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

摘要/总结 游离离子化的细胞内Mg 2+（iMg 2+）估计在0.5-1.2 mM的范围内。 在静息条件下，电离的胞质Mg 2+的浓度被磷酸代谢酶“抑制”， 核酸和蛋白质。例如，ATP结合的Kd值为50 μ M-70 μM，因此Mg 2+在 细胞质和线粒体基质主要与ATP（Mg-ATP 2-）复合。由于其丰富的（~5 mM），ATP被认为是最大的iMg 2+“库”。激素治疗后游离胞浆（cMg 2+）的波动 刺激被吹捧为Mg 2+从旺盛的Mg-ATP队伍中解离的被动调节 和其它Mg 2+的“缓冲”池。除了iMg 2+“缓冲”机制外，Mg 2+离子通道和转运蛋白 控制Mg 2+进入以及穿过质膜的流出被认为维持了 游离cMg 2+。目前，几个候选者与Mg 2+进入机制（TRPM 6、TRPM 7、MagT 1）相关，但是 仍在等待令人信服的生物物理学和生理学证据来证明这种作用。Mg 2 +/Na+交换剂 SLC 41 A1被认为有助于Mg 2+从细胞中流出，而Mrs 2被认为是线粒体膜蛋白。 Mg 2+转运蛋白。关于Mg 2+转运入/出细胞器的分子细节知之甚少 比如内质网线粒体内体和溶酶体一些研究推测ER中的游离[Mg 2 +] 而线粒体可能与[cMg 2 +]相似。然而，时间和空间动态，更不用说 iMg 2+动员的生物学相关性仍然是细胞生物学中的一个谜。然而，Mg 2+是必不可少的 阳离子控制许多生化反应。我们最近的研究表明，L-乳酸作为一种激活剂， 触发ER和线粒体之间的Mg 2+动态转移，以形成生物能量学和细胞 代谢（Cell 2020）。从机制上讲，L-乳酸盐刺激Mg 2+从ER释放，随后是Mg 2+摄取 通过线粒体。线粒体定位的Mrs 2转运蛋白被发现是负责积累 线粒体内Mg ~（2+）的含量。然而，L-乳酸诱导的ER释放的分子机制仍然不明。 我建议确定ER Mg 2+释放组件，质膜进入机制和结果 分子信号通路我将利用无偏见的RNAi筛选和靶向CRISPR/Cas9编辑 在Mg 2+信号领域，我们希望找到解决这些谜团的方法。识别这些分子机器 将有助于我们理解iMg 2+动力学和iMg 2+通量之间存在的因果关系 和细胞过程。此外，我将测试和定义Mg 2+依赖性信号事件的基础上， 细胞和小鼠模型表型。确定细胞内Mg 2+和Mg 2+之间的分子联系是令人兴奋的， 稳态和生理功能。我们对Mg 2+熔剂组分的鉴定和表征将 进一步研究这些信号通路如何以及是否影响越来越多的糖尿病患者的病理生理学。 人类镁缺乏症。总的来说，R35/MIRA资金将支持这项测试。 我的实验室将在不久的将来解决这些主要的谜团。

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.