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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. © Munksgaard 1995.

ShK toxin, a 35-residue peptide isolated from the Caribbean sea anemone Stichodactyla helianthus, is a potent inhibitor of the Kv1.3 potassium channel in lymphocytes. The natural toxin contains three disulfide bonds. The disulfide pairings of the synthetic ShK toxin were elucidated as a prerequisite for studies on its structure-function relationships. The toxin was fragmented at pH 6.5 using either thermolysin or a mixture of trypsin and chymotrypsin followed by thermolysin. The fragments were isolated by RP-HPLC and were identified by sequence analysis and MALDI-TOF mass spectrometry. The three disulfides were unambiguously identified in either proteolytic digest: Cys 3 to Cys 35, Cys ~2 to Cys 28 and Cys 17 to Cys 32. The CysLCys 35 disulfide, linking the aminoand carboxyl-termini, defines the characteristic cyclic structure of the molecule. A similar disulfide pairing motif is found in the snake venom-derived potassium channel blocker dendrotoxin and the mammalian antibiotic peptide defensins.

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