Structural and Functional Studies of Organellar Ion Channels

摘要 跨生物膜的离子转移是神经兴奋、肌肉细胞收缩、信号传递的中心 转导和激素分泌。离子通道起着至关重要的作用，因为它在 允许特定离子沿其电化学梯度向下移动的膜。巨大的生理学 离子通道的重要性反映在它们的功能障碍是导致人类各种残疾的基础上 疾病包括癫痫、耳聋、共济失调、长QT间期综合征和心律失常。有很长一段时间 四聚体阳离子通道的生理工作历史和大量功能和结构数据 包括钾、钙、钠、色氨酸和环核苷酸门控 然而，对细胞器阳离子通道的了解相对较少，部分原因是在 直接测量它们在细胞膜中的活性。目前，有一种新的研究兴趣出现在 细胞器阳离子通道在细胞器生理学和细胞中的重要作用 发信号。这项最大化调查人员研究奖的提案将集中于我们正在进行的努力 剖析了两组特定的细胞器阳离子通道的结构和功能特性： 内溶酶体阳离子通道和线粒体钙离子转运蛋白。从以下方面获得的见解： 所提出的研究将有助于我们理解这些细胞器通道是如何调节一些基本的 溶酶体和线粒体的生物学功能。 内切体和溶酶体在蛋白质和脂肪等许多生物过程中起着关键作用。 降解、分解代谢物输出、膜运输和新陈代谢感应，以及这些过程的缺陷 会导致溶酶体储存性疾病。这些酸性细胞器包含各种离子通道，它们控制着 内溶酶体pH与离子动态平衡。我实验室的一个主要研究方向是为了揭示 溶酶体内阳离子通道的门控和选择性的结构基础，包括双孔通道 (TPC)、瞬时受体势粘蛋白通道(TRPMLS)和非规范的TMEM175K 频道。线粒体可以从胞浆中摄取大量的钙，这一过程可以调节三磷酸腺苷 产生，改变细胞质的钙动力学，并触发细胞死亡。线粒体钙摄取是由 通过线粒体钙单转运体(MCU)，一种定位于内部的高选择性钙通道 线粒体膜。在人类中，单转运体的功能是一种蛋白质复合体，至少由四个 组成：成孔MCU，必要的跨膜亚单位EmRE，以及钙传感 守门蛋白MICU1和MICU2。该实验室的另一个主要项目旨在揭示 人性化MCU复合体的组装和通道调节。我们的实验方法利用单个 粒子冷冻电子显微镜(Cryo-EM)和蛋白质结晶学确定三维 这些通道的结构，以及电生理学，以阐明其生物物理特性。

ABSTRACT Ion transfer across biological membranes is central to nerve excitation, muscle cell contraction, signal transduction, and hormone secretion. Ion channels play a vital role by providing a passageway within membranes to allow specific ions to traverse down their electrochemical gradient. The immense physiological importance of ion channels is reflected in the fact that their dysfunction underlies a variety of disabling human diseases including seizures, deafness, ataxia, long QT syndrome, and cardiac arrhythmias. There is a long history of physiological work and a large body of functional and structural data on tetrameric cation channels that are localized to the plasma membrane, including the K+, Ca2+, Na+, TRP and cyclic nucleotide-gated channels; however, relatively little is known about organellar cation channels, partly because of the difficulty in directly measuring their activities in organellar membranes. Currently, there is an emerging research interest in the recently identified organellar cation channels due to their importance in organelle physiology and cell signaling. This Maximizing Investigators' Research Award proposal will be focused on our ongoing efforts to dissect the structural and functional properties of two specific groups of organellar cation channels: the endolysosomal cation channels and the mitochondrial calcium uniporters. The insights gained from the proposed studies will facilitate our understanding of how these organellar channels regulate some basic biological functions of lysosome and mitochondria. Endosomes and lysosomes play crucial roles in many biological processes such as protein and lipid degradation, catabolite export, membrane trafficking, and metabolism-sensing, and defects to these processes can result in lysosomal storage diseases. These acidic organelles contain various ion channels that control endolysosomal pH and ionic homeostasis. One major research direction in my lab is designed to reveal the structural basis of gating and selectivity in endolysosomal cation channels, including two-pore channels (TPCs), transient receptor potential mucolipin channels (TRPMLs), and the non-canonical TMEM175 K+ channels. Mitochondria can take up large amounts of Ca2+ from cytosol, a process that can modulate ATP production, alter cytoplasmic Ca2+ dynamics, and trigger cell death. Mitochondrial calcium uptake is mediated by the mitochondria calcium uniporter (MCU), a highly selective Ca2+ channel that is localized to the inner mitochondrial membrane. In humans, the uniporter functions as a protein complex consisting of at least four components: the pore-forming MCU, the essential membrane-spanning subunit EMRE, and the Ca2+-sensing gate-keeping proteins MICU1 and MICU2. Another major project in the lab aims to reveal the structural basis of the human MCU complex assembly and the channel regulation. Our experimental approach utilizes single particle cryo-electron microscopy (cryo-EM) and protein crystallography to determine the three-dimensional structures of these channels, and electrophysiology to elucidate their biophysical properties.