Biophysical mechanisms of gating and modulation in voltage-gated ion channel superfamily

项目摘要 电压门控离子通道（VGIC）的成员在整个过程中对电信号和化学信号至关重要。 生命的三个王国离子通道功能障碍是多种病理生理学的基础， 新药开发的主要目标之一。尽管它们有着共同的膜结构， 该超家族中的通道表现出令人惊讶的功能多样性。对膜的反应最开放 去极化，但一些在超极化上开放。它们中的许多也是多模态的-它们的活动受到调节 通过第二信使如环核苷酸或物理刺激如温度。主要目标 这一建议的目的是探索分子驱动力，以了解 电压门控及其通过温度和配体的调节。目前的机械方法倾向于结构化 集中到蛋白质动力学被忽略或被视为次要的程度。虽然结构 现在有许多对温度高度敏感的离子通道，我们对TEM机制的理解， 在很大程度上，由于我们不能直接探测分子力，过饱和灵敏度仍然有限。到 为了解决这一问题，我们正在采用多管齐下的方法，将新的和现有的工具相结合， 表征决定电压门控极性的分子相互作用， VGICs的异常变构。我们使用HCN通道作为模型系统来研究门控极性 和配体活化。使用零模型波导和新开发的高通量分析算法 我们能够在模型系统中探测配体结合的协同性。我们现在准备将这些 研究全长通道和受体。关于门控极性的机制，我们已经做了一个 令人惊讶的发现是，二分开关调节HCN通道中的门控极性。微秒级模拟 Anton超级计算机中的几个实例提出了一个门控模型，我们将进一步对其进行测试。我们将进行结构性 研究，并将其与电压钳荧光法结合联合收割机，以注释这些结构。接下来我们还将 使用祖先蛋白质重建方法，以确定调控门控PO的深层变构网络， 在这些渠道中。我们对温度相关门控的研究基于两个模型系统：a）Tem- 过饱和敏感的Shaker突变体和，B）古细菌MthK通道。为了确定基本要素 我们必须测量热力学性质，如热， 容量我们建议开发一种新的方法，涉及单分子力谱提取这些 能量参数总的来说，我们的“分子力”为重点的方法有潜力提供无与伦比的 深入了解VGIC中的电压门控及其温度调节机制。

Project Summary Members of the voltage-gated ion channels (VGICs) are critical for electrical and chemical signaling throughout the three kingdoms of life. Dysfunction of ion channels underlie a wide range of pathophysiology and they are one of the primary targets for new drug development. Although they share a common membrane architecture, the channels in this superfamily exhibit surprising diversity of function. Most open in response to a membrane depolarization but some open on hyperpolarization. Many of them are also polymodal- their activity is regulated by second messengers such as cyclic nucleotide or a physical stimulus such as temperature. The main objective of this proposal is to probe the molecular driving forces in order to understand the fundamental mechanisms of voltage-gating and its modulation by temperature and ligand. Current mechanistic approach tends to be structure focused to the extent that protein dynamics is either ignored or treated as secondary. Although the structures of many highly temperature-sensitive ion channels are now available, our understanding of the mechanism of tem- perature-sensitivity remains limited, in large part, due to our inability to directly probe the molecular forces. To address this issue, we are using a multi-pronged approach that combines new and existing tools to systematically characterize the molecular interactions that determine polarity of voltage-gating, exquisite temperature-sensitiv- ity and unusual allostery in VGICs. We are using the HCN channel as a model system to study gating polarity and ligand activation. Using zero model waveguides and newly developed high-throughput analysis algorithms we were able to probe the cooperativity of ligand binding in a model system. We are now poised to extend these studies to full-length channels and receptors. With regards to mechanisms of gating polarity, we have made a surprising discovery that a bipartite switch regulates gating polarity in HCN channels. Microsecond scale simu- lations in Anton supercomputer suggest a gating model which we will be tested further. We will carry out structural studies and combine it with voltage clamp fluorometry in order to annotate these structures. Next, we will also use ancestral protein reconstruction approach, to identify the deep allosteric networks that regulate gating po- larity in these channels. Our studies on temperature-dependent gating is based on two model systems: a) Tem- perature-sensitive Shaker mutant and, b) archaeal MthK channel. In order to determine the essential elements that are responsible for “sensing” temperature, we have to measure the thermodynamic properties such as heat capacity. We propose to develop a new approach involving single molecule force spectroscopy to extract these energetic parameters. Overall, our “molecular forces” focused approach has the potential to provide unparalleled insights into the mechanisms of voltage gating and its regulation by temperature in VGICs.