A 4-DISULFIDE-BRIDGED TOXIN, WITH HIGH-AFFINITY TOWARDS VOLTAGE-GATEDK+ CHANNELS, ISOLATED FROM HETEROMETRUS SPINNIFER (SCORPIONIDAE) VENOM

A new toxin, named HsTX1, has been identified in the venom of Heterome trus spinnifer (Scorpionidae), on the basis of its ability to block th e rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been puri fied and characterized as a 34-residue peptide reticulated by four dis ulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandi nus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four- disulphide-bridged toxins, whereas it is only 32-47% identical with th e other scorpion K+ channel toxins, reticulated by three disulphide br idges. The amidated and carboxylated forms of HsTX1 were synthesized c hemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C-18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pa ttern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both follow ed by mass and sequence analyses. Three of the disulphide bonds are co nnected as in the three-disulphide-bridged scorpion toxins, and the tw o extra half-cystine residues of HsTX1 are crosslinked, as in Pi1. The se results, together with those of CD analysis, suggest that HsTX1 pro bably adopts the same general folding as all scorpion K+ channel toxin s. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with I-125-apamin for binding to its r eceptor site on rat brain synaptosomal membranes, but competes efficie ntly with I-125-kaliotoxin for binding to the voltage-gated KC channel s on the same preparation (IC50 approx. 1 pM).