ShK-Dap(22), a potent Kv1.3-specific immunosuppressive polypeptide

The voltage-gated potassium channel in T lymphocytes, Kv1.3, is an importan t molecular target for immunosuppressive agents. A structurally defined pol ypeptide, ShK, from the sea anemone Stichodactyla helianthus inhibited Kv1. 3 potently and also blocked Kv1.1, Kv1.4, and Kv1.6 at subnanomolar concent rations. Using mutant cycle analysis in conjunction with complementary muta genesis of ShK and Kv1.3, and utilizing the structure of ShK, we determined a likely docking configuration for this peptide in the channel. Based upon this topological information, we replaced the critical Lys(22) in ShK with the positively charged, non-natural amino acid diaminopropionic acid (ShK- Dap(22)) and generated a highly selective and potent blocker of the T-lymph ocyte channel. ShK-Dap(22), at subnanomolar concentrations, suppressed anti -CD3 induced human T-lymphocyte [H-3]thymidine incorporation in vitro. Toxi city with this mutant peptide was low in a rodent model, with a median para lytic dose of similar to 200 mg/kg body weight following intravenous admini stration. The overall structure of ShK-Dap(22) in solution, as determined f rom NMR data, is similar to that of native ShK toxin, but there are some di fferences in the residues involved in potassium channel binding. Based on t hese results, we propose that ShK-Dap(22) or a structural analogue may have use as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.