Scorpions are predatory arthropode animals of the order Scorpiones within the class Arachnida.
All known scorpion species have venom, they use it mainly to kill or paralyze their prey, but also to defend themselves against predators. The venom is usually fast-acting, and it is composed by a mixture of neurotoxines, enzyme inhibitors, etc. The scorpion can regulate exactly the released quantity of every specific compound. The known species are 1000+ but only 25 can be lethal to human beings.
Scorpion toxins can be divided in four families which specifically interact with differen ion channels: Na+ channels, K+ channels, Cl channels and Ca2+ channels.
All known Na+ channel specific toxins are composed of 60–76 amino acid residues and stabilized by four disulfide bridges. Na+ channel specific toxins from scorpions are modifiers of the channel gating mechanism, and were initially divided into two groups: α and β toxins.
The K+channel specific toxin are composed of 31–39 amino acid residues long and stabilized by three or four disulfide bonds. K+ channel specific toxins are authentic blockers of the channels; they bind to the extracellular face of the channel and impede passage of ions. They are the best studied toxins.
Short chain scorpion toxins constitute the largest group of potassium (K+) channel blocking peptides; an important physiological role of the KCNA3 channel, also known as KV1.3, is to help maintain large electrical gradients for the sustained transport of ions such as Ca2+ that controls T lymphocyte (T cell) proliferation. Thus KV1.3 blockers could be potential immunosuppressants for the treatment of autoimmune disorders (such as rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis).
Chlorotoxin, specific for Cl − channels, has only 36 amino acid residues, but contains four disulfide bridges.
Despite the fact that the primary structure of scorpion toxins can be quite different, there is a constant structural motif or scaffold conserved among these families of proteins. Except for the Ca2+ channel specific scorpion toxins, whose threedimensional structure is still unknown, all scorpion toxic peptides have a highly conserved, dense core formed by an α helix and two to three strands of β sheet structural motifs, maintained by disulfide bridges.
FUNCTIONAL CLASSIFICATION OF Na+ CHANNEL SPECIFIC TOXIN: α and β toxin
Scorpion toxins have been divided into two principal types (α and β), based on their pharmacological effects on sodium channels, as verified by means of electrophysiological measurements and by their binding characteristics. Scorpion α toxins bind to site 3 of the Na+ channels in a voltage dependent mode, slowing or blocking the inactivation mechanism of these channels.
On the other hand, β type toxins bind to site 4 independently of membrane potential and affect sodium channel activation.
Physiological effects and allosteric interactions account for synergic activity in vivo.
The receptor sites of scorpion α and ß toxins on insect sodium channels interact allosterically. Using binding studies, it was demonstrated for the first time positive allosteric interactions between receptor sites 3 and 4 on insect sodium channels, which explain in part the synergic effects observed between α- and ß-toxins in vivo. The positive cooperativity observed on simultaneous binding of α- and ß-toxins increases the potency of the scorpion venom. The larger increase in LqhαIT binding and toxicity when combined with LqhIT2 vs. Bj-xtrIT is likely to result from the reciprocal allosteric interactions between LqhαIT and LqhIT2 receptor sites. The difference in allosteric interaction suggests that the excitatory and depressant ß toxins interact differently with receptor site 4. This view is supported by differences in the mode of action of excitatory and depressant toxins on insect neurons, differences in inhibition of their binding by antipeptide antibodies directed against various extracellular sodium channel regions, and the fact that they do not compete reciprocally in binding to fly and lepidoptera neuronal preparations.
One of the most intriguing findings is that the binding of the nontoxic ligand Bj-xtrIT-E15R to receptor site 4 (silent binding) enhances α toxin binding to receptor site 3 and its effect in vivo in a similar manner to that induced by Bj-xtrIT. This result suggests that mere binding of Bj-xtrIT-E15R induces a conformational change on the channel that can be probed by α-toxin binding. Moreover, this result raises the question whether that conformational change is related to the ß toxin physiological effect.
Since ligand binding at receptor site 4 and brevetoxin binding at receptor site 5 induce an additive increase in LqhαIT binding to receptor site 3, the conformational changes they produce are different. Receptor site 3 is mainly associated with the external loop S3-S4 in domain 4 of sodium channels, whereas receptor site 4 for ß-toxins is mainly assigned to domain 2. The allosteric interactions observed suggest cooperative interactions between domains 2 and 4 of insect sodium channels. Understanding such interactions at the molecular level requires identification of toxin receptor sites on the channel, and is an important area for future studies.
The enhancement of LqhαIT toxicity by Bj-xtrIT-E15R is considerably lower than that induced by coinjection with the unmodified Bj-xtrIT. This difference suggests that the synergic effect between α and ß toxins results from modulations of sodium channel gating that mutually enhance nerve firing in vivo, in addition to the allosteric interactions, which enhance toxin binding. α toxins induce long plateau potentials in axons attributed to an increase in the probability of sodium channels to remain in their open states due to inhibition of their fast inactivation. This effect increases neuronal excitability and neuromuscular activity. Under such conditions the activation of sodium channels at more negative membrane potentials by ß toxins could increase, as their effect is dependent and/or enhanced by membrane depolarization. ß-Toxins induce repetitive activity and depolarization of the axonal membrane potential in insect nerves, and hence may facilitate α toxin action on the channels in their open states. Thus, the enhancement in α toxin effect by ß toxin interaction with receptor site 4 results from an indirect modification of receptor site 3 and from alteration in the voltage dependence of channel activation.
SEVERAL GROUPS OF α-LIKE TOXIN ARE REVEALED BY ACTIVITY IN VIVO AND IN VITRO
The α- and α-like scorpion toxins are classified into several groups, according to their relative activity on mammals and insects.
1.The first group comprise the classical α-toxins highly active on mammals, AaH I, AaH II, AaH III, and Lqq V. These toxins demonstrate the highest affinity to vertebrate sodium channels and the lowest affinity to insect neuronal membranes.
2.The second group is represented by Lqq IV, shown to be very weakly active on insects; however, it is 54-fold less effective on mammals than AaH II. This toxin have been demonstrated to competitively inhibit the binding of AaH II to rat brain synaptosomes, as well as the binding of LqhαIT to insect sodium channels. Lqq IV may represent an intermediate scorpion toxin group, which binds with moderate affinities to both mammal and insect sodium channels but express its toxic activity mainly on mammals.
3.The third group consists of Bom III and IV, which are shown to be active on both insect and mice and compete at nanomolar concentrations for the binding of LqhαIT to insect sodium channels, but do not inhibit at all the binding of AaH II to rat brain synaptosomes. Bom III and IV are similarly active on mice and on insects and inhibit sodium current inactivation in both rat neuronal cells and in cockroach axon.
4.The fourth group consists of Lqq III and LqhαIT. These two homologous toxins demonstrate the highest affinity to insects, as opposed to the very low affinity to rat brain sodium channels. The activity of LqhαIT is very similar to that of Lqq III, but it reveals slightly higher specificity to insects versus mammals, which is also reflected by its lower ability to inhibit the binding of AaH II in rat brain membranes. Thus, LqhαIT and Lqq III are considered anti-insect α-scorpion toxins.
α Scorpion toxins receptor sites are homologous but not identical on mammal and insect sodium channels.
The existence of receptor site 3, which binds the classical α-scorpion toxins on mammalian sodium channels, could not be demonstrated on insects by direct binding studies, since no specific binding of AAH II has been detected in locust neuronal membranes, probably due to the very low affinity of this anti-mammal toxin to insects. It was demonstrated that high doses of AAH II were completely inactive when injected to fly larvae, establishing the anti-mammal specificity of AaH II. It was demonstrated that the highly active toxins on mammals, like AaH II, possess a receptor site also on insect sodium channels, as the classical α-scorpion toxins are able to compete for LqhαIT binding in insect neuronal membranes. The inhibition of sodium current inactivation by high concentration of AAH II in an isolate axon of a cockroach indicates that the α-scorpion toxin binding on insect sodium channels is pharmacologically active and its receptor site might be homologous to receptor site 3 on rat brain sodium channels.
The low affinity revealed by the α-mammal toxins on insects is in contrast to the high affinity observed on rat brain sodium channels, indicating differences in receptor site structures on mammal versus insect sodium channels. However, the complete inhibition of LqhαIT binding, especially on cockroach sodium channels and the shift in affinity detected in cockroach versus locust, which conforms with the shift in affinity of LqhαIT on these insect sodium channels, supports that the competition may result from binding to homologous, similar or overlapping receptor sites.
The specificity and differences in the insect versus mammal activity of the α- and α-like scorpion toxins may be attributed, in part, to structural differences among both the toxins and the homologous receptor sites on insect and mammalian sodium channels. Clarification of the structural basis for selectivity in action of toxins will require three-dimensional structural knowledge of the toxins coupled with molecular localization of the amino acids directly interacting with the recognition points within the receptor site structure and are important areas of future studies.
Other receptor sites are revealed by α-like toxin binding
The expanding number of selective toxin ligands with similar apparent physiological activity (inhibition of sodium current inactivation) urged us to examine their interactions with the known probes of receptor site 3 on several sodium channel preparations. However, binding experiments may reveal competitive inhibition between toxins that do not bind to the same or overlapping receptor sites, but by various criteria cannot share precisely the same binding sites. Such competition may result from steric interference (hindrance) between toxin molecules upon binding to their distinct receptor sites. Electrostatic repulsion between highly charged molecules may further contribute to this interference. The interference may be related to the three dimensional structure and flexibility of a toxin, and to the surface of its receptor site. As a practical approximation, it was suggested to refer to a toxin “binding area,” which represents the surface of projection of a toxin bound on the sodium channel surface. Such a binding area may be largely responsible for the apparent competitive inhibition observed in binding studies.