Structural energetics of voltage- and ligand-dependent gating in ion channels

摘要 离子通道是调节细胞内离子流动的精密分子机器。 膜对刺激的反应，如电压和小分子配体(例如 第二信使和神经递质)。它们是所有电兴奋性的基础 大脑和心脏，以及离子通道的缺陷是许多人类 精神错乱。尽管几十年的实验和许多高分辨率分子 结构，我们仍然不知道，对于任何通道，电压-或配体-的机制 从属门控。缺失的成分似乎是构象能量学。这个 不同通道构象的能量支配时间进程、电压- 通道孔的打开的依赖性和配体依赖性，并最终 细胞的电兴奋性。在这项提案中，我们将确定 电压依赖门控和配体依赖门控，并填补了我们的 了解离子通道生物学。我们将集中讨论环核苷酸结合。 结构域(CNBD)离子通道家族，在结构上相关，但在功能上 多种多样。虽然一些CNBD通道是通过去极化激活的，但其他通道是 被超极化激活，一些成员被cAMP激活，还有一些成员被cAMP激活 被cGMP激活。我们将利用我们为其开发的突破性FRET方法 测量分子内距离分布和构象能量学 荧光寿命成像显微镜(FLIM)，同时记录 通道功能使用膜片钳荧光法(PCF)。来自多个捐赠者的数据- 整个频道的接受者站点将被汇编成四维地图(X， Y，Z和能量)的构象重排与配体- 依赖和电压依赖的CNBD通道激活。我们的长期愿景是 了解离子通道变构调节背后的一般主题，以及 这些实验有望朝着这一目标快速发展。归根结底，方法和 我们发现的原理将对阐明所有变构的机理具有广泛的实用价值 蛋白质。

Abstract Ion channels are exquisite molecular machines that regulate the flow of ions across cell membranes in response to stimuli such as voltage and small molecule ligands (e.g. second messengers, and neurotransmitters). They underlie all electrical excitability in the brain and heart, and defects in ion channels are responsible for many human disorders. Despite decades of experiments and many high-resolution molecular structures, we still do not know, for any channel, the mechanisms for voltage- or ligand- dependent gating. The missing ingredient seems to be conformational energetics. The energetics of the different channel conformations governs the time course, voltage- dependence, and ligand-dependence of opening of the channel pore, and ultimately electrical excitability of the cell. In this proposal we will determine the mechanisms of voltage-dependent gating and ligand-dependent gating and fill important gaps in our understanding of ion channel biology. We will focus on the cyclic nucleotide-binding domain (CNBD) family of ion channels, which are structurally related, but functionally diverse. Whereas some CNBD channels are activated by depolarization, others are activated by hyperpolarization, and some members are activated by cAMP yet others are activated by cGMP. We will leverage breakthrough FRET methods we developed for measuring intramolecular distance distributions and conformational energetics using fluorescence lifetime imaging microscopy (FLIM), simultaneous with recordings of channel function using patch-clamp fluorometry (PCF). The data from multiple donor- acceptor sites throughout the channels will be compiled into a four-dimensional map (X, Y, Z, and energy) of the conformational rearrangements associated with ligand- dependent and voltage-dependent activation of CNBD channels. Our long-term vision is to understand the general themes that underlie allosteric regulation of ion channels, and these experiments promise rapid progress toward this goal. Ultimately, the methods and principles we discover will be of broad utility for elucidating mechanisms for all allosteric proteins.