Chemical biology of voltage-gated cation channels

电压门控阳离子通道的化学生物学

• 批准号: 10552311
• 负责人: Christopher A Ahern
• 金额: $ 53.51万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552311
• 关键词: Adopted; Amplifiers; Arrhythmia; Binding; Binding Sites; Biological; Biology; Calcium Channel; Cardiac; Cations; Cell membrane; Cells; Chemicals; Chemistry; Clinical; Dissection; Environment; Epilepsy; Event; Foundations; Goals; Heart Rate; Heart failure; Human; Hypertension; Individual; Ion Channel; Ion Channel Gating; Ions; Light; Link; Long QT Syndrome; Mammalian Cell; Medicine; Membrane; Methods; Modification; Molecular; Muscle; Muscle Contraction; Neurons; Pain management; Pathway interactions; Peptides; Perception; Pharmaceutical Preparations; Phenylalanine; Phosphorylation; Physiological; Potassium Channel; Process; Protein Conformation; Proteins; Research; Resolution; Second Messenger Systems; Shapes; Signal Transduction; Site; Sodium Channel; Speed; Therapeutic; Tyrosine; Vertebral column; Voltage-Gated Potassium Channel; chemical binding; experimental study; extracellular; fighting; innovation; potassium ion; programs; protein structure; response; sensor; skeletal; training opportunity; voltage; voltage gated channel

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.

电压门控离子通道塑造神经和肌肉可兴奋细胞中的电信号传导。钠 (NaV) 和钙通道 (CaV) 驱动膜去极化并通过门控激活第二信使通路 同名离子进入细胞。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 膜复极化。这些通道共同为思想、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 了解它们在细胞环境中可能采取的姿势类型。然而，了解终极 这些蛋白质的外观与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。其一，结构实验中不存在跨膜电压，因此 描绘了本质上非生理环境中的电压门控通道。因此我们正在开发 光化学“钉合”方法共价捕获活细胞膜中的高价值蛋白质构象 在纯化进行结构测定之前。此外，我们已经开始确定渠道机制 通过在跨膜片段的肽主链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 途径。例如，在战斗或战斗反应期间，CaV1.2 几乎瞬时磷酸化 通道导致通道更快、更持续地开放，从而导致更有力和更快的心率。然而 该过程的绝对速度和复杂性对于实验解析单个分子来说是一个挑战 导致通道选通修改的事件。我们描述了新验证的方法，使光 受控的位点特异性磷酸化，用于仔细解构和识别关键步骤和参与者 就是这个过程。最后，CaV 通道可以通过治疗性抑制来控制疼痛、癫痫、心律失常、高血压等。 血压，或者激活治疗心力衰竭。令人惊讶的是，这两种效应（通道 激活和失活）可以通过药物结合常见的细胞外结合位点来引发 渠道。相反，通过其他有用的方法意外阻断心脏 hERG 钾通道 90% 的药物引起的长 QT 综合征是由治疗引起的，这是一种潜在致命的心律失常。所有这些 化学结合事件依赖于苯丙氨酸侧链形成的富含芳香族的结合位点， CaV 和 hERG 通道中的酪氨酸残基。为了更好地理解这些化学相互作用，我们 开发了一种高分辨率方法，可以对这些芳香族化合物进行充满活力和细致的剖析 哺乳动物细胞环境中的 CaV 和 KV 药物结合芳香盒。执行成功 该研究计划的一部分将提供前沿培训机会，促进分子理解 通道门控的研究，将揭示具有高治疗价值的临床药物的结合模式。