Modulation of Kir Channel Function by Phosphorylation

通过磷酸化调节 Kir 通道功能

• 批准号: 8239544
• 负责人: Diomedes E. Logothetis
• 金额: $ 37.63万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8239544
• 关键词: Accounting; Acetylcholine; Affect; Amino Acids; Atrial Fibrillation; Cardiac; Cell Line; Cells; Chimera organism; Computer Simulation; Cyclic AMP-Dependent Protein Kinases; Dependence; Development; Employee Strikes; Exercise; Family member; GIRK1 subunit, G protein-coupled inwardly-rectifying potassium channel; GIRK4 subunit, G protein-coupled inwardly-rectifying potassium channel; Generations; Goals; Health; Heart; Heart Atrium; Heart Rate; In Vitro; Ion Channel; Laboratories; Literature; Maps; Mass Spectrum Analysis; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Models; Molecular Structure; Mutagenesis; Performance; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospho-Specific Antibodies; Phospholipids; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Positioning Attribute; Post-Translational Protein Processing; Potassium; Potassium Channel; Principal Investigator; Process; Protein Kinase; Proteins; PubMed; Publications; Regulation; Reporting; Research Design; Research Personnel; Rodent; Site; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Testing; Time; Transmembrane Domain; Work; base; in vivo; inorganic phosphate; insight; molecular modeling; mutant; novel; programs; tandem mass spectrometry; three dimensional structure; tool

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.

描述（由申请人提供）：蛋白质磷酸化是一种常见的细胞机制，用于调节大多数蛋白质的功能。心脏内整流钾（Kir）通道也受蛋白磷酸化调控，磷酸化改变其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通道的活性主要取决于与膜磷脂磷脂肌醇-二磷酸（PIP2）的相互作用。此外，在过去五年中，在解决具有代表性的基尔家族成员的三维结构方面取得了巨大进展。总的来说，我们实验室的长期目标是了解离子通道的功能和分子结构方面的调控，特别是获得对Kir活性对PIP2依赖性的机制见解。我们发现许多不同类型的Kir通道调节，包括磷酸化，依赖于通道- pip2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PIP2激活的敏感性。对参与磷酸化作用的假定位点的三维结构进行检查，揭示了影响对PIP2敏感性的氨基酸残基周围的惊人聚类。因此，我们制定了以下假设，我们建议在本应用中进行测试：“磷酸化可以通过调节通道- pip2相互作用对心脏Kir通道发挥其功能影响”。尽管蛋白质磷酸化及其作用机制的问题已经吸引了许多杰出研究者的巨大努力，但我们必须明确确定单个磷酸化位点的实验工具一直受到限制。因此，在离子通道领域，我们还没有对磷酸化如何影响通道活性的机制结构的理解。在这里，我们建议使用质谱法鉴定Kir3通道中的磷酸化位点，以便在三维环境中验证我们的假设。我们的初步结果已经确定了一个蛋白激酶a靶向磷酸化位点（Kir3.1-S385），使用组合质谱方法（MALDI-TOF和串联质谱）。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。我们建议通过电生理学测试特定磷酸化位点是否会影响对PIP2的敏感性。对不同蛋白激酶所使用的位点的综合描述，对哪些位点通过PIP2发挥作用的评估，以及实验可测试的计算模型的发展，应该会给我们提供关于磷酸化如何调节通道活性的良好机制见解。PHS 398/2590（09/04修订版，4/2006修订版）第1页延续格式页。公共卫生相关性：在包括运动在内的许多条件下，磷酸化过程调节心脏功能，如心率和收缩强度。本项目旨在鉴定被磷酸化的心脏钾通道蛋白的氨基酸残基。在蛋白质的三维背景下要测试的假设是，磷酸化残基可以通过改变这些通道与关键膜磷脂PIP2的直接或变构相互作用来发挥其功能作用。如果这是真的，这一假设将提供一个框架，磷酸化对通道活性的影响可以机械地解释。