Chemical biology of voltage-gated cation channels

电压门控离子通道在神经和肌肉的可兴奋细胞中塑造电信号。钠(NAV) 钙通道(Cav)驱动膜去极化，通过门控激活第二信使通路 同名离子的细胞进入。在骨骼和心肌细胞中，Cav通道触发肌肉收缩。 电压门控钾通道(KV)允许从细胞内释放钾离子以驱动 膜复极。总的来说，这些通道为思维、感知、 和收缩。人类电压门控通道的高分辨率蛋白质结构现在提供了第一个 他们在蜂窝环境中可能采取的姿势类型的一瞥。然而，理解终极 这些蛋白质的外观和它们如何支持生理机制之间的联系是一个重大挑战， 将需要创新的方法。首先，在结构性实验中没有跨膜电压。 描述了在基本上非生理环境中的电压门控通道。因此，我们正在开发 在活细胞膜中共价捕获高价蛋白质构象的光化学‘装订’方法 在纯化进行结构测定之前。此外，我们已经开始确定渠道的机制 通过在形成的跨膜片段的肽骨架上引入修饰的化学物质来发挥作用 电压传感器和通道门。在细胞环境中，离子通道也是转导的关键放大器 小路。例如，在战斗或战斗反应中，CaV1.2几乎瞬间的磷酸化 经络导致更快和持续的经络开放，导致更有力和更快的心率。还没有 这一过程的绝对速度和复杂性是对单个分子进行实验性解析的挑战 导致通道门控修改的事件。我们描述了新验证的启用光的方法 受控的特定部位的磷酸化，用于仔细地解构和识别关键步骤和角色 就是这个过程。最后，CAV通道可以在治疗上被抑制，以控制疼痛、癫痫、心律失常、高 降血压，或者被激活来治疗心力衰竭。令人惊讶的是，这两种效果(通道 激活和失活)可以通过药物结合共同的细胞外结合部位而引起 频道。相反，意外地阻断心脏Herg钾通道是有用的 药物引起的长QT综合征有90%是由治疗引起的，这是一种潜在的致命性心律失常。所有这些都是 化学结合事件依赖于苯丙氨酸和苯丙氨酸侧链形成的芳香族丰富的结合部位 Cav和HERG通道中的酪氨酸残留量。为了更好地了解这些化学相互作用，我们有 开发了一种高分辨率的方法，允许对这些芳烃进行能量和细微差别的剖析 在哺乳动物细胞环境中，CAV和KV药物结合芳香盒。成功的执行 这项研究计划将提供尖端的培训机会，增进对分子的理解 并将揭示具有较高治疗价值的临床药物的结合模式。

Voltage-gated ion channels shape electrical signaling in the excitable cells of nerve and muscle. Sodium (NaV) and calcium channels (CaV) drive membrane depolarization and activate second messenger pathways via gated cellular entry of their namesake ions. In skeletal and cardiac cells, CaV channels trigger muscle contraction. Voltage-gated potassium channels (KV) allow the release of potassium ions from within the cell to drive membrane repolarization. In concert, these channels provide the molecular foundation for thought, perception, and contraction. High-resolution protein structures of human voltage-gated channels are now providing the first glimpses of the types of poses they may adopt in cellular environments. However, understanding the ultimate link between how these proteins look and how they support physiological mechanisms is a major challenge that will require innovative approaches. For one, transmembrane voltage is absent in a structural experiment thus depicting voltage-gated channels in an essentially non-physiological environment. We are therefore developing photochemical `stapling' approaches to covalently trap high-value protein conformations in live cell membranes prior to purification for structural determination. Further, we have begun to identify mechanisms of channel function by introducing modified chemistries at the peptide backbone in the transmembrane segments that form voltage-sensors and channel gates. In cellular settings, ion channels are also critical amplifiers of transduction pathways. During the fight-or-fight response, for instance, the near instantaneous phosphorylation of CaV1.2 channels results in faster and sustained channel opening, leading to a more forceful and rapid heart rate. Yet the absolute speed and complexity of the process is a challenge to experimentally parse individual molecular events that result in channel gating modifications. We describe newly validated methods that enable light controlled, site-specific phosphorylation, for the careful deconstruction and identification of key steps and players is this process. Lastly, CaV channels can be therapeutically inhibited to manage pain, epilepsy, arrythmia, high blood pressure, and alternatively, activated to treat heart failure. Surprisingly, both of these effects (channel activation and inactivation) can be elicited by medicines binding a common extracellular binding site on the channel. Conversely, unintended blockade of cardiac hERG potassium channels by otherwise useful therapeutics cause 90% of drug induced long-QT syndrome, a potentially lethal cardiac arrhythmia. All of these chemical binding events rely on aromatic rich binding sites formed by the side-chains of phenylalanine and tyrosine residues in CaV and hERG channels. To better understand these chemical interactions, we have developed a high-resolution method that allows for energetic and nuanced dissection of these aromatics within the CaV and KV drug binding aromatic boxes in the environment of mammalian cells. The successful execution of this research program will provide cutting edge training opportunities, advance the molecular understanding of channel gating, and will reveal the binding modes of clinical drugs with high therapeutic value.