An Essential Binding Surface for ShK Toxin Interaction with Rat Brain Potassium Channels

Abstract

An "Ala scan" analysis of ShK toxin, a 35-residue basic peptide possessing three disulfide bonds, identifies seven side chains which influence binding to brain delayed rectifier potassium channels. Additional analogs were synthesized and tested to further decipher the roles of these residues, particularly Tyr23. The inhibitory effects of these analogs on 125I-labeled dendrotoxin binding to rat brain membranes showed that replacement of Tyr23 with Ala drastically lowered the affinity of the toxin for the Kv1.2 channels. Ala substitution of Phe27 reduced potency more than 15-fold. Monosubstituted Ala analogs for Ile7, Ser20, or Lys30 each displayed 5-fold reductions in potency. Thus, aromaticity at position 23 is important for effective delayed rectifier brain K channel binding. In contrast, the aromatic residue at position 27 was not critical, since cyclohexylalanine substitution increased affinity. The solution structure of ShK toxin clusters Ile7, Arg11, Ser20, Lys22, Tyr23, and Phe27 in close proximity, forming the potassium channel binding surface of the toxin. We propose an essential binding surface on the toxin in which Lys22 and Tyr23 are major contributors, through ionic and aromatic (hydrophobic) interactions, with the potassium channel.

Developmental Biology of Neoplastic Growth

“…Each peptide contains two small alpha-helical segments that flank an essential lysyl sidechain that enters and blocks the pore. The mechanism of channel block is nearly identical to what had previously been demonstrated for a scorpion toxin, charybdotoxin (Pennington et al 1996a; Dauplais et al 1997). These anemone toxins mainly block "delayed rectifier" K channels that are activated during the repolarization phase of action potential, but they also block an intermediate conductance Ca 2+ activated K + channel found in certain blood cells.…”

Delineation of the Functional Site of α-Dendrotoxin

“…Membranes-Rat brain synaptosomal membranes are frequently used to study the binding properties of various ligands toward Kv1 channels, using radiolabeled ␣DTX as a tracer (11,25,32,44). High proportions of synaptosomal membranes are usually used in such experiments.…”

“…Four residues were identified as playing a major role in the toxin binding function: Lys 3 and Lys 6 in the N-terminal part of the toxin, and Trp 25 and Lys 26 , which are located in the ␤-turn that joins the two antiparallel ␤-strands. Comparison of the functional sites of ␣DTX and DTX-K (Fig.…”

“…The answer to such a question may not only shed light on the evolution of these toxins but should also help characterize the surface by which Kv1 channels interact with these toxins. Mutational analyses have finely delineated the functional sites of scorpion toxins (26) and sea anemone toxins (11,25). Although the sea anemone and scorpion toxins are not structurally related, their functional sites share some similarities.…”

“…This diad in both toxins comprises a lysine, which always plays a predominant part in binding, and an aromatic residue separated from the lysine by 6.6 Å. It was proposed that the lysine of scorpion toxins projects into the conduction pore of the channel (26 -29), and the most critical lysine of sea anemone toxins was predicted to play a similar role (11,25). Invertebrate toxins that have unrelated architectures but commonly block voltage-gated potassium channels therefore seem to display quite similar functional sites, suggesting that they underwent a convergent functional evolution.…”