Legros C, Pollmann V, Knaus H G, Farrell A M, Darbon H, Bougis P E, Martin-Eauclaire M F, Pongs O
Institut für Neurale Signalverarbeitung, Zentrum für Molekulare Neurobiologie Hamburg, Universität Hamburg, D-20246 Hamburg, Germany.
J Biol Chem. 2000 Jun 2;275(22):16918-24. doi: 10.1074/jbc.275.22.16918.
The crystal structure of the bacterial K(+) channel, KcsA (Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77), and subsequent mutagenesis have revealed a high structural conservation from bacteria to human (MacKinnon, R., Cohen, S. L., Kuo, A., Lee, A., and Chait, B. T. (1998) Science 280, 106-109). We have explored this conservation by swapping subregions of the M1-M2 linker of KcsA with those of the S5-S6 linker of the human Kv-channel Kv1.3. The chimeric K(+) channel constructs were expressed in Escherichia coli, and their multimeric state was analyzed after purification. We used two scorpion toxins, kaliotoxin and hongotoxin 1, which bind specifically to Kv1.3, to analyze the pharmacological properties of the KcsA-Kv1.3 chimeras. The results demonstrate that the high affinity scorpion toxin receptor of Kv1.3 could be transferred to KcsA. Our biochemical studies with purified KcsA-Kv1.3 chimeras provide direct chemical evidence that a tetrameric channel structure is necessary for forming a functional scorpion toxin receptor. We have obtained KcsA-Kv1.3 chimeras with kaliotoxin affinities (IC(50) values of approximately 4 pm) like native Kv1.3 channels. Furthermore, we show that a subregion of the S5-S6 linker may be an important determinant of the pharmacological profile of K(+) channels. Using available structural information on KcsA and kaliotoxin, we have developed a structural model for the complex between KcsA-Kv1.3 chimeras and kaliotoxin to aid future pharmacological studies of K(+) channels.
细菌钾离子通道KcsA的晶体结构（多伊尔，D.A.，莫赖斯，C.J.，普费茨纳，R.A.，郭，A.，古尔比斯，J.M.，科恩，S.L.，蔡特，B.T.，以及麦金农，R.（1998年）《科学》280卷，69 - 77页），以及随后的诱变研究揭示了从细菌到人类在结构上的高度保守性（麦金农，R.，科恩，S.L.，郭，A.，李，A.，以及蔡特，B.T.（1998年）《科学》280卷，106 - 109页）。我们通过将KcsA的M1 - M2连接子的亚区域与人类钾通道Kv1.3的S5 - S6连接子的亚区域进行交换，来探究这种保守性。嵌合钾离子通道构建体在大肠杆菌中表达，并在纯化后分析其多聚体状态。我们使用了两种蝎毒素，即钾通道毒素和红藻毒素1，它们特异性结合Kv1.3，来分析KcsA - Kv1.3嵌合体的药理学特性。结果表明，Kv1.3的高亲和力蝎毒素受体可以转移到KcsA上。我们对纯化的KcsA - Kv1.3嵌合体进行的生化研究提供了直接的化学证据，表明四聚体通道结构对于形成功能性蝎毒素受体是必要的。我们已经获得了与天然Kv1.3通道具有相似钾通道毒素亲和力（IC50值约为4皮摩尔）的KcsA - Kv1.3嵌合体。此外，我们表明S5 - S6连接子中的一个亚区域可能是钾离子通道药理学特性的一个重要决定因素。利用现有的关于KcsA和钾通道毒素的结构信息，我们构建了KcsA - Kv1.3嵌合体与钾通道毒素之间复合物的结构模型，以辅助未来对钾离子通道的药理学研究。
