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Abstract：
Studies on the interaction sites of peptide toxins and ion channels typically involve site-directed mutations in toxins. However, natural mutant toxins exist among them, offering insights into how the evolutionary process has conserved crucial sequences for activities and molecular target selection. In this study, we present a comparative investigation using electrophysiological approaches and computational analysis between two alpha toxins from evolutionarily close scorpion species of the genus Tityus, namely, Tst3 and Ts3 from T. stigmurus and T. serrulatus, respectively. These toxins exhibit three natural substitutions near the C-terminal region, which is directly involved in the interaction between alpha toxins and Nav channels. Additionally, we characterized the activity of the Tst3 toxin on Nav1.1-Nav1.7 channels. The three natural changes between the toxins did not alter sensitivity to Nav1.4, maintaining similar intensities regarding their ability to alter opening probabilities, delay fast inactivation, and induce persistent currents. Computational analysis demonstrated a preference for the down conformation of VSD4 and a shift in the conformational equilibrium towards this state. This illustrates that the sequence of these toxins retained the necessary information, even with alterations in the interaction site region. Through electrophysiological and computational analyses, screening of the Tst3 toxin on sodium isoform revealed its classification as a classic α-NaTx with a broad spectrum of activity. It effectively delays fast inactivation across all tested isoforms. Structural analysis of molecular energetics at the interface of the VSD4-Tst3 complex further confirmed this effect.
摘要：
对肽毒素和离子通道的相互作用位点的研究通常涉及毒素中的位点定向突变。然而,其中存在天然突变毒素，提供有关进化过程如何为活动和分子靶标选择保守关键序列的见解。在这项研究中,我们使用电生理学方法和计算分析对来自Tityus属进化上接近的蝎种的两种α毒素进行了比较研究，即,来自T.stigmurus和T.serrulatus的Tst3和Ts3，分别。这些毒素在C末端区域附近表现出三个天然取代，它直接参与α毒素和Nav通道之间的相互作用。此外,我们表征了Tst3毒素对Nav1.1-Nav1.7通道的活性。毒素之间的三个自然变化并没有改变对Nav1.4的敏感性，它们改变打开概率的能力保持相似的强度。延迟快速失活，并诱发持续的电流。计算分析表明对VSD4的下构象的偏好以及构象平衡向该状态的转变。这说明这些毒素的序列保留了必要的信息，即使相互作用位点区域发生了变化。通过电生理学和计算分析，对钠同种型Tst3毒素的筛选显示其分类为具有广谱活性的经典α-NaTx。它有效地延迟了所有测试的同种型的快速失活。在VSD4-Tst3复合物的界面处的分子能量学的结构分析进一步证实了这种效应。
