Investigating the molecular mechanisms of Ca2+ selectivity and Ca2+ potentiation of the mitochondrial calcium uniporter

项目概要/摘要 线粒体钙单向转运蛋白介导Ca 2+摄取进入线粒体，从而调节代谢， 细胞死亡和细胞质Ca 2+信号传导。Uniporter如何催化离子渗透并实现离子 选择性还没有被很好地理解。Uniporter是蛋白质亚基的超复合物，至少含有： MCU、EMRE、MICU 1-3、MCUR 1和MCUb。Ca 2+选择性的机制和亚基如何控制 通道的门控问题尚未完全解决，部分原因是与研究有关的困难。 单向转运蛋白在纯净环境中的功能本提案的目的是了解 钙离子选择性和MCUR 1调控的Uniporter。我将采取简化的方法来研究这些问题 使用纯化组分的方法。通过结构（cryo-EM）和功能 （电生理学）的方法，我将确定关键残基的选择性钙离子的孔和我 将确定孔是否以多离子方式操作（目标1）。此外，我将确定 MCU-EMRE-MCUR 1（MER）全复合物参与门控（目标2）。我假设高钙 通过孔的选择性由多离子孔机制介导（Aim 1），并且MCUR 1促进Ca 2 + 通过抑制Ca 2+依赖性失活（目的2）的摄取。单粒子冷冻的补充使用， EM和平面脂质双层电生理学将定义强调离子选择性和门控的原则。

Project Summary/ Abstract The mitochondrial calcium uniporter mediates Ca2+ uptake into mitochondria and thereby regulates metabolism, cell death, and cytoplasmic Ca2+ signaling. How the Uniporter catalyzes ion permeation and achieves ion selectivity are not well understood. The Uniporter is a supercomplex of protein subunits, containing at least: MCU, EMRE, MICUs 1-3, MCUR1, and MCUb. The mechanism of Ca2+ selectivity and how the subunits control gating of the channel have not been fully resolved, in part, because of the difficulties associated with studying the function of the Uniporter in a purified context. The goal of this proposal is to understand the mechanisms of Ca2+ selectivity and MCUR1 regulation of the Uniporter. I will take a reductionist approach to investigate these processes using purified components. Through a blend of structural (cryo-EM) and functional (electrophysiological) approaches, I will determine critical residues for selectivity of Ca2+ ions in the pore and I will determine if the pore operates in a multi-ion fashion (Aim 1). Additionally, I will determine interactions of MCU-EMRE-MCUR1 (MER) holocomplex that are involved in gating (Aim 2). I hypothesize that high Ca2+ selectivity through the pore is mediated by a multi-ion pore mechanism (Aim 1) and that MCUR1 promotes Ca2+ uptake through inhibition of Ca2+ dependent inactivation (Aim 2). The complementary use of single-particle cryo- EM and planar lipid bilayer electrophysiology will define principles that underline ion selectivity and gating.