PURIFICATION, CHARACTERIZATION, AND SYNTHESIS OF 3 NOVEL TOXINS FROM THE CHINESE SCORPION BUTHUS-MARTENSI, WHICH ACT ON K+ CHANNELS

Three novel toxins belonging to the scorpion K+ channel-inhibitor fami ly were purified to homogeneity from the venom of the;Chinese scorpion Buthus martensi. They have been identified according to their molecul ar mass (3800-4300 Da) and their neurotoxicity in mice and characteriz ed as 37-amino acid peptides. One of them shows 81-87% sequence identi ty with members of the kaliotoxin group (named BmKTX), whereas the oth er two, named BmTX1 and BmTX2, show 65-70% identity with toxins of the charybdotoxin group. Their chemical synthesis by the Fmoc methodology allowed us to show that BmKTX, unlike BmTX1 and BmTX2, possesses an a midated C-terminal extremity. Toxicity assays in vivo established that they are lethal neurotoxic agents in mice (LD(50)s of 40-95 ng per mo use). Those toxins proved to be potent inhibitors of the voltage-gated K+ channels, as they were able to compete with [I-125]kaliotoxin for its binding to rat brain synaptosomes (IC(50)s of 0.05-1 nM) and to bl ock the cloned voltage-gated K+ channel Kv1.3 from rat brain, expresse d in Xenopus oocytes (IC(50)s of 0.6-1.6 nM). BmTX1 and BmTX2 were als o shown to compete with [I-125]charybdotoxin for its binding to the hi gh-conductance Ca2+-activated K+ channels present on bovine aorta sarc olemmal membranes (IC(50)s of 0.3-0.6 nM). These new sequences show mu ltipoint mutations when compared to the other related scorpion K+ chan nel toxins and should prove to be useful probes for studying the diver se family of K+ channels.