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

摘要 离子通道是一种精密的分子机器，它调节细胞内离子的流动 膜响应刺激，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，以及离子通道缺陷是许多人 紊乱尽管几十年的实验和许多高分辨率的分子 结构，我们仍然不知道，对于任何通道，电压或配体的机制， 相关门控缺少的成分似乎是构象能量学。的 不同通道构象的能量学控制时间过程，电压， 依赖性，以及通道孔的打开的配体依赖性，并且最终 细胞的电兴奋性。在本提案中，我们将确定 电压依赖性门控和配体依赖性门控，并填补了我们的重要空白， 了解离子通道生物学。我们将重点讨论环核苷酸结合 结构域（CNBD）离子通道家族，其在结构上相关，但在功能上 多样化。尽管一些CNBD通道通过去极化激活，但其他通道通过去极化激活。 由超极化激活，一些成员由cAMP激活，还有一些成员由cAMP激活， 被cGMP激活。我们将利用我们开发的突破性FRET方法， 测量分子内距离分布和构象能量， 荧光寿命成像显微镜（FLIM），同时记录 使用膜片钳荧光测定法（PCF）检测通道功能。多个捐赠者的数据- 整个通道的受体位点将被编译成四维图（X， Y，Z和能量）的构象重排与配体- CNBD通道的依赖性和电压依赖性激活。我们的长期愿景是 了解离子通道变构调节的基本主题， 这些实验有望在实现这一目标方面取得迅速进展。最后，方法和 我们发现的原则将广泛用于阐明所有变构的机制， proteins.

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.