Regulation of the Na/K Pump by RNA Editing

项目描述（申请人提供）：本项目主要研究RNA编辑如何调控Na/K泵。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引导我们找到Na/K泵的重要功能区域。它们也可能被用作补偿快速发作的肌张力障碍帕金森病的工具，这是一种与人类Na/Ka3亚基相关的遗传疾病。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点会导致功能的增加，这是一种罕见的突变现象。初步数据显示，Na/K泵mrna可以在三个密码子上进行编辑，两个在磷酸化结构域(P)，另一个在第7跨膜跨越的顶部（M7）。这些变化会影响泵送循环的关键组成部分。例如，P结构域的编辑增加了对ATP的明显亲和力，M7中的编辑调节Na如何释放到外部。利用电生理学方法，该提案的前两个目标将描述这些编辑发挥其功能的机制。最后一个目标是观察鱿鱼的RNA编辑是否可以补偿人类Na/Ka3的低周转率，这种低周转率是由与快速发作性肌张力障碍帕金森病相关的突变引起的。此外，我们不仅将研究它们是否可以补偿，我们还将尝试开发一种方法，将这些编辑引入mRNA水平的人体泵。初步数据表明，人类编辑酶ADAR2能够编辑鱿鱼mrna，因为它识别合适的二级结构。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欺骗人类ADAR2来编辑人类泵。从公共卫生的角度来看，这项工作在几个方面意义重大。钠钾泵产生离子梯度，这是兴奋性和大多数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，这是一个了解更多关于Na/K泵如何运作的好时机。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，两种神经疾病与Na/K泵的突变直接相关：与Na/Ka2亚基相关的家族性偏瘫偏头痛，以及与Na/Ka3亚基相关的快速发作性肌张力障碍帕金森病。这项提议的结果将直接关系到治疗快速发作性肌张力障碍帕金森病的发展。一般方法也可能证明与各种遗传疾病相关。

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. PUBLIC HEALTH RELEVANCE: The Na/K pump plays a vital role in establishing ion gradients across cells. Preliminary data shows that its ability to pump can be regulated by RNA editing. This proposal focuses on understanding how RNA editing regulates the Na/K pump and how it might be used to treat genetic disorders that affect the Na/K pump.