Magnesium Channel Cation Selectivity

说明（申请人提供）：Mg 2+是必需元素。Mg 2+选择性、与酶和小分子的结合和配位的基础是很好理解的，但转运系统实现Mg 2+选择性超过其他阳离子的分子基础在任何系统中都不清楚。CorA和MgtE Mg 2+通道的结构清楚地表明，通道对Mg 2+的选择性不能通过类比任何酶系统中选择性Mg 2+位点的化学来解释。CorA Mg 2+通道是约50%的所有细菌和细菌的Mg 2+的主要来源，而MgtE是每个界的另一半的主要来源。两者在包括人类在内的真核生物中都有多个同源物。电生理学数据显示，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，通量速率>107 Mg 2+离子/秒。这一非常高的比率造成了严重的机械问题。Mg 2+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Mg 2+离子，这必须有助于通道对Mg 2+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，静电相互作用并不涉及这个过程或离子通量。这两个离子通道，具有相当不同的结构的研究，提供了一个机会，解剖不同的化学参与Mg 2+的选择性。目的1将通过i）在巨细胞中表达或在脂质中重构的定点突变体的电生理学分析，ii）主要是环和孔残基的氢-氘交换，和iii）使用具有非常慢的脱水速率的阳离子和CorA跨膜区段2中的半胱氨酸突变的X射线晶体学方法来研究CorA对Mg 2+的选择性的机制。后者产生交联的低聚物，似乎被困在一个构象不同的封闭状态，以前解决。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讨MgtE的类似问题。 公共卫生相关性：Mg 2+是一种必需元素。一些人类Mg 2+转运系统是必需的基因，而其他基因的突变会导致毁灭性的疾病。Mg 2+的选择性、与酶和小分子的结合和配位的化学基础是很好理解的。然而，运输系统实现Mg 2+选择性超过其他阳离子的分子基础是完全未知的。CorA和MgtE Mg 2+通道的结构清楚地表明，通道对Mg 2+的选择性不能通过类比任何酶系统中选择性Mg 2+位点的化学来解释。CorA Mg 2+通道是约50%的细菌和细菌的Mg 2+的主要来源。它的主要真核同源物是普遍存在的线粒体Mg 2+通道Mrs 2，一个必需的基因。MgtE Mg 2+通道是其他50%细菌和细菌的Mg 2+的主要来源。它的主要人类同源物是运输系统的SLC 41 A家族。虽然两个通道的Mg 2+的物理基础，唯一的离子通道，完全水合的Mg 2+离子，这种选择性的结构基础是不同的两个通道。这两个结构相关的运输系统的机制的调查，预计将提供重要的洞察机制，通过该机制Mg 2+的选择性运输。最终，这些信息可能有助于理解和治疗Mg 2+稳态紊乱。

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. PUBLIC HEALTH RELEVANCE: Mg2+ is an essential element. Several human Mg2+ transport systems are essential genes while mutations in others lead to devastating diseases. The chemical basis of Mg2+ selectivity, binding and coordination with enzymes and small molecules is well understood. However, the molecular basis by which transport systems achieve Mg2+ selectivity over other cations is completely unknown. 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. Its primary eukaryotic homolog is the ubiquitous mitochondrial Mg2+ channel Mrs2, an essential gene. The MgtE Mg2+ channel is the primary source of Mg2+ for the other 50% of Bacteria and Archaea. Its primary human homologs are the SLC41A family of transport systems. While the physical basis of Mg2+ for both channels involved, uniquely for ion channels, the fully hydrated Mg2+ ion, the structural basis for this selectivity is different in the two channels. Investigation of the mechanisms of these two structurally related transport systems is expected to provide important insight into the mechanisms through which Mg2+ is selectively transported. Ultimately, this information may be of use in understanding and possibly treating disorders of Mg2+ homeostasis.