Depressant insect selective neurotoxins from scorpion venom: Chemistry, action, and gene cloning

Abstract

The present study examines the similarity in the symptoms and binding properties between the depressant and excitatory insect-selective neurotoxins, derived from scorpion venom. A comparison of their primary structures and neuromuscular effects is presented. A new depressant toxin (LqhIT2) was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic its effects on the intact insect, i.e, a brief period of repetitive bursts of regular junction potentials (JPs) is followed by reduced amplitude JPs ending with a block of the neuromuscular transmission. "Loose" patch clamp recordings indicate that the repetitive activity has a presynaptic origin (the motor nerve) and resembles the effect of the excitatory toxin AaIT. The final synaptic block is supposed to be the end result of neuronal membrane depolarization. Such an effect is not caused by an excitatory toxin, which induces long "trains" of repetitive firing. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation indicating a high degree of sequence homology. This conservation differs from those of other groups of scorpion toxins. The opposing pharmacological effects of depressant toxins are discussed in light of the above neuromuscular effects and sequence analysis. A genetic approach in the study of the structure-function relationships of the depressant toxins was initiated by isolating cDNA clones encoding the LqhIT2 and BjIT2 toxins. Their sequence analysis revealed the precursor form of these toxins: A 21 amino acid residue signal peptide followed by a 61 amino acid region of the mature toxin, and three additional amino acids at the carboxy terminus.

Mammalian Skeletal Muscle Voltage-Gated Sodium Channels Are Affected by Scorpion Depressant “Insect-Selective” Toxins when Preconditioned

“…The classification of depressant toxins as insectselective relied on the lack of toxicity when injected to mice and inability to bind rat brain synaptosomes (Zlotkin et al, 1993;Gordon et al, 1998;Gurevitz et al, 2007) or affect mammalian Na v s expressed in X. laevis oocytes (Gordon et al, 2003;Bosmans et al, 2005). However, a number of recent results have suggested that the issue of selectivity of depressant toxins toward insects deserves reexamination: 1) scorpion ␤-toxins share a common pharmacophore Karbat et al, 2007), which explains their ability to compete in binding; 2) receptor site 4 on rat skeletal muscle Na v s was suggested to differ from those of various mammalian neuronal and cardiac Na v s (Marcotte et al, 1997;Cestèle et al, 1998;Leipold et al, 2006;Shciavon et al, 2006;Cohen et al, 2007); and 3) scorpion ␣-and ␤-toxins exert synergistic effects as a e result of allosteric interactions between receptor sites 3 and 4 on insect Na v s (Cohen et al, 2006).…”

“…2). The ability of depressant toxins to bind with an apparent high affinity to rat skeletal muscle Na v s raised the inevitable question of why these toxins were inactive when injected subcutaneously into mice (Lester et al, 1982;Zlotkin et al, 1991Zlotkin et al, , 1993. Therefore, we analyzed the effects of LqhIT2 and Lqh-dprIT3 on the rat muscle channel rNa v 1.4 and compared them with those obtained on the Drosophila melanogaster channel DmNa v 1, expressed in X. laevis oocytes.…”

Genetic Polymorphism and Expression of a Highly Potent Scorpion Depressant Toxin Enable Refinement of the Effects on Insect Na Channels and Illuminate the Key Role of Asn-58

“…Toxicity assays on blowfly larvae provided typical poisoning symptoms normally obtained with depressant toxins, i.e., an immediate transient contraction paralysis that briefly turns into a gradually increasing flaccidity and immobilization (14). Strikingly, the activity of the new toxin on insects was ∼10-fold higher (ED 50 ) 3-5 ng/100 mg body weight) compared to those of other known depressant toxins (10,12,14,(16)(17)(18)(19)34). On the basis of its effect and the similarity to the sequence of 30 amino acid residues of the N-terminal region of the depressant toxin LqhIT 2 (Figure 1; 14), the new toxin was named Lqh-dprIT 3 .…”

The Sophisticated Peptide Chemistry of Venomous Animals as a Source of Novel Insecticides Acting on Voltage-Gated Sodium Channels

“…Current clamp experiments have shown that peptides belonging to this group suppress the evoked action potentials as a result of strong depolarization of the membrane, causing an inability of axons to generate action potentials (Strugatsky et al, 2005). Representatives of this group are LqqIT2 from Leiurus quinquestriatus quinquestriatus, BjIT2 from Buthotus judaicus and the highly potent and insecticidal toxins lqh-dprIT3 and LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus (Zlotkin et al, 1993). It is interesting to note that LqqIT2 did not only cause a hyperpolarizing shift in the activation of channels, it also affected the inactivation and the ion selectivity .…”