INTERACTION OF CA2-ACTIVATED K+ CHANNELS WITH REFOLDED CHARYBDOTOXINSMUTATED AT A CENTRAL INTERACTION RESIDUE()

Charybdotoxin is a small peptide blocker of K+ channels, rigidly held in an active conformation by three disulfide bonds. The toxin blocks K + channels by binding to a receptor site located at the external ''ves tibule'', and thus physically occluding the outer opening of the K+ co nduction pore. In the blocked complex, K27, a residue on the toxin's m olecular surface, projects its epsilon-amino group into the K+-selecti ve pore. The results here show that CTX, produced by heterologous expr ession in E. coli, may be manipulated to place unnatural positively ch arged residues at position 27. The toxin folds faithfully to its nativ e conformation when the crucial lysine at position 27 is replaced by a cysteine residue, a maneuver that allows specific chemical modificati on of this side-chain,Replacements of K27 by side-chains slightly shor ter or slightly longer than lysine yield active toxins. The toxin vari ant with ornithine at this position interacts much less strongly with K+ ions in the pore of slowpoke-type Ca2+-activated K+ channels than d oes wild-type toxin. This result argues that the epsilon-amino group o f K27 in bound toxin lies only a few angstroms away from a K+ ion occu pying the blocked pore. The peptide folds with high efficiency to form the correct disulfides even in the presence bf strong denaturants. Co pyright (C) 1996 Published by Elsevier Science Ltd