THE KALIOTOXIN FAMILY ENLARGED - PURIFICATION, CHARACTERIZATION, AND PRECURSOR NUCLEOTIDE-SEQUENCE OF KTX(2) FROM ANDROCTONUS-AUSTRALIS VENOM

Kaliotoxin (KTX) has been originally described as an inhibitor of the intermediate conductance Ca2+-activated K+ channel (Crest, M., Jacquet , G., Cola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P. , and Martin-Eauclaire, M.-F. (1992) J. Biol. Chem. 267, 1640-1647). H owever, the radioiodinated I-125-KTX-(1-37) was also able to bind to t he dendrotoxin sensitive voltage-dependent K+ channel (Romi, R., Crest , M., Cola, M., Sampieri, El, Jacquet, G., Zerrouk, H., Mansuelle, P., Sorokine, O., Van Dorsselaer, A., Rochat, H., Martin Eauclaire, M.-F. , and Van Rietschoten, J. (1993) J. Biol. Chem. 268, 26302-26309). By following the ability to compete with I-125-KTX-(1-37) for binding to its receptor on rat brain synaptosomes, a new kaliotoxin-like peptide, KTX(2), was isolated from Androctonus australis scorpion venom. It is a 37-amino acid residue peptide, and its sequence shares 76% identity with KTX. The differences between the two peptides concern the NH2-te rminal region and the residues 31 and 34 located in the region involve d in the channel recognition. These differences may explain the 5-fold decrease of the molluscan Ca2+-activated K+ channel blocage by KTX(2) (k(d) = 135 nM) as well as of its binding affinity to rat brain synap tosomes (IC50 = 50 pM), compared with KTX. Specific antibodies raised against KTX-(1-37) were not able to recognize KTX(2). Using degenerate primers, a 370-base pair cDNA encoding the KTX(2) precursor was ampli fied by polymerase chain reaction from a cDNA library of A. australis venom glands. It encoded a presumed signal peptide of 22 residues foll owed by the sequence of the mature peptide.