Proteomic Dissection of M-channel Phosphorylation

M通道磷酸化的蛋白质组学解析

• 批准号: 6529992
• 负责人: EDWARD C COOPER
• 金额: $ 19.81万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6529992
• 关键词: SDS polyacrylamide gel electrophoresis; active sites; affinity chromatography; brain mapping; cell line; developmental neurobiology; epilepsy; gene expression; human tissue; immunocytochemistry; immunofluorescence technique; laboratory mouse; mass spectrometry; method development; muscarinic receptor; neurotransmitter receptor; organ culture; phosphorylation; potassium channel; protein protein interaction; protein signal sequence; proteomics; regulatory gene; voltage /patch clamp

DESCRIPTION (provided by applicant): Careful study of genes responsible for rare Mendelian forms of human neurological disorders is an powerful approach for gaining insight into the causes, treatment, and potential cure for common, related diseases. The KCNQ genes were recently discovered as the result of the search for mutant genes causing Benign Neonatal Familial Convulsions, an autosomal dominant epileptic syndrome associated with seizures in infancy and throughout life. KCNQ genes encode voltage-dependent potassium channels called M-channels. M-channels regulate neuronal excitability by serving as effectors for neurotransmitter receptors and intracellular signaling pathways. Determining how these receptors and pathways modulate M-channels will enhance our ability to exploit M-channels as therapeutic targets in conditions involving excessive excitability or alterations and imbalances in modulatory neurotransmission, such as epilepsy, pain syndromes, and Alzheimer's disease. Because M-channels are large, oligomeric proteins and appear to be regulated by several distinct intracellular pathways, a novel, comprehensive approach to understanding their regulatory mechanisms is warranted. This proposed research approach exploits new instrumentation and analytical methods in mass spectrometry to identify sites of in vivo phosphorylation on KCNQ subunits. A combination of electrophysiological and cellular and molecular biological methods will then be used to determine the consequences of phosphorylation at the identified sites. The proposal involves a new collaboration between individuals with expertise in the clinical and genetic aspects of epilepsy, ion channel physiology, and proteomics. In addition to addressing specific questions concerning M-channel regulation, the proposed research will contribute to the development of mass spectrometric methods and the adaptation of these methods to the study of signaling proteins that are large, complex, and expressed either transiently, locally, or at low abundance in the brain. Because such signaling proteins play essential roles in brain development and in the maintenance of normal brain structure and activity, the proposed research may result in enhanced methodologies of broad usefulness for research into anomalous neurodevelopment, degeneration, and injury.

描述（由申请人提供）：仔细研究负责的基因 罕见的孟德尔形式的人类神经疾病是一种强大的方法 为了深入了解共同的原因，治疗和潜在的治疗 相关疾病。最近发现KCNQ基因是 寻找引起良性新生儿家族性抽搐的突变基因 常染色体显性癫痫综合征与婴儿期癫痫发作有关 一生。 KCNQ基因编码依赖电压的钾通道称为 M通道。 M通道通过用作效应子来调节神经元兴奋性 用于神经递质受体和细胞内信号通路。 确定这些受体和途径如何调节M通道会增强 我们在条件下利用M通道作为治疗目标的能力 涉及过度兴奋性或调节性的变化和失衡 神经传递，例如癫痫，疼痛综合征和阿尔茨海默氏病。 由于M通道很大，因此寡聚蛋白，似乎受 几种不同的细胞内途径，一种新颖的，全面的方法 有必要了解其监管机制。这项拟议的研究 方法利用质量的新仪器和分析方法 识别KCNQ亚基上体内磷酸化位点的光谱法。一个 电生理，细胞和分子生物学的结合 然后，方法将用于确定磷酸化的后果 已确定的站点。该提案涉及在 在癫痫的临床和遗传方面具有专业知识的个人 渠道生理学和蛋白质组学。除了解决特定 有关M通道法规的问题，拟议的研究将 有助于质谱法的发展和适应性 这些方法用于研究大型，复杂的信号蛋白 并在大脑中瞬时，局部或低丰度表示。 因为这种信号蛋白在大脑发育和 在维持正常的大脑结构和活动时，提出了 研究可能会导致对研究的广泛实用性的增强 进入异常的神经发育，变性和损伤。