Magnesium Channel Cation Selectivity

说明(申请人提供)：镁是必需的元素。镁离子的选择性、与酶和小分子的结合和配位的基础是众所周知的，但运输系统实现对其他阳离子的选择性的分子基础在任何体系中都不是很清楚。CORA和镁离子通道的结构清楚地表明，通道对镁离子的选择性不能用任何酶系统中选择性镁离子位点的化学来解释。珊瑚镁离子通道是~50%的细菌和古生菌的主要镁离子来源，而镁离子通道是每个王国的另一半的主要来源。这两种基因在包括人类在内的真核生物中都有多种同源基因。电生理数据表明，CORA和MgTE都是真正的离子通道，具有惊人的高电导，-gt；100ps，通量速率&gt；107mg2+/s。这一非常高的比率带来了严重的机械问题。镁离子的脱水速率仅为105/秒，比通量速率慢100-1000倍。在所有已知的离子通道中独一无二的是，CORA和MgtE最初都结合了完全水合的镁离子，这必须有助于该通道对镁离子的选择性。为了调节大量脱水阳离子的流量，通道必须以某种方式加速脱水速度。然而，静电相互作用并不涉及这一过程或离子通量。对这两个结构完全不同的离子通道的研究，提供了一个剖析与镁离子选择性有关的不同化学成分的机会。目的1通过对巨细胞中表达的或在脂质中重组的定点突变体的电生理分析，主要是环和孔残基的氢-氚交换，以及利用极慢脱水速率的阳离子和CORA跨膜片段2上的半胱氨酸突变的X射线结晶学方法，来研究CORA对镁离子的选择性机制。后者产生似乎被捕获在与先前解决的闭合状态不同的构象中的交联型低聚物。它们的结晶可能为CORA提供了额外的构象信息。目标2将使用相同的方法为管理企业探索类似的问题。

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.