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、信号传导和细胞外基质的形成是至关重要的。 转导和激素分泌。离子通道通过在细胞内提供通道， 膜，以允许特定的离子穿越其电化学梯度。巨大的生理 离子通道的重要性反映在它们的功能障碍是各种致残性人类疾病的基础这一事实中。 疾病包括癫痫、耳聋、共济失调、长QT综合征和心律失常。还有很长 四聚体阳离子通道生理学研究历史和大量功能与结构数据 细胞膜上的K+、Ca2+、Na+、TRP和环核苷酸门控的 通道;然而，对细胞器阳离子通道知之甚少，部分原因是难以在 直接测量它们在细胞器膜中的活性。目前，有一个新兴的研究兴趣， 最近发现细胞器阳离子通道由于其在细胞器生理学和细胞 发信号。这个最大限度地提高研究人员的研究奖的建议将集中在我们正在进行的努力， 剖析两个特定的细胞器阳离子通道组的结构和功能特性： 内溶酶体阳离子通道和线粒体钙单向转运体。从《经济学人》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生物过程中起着重要的作用 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 会导致溶酶体贮积病这些酸性细胞器含有各种离子通道， 溶酶体内pH和离子稳态。我实验室的一个主要研究方向是揭示 内溶酶体阳离子通道（包括双孔通道）门控和选择性的结构基础 瞬时受体电位粘磷脂通道（TRPML）和非经典TMEM 175 K+通道 渠道线粒体可以从细胞质中摄取大量的Ca2+，这是一个可以调节ATP的过程。 产生，改变细胞质Ca2+动力学，并触发细胞死亡。线粒体钙摄取是由 通过线粒体钙单向转运体（MCU），一种高度选择性的Ca2+通道，定位于内 线粒体膜在人类中，单向转运蛋白的功能是一种由至少四个蛋白质组成的蛋白质复合物 组成部分：成孔MCU，必需的跨膜亚基EMRE和Ca2+传感 门控蛋白MICU1和MICU2。实验室的另一个主要项目旨在揭示 人体MCU复杂的装配和通道调节。我们的实验方法利用单一的 颗粒冷冻电子显微镜（cryo-EM）和蛋白质晶体学，以确定三维 这些通道的结构和电生理学，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.