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Ubiquitylation as a regulator of potassium channel function

泛素化作为钾通道功能的调节剂

基本信息

批准号：
8496157
负责人：
ANTHONY D MORIELLI
金额：
$ 7.36万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8496157
关键词：
Address Affect Anesthetics Applications Grants Arousal Brain Cardiovascular system Cell Culture System Cell model Cell surface Cerebellar Ataxia Cerebellar Nuclei Cerebellar cortex structure Cerebellum Data Dendrites Employee Strikes Endocytosis Equilibrium Gated Ion Channel Goals Health Hippocampus (Brain)Human Inherited Ion Channel Kv1.2 potassium channel Learning Lysine Mass Spectrum Analysis Membrane Memory Methods Molecular Mutagenesis Mutation Nervous system structure Neurologic Neurons Output Pathology Pattern Phosphorylation Phosphorylation Site Post-Translational Protein Processing Potassium Channel Presynaptic Terminals Proteins Proteome Purkinje Cells Recycling Regulation Reporting Rest Role Serine/Threonine Phosphorylation Signaling Protein Site Spinal Ganglia Stimulus System Testing Thalamic structure Tissues Tyrosine Tyrosine Phosphorylation Ubiquitin Voltage-Gated Potassium Channel brain tissue density multicatalytic endopeptidase complex mutant painful neuropathy protein degradation protein function response trafficking voltage

项目摘要

DESCRIPTION (provided by applicant): Voltage gated ion channels are major determinants of membrane excitability. The Kv1.2 potassium channel is expressed widely throughout the brain, but little is known about Kv1.2 regulation in the brain. Using model cell systems, Kv1.2 was found to be regulated by tyrosine and serine/threonine phosphorylation. More recently, we have identified ubiquitylation as being capable of modulating Kv1.2 function. In preliminary studies we show that ubiquitylation affects Kv1.2 endocytosis and recycling and that it does so independently of Kv1.2 degradation. Intriguingly, mutagenesis of ubiquitylation sites within Kv1.2 revealed that distinct patterns of ubiquitylation within Kv1.2 affect channel trafficking in dramatically different ways, some decreasing and some increasing Kv1.2 levels at the cell surface. These preliminary studies employed model cell systems and ectopically expressed wild type or mutant forms of Kv1.2 to reveal a nuanced role for ubiquitylation in Kv1.2 regulation. As informative as it is, however, this approach only suggests how ubiquitylation affects Kv1.2 endogenously expressed in the brain. Given the key role of Kv1.2 in the brain, the complete lack of information on the occurrence and effects of Kv1.2 ubiquitylation there is a striking gap. The current challenge, and goal of this grant proposal, is to determine whether the types of ubiquitylation predicted by our preliminary studies with ectopically expressed Kv1.2 occur in Kv1.2 expressed endogenously within the brain. To do so, we will use AQUA and SILAC mass spectrometry methods to quantitatively determine the amount and intra-molecular patterns of stimulus-induced ubiquitylation of Kv1.2 expressed endogenously within the brain.

描述（由申请人提供）：电压门控离子通道是膜兴奋性的主要决定因素。Kv1.2钾通道在整个大脑中广泛表达，但对大脑中Kv1.2的调节知之甚少。使用模型细胞系统，Kv1.2被发现是由酪氨酸和丝氨酸/苏氨酸磷酸化。最近，我们已经确定泛素化能够调节Kv1.2功能。在初步研究中，我们发现，泛素化影响Kv1.2的内吞和回收，它这样做独立于Kv1.2降解。有趣的是，Kv1.2内泛素化位点的诱变揭示了Kv1.2内泛素化的不同模式以显著不同的方式影响通道运输，一些降低和一些增加细胞表面的Kv1.2水平。这些初步研究采用模型细胞系统和异位表达的野生型或突变形式的Kv1.2，揭示了Kv1.2调控中的泛素化的微妙作用。然而，尽管这种方法提供了大量信息，但它只表明了泛素化如何影响Kv1.2在大脑中的内源性表达。鉴于Kv1.2在大脑中的关键作用，完全缺乏关于Kv1.2泛素化的发生和影响的信息，这是一个惊人的差距。目前的挑战和这项拨款提案的目标是确定我们对异位表达Kv1.2的初步研究所预测的泛素化类型是否发生在大脑内内源性表达的Kv1.2中。为此，我们将使用AQUA和SILAC质谱法定量测定刺激诱导的Kv1.2内源性表达的泛素化在脑内的量和分子内模式。