Magnesium flux compendium: Discover ligands, channels, and metabolic signals

摘要/总结 游离离子化细胞内 Mg2 (iMg2) 估计在 0.5–1.2 mM 范围内。总体来说还是可以接受的 在静息条件下，离子化胞质 Mg2 的浓度被磷酸代谢物“抑制”， 核酸和蛋白质。例如，ATP 的结合 Kd 值为 50 μM-70 μM，因此 Mg2 在 细胞质和线粒体基质主要与 ATP (Mg-ATP2-) 复合。由于其丰富（〜5 mM)，ATP 被认为是最大的 iMg2“储存库”。激素后游离胞质 (cMg2) 的波动 刺激被认为是 Mg2 从旺盛的 Mg-ATP 队伍中解离出来的被动调节 和其他 Mg2 的“缓冲”池。除了 iMg2“缓冲”机制之外，Mg2 离子通道和转运蛋白 控制 Mg2 进入以及穿过质膜的流出被认为可以维持平衡 游离cMg2。目前，有几个候选者与 Mg2 进入机制相关（TRPM6、TRPM7、MagT1），但是 仍在等待此类作用的令人信服的生物物理和生理学证据。 Mg2/Na交换器 SLC41A1 被认为有助于从细胞中流出 Mg2，而 Mrs2 被认为是线粒体 镁转运蛋白。关于 Mg2 进出细胞器的分子细节知之甚少 如内质网、线粒体、内体和溶酶体。一些研究推测 ER 中的游离 [Mg2] 线粒体可能与[cMg2]相似。然而，时空动态，更不用说 iMg2 动员的生物学相关性仍然是细胞生物学中的一个谜。然而，Mg2+是一种必需的元素。 阳离子控制许多生化反应。我们最近的工作表明，L-乳酸作为一种激活剂， 触发内质网和线粒体之间 Mg2 的动态转移，从而塑造生物能学和细胞学 新陈代谢（Cell 2020）。从机制上讲，L-乳酸刺激内质网释放 Mg2，然后吸收 Mg2 通过线粒体。发现线粒体局部 Mrs2 转运蛋白负责积累 线粒体中的 Mg2。然而，L-乳酸诱导的 ER 释放分子机制仍不清楚。 我建议确定 ER Mg2 释放成分、质膜进入机制以及由此产生的结果 分子信号传导途径。我将利用公正的 RNAi 筛选和靶向 CRISPR/Cas9 编辑 解开 Mg2 信号传导领域这些谜团的方法。这些分子机器的识别 有助于我们理解 iMg2 动力学以及 iMg2 通量之间存在的因果关系 和细胞过程。此外，我将根据以下内容测试和定义 Mg2 相关信号事件 细胞和小鼠模型表型。定义细胞 Mg2 之间的分子联系是令人兴奋的 体内平衡和生理功能。我们对 Mg2 助熔剂成分的识别和表征将 进一步研究这些信号通路如何以及是否影响越来越多的病理生理学 人类缺镁疾病。总体而言，R35/MIRA 资金将支持此测试 非常规假设和我的实验室将在不久的将来解决这些重大谜团。

ABSTRACT/SUMMARY Free ionized intracellular Mg2+ (iMg2+) is estimated to be in the range of 0.5–1.2 mM. In general, it is accepted that under resting conditions, the concentration of ionized cytosolic Mg2+ is `muffled' by phosphometabolites, nucleic acids and proteins. For example, ATP binds with a Kd value of 50 M-70 μM and therefore Mg2+ in the cytosol and the mitochondrial matrix is primarily complexed with ATP (Mg-ATP2-). Because of its abundance (~5 mM), ATP is considered to be the largest iMg2+ `store'. Fluctuations in free cytosolic (cMg2+) following hormonal stimuli have been touted as passive adjustments of Mg2+ dissociating from the exuberant Mg-ATP contingent and other `buffered' pools of Mg2+. Apart from iMg2+ `buffering' mechanism, Mg2+ ion channels and transporters controlling Mg2+ entry as well as efflux across the plasma membrane are thought to maintain the equilibrium of free cMg2+. Currently, several candidates are correlated to Mg2+ entry machinery (TRPM6, TRPM7, MagT1), but are still awaiting convincing biophysical and physiological evidence for such roles. The Mg2+/Na+ exchanger SLC41A1 was proposed to contribute Mg2+ efflux from the cell, whereas Mrs2 was proposed as a mitochondrial Mg2+ transporter. Very little is known about the molecular details of Mg2+ transport into/from cellular organelles like the ER, mitochondria, endosomes and lysosomes. A few studies have speculated that free [Mg2+] in the ER and mitochondria are likely to be similar to [cMg2+]. However, the temporal and spatial dynamics, let alone the biological relevance of iMg2+ mobilization, remain a mystery in cell biology. Nevertheless, Mg2+ is an essential cation controlling many biochemical reactions. Our recent work has shown that L-lactate acts as an activator that triggers a dynamic transfer of Mg2+ between the ER and mitochondria to shape bioenergetics and cellular metabolism (Cell 2020). Mechanistically, L-lactate stimulates Mg2+ release from the ER followed by Mg2+ uptake by mitochondria. The mitochondrial localized Mrs2 transporter was found to be responsible for the accumulation of Mg2+ in mitochondria. However, the L-lactate-induced ER release molecular machinery remains unidentified. I propose to identify ER Mg2+ release component, plasma membrane entry machinery and the resultant molecular signaling pathways. I will take advantage of unbiased RNAi screen and targeted CRISPR/Cas9 editing approaches to answer these mysteries in the Mg2+ signaling field. Identification of these molecular machineries would aid in our understanding of iMg2+ dynamics and the cause-effect relationships that exist between iMg2+ flux and cellular processes. Additionally, I will test and define the Mg2+-dependent signaling events based on the cellular and mouse model phenotypes. It is thrilling to define the molecular link between cellular Mg2+ homeostasis and physiological function. Our identification and characterization of the Mg2+ flux components will further investigate how, and if, these signaling routes impinge on the pathophysiology of a growing number of Mg2+ deficiency diseases in humankind. Overall, the R35/MIRA funding will support the testing of this unconventional hypothesis and my laboratory will address these major mysteries in the near future.