Expression of recombinant α-toxin BmKM9 from scorpion Buthus martensii Karsch and its functional characterization on sodium channels

Scorpion toxins are invaluable pharmacological tools for studying ion channels and potential drugs for channelopathies. The long-chain toxins from scorpion venom with four disulfide bridges exhibit their unusual bioactivity or biotoxicity by acting on the sodium channels. However, the functional properties of most toxins are still unclear due to their tiny amounts in crude venom and their challenging production by chemical and gene engineering techniques. Here, we expressed one of the long-chain a-toxins, BmKM9, found in the venom of the scorpion Buthus martensii Karsch and characterized its pharmacological properties on sodium channels. Unlike previous toxin production, the recombinant BmKM9 (rBmKM9) possessed no additional amino acid residues such as the His-tag and thrombin cleavage site. The refolded toxin could inhibit the inactivation of rNa(v)1.4, hNa(v)1.5 and hNa(v)1.7 sodium channels. Dose-response experiments were further conducted on these channels. The calculated EC50 values were 131.7 +/- 6.6 nM for rNa(v)1.4, 454.2 +/- 50.1 nM for hNa(v)1.5 and 30.9 +/- 10.3 mu M for hNa(v)1.7. The channel activation experiments indicated that the rBmKM9 toxin could shift the activation curves of rNa(v)1.4 and hNa(v)1.5 channels toward a more negative direction and present the typical features of a beta-toxin. However, instead of the same activation property on sodium channels, the rBmKM9 toxin could result in different inactivation shift capabilities on rNa(v)1.4 and hNa(v)1.5 channels. The V-1/2 values of the steady-state inactivation were altered to be more positive for rNa(v)1.4 and more negative for hNa(v)1.5. Moreover, the recovery of the hNa(v)1.5 channel from inactivation was more significantly delayed than that of the rNa(v)1.4 channel by exposure to rBmKM9. Together, these findings highlighted that the rBmKM9 toxin presents the pharmacological properties of both alpha- and beta-toxins, which would increase the challenge to the classical classification of scorpion toxins. Furthermore, the expression method and functional information on sodium channels would promote the potential application of toxins and contribute to further channel structural and functional studies.