Regulation of the Na/K Pump by RNA Editing

描述（由申请人提供）：该项目的重点是Na/K泵如何通过RNA编辑进行调节。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与人类Na/Ka 3亚基相关的遗传性疾病--快速发作的肌张力障碍性帕金森综合征的工具。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，并且因为我们已经确定的编辑位点导致功能的获得，这是突变的罕见现象。初步数据显示，Na/K泵mRNA可以在三个密码子处进行编辑，两个在磷酸化结构域（P）中，另一个在第7跨膜跨度（M7）的顶部。这些变化影响泵送循环的关键部件。例如，P结构域中的编辑增加了对ATP的表观亲和力，M7中的编辑调节Na如何释放到外部。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。最后一个目标的目标是看看鱿鱼RNA编辑是否可以补偿由与快速发作的肌张力障碍帕金森症相关的突变引起的人类Na/Ka 3的转换率降低。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，在mRNA水平上将这些编辑引入人类泵。初步数据显示，人类编辑酶ADAR 2能够编辑鱿鱼mRNA，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欺骗人类ADAR 2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泵产生兴奋性和大部分溶质跨细胞膜转运所需的离子梯度。随着最近发表的第一个晶体结构，这是一个适当的时间来了解更多关于Na/K泵如何工作。在临床上，Na/K泵很重要，因为它是地高辛的受体，地高辛是一种广泛用于控制许多心律失常的心脏类固醇。此外，有两种神经疾病与Na/K泵突变直接相关：家族性偏瘫性偏头痛，与Na/Ka 2亚基有关，以及速发型肌张力障碍性帕金森病，与Na/Ka 3亚基有关。从这个建议的结果将直接相关的治疗快速发作的肌张力障碍帕金森综合征的发展。一般的方法也可能证明与各种遗传性疾病有关。 公共卫生相关性：Na/K泵在建立跨细胞离子梯度方面起着至关重要的作用。初步数据显示，它的泵送能力可以通过RNA编辑来调节。该提案的重点是了解RNA编辑如何调节Na/K泵，以及如何用于治疗影响Na/K泵的遗传疾病。

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.