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Introduction and Objectives: Potassium channel play a key role in cellular excitability and signal transduction pathways. Most pharmacological and structural characteristics of K+ channels have been elucidated by toxins isolated from scorpion venoms that modulate the flow of K+ ions while interacting with the channel pore. The compact small size and the strong specificity of scorpion toxins make them the ideal support for the analysis of protein folds as well as the study of the mechanisms involved in the toxin-channel interaction. Material and Methods: Buthus occitanus tunetanus scorpion venom was provided by electric stimulation of the scorpion's post-abdomen. Sephadex G50 fractionization was followed by an HPLC purification step using a Hewlett-Packard Series II 1100 chromatograph with diode array UV detector. Molecular weight of Kbot55 was first estimated by SDS-PAGE analysis under non-reducing conditions and confirmed by MALDI-TOF mass spectrometry. Amino-acid sequence was identified by automated Edman degradation in an applied Biosystems 476A protein sequence. Kbot55 has been subjected to a screening on a wide range of 7 cloned voltage-gated potassium channels Kv1.1-Kv1.6, Shaker IR insect channel and Nav1.4, expressed in Xenopus laevis ovocytes in order to evaluate its electrophysiological properties. Results and Conclusion: Herein, we isolated, identified and characterized a new peptide from Buthus occitanus tunetanus scorpion venom. The 39 amino acid sequence of Kbot55is crosslinked by 3 disulfide bridges and has a molecular mass of 4128.65 Da. Kbot55 is very weakly represented in the venom and remains a challenge for biochemical characterization. This molecule targets voltage-gated potassium channels with high affinity. In term of sequence homology, Kbot55 shows very low amino acid similarity with other scorpion potassium toxins and therefore was considered as novel type of scorpion toxins. It is thus considered as the first representative toxin of the new a-Ktx31 subfamily and therefore was classified as a-Ktx31.1. [ABSTRACT FROM AUTHOR]