Characterization of Amm VIII from Androctonus mauretanicus mauretanicus: a new scorpion toxin that discriminates between neuronal and skeletal sodium channels

Abstract

The venom of the scorpion Androctonus mauretanicus mauretanicus was screened by use of a specific serum directed against AaH II, the scorpion α-toxin of reference, with the aim of identifying new analogues. This led to the isolation of Amm VIII (7382.57 Da), which gave a highly positive response in ELISA, but was totally devoid of toxicity when injected subcutaneously into mice. In voltage-clamp experiments with rat brain type II Na+ channel rNav1.2 or rat skeletal muscle Na+ channel rNav1.4, expressed in Xenopus oocytes, the EC50 values of the toxin-induced slowing of inactivation were: 29±5 and 416±14 nM respectively for AmmVIII and 2.6±0.3 nM and 2.2±0.2 nM, respectively, for AaH II interactions. Accordingly, Amm VIII clearly discriminates neuronal versus muscular Na+ channel. The Amm VIII cDNA was amplified from a venom gland cDNA library and its oligonucleotide sequence determined. It shows 87% sequence homology with AaH II, but carries an unusual extension at its C-terminal end, consisting of an additional Asp due to a point mutation in the cDNA penultimate codon. We hypothesized that this extra amino acid residue could induce steric hindrance and dramatically reduce recognition of the target by Amm VIII. We constructed a model of Amm VIII based on the X-ray structure of AaH II to clarify this point. Molecular modelling showed that this C-terminal extension does not lead to an overall conformational change in Amm VIII, but drastically modifies the charge repartition and, consequently, the electrostatic dipole moment of the molecule. At last, liquid-phase radioimmunassays with poly- and monoclonal anti-(AaH II) antibodies showed the loss of conformational epitopes between AaH II and Amm VIII.