Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Av. Universidad, 2001, P. O. Box 510-3, Cuernavaca 62210, Mexico.
Toxicon. 2012 May;59(6):633-41. doi: 10.1016/j.toxicon.2012.02.001. Epub 2012 Feb 16.
Peptides purified from scorpion venoms were shown to interact with specific amino acid residues present in the outer vestibule of various sub-types of potassium channels, occluding the pore and causing a decrement of K(+) permeability through the membrane of excitable and non excitable cells. This communication describes the identification of several interacting sites of toxin ErgTx1, a toxin purified from the venom of the scorpion Centruroides noxius, with the human ERG1 K(+) channels, by means of site-directed mutagenesis of specific residues of the toxin. Recombinant mutants of the gene coding for ErgTx1 were expressed heterologously in Escherichia coli, properly folded and their affinities and interactions with hERG1 channels were determined by patch-clamp techniques. Residues in position Y14, Y17 and F37 of the solvent exposed hydrophobic surface, and charged residues at the position K13 and K38 of ErgTx1 were shown to cause a decrement of the affinity from 20 folds to 3 orders of magnitude, thus suggesting that they are certainly participating on the binding surface of this toxin towards the hERG1 channels. Double mutants at positions K13 and F37, Y14 and F37, Y17 and F37 and K13 and K38 were also prepared and assayed, but the results obtained are not much different from the single point mutants of ErgTx1. The results of the present work indicate the most probable surface area of ErgTx1 that makes contact with the hERG channels.
从蝎子毒液中分离出来的肽被证明可以与各种钾通道亚型外腔中存在的特定氨基酸残基相互作用，从而阻塞孔并导致可兴奋和非兴奋细胞的膜透过性 K(+) 通透性降低。本通讯描述了通过对毒素 ErgTx1 的特定残基进行定点突变，鉴定出从 Centruroides noxius 蝎子毒液中分离出的毒素与人类 ERG1 K(+) 通道相互作用的几个结合位点。编码 ErgTx1 的基因的重组突变体在大肠杆菌中异源表达，正确折叠，其与 hERG1 通道的亲和力和相互作用通过膜片钳技术确定。溶剂暴露的疏水面上的位置 Y14、Y17 和 F37 的残基，以及 ErgTx1 位置 K13 和 K38 的带电残基，导致亲和力降低 20 倍到 3 个数量级，这表明它们肯定参与了这种毒素与 hERG1 通道的结合表面。还制备和检测了位置 K13 和 F37、Y14 和 F37、Y17 和 F37 以及 K13 和 K38 的双突变体，但获得的结果与 ErgTx1 的单点突变体没有太大区别。本工作的结果表明 ErgTx1 与 hERG 通道接触的最可能的表面积。
