Modulation of Kir Channel Function by Phosphorylation

描述（由申请人提供）：蛋白质磷酸化是用于调节大多数蛋白质功能的常见细胞机制。心脏内向整流钾（Kir）通道也受到蛋白磷酸化的调节，蛋白磷酸化改变了它们的活性并调节心脏兴奋性。在过去的十年中，人们已经认识到，所有Kir通道的活性关键取决于与膜磷脂磷脂酰肌醇二磷酸（PIP 2）的相互作用。此外，在过去五年中，在解决具有代表性的Kir家族成员的三维结构方面取得了很大进展。我们实验室的长期目标一般是了解离子通道的功能和调节的分子结构，特别是获得机制的洞察Kir活性对PIP 2的依赖性。我们已经发现许多不同类型的Kir通道调节，包括磷酸化，依赖于通道-PIP 2相互作用，我们的目标是了解这种依赖性的分子基础。来自文献和我们自己的初步研究的证据表明，磷酸化改变了通道对PIP 2激活的敏感性。在三维结构的位置的推定的网站，已牵连到参与磷酸化作用的检查揭示了一个惊人的聚类周围的氨基酸残基，影响敏感性PIP 2。因此，我们提出了以下假设，我们提出在本申请中进行测试：“磷酸化可以通过调节通道-PIP 2相互作用对心脏Kir通道发挥其功能作用”。虽然蛋白质磷酸化及其作用机制的问题已经吸引了许多杰出的研究人员的巨大努力，我们已经明确地确定单个磷酸化位点的实验工具一直是有限的。因此，在离子通道领域，我们还没有磷酸化如何影响通道活性的机械结构的理解。在这里，我们建议使用质谱来鉴定Kir 3通道中的磷酸化位点，以便在三维背景下测试我们的假设。我们的初步结果已经确定了蛋白激酶A靶向磷酸化位点（Kir3.1-S385），使用质谱方法（MALDI-TOF和串联质谱）的组合。这一结果向我们证明了这种方法在鉴定磷酸化位点方面的可行性。我们建议测试电生理是否特定的磷酸化位点影响敏感性PIP 2。一个全面的帐户网站使用不同的蛋白激酶，评估哪些网站发挥其作用，通过PIP 2，和实验测试的计算模型的发展应该给我们很好的机制的见解，磷酸化如何调节通道活性。PHS 398/2590（2004年9月修订，2006年4月重新发布）第1页续格式页。公共卫生相关性：磷酸化过程在许多条件下（包括运动）调节心脏性能，如心率和收缩强度。本研究的目的是鉴定心脏钾通道蛋白磷酸化的氨基酸残基。在蛋白质的三维背景下测试的假设是，磷酸化残基可以通过直接改变或变构改变这些通道与关键膜磷脂PIP 2的相互作用来发挥其功能作用。如果是真的，这一假设将提供一个框架，磷酸化对通道活性的影响可以解释机制。

DESCRIPTION (provided by applicant): Protein phosphorylation is a common cellular mechanism used to regulate the function of most proteins. Cardiac inwardly rectifying potassium (Kir) channels are also regulated by protein phosphorylation that changes their activity and modulates cardiac excitability. Over the past ten years it has been appreciated that the activity of all Kir channels depends critically on interactions with the membrane phospholipid phosphatidylinositol-bis-phosphate (PIP2). Moreover great advances over the past five years have been made in solving the three-dimensional structures of representative Kir family members. The long term goal of our laboratory in general is to understand ion channel function and regulation in terms of molecular structure and in particular to gain mechanistic insight for the dependence of Kir activity on PIP2. We have found that many different types of Kir channel modulation, including phosphorylation, depend on channel-PIP2 interactions and we aim to understand the molecular basis of such dependence. Evidence from the literature and from our own preliminary studies suggest that phosphorylation changes the sensitivity of the channel to activation by PIP2. Examination in the three-dimensional structures of the position of putative sites that have been implicated to be involved in phosphorylation effects reveal a striking clustering around amino acid residues that affect sensitivity to PIP2. We have thus formulated the following hypothesis that we propose to test in this application: "Phosphorylation can exert its functional effects on the cardiac Kir channels by modulating channel-PIP2 interactions". Although the problem of protein phosphorylation and its mechanism of action has attracted great effort from many outstanding investigators, the experimental tools we have had to unequivocally identify single phosphorylation sites have been limiting. Thus, in the ion channel field we do not yet have mechanistic structural understanding of how phosphorylation affects channel activity. Here, we propose to use Mass Spectrometry to identify phosphorylation sites in Kir3 channels in order to test our hypothesis in a three- dimensional context. Our preliminary results have identified a protein kinase A-targeted phosphorylation site (Kir3.1-S385), using a combination of Mass Spectrometry methods (MALDI-TOF and tandem Mass Spectrometry). This result has demonstrated to us the feasibility of this approach in identifying phosphorylation sites. We propose to test electrophysiologically whether specific phosphorylation sites affect sensitivity to PIP2. A comprehensive account of sites used by different protein kinases, the assessment of which sites exert their effects through PIP2, and development of experimentally testable computational models ought to give us good mechanistic insights as to how phosphorylation regulates channel activity. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 1 Continuation Format Page. PUBLIC HEALTH RELEVANCE: Phosphorylation processes regulate cardiac performance, such as heart rate and strength of contraction, under many conditions, including exercise. This project aims to identify amino acid residues of cardiac potassium channel proteins that are phosphorylated. The hypothesis to be tested in the three-dimensional context of the proteins is that phosphorylated residues can exert their functional effects by altering directly or allosterically interactions of these channels with the key membrane phospholipid PIP2. If true, this hypothesis will provide a framework on which phosphorylation effects on channel activity could be explained mechanistically.