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.
We isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus an extremely active anti-insect selective depressant toxin, Lqh-dprIT(3). Cloning of Lqh-dprIT(3) revealed a gene family encoding eight putative polypeptide variants (a-h) differing at three positions (37A/G, 50D/E, and 58N/D). All eight toxin variants were expressed in a functional form, and their toxicity to blowfly larvae, binding affinity for cockroach neuronal membranes, and CD spectra were compared. This analysis links Asn-58, which appears in variants a-d, to a toxin conformation associated with high binding affinity for insect sodium channels. Variants e-h, bearing Asp-58, exhibit a different conformation and are less potent. The importance of Asn-58, which is conserved in other depressant toxins, was further validated by construction and analysis of an N58D mutant of the well-characterized depressant toxin, LqhIT(2). Current and voltage clamp assays using the cockroach giant axon have shown that despite the vast difference in potency, the two types of Lqh-dprIT(3) variants (represented by Lqh-dprIT(3)-a and Lqh-dprIT(3)-e) are capable of blocking the action potentials (manifested as flaccid paralysis in blowfly larvae) and shift the voltage dependence of activation to more negative values, which typify the action of beta-toxins. Moreover, the stronger and faster shift in voltage dependence of activation and lack of tail currents observed in the presence of Lqh-dprIT(3)-a suggest an extremely efficient trapping of the voltage sensor compared to that of Lqh-dprIT(3)-e. The current clamp assays revealed that repetitive firing of the axon, which is reflected in contraction paralysis of blowfly larvae, can be obtained with either the less potent Lqh-dprIT(3)-e or the highly potent Lqh-dprIT(3)-a at more negative membrane potentials. Thus, the contraction symptoms in flies are likely to be dominated by the resting potential of neuronal membranes. This study clarifies the electrophysiological basis of the complex symptoms induced by scorpion depressant toxins in insects, and highlights for the first time molecular features involved in their activity.
Strugatsky
David
D
Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.
Zilberberg
Noam
N
Stankiewicz
Maria
M
Ilan
Nitza
N
Turkov
Michael
M
Cohen
Lior
L
Pelhate
Marcel
M
Gilles
Nicolas
N
Gordon
Dalia
D
Gurevitz
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003058:Cockroaches; D004594:Electrophysiology; D015870:Gene Expression / Q000235:genetics; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D011110:Polymorphism, Genetic; D012604:Scorpion Venoms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012605:Scorpions / Q000235:genetics; D016415:Sequence Alignment; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism*
D012604:Scorpion Venoms; D015222:Sodium Channels; D001216:Asparagine