Modulation of Kir Channel Function by Phosphorylation

通过磷酸化调节 Kir 通道功能

• 批准号: 7653214
• 负责人: Diomedes E. Logothetis
• 金额: $ 38.84万
• 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/7653214
• 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; 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; Structure; Testing; Time; Transmembrane Domain; Work; base; in vivo; inorganic phosphate; insight; molecular modeling; mutant; novel; programs; public health relevance; 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(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。心脏内向整流钾(KIR)通道也受蛋白磷酸化的调节，从而改变其活性并调节心脏的兴奋性。在过去的十年里，人们认识到所有KIR通道的活性都严重依赖于与膜磷脂磷脂酰肌醇-双磷酸盐(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庭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是从分子结构的角度了解离子通道的功能和调节，特别是从机理上了解KIR活性对PIP2的依赖。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。对与磷酸化效应有关的假定位置的三维结构的研究表明，影响对PIP2敏感性的氨基酸残基周围有一个显着的聚集。因此，我们提出了以下假设，我们打算在这一应用中进行测试：“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大量努力，但我们所拥有的明确识别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，我们还没有对磷酸化如何影响通道活性的机械性结构理解。在这里，我们建议使用质谱学来确定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蛋白激酶A靶向磷酸化位点(Kir3.1-S385)，结合使用了质谱学方法(MALDI-TOF和串联质谱学)。这一结果向我们证明了该方法在识别磷酸化位点方面的可行性。我们建议从电生理学的角度测试特定的磷酸化位点是否会影响对PIP2的敏感性。全面介绍不同蛋白激酶使用的位点，评估哪些位点通过PIP2发挥作用，以及开发可实验测试的计算模型，应该会给我们很好的机制洞察磷酸化如何调节通道活性。PHS 398/2590(09/04版，2006年4月4日重新发布)第1页延续格式页。与公共健康相关：在包括运动在内的许多情况下，磷酸化过程调节心脏表现，如心率和收缩强度。该项目旨在鉴定被磷酸化的心肌钾通道蛋白的氨基酸残基。在蛋白质的三维背景下需要检验的假设是，磷酸化残基可以通过改变这些通道与关键的膜磷脂PIP2的直接或变构相互作用来发挥其功能作用。如果是真的，这个假说将提供一个框架，在这个框架上，磷酸化对通道活性的影响可以从机制上得到解释。