Phosphorylation as a Determinant of BK Channel Expression and Localization

磷酸化作为 BK 通道表达和定位的决定因素

• 批准号: 7843641
• 负责人: James S Trimmer
• 金额: $ 19.13万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7843641
• 关键词: Abbreviations; Alanine; Amino Acids; Arts; Binding; Binding Sites; Brain; COS Cells; Calcium; Canis familiaris; Cell membrane; Cells; Code; Consensus; D Aspartate; Data; Data Set; Dendrites; Development; Embryo; Employee Strikes; Endoplasmic Reticulum; Enzymes; Fibroblasts; Grant; Hippocampus (Brain); Human; Immunofluorescence Immunologic; Ion Channel; Kidney; Laboratories; Letters; Link; Liquid Chromatography; Lysine; Mass Spectrum Analysis; Mediating; Membrane; Mental disorders; Molecular; Monkeys; Monoclonal Antibodies; Mutate; Neurologic; Neurons; Phosphorylation; Phosphorylation Site; Phosphoserine; Phosphothreonine; Phosphotransferases; Physiological; Play; Potassium; Potassium Channel; Presynaptic Terminals; Proline; Protein Binding; Proteins; Proteomics; Publications; Rattus; Regulation; Role; Serine; Shapes; Signal Transduction; Site; Synapses; Synaptic Transmission; Testing; Threonine; base; in vivo; insight; intervention effect; large-conductance calcium-activated potassium channels; mind control; neuronal excitability; new therapeutic target; novel; novel therapeutics; protein purification; public health relevance; research study; trafficking; voltage

DESCRIPTION (provided by applicant): BK channels are fundamental components of neuronal signaling through effects on neuronal excitability and synaptic transmission. This proposal is aimed at determining the fundamental mechanisms that govern expression and localization of BK channels in mammalian hippocampus. Using novel, state-of- the art mass spectrometric approaches we have made great inroads in defining the in vivo phosphorylation sites on the primary or BK1 subunit of BK channels purified from rat brain. We find that most in vivo phosphorylation sites are Pro-associated pSer and conform to consensus binding sites for proteins containing pSer-binding modules. Moreover, these sites are likely phosphorylated by proline- directed kinases (ProDKs), whose importance in neuronal function and as targets for new therapeutics is just now being appreciated. These data provide the first opportunity to investigate the role of bona fide and unambiguously identified in vivo brain phosphosites on BK channels in governing their expression levels and subcellular localization in hippocampus. We will test the overall hypothesis of this proposal that these sites are crucial to neuronal function and plasticity as mediated by ProDKs acting on native BK channels. In aim 1 we will accomplish this by examining the effects of interventions that alter the phosphorylation state of BK1. We will mutate identified in vivo ProDK phosphorylation sites, and will intervene in ProDK expression levels in heterologous cells and hippocampal neurons, and determine effects on BK channel expression. In Aim 2 we will use similar approaches to determine the role of these phosphorylation sites in polarized localization of BK channels. These studies will yield important insights into the physiological and pathological regulation of BK channels, which are key regulators of neuronal excitability and synaptic transmission in mammalian hippocampus. PUBLIC HEALTH RELEVANCE: This study aims to better understand basic mechanisms controlling brain function. It focuses on neuronal ion channels and their regulatory enzymes that are important targets for developing new therapeutics for neurological and psychiatric disorders.

描述（由申请人提供）：BK通道是通过影响神经元兴奋性和突触传递的神经元信号传导的基本组分。该建议旨在确定哺乳动物海马BK通道表达和定位的基本机制。使用新的，国家的最先进的质谱方法，我们已经取得了很大的进展，在确定在体内磷酸化位点的主要或BK 1亚基的BK通道纯化从大鼠大脑。我们发现，大多数在体内磷酸化位点是Pro相关的pSer和符合共识的蛋白质含有pSer结合模块的结合位点。此外，这些位点可能被脯氨酸定向激酶（ProDK）磷酸化，其在神经元功能中的重要性以及作为新治疗剂的靶点的重要性现在才被认识到。这些数据提供了第一次机会，调查真正的和明确确定的BK通道在体内脑磷酸化位点的作用，在管理他们的表达水平和亚细胞定位在海马。我们将测试这一建议的总体假设，这些网站是至关重要的神经元功能和可塑性介导的ProDK作用于天然BK通道。在目标1中，我们将通过检查改变BK 1磷酸化状态的干预措施的效果来实现这一目标。我们将突变体内鉴定的ProDK磷酸化位点，并将干预异源细胞和海马神经元中的ProDK表达水平，并确定对BK通道表达的影响。在目标2中，我们将使用类似的方法来确定这些磷酸化位点在BK通道极化定位中的作用。BK通道是哺乳动物海马神经元兴奋性和突触传递的关键调节因子，这些研究将对BK通道的生理和病理调节产生重要的影响。公共卫生相关性：这项研究旨在更好地了解控制大脑功能的基本机制。它专注于神经元离子通道及其调节酶，这是开发神经和精神疾病新疗法的重要目标。