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.
Subject(s)
Brain/metabolism , Cnidarian Venoms/metabolism , Potassium Channel Blockers , Potassium Channels/metabolism , Amino Acid Sequence , Animals , Binding Sites , Brain/drug effects , Cnidarian Venoms/chemical synthesis , Cnidarian Venoms/chemistry , In Vitro Techniques , Membranes/metabolism , Models, Molecular , Protein Conformation , Protein Folding , Rats , ThermodynamicsABSTRACT
Eighteen synthetic analogs of ShK toxin, a thirty-five residue K channel blocker derived from the sea anemone Stichodactyla helianthus, were prepared in order to identify functionally important residues. CD spectra of sixteen of the analogs were virtually identical with the spectrum of wild-type toxin, indicating that the conformations were not affected by the substitutions. A conserved residue, Lys22, is essential for ShK binding to rat brain K channels which are primarily of the Kv1.2 type. However, a cationic side chain at position 22 is not essential for binding to the human Jurkat T-lymphocyte Kv1.3 channel. While decreasing bulkiness at this position affected toxin affinity for the brain K channels, increasing bulkiness decreased toxin affinity for both brain and lymphocyte K channels. In contrast to the rat brain channels, ShK binding to Kv1.3 was sensitive to substitution at Lys9 and Arg11.
Subject(s)
Brain/metabolism , Cnidarian Venoms/chemistry , Cnidarian Venoms/pharmacology , Potassium Channel Blockers , T-Lymphocytes/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Cnidarian Venoms/chemical synthesis , Humans , Membrane Potentials/drug effects , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Potassium Channels/physiology , Rats , Sequence Homology, Amino Acid , Structure-Activity RelationshipABSTRACT
ShK-toxin, a 35 residue peptide isolated from the sea anemone Stichodactyla helianthus, was synthesized using an Fmoc strategy and successfully folded to the biologically active form containing three intramolecular disulfide bonds. The ability of synthetic ShK toxin to inhibit specific [125I]-dendrotoxin I binding to rat brain membranes slightly exceeded (was more potent than) that of the natural ShK toxin sample, but was comparable with previously reported data for ShK toxin. The peptide toxin inhibited [125I]-charybdotoxin binding to Jurkat T lymphocytes with an IC50 value of 32 pM. In addition, Jurkat T lymphocytes Kv1.3 potassium channels were inhibited with an IC50 value of 133 pM. Owing to their unique structure and high affinity for at least some potassium channels, ShK toxin and related sea anemone potassium channel toxins may become useful molecular probes for investigating potassium channels.
