We systematically predict the internal flexibility of the S3 segment, one of the most mobile elements in the voltage-sensor domain. By analyzing the primary amino acid sequences of V-sensor containing proteins, including Hv1, TPC channels and the voltage-sensing phosphatases, we established correlations between the local flexibility and modes of activation for different members of the VGIC superfamily. Taking advantage of the structural information available, we also assessed structural aspects to understand the role played by the flexibility of S3 during the gating of the pore. We found that S3 flexibility is mainly determined by two specific regions: (1) a short NxxD motif in the N-half portion of the helix (S3a), and (2) a short sequence at the beginning of the so-called paddle motif where the segment has a kink that, in some cases, divide S3 into two distinct helices (S3a and S3b). A good correlation between the flexibility of S3 and the reported sensitivity to temperature and mechanical stretch was found. Thus, if the channel exhibits high sensitivity to heat or membrane stretch, local S3 flexibility is low. On the other hand, high flexibility of S3 is preferentially associated to channels showing poor heat and mechanical sensitivities. In contrast, we did not find any apparent correlation between S3 flexibility and voltage or ligand dependence. Overall, our results provide valuable insights into the dynamics of channel-gating and its modulation.

我们系统地预测S3段的内部灵活性，S3段是电压传感器领域中移动性最强的元素之一。通过分析包含Vv传感器的蛋白质的主要氨基酸序列，包括Hv1，TPC通道和电压感应磷酸酶，我们建立了VGIC超家族不同成员的局部柔性和激活方式之间的相关性。利用可用的结构信息，我们还评估了结构方面，以了解孔门控期间S3的柔韧性所起的作用。我们发现，S3的柔韧性主要由两个特定区域决定：（1）螺旋（S3a）的N半部分中的短NxxD基序，以及（2）所谓桨的开始处的短序列该段具有扭结的主题，在某些情况下，该扭结将S3分为两个不同的螺旋（S3a和S3b）。发现S3的柔韧性与所报道的对温度和机械拉伸的敏感性之间具有良好的相关性。因此，如果通道对热或膜拉伸表现出高敏感性，则局部S3柔韧性低。另一方面，S3的高柔韧性优选与显示不良的热和机械敏感性的通道相关。相反，我们未发现S3柔韧性与电压或配体依赖性之间有任何明显的相关性。总体而言，我们的结果提供了有关通道门控及其调制的动态的宝贵见解。