Structural and Functional Studies of Organellar Ion Channels

摘要 跨生物膜的离子转移对于神经兴奋、肌肉细胞收缩、信号传递至关重要 转导和激素分泌。离子通道通过在内部提供通道而发挥着至关重要的作用 膜允许特定离子沿着其电化学梯度移动。巨大的生理 离子通道的重要性反映在以下事实：它们的功能障碍是人类多种残疾的基础 疾病包括癫痫、耳聋、共济失调、长 QT 综合征和心律失常。有一个长 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 定位于质膜，包括 K 、 Ca2 、 Na 、 TRP 和环核苷酸门控 渠道；然而，人们对细胞器阳离子通道知之甚少，部分原因是难以确定 直接测量它们在细胞器膜中的活性。目前，人们对以下方面的研究兴趣正在兴起 最近发现的细胞器阳离子通道由于其在细胞器生理学和细胞中的重要性 发信号。这项最大化研究人员研究奖提案将重点关注我们不断努力 剖析两组特定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单向转运蛋白。从中获得的见解 拟议的研究将有助于我们了解这些细胞器通道如何调节一些基本的 溶酶体和线粒体的生物学功能。 内体和溶酶体在蛋白质和脂质等许多生物过程中发挥着至关重要的作用 降解、分解代谢物输出、膜运输和代谢传感以及这些过程的缺陷 可导致溶酶体贮积病。这些酸性细胞器含有各种控制离子通道 内溶酶体 pH 值和离子稳态。我实验室的一个主要研究方向旨在揭示 内溶酶体阳离子通道（包括双孔通道）的门控和选择性的结构基础 (TPC)、瞬时受体电位粘脂蛋白通道 (TRPML) 和非规范 TMEM175 K 渠道。线粒体可以从细胞质中吸收大量 Ca2，这一过程可以调节 ATP 产生，改变细胞质 Ca2 动力学，并引发细胞死亡。线粒体钙摄取是介导的 由线粒体钙单向转运蛋白 (MCU) 实现，这是一种高度选择性的 Ca2 通道，位于线粒体内部 线粒体膜。在人类中，单向转运蛋白作为蛋白质复合物发挥作用，该蛋白质复合物由至少四个组成 组成部分：成孔 MCU、重要的跨膜亚基 EMRE 和 Ca2 感应 看门蛋白 MICU1 和 MICU2。实验室的另一个主要项目旨在揭示 人类MCU复杂的组装和通道调节。我们的实验方法利用单一 粒子冷冻电子显微镜（cryo-EM）和蛋白质晶体学以确定三维 这些通道的结构，以及电生理学来阐明它们的生物物理特性。

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.