The binding site of the nicotinic acetylcholine receptor in animal species resistant to alpha-bungarotoxin.

The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the alpha-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193. We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to alpha-bungarotoxin (alpha-BTX). Our earlier work on the snake and mongoose AChR, both of which do not bind alpha-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR alpha-subunit are important in determining the resistance of these AChRs to alpha-BTX. In the present study, we have examined the correlation between alpha-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human. Fragments of the AChR alpha-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli. The hedgehog fragment does not bind alpha-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder. The shrew and cat fragments bind alpha-BTX to a similar extent as the mouse fragment. The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the alpha-subunit, as is seen with the "toxin resistant" snake and mongoose, and in contrast with the "toxin binders", which have aromatic residues at these two positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological activities of [Thr2]sarafotoxin-b, a synthetic analogue of sarafotoxin-b.

The 21 amino acid sarafotoxins (SRTX) c and d/e as well as endothelin-3 (ET-3) are known to be less toxic and weaker pharmacologically than the other isopeptides SRTX-a, SRTX-b and ET-1. Since SRTX-c, SRTX-d/e and ET-3 possess a Thr instead of a Ser at position 2, we investigated the possibility that this mutation could be responsible for the observed biological differences. Here we show that the synthetic [Thr2]SRTX-b has indeed a lower vasoconstriction efficacy (approximately 35%) in the rabbit aorta, but it is nearly as potent as SRTX-b in toxicity tests and in influencing contraction of the rat uterus. Using monoclonal antibodies directed against the structurally related endothelin-1, we also show that the antigenicity of the analogue is comparable to that of SRTX-b, suggesting that the overall structure of the two peptides is similar, despite the substitution at position 2. We suggest that the Thr2 substitution contributes to the lower activity of the 'weak' peptides in some systems; however, additional substitutions found in the 'weak' peptides of the ET/SRTX family most probably contribute to their low pharmacological activity.

How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well.

Endothelin and sarafotoxin: influence on steroid-regulated motility of rat uterus.

The sarafotoxins (SRTX) and endothelins (ET) were shown to influence the motility of the isolated rat uterus by inducing an increase in the rate and in the maximum tension of the spontaneous rhythmic contractions and a suppression of the relaxation phase of these contractions. Ovariectomized rats, 24 weeks post-operation, show no spontaneous motility of their uteri and the SRTX/ET peptides induce only a slight tonic increase in the uterine tension. Treatment with 17 beta estradiol restores spontaneous motility and sensitivity to the SRTX/ET peptides in all three contraction modes. It is concluded that the influence of the SRTXs and ETs on uterine motility depends on the hormonal status of the animal.

Neutralization by homologous plasma of Crotalus durissus terrificus (South American rattlesnake) venom and crotoxin

A resistência de certos mamíferos à açäo tóxica de venenos de serpentes é bem conhecida, näo só na literatura especializada mas também popularmente. Essa resistência estende-se também a algumas serpentes venenosas e näo venenosas. O mecanismo responsável pelo fenômeno näo é único em todos os casos mas, em alguns, deve-se à presença de fatores antitóxicos no sangue circulante desses animais. Neste trabalho mostramos que o plasma de casvavel é capaz de neutralizar o efeito letal do veneno crotálico e de seu principal componente tóxico (crotoxina), em camundongos. O plasma crotálico inibe também a atividade fosfolipásica A2 da crotoxina in vitro, geralmente associada à sua toxicidade. A açäo neutralizante do plasma crotálico está associada á sua fraçäo alfa1-globulina, provavelmente devido à presença de um fator anti-tóxico na sua composiçäo

SRTX-d, a new native peptide of the endothelin/sarafotoxin family.

The primary structure of a new sarafotoxin, SRTX-d, from the venom of Atractaspis engaddensis is described. SRTX-d differs from SRTX-b in two substitutions: Ile19 instead of Val and Thr2 instead of Ser. The toxicity of SRTX-d and its vasoconstriction potency are very low in comparison to SRTX-a and SRTX-b, whereas its IC50 for 125I-SRTX-b binding is similar to that of SRTX-b. It is suggested that the Thr to Ser substitution, which is shared by two additional weak members of the endothelin/sarafotoxin family, SRTX-c and ET-3, affects the biological activity of SRTX-d as well.

Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake AcChoR.

Functional endothelin/sarafotoxin receptors in the rat uterus.

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) families were detected in the rat uterus. These receptors specifically bind 125I-SRTX-b (Bmax = 220 fmol/mg protein), as well as ET-1, ET-3 and SRTX-c (IC50's 10, 5, 300 and 780 nM, respectively). Activation of the uterine ET/SRTX receptors induced dose-dependent phosphoinositide (PI) hydrolysis and three typical contractile responses: 1) increase in the muscle tonic tension; 2) increase in frequency of the spontaneous rhythmic contractions; 3) decrease of relaxation in each spontaneous rhythmic cycle. All three effects appeared at doses as low as 0.5-1 nM. Dose responses yield ED50 values of 5.5, 30 and 680 nM for ET-1, SRTX-b and ET-3, respectively. SRTX-c was the least effective peptide in achieving decrease in relaxation. In view of these results, and since the uterine responses to the peptides were almost immediate and reversible, we suggest that the functional ET/SRTX receptor of the rat uterus that is coupled to PI hydrolysis may be of physiological significance.

Vasoconstrictor effects of sarafotoxins in rabbit aorta: structure-function relationships.

The sarafotoxins SRTX-a, b and c from the venom of the snake Atractaspis engaddensis are 21-amino acid peptides that affect the cardiovascular system. They are strong vasoconstrictors, the potency of which may be in correlation with their primary structure: SRTX-a, which differs from SRTX-b in a single amino acid residue (Asn instead of Tyr), shows about half of its maximal vasoconstriction, while SRTX-c, which differs in 3 additional residues is a very weak vasoconstrictor and, at high doses, shows vasodilatory effects. Sequential application of the three isotoxins result in a summated response.

Sarafotoxin, a novel vasoconstrictor peptide: phosphoinositide hydrolysis in rat heart and brain.

Sarafotoxins, a group of 21-residue cardiotoxic peptides from snake venom that induce coronary vasoconstriction, show high-affinity binding to rat atrial and brain membranes and activate the hydrolysis of phosphoinositides. Neither their binding nor their activity is affected by blockers or activators of known receptors and ion channels, suggesting that sarafotoxins act either directly on the phosphoinositide phosphodiesterase system or on a novel receptor. Their amino acid sequence shows a high degree of homology with that of endothelin, a recently described 21-residue vasoconstrictor peptide found in porcine aortic endothelium. This is remarkable, since endothelin is a natural compound of the mammalian vascular system while sarafotoxins are highly toxic components of snake venom.

A novel cardiotoxic polypeptide from the venom of Atractaspis engaddensis (burrowing asp): cardiac effects in mice and isolated rat and human heart preparations.

A new cardiotoxic polypeptide isolated from the venom of the snake Atractaspis engaddensis has an LD50 of 15 micrograms/kg body weight in white mice. Intravenous administration in mice of lethal doses of the toxin causes, within seconds, marked changes in the ECG, consisting primarily of a transient slope elevation of the S-T segment, a temporary diminution of the S-wave and an increase in the amplitudes of the R- and T-waves. Concomitantly, and apparently unrelated to these changes, a severe A-V block develops and leads to complete cardiac arrest within a few min. Studies with rat and human isolated heart preparations showed that the toxin exerts a powerful coronary vasoconstriction (rats), and positive inotropic effects (rats and humans).