Characterization of the first K+ channel blockers from the venom of the Moroccan scorpion Buthus occitanus Paris

• Published in: Toxicon (Oxford) 2013-12, Vol.75, p.168-176
• Main Authors: Martin-Eauclaire, Marie-France, Céard, Brigitte, Belghazi, Maya, Lebrun, Régine, Bougis, Pierre E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Amino acids; Animals; Antibodies; Binding Sites; Blocking; brain; Brain - drug effects; Brain - metabolism; Buthus martensii; Buthus occitanus; Channels; Chromatography, High Pressure Liquid; ELISA; enzyme-linked immunosorbent assay; high performance liquid chromatography; HPLC; Inhibitory Concentration 50; insulin-dependent diabetes mellitus; MALDI-TOF/MS; Male; Mice; Molecular Sequence Data; Multiple sclerosis; mutation; Paris; Peptides; Peptides - chemistry; Peptides - genetics; potassium; Potassium Channel Blockers - isolation & purification; Potassium Channel Blockers - pharmacology; Potassium channels; Protein Conformation; Radioimmunoassay; Rats; rheumatoid arthritis; sclerosis; Scorpion toxins; Scorpion Venoms - classification; Scorpion Venoms - isolation & purification; Scorpion Venoms - pharmacology; Scorpions; Sequence Alignment; sequence analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; structure-activity relationships; synaptosomes; Synaptosomes - drug effects; Synaptosomes - metabolism; toxicity; Toxins; venoms
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2013.03.004

The availability of a large variety of specific blockers, which inhibit different K+ currents, would help to elucidate their differences in physiological function. Short peptide toxins isolated from scorpion venoms are able to block voltage-dependent or Ca2+-activated K+ channels. Here, we have studied the venom of the Moroccan scorpion Buthus occitanus Paris (BoP) in order to find new peptides, which could enlarge our structure–function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) that belongs to the α-KTx3.1 family. Indeed and since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channel, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. The BoP venom was first investigated using HPLC and MALDI-TOF/MS. Further, the HPLC fractions were screened by ELISA with antibodies raised against KTX. These antibodies recognized at least three components toxic in mice by intracerebroventricular injection. They were further pharmacologically characterized by competition using 125I-KTX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited totally the 125I-KTX binding and with high-affinity (IC50 = 0.1 nM), while the two other components poorly competed with (IC50 > 100 nM). These toxins were sequenced in full by Edman's degradation. The high-affinity ligand (BoPKTX) shares 86% sequence identity with KTX and was classified as toxin α-KTx3.17. The two others peptides (BoP1 and BoP2, 4093 Da and 4121 Da, respectively) only differ by a Lys/Arg mutation. Their amino acid sequences were related to Martentoxin, which has been characterized from the Chinese scorpion Buthus martenzi Karch and described as both a BKCa and Kv1.3 blocker. Accordingly, they belong to the α-KTx16 family. ► We characterized 3 novel peptides from the Buthus occitanus Paris scorpion venom. ► BoPKTX behaves as the Kv1.3 channel blocker KTX and is designated α-KTx3.17. ► BoP1 and BoP2 relate to the Martentoxin and thus belong to the α-KTx16 family.