Kalman K, Pennington M W, Lanigan M D, Nguyen A, Rauer H, Mahnir V, Paschetto K, Kem W R, Grissmer S, Gutman G A, Christian E P, Cahalan M D, Norton R S, Chandy K G
Departments of Physiology & Biophysics, and Microbiology and Molecular Genetics, University of California, Irvine, California 92697-4560, USA.
J Biol Chem. 1998 Dec 4;273(49):32697-707. doi: 10.1074/jbc.273.49.32697.
The voltage-gated potassium channel in T lymphocytes, Kv1.3, is an important molecular target for immunosuppressive agents. A structurally defined polypeptide, ShK, from the sea anemone Stichodactyla helianthus inhibited Kv1.3 potently and also blocked Kv1.1, Kv1.4, and Kv1.6 at subnanomolar concentrations. Using mutant cycle analysis in conjunction with complementary mutagenesis 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 Lys22 in ShK with the positively charged, non-natural amino acid diaminopropionic acid (ShK-Dap22) and generated a highly selective and potent blocker of the T-lymphocyte channel. ShK-Dap22, at subnanomolar concentrations, suppressed anti-CD3 induced human T-lymphocyte [3H]thymidine incorporation in vitro. Toxicity with this mutant peptide was low in a rodent model, with a median paralytic dose of approximately 200 mg/kg body weight following intravenous administration. The overall structure of ShK-Dap22 in solution, as determined from NMR data, is similar to that of native ShK toxin, but there are some differences in the residues involved in potassium channel binding. Based on these results, we propose that ShK-Dap22 or a structural analogue may have use as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.
T淋巴细胞中的电压门控钾通道Kv1.3是免疫抑制剂的重要分子靶点。来自海葵壮丽海葵的一种结构明确的多肽ShK，能有效抑制Kv1.3，并且在亚纳摩尔浓度下还能阻断Kv1.1、Kv1.4和Kv1.6。通过将突变循环分析与ShK和Kv1.3的互补诱变相结合，并利用ShK的结构，我们确定了该肽在通道中可能的对接构型。基于这一拓扑学信息，我们用带正电荷的非天然氨基酸二氨基丙酸取代了ShK中关键的赖氨酸22（ShK-Dap22），并生成了一种对T淋巴细胞通道具有高选择性和强效的阻滞剂。ShK-Dap22在亚纳摩尔浓度下，能在体外抑制抗CD3诱导的人T淋巴细胞[3H]胸苷掺入。在啮齿动物模型中，这种突变肽的毒性较低，静脉注射后的中位麻痹剂量约为200mg/kg体重。根据核磁共振数据确定，溶液中ShK-Dap22的整体结构与天然ShK毒素相似，但在与钾通道结合的残基上存在一些差异。基于这些结果，我们提出ShK-Dap22或其结构类似物可能用作免疫抑制剂，用于预防移植排斥和治疗自身免疫性疾病。
