STRUCTURE-ACTIVITY STUDIES ON SCORPION TOXINS THAT BLOCK POTASSIUM CHANNELS

Scorpion venoms contain toxins that block different types of potassium channels. Some of these toxins have affinity for high conductance Ca2+-activated K+ channels and for dendrotoxin-sensitive voltage-dependent K+ channels. The structural features that determine the specificity of binding to different channel types are not known, We investigated this using natural and synthetic scorpion toxins. We have tested the effects of charybdotoxin (CTX) and two homologues (Lqh 15-1 and Lqh 18-2), iberiotoxin (IbTX), and kaliotoxin (KTX) from the scorpions Leiurus quinquestriatus hebreus, Buthus tamulus and Androctonus mauretanicus mauretanicus, respectively, and synthetic variants of CTX, namely CTX(2-37), CTX(3-37), CTX(4-37), and CTX(7-37), on a Ca2+-activated K+ current (I-K-Ca) at a mammalian motor nerve terminal, and on the binding of a radiolabelled dendrotoxin, I-125-DpI, to voltage-dependent K+ channels on rat brain synaptosomal membranes. The native toxins contain 37-38 amino acid residues, they are over 30% identical in sequence (CTX and IbTX are 68% identical), and they have similar three-dimensional conformations, All toxins, except IbTX, displaced I-125-DpI from its synaptosomal binding sites: Lqh 18-2 (K-i = 0.25 nM), KTX (K-i = 2.1 nM), CTX (K-i = 3.8 nM), CTX(2-37), (K-i = 30 nM), Lqg 15-1 (K-i = 5O nM), CTX(3-37) (K-i = 60 nM), CTX(4-37) (K-i = 50 nM), CTX(7-37) (K-i = 105 nM). IbTX had no effect at 3 mu M. When variants of CTX with deletions at the N-terminal portion were tested for their activity on I-K-Ca on motor nerve terminals in mouse triangularis sterni nerve-muscle preparations, CTX(3-37) and CTX(4-37) were ineffective at 100 nM; and CTX7-37 was ineffective at up to 1 mu M. IbTX and CTX (100 nM) completely blocked I-K-Ca, but KTX (100 nM) did not affect the nerve terminal I-K-Ca. Different residues appear to be important for interactions of the toxins with different K+ channels. IbTX did not displace dendrotoxin binding, but it did block I-K-Ca, whereas KTX was as active as CTX against dendrotoxin binding but it did not affect the I-K-Ca of the motor nerve terminals. The N-terminal section of the toxins appears to be particularly involved in block of I-K-Ca at the motor nerve terminal: it is truncated in the inactive synthetic CTX variants; and it is positively charged at lysine-6 in KTX (which is inactive), but negatively charged in IbTX and neutral in CTX. Phenylalanine at position 2 seems to be essential: there is a marked loss in activity between CTX(2-37) and CTX(3-37), and KTX has valine-2. Phenylalanine may be important in the beta-sheet region of charybdotoxin and iberiotoxin. For binding to dendrotoxin sites, the inactive IbTX lacks the conserved asparagine residues at positions 4 and 30, and contains additional negatively charged residues at positions 4, 6 and 24. The side-chains of these residues are on the opposite face of the molecule from the positively charged residues in the beta-sheet region (namely Arg 25, Lys 27 and Arg 34).