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The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane and is inhibited by toxins from various venomenous animals.We used a combination of high-resolution solid-state nuclear magnetic resonance (ss NMR) spectroscopy and electrophysiological studies to show that high-affinity binding of the scorpion toxin kaliotoxin to the pore of the K+ channel (KcsA-Kvl.3) is associated with significant structural rearrangements in both molecules.Our results show that conformational entropy,related to structural flexibility of both the K+ channel binding site and the toxin,tunes high affinity toxin-K+ channel interactions.Based on these data we designed synthetic K+ channel blockers (syntoxins).The syntoxin structural fold matched the fourfold symmetry of the K+ channel pore.We find that syntoxin Ⅰ bound to the extemal K+ channel pore and that syntoxin I inhibits kaliotoxin binding.Syntaxin Ⅰ makes contacts with several residues inside the selectivity filter.However,the affinity of syntoxin Ⅰ was about three magnitudes lower than the one of kaliotoxin.Structural analysis of the syntoxin Ⅰ-KcsA-Kvl.3 complex showed that matching symmetries were unimportant for syntoxin Ⅰ binding to the K+ channel pore.Syntoxin Ⅰ inhibits specifically some members of the Shaker related Kvl family of voltage-gated K+ channels.Importantly,cardiac Kv channels are insensitive to syntoxin Ⅰ block.This opens the possibility to design a novel class of specific K+ channel blockers without cardiac side effects.Our findings highlight conformational entropy as an important principle to tune high-affinity binding of compounds to K+ channel pores,and advance current knowledge on K+ channel-ligand complexes leading to novel therapeutic agents in K+ channel pharmacology.This work was supported by grants of the DFG to M.B.,S.B.,O.P.,and D.T.