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 CTX2-37, CTX3-37, CTX4-37, and CTX7-37, on a Ca2+-activated K+ current (ik---ca) at a mammalian motor nerve terminal, and on the binding of a radiolabelled dendrotoxin, 125I-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 125I-DpI from its synaptosomal binding sites: Lqh 18-2 (Ki = 0.25 nM), KTX (Ki = 2.1 nM), CTX (Ki = 3.8 nM), CTX2-37, (Ki = 30nM), Lqg 15-1 (Ki = 50nM), CTX3-37 (Ki = 60 nM), CTX4-37 (Ki= 50 nM), CTX7-37 (Ki = 105 nM). IbTX had no effect at 3μM. When variants of CTX with deletions at the N-terminal portion were tested for their activity on IK---Ca on motor nerve terminals in mouse triangularis sterni nerve-muscle preparations, CTX3-37 and CTX4-37 were ineffective at 100nM; and CTX7-37 was ineffective at up to 1 μM. IbTX and CTX (100 nM) completely blocked IK---Ca but KTX (100 nM) did not affect the nerve terminal IK---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 IK---Ca, whereas KTX was as active as CTX against dendrotoxin binding but it did not affect the IK---Ca of the motor nerve terminals. The N-terminal section of the toxins appears to be particularly involved in block of IK---Caat 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 CTX2-37 and CTX3-37, and KTX has valine-2. Phenylalanine may be important in the β-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 β -sheet region (namely Arg 25, Lys 27 and Arg 34)

Neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion Leiurus quinquestriatus hebreus

The scorpion venom Leiurus quinquestriatus hebreus was fractionated by chromatography in order to isolate toxins that affected binding of radiolabelled dendrotoxin to K+ channel proteins on synaptosomal membranes and that facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations. In addition to the previously characterized charybdotoxin, three toxins were isolated: 14-2, 15-1 and 18-2. Toxin 14-2 has a blocked N-terminus and because of low quantities, it has not been sequenced; 15-1 is a newly sequenced toxin of 36 residues with some overall homology to charybdotoxin and noxiustoxin; 18-2 is identical to charybdotoxin-2. The apparent Ki against dendrotoxin binding were: charybdotoxin, 3.8 nM; 14-2, 150 nM; 15-1, 50 nM; and 18-2, 0.25 nM. Toxin 14-2 (75 nM-1.5 microM) had a presynaptic facilitatory effect on neuromuscular preparations. Toxin 15-1 augmented responses to direct muscle stimulation, probably because it blocked Ca(2+)-activated K+ currents in muscle fibres. Toxin 18-2 (charybdotoxin-2) had a potent presynaptic facilitatory action, with less effect on direct muscle stimulation. This contrasts with the relatively weak neuromuscular effects of the highly homologous charybdotoxin. On a Ca(2+)-activated K+ current in mouse motor nerve endings, charybdotoxin and toxin 18-2 produced maximal block at around 100 nM, whereas 15-1 was inactive at 300 nM. Charybdotoxin can increase quantal content, but this is more likely to result from block of voltage-dependent K+ channels than Ca(2+)-activated channels: the increase in transmitter release occurred in conditions in which little IKCa would be present; higher concentration of charybdotoxin and longer exposure times were required to increase transmitter release than those needed to block IKCa, and the facilitatory effects of charybdotoxin and toxin 18-2 correlated more with their effects on dendrotoxin binding than on block of IKCa