Synthesis and characterization of kaliotoxin. Is the 26-32 sequence essential for potassium channel recognition?

Abstract

Kaliotoxin (KTX), a scorpion toxin characterized as a 37-residue inhibitor of the neuronal high conductance Ca(2+)-activated K+ channels (KCa channels), has been chemically synthetized. Differences were observed between natural toxin and the two peptides, KTX(1-37) and KTX(1-37)-amide. Re-examination of the KTX sequence showed that an extra lysine residue was present at the C-terminal end. The 38-residue synthetic peptide was found identical with natural toxin. All three peptides had comparable activities, with LD50 values of 6-9 pmol/mouse after intracerebroventricular injection, and Kd = 2-8 nM for blockage of the whole cell and unitary molluscan KCa currents. Pairing of the disulfide bonds in synthetic KTX corresponded to that in charybdotoxin and iberiotoxin. A competition assay between 125I-KTX(1-37) and different toxins (KTX, dendrotoxin, charybdotoxin, MCD peptide, and iberiotoxin) for binding to rat brain synaptosomal membranes suggested that KTX interacts also with voltage-gated K+ channels. Shorter peptides, KTX(25-35)-amide and KTX(26-32)-amide, expressed no KTX activity, but were able to compete in binding. They were further shown to antagonize KTX in both its toxicity and blocking activity. The (26-32) sequence of KTX, which is a highly conserved region, may contain a low affinity binding subsite essential for potassium channel recognition.