Regulation of the Na/K Pump by RNA Editing

描述(申请人提供)：本项目重点研究Na/K泵如何通过RNA编辑来调节。我们方法的一个中心前提是，由RNA编辑引起的自然发生的密码子变化可以将我们带到Na/K泵的功能重要区域。它们也可能被用作工具来补偿快速发作的肌张力障碍帕金森综合症，这是一种与人类Na/Ka3亚单位相关的遗传病。鱿鱼神经系统将被用作模型，因为RNA编辑在头足类动物中广泛存在，而且我们已经确定的编辑位置会导致功能增强，这对于突变来说是一种罕见的现象。初步数据显示，Na/K泵mRNAs可以编辑在三个密码子上，其中两个在磷酸化结构域(P)，另一个在第7跨膜跨膜(M7)的顶部。这些变化影响到抽水循环的关键部件。例如，P结构域中的编辑增加了对ATP的表观亲和力，而M7中的编辑调节了Na向外释放的方式。使用电生理学方法，该提案的前两个目标将描述这些编辑发挥其功能的机制。最后一个目标是看看鱿鱼RNA编辑是否可以弥补由于与快速发作的帕金森障碍症相关的突变而导致的人类Na/Ka3的低周转率。此外，我们不仅将研究它们是否能够补偿，我们还将尝试开发一种方法，将这些编辑引入到人类泵的mRNA水平上。初步数据显示，人类编辑酶ADAR2能够编辑鱿鱼mRNAs，因为它识别适当的二级结构。我们假设我们可以通过用反义RNA寡核苷酸模仿鱿鱼的二级结构来诱骗人类ADAR2编辑人类泵。从公共卫生的角度来看，这项工作在几个方面具有重要意义。Na/K泵产生的离子梯度是兴奋性和大部分溶质跨细胞膜运输所必需的。随着最近发表的第一个晶体结构，这是一个很好的时机来了解更多关于Na/K泵的工作原理。临床上，钠钾泵很重要，因为它是地高辛的受体，地高辛是一种广泛使用的心脏类固醇，用于控制许多心律失常。此外，有两种神经疾病与Na/K泵的突变直接相关：与Na/Ka2亚单位有关的家族性偏瘫偏头痛，以及与Na/Ka3亚单位有关的速发型肌张力障碍帕金森综合症。这项建议的结果将直接关系到快速发作的帕金森氏肌张力障碍的治疗方法的发展。一般的方法也可能被证明与多种遗传性疾病有关。

DESCRIPTION (provided by applicant): This project focuses on how the Na/K pump is regulated by RNA editing. A central premise to our approach is that naturally occurring codon changes, caused by RNA editing, can lead us to functionally important regions of the Na/K pump. They might also be used as tools to compensate for rapid-onset dystonia parkinsonism, genetic disease associated with the human Na/Ka3 subunit. The squid nervous system will be used as a model because RNA editing is extensive in cephalopods and because the editing sites that we have identified cause a gain of function, a rare phenomenon for a mutation. Preliminary data show that Na/K pump mRNAs can be edited at three codons, two in the phosphorylation domain (P), and the other at the top of the 7th transmembrane span (M7). These changes affect critical components of the pumping cycle. For example, the edits in the P domain increase the apparent affinity for ATP and the edit in M7 regulates how Na is released to the outside. Using an electrophysiological approach, the proposal's first two aims will characterize the mechanism by which these edits exert their function. The goal of the last aim is to see whether squid RNA edits can compensate for depressed turnover rates in human Na/Ka3 caused by mutations associated with rapid-onset dystonia parkinsonism. In addition, not only will we study if they can compensate, we will also try to develop a method to introduce these edits into human pumps at the level of mRNA. Preliminary data show that the human editing enzyme ADAR2 is capable of editing squid mRNAs because it recognizes the appropriate secondary structure. We hypothesize that we can trick human ADAR2 into editing human pumps by mimicking the squid secondary structure with an antisense RNA oligo. From the standpoint of public health, this work is significant on several fronts. The Na/K pump creates the ion gradient that is required for excitability and the majority of solute transport across cell membranes. With the first crystal structure recently published, this is an opportune time to learn more about how the Na/K pump operates. Clinically, the Na/K pump is important because it is the receptor of digoxin, a widely prescribed cardiac steroid used to control many cardiac arrhythmias. Further, two neural disorders have been directly correlated with mutations to the Na/K pump: familial hemiplegic migraine, which is linked to the Na/Ka2 subunit, and rapid-onset dystonia parkinsonism, which is linked to the Na/Ka3 subunit. Results from this proposal will be directly relevant to the development of therapeutics for rapid-onset dystonia parkinsonism. The general approach may also prove relevant for a wide variety of genetic disorders.