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

点击翻译按钮获取中文摘要

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.