also caused signs of denervation in the extensor muscle 7 days after injection, while spontaneous transmitter release was still present. Some structural and and most electrophysiologic alterations after batrachotoxin injections were reversible.
Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.
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.
The neurotoxins, batrachotoxin and veratridine, are specific activators of sodium channels and cause an increase in the rate of 22Na uptake in neuroblastoma cells. Yohimbine, an indolakylamine alkaloid, inhibits this batrachotoxin-induced 22Na uptake. The dose-response curve of yohimbine suggest that the inhibitor acts reversibly on a single class of binding sites with dissociation constant of 3--4 x 10(-5) M. The dissociation constant is not affected by depolarization from--41 to 0 mV. Kinetic and equilibrium experiments indicate that yohimbine is a competitive inhibitor of the action of batrachotoxin. These results support the conclusion that yohimbine inhibitis the sodium flux by acting on the channel gating mechanism rather than by occluding the channels.
Batrachotoxins are neurotoxic steroidal alkaloids first isolated from a Colombian poison-dart frog and later found in certain passerine birds of New Guinea. Neither vertebrate group is thought to produce the toxins de novo, but instead they likely sequester them from dietary sources. Here we describe the presence of high levels of batrachotoxins in a little-studied group of beetles, genus Choresine (family Melyridae). These small beetles and their high toxin concentrations suggest that they might provide a toxin source for the New Guinea birds. Stomach content analyses of Pitohui birds revealed Choresine beetles in the diet, as well as numerous other small beetles and arthropods. The family Melyridae is cosmopolitan, and relatives in Colombian rain forests of South America could be the source of the batrachotoxins found in the highly toxic Phyllobates frogs of that region.