Gating Mechanisms of Retinal Cyclic Nucleotide-Regulated Ion Channels

摘要 超极化激活的环核苷酸调节（HCN）离子通道是第一个 在光感受器中发现，它们塑造光反应。他们展示了几个 使它们专门用于视网膜信号传导的特性：1）它们被激活， 膜超极化而不是去极化，2）它们由直接的 环核苷酸与细胞内结构域的结合，以及3）它们在细胞内表达。 神经元的远端树突。最近，HCN通道的辅助亚基在 光感受器和其他神经元被发现，称为TRIP 8b，具有深刻的 影响这些重要的通道特性。我们的长期目标是了解 这些特性的分子机制。在过去的融资周期中， 在实现这一目标方面取得了很大进展。我们已经解决了X射线晶体 HCN 2的环核苷酸结合结构域的结构和TRIP 8b的结构 与HCN 2结合。我们还发明了三种突破性的新荧光 这些方法可以让我们记录完整通道中的分子重排 与电生理记录同时进行。在这段拨款期内，我们建议 联合收割机将这些方法与双电子-电子共振（DEER）结合起来， 磁共振为基础的方法，和分子动力学模拟，以测量和 模拟HCN通道的结构和动力学及其与TRIP 8b的相互作用。 这些实验将导致HCN通道如何调节的第一个动态图像 光感受器和其他神经元的兴奋性。

Abstract Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels were first discovered in photoreceptors where they shape the light response. They exhibit several properties that make them specialized for retinal signaling: 1) they are activated by membrane hyperpolarization instead of depolarization, 2) they are regulated by the direct binding of cyclic nucleotides to an intracellular domain, and 3) they are expressed in the distal dendrites of neurons. Recently an accessory subunit of HCN channels in photoreceptors and other neurons was discovered, called TRIP8b, that has a profound effect on each of these important channel properties. Our long term goal is to understand the molecular mechanisms for these properties. In previous funding periods we have made great progress toward achieving this goal. We have solved the X-ray crystal structure of the cyclic nucleotide-binding domain of HCN2 and the structure of TRIP8b bound to HCN2. We have also invented three ground-breaking new fluorescence methods that allow us to record molecular rearrangements in intact channels simultaneous with electrophysiological recording. In this funding period, we propose to combine these methods with double electron-electron resonance (DEER), a powerful magnetic resonance-based method, and molecular dynamics simulations, to measure and model the structure and dynamics of the HCN channel and its interaction with TRIP8b. These experiments will lead to the first dynamic picture for how HCN channels regulate the excitability of photoreceptors and other neurons.