Pumiliotoxin B binds to a site on the voltage-dependent sodium channel that is allosterically coupled to other binding sites.

Abstract

Pumiliotoxin B (PTX-B), an alkaloid that has cardiotonic and myotonic activity, increases sodium influx in guinea pig cerebral cortical synaptoneurosomes. In the presence of scorpion venom (Leiurus) or purified a-scorpion toxin, the PTX-B-induced sodium influx is enhanced severalfold. PTX-B alone has no effect on sodium flux in N18 neuroblastoma cells but, in the presence of a-scorpion toxin, stimulation of sodium influx by PTX-B reaches levels comparable to that attained with the sodium channel activator veratridine. In neuroblastoma LV9 cells, a variant mutant that lacks sodium channels, neither veratridine nor PTX-B induces sodium fluxes in either the presence or absence of a-scorpion toxin. In synaptoneurosomes and in N18 cells, the sodium influx induced by the combination of PTX-B and a-scorpion toxin is inhibited by tetrodotoxin and local anesthetics. PTX-B does not interact with two of the known toxin sites on the sodium channel, as evidenced by a lack of effect on binding of [3Hlsaxitoxin or [3Hlbatrachotoxinin A benzoate to brain synaptoneurosomes. Synergistic effects on sodium influx with a-scorpion toxin, ,8-scorpion toxin, and brevetoxin indicate that PTX-B does not interact directly with three other toxin sites on the sodium channel. Thus, PTX-B appears to activate sodium influx by interacting with yet another site on the voltage-dependent sodium channel, a site that is coupled allosterically to sites for a-scorpion toxin, ,8-scorpion toxin, and brevetoxin.The alkaloid pumiliotoxin B (PTX-B) has positive inotropic and chronotropic effects in isolated spontaneously beating atria (1-3). In neuromuscular preparations, PTX-B prolongs and potentiates directly elicited muscle twitches (4) MATERIALS AND METHODSBuffers. The following buffers were used: sodium-free incubation buffer, 50 mM Hepes (adjusted to pH 7.4 at 36°C with 50 mM Tris buffer)/130 mM choline chloride/5.4 mM KCI/0.8 mM MgSO4/5.5 mM glucose containing bovine serum albumin at 1 mg/ml; sodium influx buffer, 2.66 mM NaCl/50 mM Hepes-Tris (pH 7.4)/128 mM choline chloride/5.4 mM KCI/0.8 mM MgSO4/5.5 mM glucose containing 22NaCl at 1.3 ,uCi/ml (1 Ci = 37 GBq), bovine serum albumin at 1 mg/ml, and 5 mM ouabain; washing buffer, 5 mM Hepes-Tris (pH 7.4)/163 mM choline chloride/0.8 mM MgSO4/1.8 mM CaCl2 containing bovine serum albumin at 1 mg/ml. Synaptoneurosome Preparation. Guinea pig cerebral cortical synaptoneurosomes were obtained as described by Hollingsworth et al. (7). Briefly, the cortex of one brain was homogenized in 7-10 vol of sodium-free incubation buffer (pH 7.4) in a glass-glass homogenizer (5 strokes). The suspension was centrifuged at this point at 1000 x g for 10 min; then the supernatant was decanted and the pellet was suspended in an appropriate volume of sodium-free incubation buffer to a concentration of =3 mg of protein/ml. Sodium Influx into Synaptoneurosomes. Sodium flux studies were conducted essentially as described by Tamkun and Catterall (8) for synaptosomes. Briefly, aliquots of synaptoneurosome suspensions con...

Constraint shapes convergence in tetrodotoxin-resistant sodium channels of snakes

“…However, these toxins do not bind to the outer pore but instead to the inner pore (BTX) (48)(49)(50) or to other transmembrane helices (PTX-B) (47,51). Thus, although overall Na + channel structure in L. epinephelus likely is influenced by multiple prey toxins, we are confident that the P-loop replacements we discuss have been shaped by selection from TTX, because the outer pore is the target of TTX, and these functional mutations are at sites critical to TTX binding but uninvolved in BTX or PTX-B binding (47)(48)(49)(50)(51).…”

“…L. epinephelus also is one of the few predators of deadly dart-poison frogs (25), some of which possess batrachotoxin (BTX) and pumiliotoxin (PTX-B) (46). Both toxins interfere with Na + channel inactivation, causing the channels to remain persistently open (16,47,48). However, these toxins do not bind to the outer pore but instead to the inner pore (BTX) (48)(49)(50) or to other transmembrane helices (PTX-B) (47,51).…”

Evidence for an enantioselective pumiliotoxin 7-hydroxylase in dendrobatid poison frogs of the genus Dendrobates

“…Such high toxicity does not seem to correlate with the only known mechanism of action of PTXs, namely as positive modulators of sodium channels (40,41), where PTX 251D and aPTX 267A seemed ineffective and only weakly active, respectively (42)(43)(44). It is noteworthy that, in initial experiments with anticonvulsants (45), the toxic effects of PTX (ϩ)-251D were reduced by carbamazepine, an anticonvulsant that does target sodium channels, whereas diazepam, which targets ␥-aminobutyric acid type A (GABA A ) receptors, and dizocilpine, which targets N-methyl-D-aspartate (NMDA) receptors, were relatively ineffective.…”

Effects of newt chemical cues on the distribution and foraging behavior of stream macroinvertebrates

“…Voltage-gated sodium channels, which are the target of TTX, control action potentials (Catterall, 1980). These channels are also the targets of numerous poisons known to affect macroinvertebrate physiology (Catterall, 1980;Catterall et al, 1981;Gusovsky et al, 1988;Olivera et al, 1990;Narahashi et al, 1992;Song et al, 1996;Cestele et al, 1998;Dechraoui et al, 1999;Li et al, 2001;Yotsu-Yamashita et al, 2004;Du et al, 2013). Some taxa buffer the effects of such poisons by investing energetic resources into cuticle deposition, thereby decreasing the need to upregulate transcription of more enzymes to catalyze the metabolism of poisons (Wood et al, 2010).…”