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

项目摘要/摘要 线粒体钙单转运体介导钙摄取进入线粒体，从而调节代谢， 细胞死亡和胞质钙信号转导。单一转运体如何催化离子渗透和实现离子 选择性还没有被很好地理解。Uniporter是一种蛋白质亚基的超复合体，至少包含： MCU、EMRE、MICUS 1-3、MCUR1和MCUb。钙选择性的机制及其亚基的调控 通道的选通尚未完全解决，部分原因是与学习相关的困难 Uniporter在纯净的背景下的功能。这项建议的目标是了解 单一转运体的CA2选择性和MCUR1调节。我将采取简化论的方法来调查这些 使用提纯组件的工艺。通过结构(冷冻-EM)和功能的混合 (电生理)方法，我将确定孔中和I中钙离子选择性的临界残留量 将确定孔是否以多离子方式运行(目标1)。此外，我还将确定 参与门控的MCU-EMRE-MCUR1(MER)全息复合体(目标2)。我假设高钙 通过孔的选择性是由多离子孔机制介导的(目标1)，并且MCUR1促进钙 通过抑制钙依赖的失活来摄取(目标2)。单粒子低温的互补用途-- 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.