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

摘要/摘要 游离的胞内电离镁(IMG2)估计在0.5-1.2毫米范围内。一般来说，它是被接受的。 在静息状态下，细胞内游离镁的浓度被磷代谢产物“抑制”， 核酸和蛋白质。例如，三磷酸腺苷与Kd值50M-70μM结合，因此在 胞浆和线粒体基质主要与三磷酸腺苷(mg-ATP2-)络合。因为它的丰度(~5 (Mm)，ATP被认为是iMG2最大的‘商店’。激素性激素对游离胞浆(CMG2)的影响 刺激被吹捧为从旺盛的镁-三磷酸腺苷状态中分离出来的镁的被动调整 以及其他有缓冲的镁2池。除了IMG2的“缓冲”机制，镁离子通道和转运体 控制质膜上镁的进入和流出被认为是维持细胞内镁离子的平衡。 免费CMG2。目前，有几个候选者与mg2进入机制(TRPM6、TRPM7、MagT1)相关，但 仍在等待这种作用的令人信服的生物物理和生理证据。镁钠交换器 Slc41a1被认为有助于镁从细胞外流，而mrs2被认为是线粒体。 MG2传送器。关于镁离子转运进出细胞器的分子细节还知之甚少。 如内质网、线粒体、内切体和溶酶体。一些研究推测，内质网中的游离[Mg2] 线粒体很可能类似于[CMG2]。然而，时间和空间动态，更不用说 IMg2动员的生物学意义，在细胞生物学中仍然是一个谜。尽管如此，mg2是一种必不可少的 控制许多生化反应的阳离子。我们最近的工作表明，L-乳酸作为激活剂， 触发内质网和线粒体之间的镁动态转移，以形成生物能量学和细胞 代谢(细胞2020)。L乳酸盐促进内质网镁离子释放和镁摄取的作用机制 通过线粒体。线粒体定位的mrs2转运蛋白被发现是导致这种积累的原因。 线粒体中的镁含量。然而，L-乳酸诱导内质网释放的分子机制尚不清楚。 我建议鉴定内质网mg2释放组分、质膜进入机制以及由此产生的 分子信号通路。我将利用无偏见的RNAi屏幕和有针对性的CRISPR/Cas9编辑 在MG2信令领域，解答这些谜团的方法。这些分子机制的鉴定 将有助于我们理解iMg2的动力学以及iMg2通量之间存在的因果关系 和细胞过程。此外，我将测试和定义依赖于MG2的信号事件 细胞和小鼠模型表型。这是令人兴奋的定义之间的分子联系细胞镁 动态平衡和生理功能。我们对镁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会影响越来越多的 人类中的镁缺乏疾病。总体而言，R35/Mira的资金将支持这一测试 非常规假说和我的实验室将在不久的将来解开这些重大谜团。

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.