Hu Pu, Sun Lei, Zhu Zhi-Qiang, Hou Xiao-Wei, Wang Shu, Yu Shan-Shan, Wang Hui-Li, Zhang Ping, Wang Ming, Niu Li-Wen, Teng Mai-Kun, Ruan Di-Yun
Department of Neurobiology and Biophysics, School of Life Sciences, University of Science and Technology of China, HeFei, Anhui, 230027, People's Republic of China.
Proteins. 2008 Aug;72(2):673-83. doi: 10.1002/prot.21964.
Snake secreted phospholipasesA2 (sPLA2s) are widely used as pharmacological tools to investigate their role in diverse pathophysiological processes. Some members of snake venom sPLA2s have been found to block voltage-activated K(+) channels (K(v) channels). However, most studies involved in their effects on ion channels were indirectly performed on motor nerve terminals while few studies were directly done on native neurons. Here, a novel snake sPLA2 peptide neurotoxin, Natratoxin, composed of 119 amino acid residues and purified from Naja atra venom was reported. It was characterized using whole-cell patch-clamp in acutely dissociated rat dorsal root ganglion (DRG) neurons. It was found to effectively inhibit A-type K(+) currents and cause alterations of channel gating characters, such as the shifts of steady-state activation and inactivation curves to hyperpolarization direction and changes of V(1/2) and slope factor. Therefore, Natratoxin was suggested to be a gating modifier of K(v) channel. In addition, this inhibitory effect was found to be independent of its enzymatic activity. These results suggested that the toxin enacted its inhibitory effect by binding to K(v) channel. To further elucidate the structural basis for this electrophysiological phenomenon, we determined the crystal structure of Natratoxin at 2.2 A resolution by molecular replacement method and refined to an R-factor of 0.190. The observed overall fold has a different structural organization from other K(+) channel inhibitors in animal toxins. Compared with other K(v) channel inhibitors, a similar putative functional surface in its C-terminal was revealed to contribute to protein-protein interaction in such a blocking effect. Our results demonstrated that the spatial distribution of key amino acid residues matters most in the recognition of this toxin towards its channel target rather than its type of fold.
蛇分泌的磷脂酶A2（sPLA2s）被广泛用作药理学工具，以研究它们在各种病理生理过程中的作用。已发现蛇毒sPLA2s的一些成员可阻断电压激活的钾通道（K（v）通道）。然而，大多数关于它们对离子通道影响的研究是在运动神经末梢间接进行的，而直接对原代神经元进行的研究很少。在此，报道了一种新型的蛇sPLA2肽神经毒素——眼镜蛇毒素，它由119个氨基酸残基组成，从眼镜蛇毒液中纯化得到。利用全细胞膜片钳技术对急性分离的大鼠背根神经节（DRG）神经元进行了表征。发现它能有效抑制A型钾电流，并引起通道门控特性的改变，如稳态激活和失活曲线向超极化方向移动以及V（1/2）和斜率因子的变化。因此，眼镜蛇毒素被认为是K（v）通道的门控修饰剂。此外，发现这种抑制作用与其酶活性无关。这些结果表明，该毒素通过与K（v）通道结合发挥其抑制作用。为了进一步阐明这种电生理现象的结构基础，我们通过分子置换法以2.2埃的分辨率测定了眼镜蛇毒素的晶体结构，并将其精修至R因子为0.190。观察到的整体折叠结构与动物毒素中的其他钾通道抑制剂具有不同的结构组织。与其他K（v）通道抑制剂相比，其C末端类似的假定功能表面被揭示在这种阻断作用中有助于蛋白质-蛋白质相互作用。我们的结果表明，关键氨基酸残基的空间分布在该毒素对其通道靶点的识别中最为重要，而不是其折叠类型。
