Cobra ( Naja spp. ) nicotinic acetylcholine receptor exhibits resistance to Erabu sea snake ( Laticauda semifasciata) short-chain alpha-neurotoxin.

Snake alpha-neutotoxins of Elapidae venoms are grouped into two structural classes, short-chain and long-chain alpha-neutotoxins. While these two classes share many chemical and biological characteristics, there are also distinct dissimilarities between them, including their binding site on the nicotinic acetylcholine receptor (nAChR), specificity among species of Chordata, and the associated pharmacological effects. In the present study we test the hypothesis that structural motifs that evolved to confer natural resistance against conspecific long-chain alpha-neurotoxins in Elapidae snakes also interfere with the biological action of short-chain alpha-neurotoxins. We expressed functional nAChRs that contains segments or single residues of the Elapidae nAChR ligand binding domain and tested the effect of short-chain alpha-neurotoxin erabutoxin-a (ETX-a) from the Erabu sea snake Laticauda semifasciata on the acetylcholine-induced currents as measured by two-microelectrode voltage clamp. Our results show that the Elapidae nAChR alpha subunit segment T(154)-L(208) ligand binding domain has an inhibitory effect on the pharmacological action of ETX-a. This effect is primarily attributed to the presence of glycosylation at position N(189). If the glycosylation is removed from the T(154)-L(208) segment, the nAChR will be inhibited, however, to a lesser extent than seen in the mouse. This effect correlates with the variations in alpha-neurotoxin sensitivity of different species and, importantly, reflects the evolutionary conservation of the binding site on the nAChR polypeptide backbone per se. Phylogenetic analysis of alpha-neurotoxin resistance suggests that alpha-neurotoxin-resistant nAChR evolved first, which permitted the evolution of snake venom alpha-neurotoxins. A model describing alpha-neurotoxin resistance in Elapidae snakes is presented.

Effects of alpha-erabutoxin, alpha-bungarotoxin, alpha-cobratoxin and fasciculin on the nicotine-evoked release of dopamine in the rat striatum in vivo.

Snake neurotoxins (NTX) have proven to be valuable tools for the characterisation of muscular nicotinic acetylcholine receptor structure and function. It is very likely that they could also be utilised to identify subtypes of neuronal nicotinic receptors controlling specific functions within the central nervous system. In this study we examined the effects of long alpha NTX (alpha-bungarotoxin, alpha-Bgt, and alpha-cobratoxin, alpha-Cbt) and short alpha NTX (alpha-erabutoxin a, alpha-Ebt) as well as the anticholinesterase toxin fasciculin-2 (FAS), on the nicotine-evoked release of dopamine (DA) in the striatum, using the in vivo push-pull technique. The short toxins alpha-Ebt and FAS blocked the extracellular increase of DA evoked by nicotine at 4.2 microM concentrations and alpha-Ebt was more potent, as reflected by the blockade at the lower dose of 0.42 microM. In contrast, the long toxins showed a different profile of action. Alpha-Cbt did not show any blockade of the nicotine-evoked release of DA at the doses studied while alpha-Bgt did block it only at the higher dose (4.2 microM) These results indicate that short neurotoxins show a stronger interaction with striatal nicotinic receptors subtypes controlling DA release when compared to the long ones. This interaction of short neurotoxin polypeptides and presynaptic receptors may permit the further elucidation of the particular nicotinic receptor populations responsible for the modulation of striatal DA release.

Functional determinants by which snake and cone snail toxins block the alpha 7 neuronal nicotinic acetylcholine receptors.

Snakes and cone snails produce toxins which block muscular and/or neuronal nicotinic acetylcholine receptors (AChRs). This paper mostly focuses on the determinants by which a snake long chain curaremimetic toxin and the cone snail toxin ImI bind specifically to the alpha 7 neuronal receptor. In both cases, the site involves a small turn-like structure constrained by two half-cystines.

Partial inhibition of the in vitro infection of adult mouse dorsal root ganglion neurons by rabies virus using nicotinic antagonists.

The infection of target cells by rabies virus is effected through membrane receptors. Several authors have suggested that nicotinic receptors could be used by this virus, but no direct experimental evidence is available. In this study mouse dorsal root ganglia cells were treated with various nicotinic antagonists (dihydro-beta-eritroidine, mecamilamine, d-tubocurarin, hexametonium, alpha-bungarotoxin and erabutoxin). After incubation, the cultures were infected with rabies virus. Cells were fixed, and processed for immunodetection of rabies virus. Treatment with mecamilamine or d-tubocurarine reduced the percentage of infected neurons. None of the antagonists tested changed the percentage of infected non-neuronal cells.

The alpha-neurotoxin erabutoxin b causes fade at the rat end-plate.

d-Tubocurarine and the alpha-neurotoxins from snake venom are antagonists at the nicotinic acetylcholine receptor. It is well established that d-tubocurarine causes fade in neuromuscular transmission during repetitive nerve stimulation but paradoxically there are many reports which indicate that the alpha-neurotoxins do not cause such fade. We found that high concentrations of erabutoxin b (100-150 nM) from the venom of Laticauda semifasciata did not cause much fade in the rat diaphragm preparation. However, low concentrations of toxin (5 nM) caused severe fade which was similar to the effects of d-tubocurarine. The data suggest that fade may be caused by toxin binding to a high-affinity site on the postsynaptic acetylcholine receptor.

Dissociation of the end-plate potential run-down and the tetanic fade from the postsynaptic inhibition of acetylcholine receptor by alpha-neurotoxins.

The effects of so-called postsynaptic snake alpha-neurotoxins (alpha-bungarotoxin, cobratoxin, erabutoxin b) on the wanings of tetanic contraction (tetanic fade) and the run-down of end-plate potentials during stimulation at 100 Hz were studied, respectively, in intact and cut mouse phrenic nerve-diaphragm preparations. No tetanic fade was evident with high concentrations of toxins until the complete failure of contractile response whereas the tetanic fade was evident after prolonged incubation with lower concentrations of toxins. The proportion of junctions exhibiting end-plate potential run-down increased progressively during toxin incubation. However, depression of end-plate potential amplitude by the toxins was not necessarily accompanied by run-down. The tetanic fade and the run-down became more pronounced for a time shortly after washout of toxins despite the restoration of single twitches and end-plate potential amplitudes, indicating the presynaptic origin of these effects induced by alpha-neurotoxins. We demonstrated that the functions of the pre- and postsynaptic acetylcholine receptors can be dissociated by using the different kinetics of toxin-receptor interactions. The results also implicate that a positive feedback enhancement of transmitter release operates via the presynaptic acetylcholine receptor in the neuromuscular junction in normal physiological conditions during repetitive pulses.

Nicotinic antagonists produce differing amounts of tetanic fade in the isolated diaphragm of the rat.

The effects of nicotine antagonists on single twitches, trains of four twitches and tetanic contractions of the isolated diaphragm of the rat were examined. Different drugs were found to produce different amounts of tetanic fade relative to depression of twitch tension. The order of activity from most able, to least able to produce fade was: hexamethonium greater than trimetaphan=atracurium=tubocurarine greater than pancuronium greater than erabutoxin b. The effect of erabutoxin b was distinctive for its almost complete lack of tetanic fade. 3,4-Diaminopyridine increased tetanic fade produced by tubocurarine, atracurium and hexamethonium, but not that produced by erabutoxin b. It is concluded that nicotinic antagonists act at more than one site at the neuromuscular junction. Assuming block of the postjunctional acetylcholine receptor produces tension depression, a second or third site must be involved in producing tetanic fade. The possibility that tetanic fade results from block of the ion channel associated with the postjunctional acetylcholine receptor or from the block of a prejunctional nicotinic receptor is discussed.

Acetylcholine receptors of skeletal muscle cells in cultures of rat thymus glands.

Skeletal muscle fibres develop in cultures established from dissociated rat thymus glands. The properties of their acetylcholine receptors were compared with those on fibres in skeletal muscle cultures. Acetylcholine depolarized the thymus-derived muscle cells in a concentration-dependent manner. Under voltage clamp the reversal potential was determined to be -0.4 mV, which was not significantly different from the reversal potential of cells in the muscle cultures. In both types of cultures, sensitivity to ionophoretically applied acetylcholine decreased during the period when fibres were growing rapidly and then increased. The mean channel lifetime of acetylcholine-activated channels was similar in both types of muscle fibre (3-4 ms at -40 mV), but the single channel conductance was significantly higher in the thymus-derived cells (70-75 pS compared to 55-60 pS). The cholinoceptors on both types of muscle fibre had nicotinic properties as judged by their preferential activation by acetylcholine rather than by acetyl-beta-methylcholine and oxotremorine and by the selective blockade by tubocurarine and erabutoxin b rather than by atropine.

Histamine as an endogenous antagonist of nicotinic ACh-receptor.

The mechanism of an inhibitory effect of histamine on the sensitivity of frog skeletal muscle endplate was analyzed by studying the dose-response relation between the quantity of ACh applied iontophoretically and the ACh-induced postsynaptic current (ACh current), and also the interaction between histamine and erabutoxin-b (ETX-b). The results obtained show that histamine, like curare, decreased the sensitivity of ACh-receptor in a competitive manner.

Pre-and post-junctional effects of tubocurarine and other nicotinic antagonists during repetitive stimulation in the rat.

The effects of tubocurarine and trimetaphan have been examined at voltage-clamped rat diaphragm neuromuscular junctions during (a) single and repetitive stimulation of the phrenic nerve in cut muscles and (b) repetitive ionophoretic application of acetylcholine (ACh). Tubocurarine (2.5 X 10(-7)-10(-6)M) produced a concentration-dependent reduction in the amplitude of neurally evoked end-plate currents (e.p.c.s). It also reduced their time constant of decay (tau e.p.c.) in a manner that was independent of membrane potential, and not markedly dependent on the tubocurarine concentration. Likewise the snake alpha-neurotoxin, erabutoxin b, reduced the e.p.c. amplitude and produced a voltage-independent shortening of tau e.p.c. Estimates of mean channel lifetime (tau noise) from ACh-induced e.p.c. fluctuations revealed that (a) tau noise was 46.4 +/- 3.7% shorter than tau e.p.c. measured at the same end-plate. At these same end-plates in the presence of tubocurarine (2.5 X 10(-7)M) tau e.p.c. was 32.6 +/- 1.0% shorter than the control tau e.p.c. but tubocurarine did not change tau noise, (b) trimetaphan (2.5 X 10(-5)-2 X 10(-4)M) produced a concentration-dependent and voltage-dependent reduction of tau e.p.c., and a concentration-dependent reduction of peak e.p.c. amplitude. Trimetaphan (2.5 X 10(-5)M) produced a 50% reduction of tau noise. (a) Both tubocurarine and trimetaphan produced concentration-dependent increases in the run-down of trains of neurally evoked e.p.c.s (50 Hz, 0.4 s). This effect did not vary with membrane potential in tubocurarine, but was voltage dependent when induced by trimetaphan. (b) Erabutoxin b reduced the e.p.c. amplitude but did not produce any increase in the run-down of trains of neurally evoked e.p.c.s. During 50 Hz repetitive ionophoretic application of ACh, tubocurarine (2.5 X 10(-7)M) reduced the amplitude of each current in the train without inducing any run-down of the current amplitudes. This effect was not dependent on the membrane potential. In contrast trimetaphan (2.5 X 10(-5)M) induced a voltage-dependent run-down of trains of ionophoretically evoked e.p.c.s. We conclude that tubocurarine and erabutoxin b reduce the e.p.c. amplitude by blocking the post-junctional ACh receptor. Tubocurarine produces tetanic rundown of e.p.c.s. by a prejunctional mechanism, whereas the effects of trimetaphan during single and repetitive stimulation are at least partly due to block of the open ion channel associated with the ACh receptor.

Biogenic antagonists of the nicotinic receptor: their interactions with erabutoxin.

The hypothesis that the sensitivity of the nicotinic ACh-receptor is reduced by some neurotransmitters was evaluated by studying the interaction between these neurotransmitters and erabutoxin-b (ETX-b), known to bind irreversibly with the specific ACh-receptor site. It was found that the blocking action of ETX-b was apparently prevented by previous application of 5-HT, whereas it was not prevented by application of catecholamine (CA). These results indicate that 5-HT blocks the nicotinic ACh-receptor by interacting with the specific ACh binding site, whereas CA blocks it by interacting with an allosteric site of the ACh-receptor ionic channel complex.

Fluorescent staining of neuromuscular junctions by using the antibody against acetylcholine receptors of Narke japonica, and double staining with the antibody and erabutoxin b.

The neuromuscular junctions of various vertebrates were visualized by indirect immunofluorescence microscopy using antibody against purified acetylcholine receptors (AChRs) of the electric organ from Narke japonica. Further, by using rhodamine-labeled erabutoxin b (TMR-Eb), we showed that AChRs at the neuromuscular junctions of frog, chick and mouse muscle could not be doubly stained with the antibody and erabutoxin b. AChRs of snake muscle could not be stained with TMR-Eb, while they were stained with the antibody against AChR. Moreover, the antibody did not inhibit the binding of 3H-labeled erabutoxin b to the solubilized AChRs from the mouse muscle. These results indicate that, as far as the antibodies against Narke AChRs are concerned, most antibody-binding sites in the molecules of muscle AChR in situ are different from those responsible for binding of the snake neurotoxin.

Effect of erabutoxin B on acetylcholine release and root potentials of the frog spinal cord.

Erabutoxin b (ETX), a sea snake neurotoxin with neuromuscular blocking properties, slightly reduced resting ACh release from the isolated fron spinal cord. ETX did not affect ACh release evoked by antidromic stimulation of ventral roots but reduced the ventral-dorsal root potential and prolonged the dorsal-ventral root potential. The latter effect was associated with a reduction in the orthodromically evoked ACh release. It is suggested that ETX did not affect the presynaptic terminals of the motor axon collaterals and that its effects occurred at postsynaptic sites.

Effect of erabutoxin on respiration of rabbits.

The effect of erabutoxin (ETX) was examined on respiration of rabbits. A single dose of 15 microgram/kg or 25 microgram/kg of ETX did not cause respiratory paralysis, but repeated administration of 15 microgram/kg of ETX at 3 hour intervals (three times) or 25 microgram/kg at 4 hour intervals (twice) caused fatal respiratory paralysis. Furthermore, a single administration of 50 microgram/kg of ETX caused fatal respiratory paralysis within 2 hours after the administration in all animals. This respiratory paralysis caused by ETX was reversible and of long duration (10-20 hour). This respiratory paralysis was not blocked by the administration of anticurare agents (neostigmine, edrophonium) or respiratory stimulants (nikethamide, picrotoxin, dimorpholamine). Since ETX had no influence on the circulatory system, death caused by ETX was prevented with application of artificial respiration.