Magnesium Channel Cation Selectivity

镁通道阳离子选择性

• 批准号: 8550094
• 负责人: MICHAEL E MAGUIRE
• 金额: $ 28.79万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-24 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8550094
• 关键词: Archaea; Bacteria; Binding; Binding Sites; Catalysis; Cations; Cells; Chemicals; Chemistry; Crystallization; Cysteine; Data; Dehydration; Deuterium; Disease; Dissociation; Divalent Cations; Electrostatics; Elements; Endoplasmic Reticulum; Enzymes; Essential Genes; Eukaryota; Extracellular Domain; Family; Giant Cells; Homeostasis; Homologous Gene; Human; Hydration status; Hydrogen; Investigation; Ion Channel; Ions; Laboratories; Lead; Lipids; Magnesium; Maintenance; Mediating; Mitochondria; Molecular; Molecular Conformation; Movement; Mutation; Organism; Physiological; Process; Rehydrations; Relative (related person); Roentgen Rays; Role; Series; Signal Transduction; Site; Solutions; Source; Structure; System; Thermotoga maritima; Vertebral column; analog; base; crosslink; extracellular; insight; mutant; novel; periplasm; prevent; reconstitution; sensor; small molecule; uptake

DESCRIPTION (provided by applicant): Mg2+ is an essential element. The basis of Mg2+ selectivity, binding and coordination with enzymes and small molecules is well understood but the molecular basis by which transport systems achieve Mg2+ selectivity over other cations is not understood in any system. The structures of the CorA and MgtE Mg2+ channels demonstrate clearly that channel selectivity for Mg2+ cannot be explained by analogy to the chemistry of selective Mg2+ sites in any enzyme system. The CorA Mg2+ channel is the primary source of Mg2+ for ~50% of all Bacteria and Archaea while MgtE is the primary source for the other half of each kingdom. Both have multiple homologs in eukaryotes, including humans. Electrophysiological data show that both CorA and MgtE are true ion channel with surprisingly high conductance, >100 pS, a flux rate >107 Mg2+ ions/sec. This very high rate poses serious mechanistic issues. The rate of dehydration of Mg2+ is only 105/sec, 100-1000-fold slower than the flux rate. Uniquely among all known ion channels, both CorA and MgtE initially bind a fully hydrated Mg2+ ion, which must contribute to the channel's selectivity for Mg2+. To mediate flux of a largely dehydrated cation, the channel must in some manner accelerate the rate of dehydration. However, electrostatic interactions are not involved in this process or in ion flux. The study of these two ion channels, which have quite different structures, provides an opportunity to dissect the distinct chemistry involved in Mg2+ selectivity. Aim 1 will investigate the mechanism of selectivity of CorA for Mg2+ through i) electrophysiological analysis of site-directed mutants expressed in giant cells or reconstituted in lipid, ii) hydrogen-deuterium exchange primarily of loop and pore residues, and iii) X-ray crystallographic approaches using cations with very slow rates of dehydration and cysteine mutations in transmembrane segment 2 of CorA. The latter generate crosslinked oligomers which appear to be trapped in a conformation different from the closed state, previously solved. Their crystallization may provide additional conformational information for CorA. Aim 2 will explore analogous issues for MgtE using the same approaches.

描述（由申请人提供）： Mg2+ 是必需元素。 Mg2+ 选择性、与酶和小分子的结合和配位的基础已被充分了解，但在任何系统中，转运系统相对于其他阳离子实现 Mg2+ 选择性的分子基础尚不清楚。 CorA 和 MgtE Mg2+ 通道的结构清楚地表明，Mg2+ 通道的选择性不能通过类比任何酶系统中选择性 Mg2+ 位点的化学来解释。 CorA Mg2+ 通道是约 50% 的细菌和古细菌的 Mg2+ 的主要来源，而 MgtE 是每个王国另一半的主要来源。两者在真核生物（包括人类）中都有多种同源物。电生理学数据表明，CorA 和 MgtE 都是真正的离子通道，具有令人惊讶的高电导率，>100 pS，通量率>107 Mg2+ 离子/秒。这种非常高的比率带来了严重的机械问题。 Mg2+的脱水速率仅为105/秒，比通量速率慢100-1000倍。在所有已知的离子通道中，CorA 和 MgtE 最初都结合完全水合的 Mg2+ 离子，这一定有助于通道对 Mg2+ 的选择性。为了调节大量脱水阳离子的通量，通道必须以某种方式加速脱水速率。然而，静电相互作用不参与该过程或离子通量。对这两个结构截然不同的离子通道的研究为剖析 Mg2+ 选择性所涉及的不同化学提供了机会。目标 1 将通过 i) 对在巨细胞中表达或在脂质中重构的定点突变体进行电生理分析，ii) 主要针对环和孔残基的氢-氘交换，以及 iii) X 射线来研究 CorA 对 Mg2+ 的选择性机制晶体学方法使用脱水速率非常慢的阳离子以及 CorA 跨膜片段 2 中的半胱氨酸突变。后者产生交联低聚物，其似乎被困在与先前解决的闭合状态不同的构象中。它们的结晶可以为 CorA 提供额外的构象信息。目标 2 将使用相同的方法探索 MgtE 的类似问题。