Structure-activity studies on scorpion toxins that block potassium channels

Abstract

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, 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 (K_i = 0.25 nM), KTX (K_i = 2.1 nM), CTX (K_i = 3.8 nM), CTX_2–37, (K_i = 30nM), Lqg 15-1 (K_i = 50nM), 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 3μM. 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 100nM; and CTX_7–37 was ineffective at up to 1 μM. 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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 CTX_2–37 and CTX_3–37, 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).