Altered phosphorylation of connexin 43 contributes to increased gap-junction coup

连接蛋白 43 磷酸化的改变有助于增加间隙连接突变

基本信息

批准号：
8727124
负责人：
Heidi Grabenstatter
金额：
$ 9.72万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8727124
关键词：
Acute Address Agonist Animals Antibodies Astrocytes Biological Assay Buffers Calcium Signaling Cells Chronic Communication Connexin 43 Connexins Coupling Data Development Diffusion Disease Disease Progression Early treatment Electroencephalography Enzymes Epilepsy Epileptogenesis Equilibrium Extracellular Space Freeze Fracturing Frequencies Gap Junctions Gliosis Glutamate Transporter Glutamates Goals Hippocampus (Brain)Immunohistochemistry Knock-in Mouse Label Measurement Measures Mediating Membrane Methods Modeling Modification Molecular Monitor Mus Neuroglia Neurons Phase Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Pilocarpine Potassium Potassium Glutamate Predisposition Probability Protein Dephosphorylation Protein-Serine-Threonine Kinases Proteins Rat Protein Rattus Regulation Relative (related person)Resistance Role Seizures Severities Signal Transduction Slice Status Epilepticus Temporal Lobe Epilepsy Time Tissues Transgenic Organisms Western Blotting Wild Type Mouse biocytin inhibitor/antagonist kinase inhibitor patch clamp phosphatase inhibitor prevent public health relevance research study response

项目摘要

DESCRIPTION (provided by applicant): Pilocarpine-induced status epileptics (SE) induce reactive gliosis and activate calcium signaling in astrocytes of the hippocampus in a time-dependent manner (1, 2). Primary studies demonstrate significant decreases in connexin phosphorylation 48 h after SE and in chronically epileptic rats 30 d after SE. This data combined with previous studies showing that paroxysmal depolarization shifts can be initiated by release of glutamate from astrocytes3 suggests that increased open probability of gap- junction hemi channels via dephosphorylating of connexin 43 in hippocampal astrocytes may contribute to the progression of epileptogenesis. The major goal of this proposal is to characterize the role of serine/threonine kinases and phosphatases in seizure-induced dephosphorylating of connexin 43 and specifically, to evaluate the hypothesis that status epileptics acutely activates phosphatases (and may decrease kinases at specific time points) in glyptic astrocytes which dephosphorylates connexin 43 to subsequently increase connexin 43-containing hemi channel open probability and astrocytes gap- junction coupling during epileptogenesis in the hippocampus. Molecular methods will be used to characterize the time course of phosphatase and kinase activation. Alterations in gap-junction coupling will be quantitatively measured in response to equilibrium shifts between kinase and phosphatase activity in hippocampal slices at multiple time points following SE. Kinase and phosphatase activity will be pharmacologically and genetically modulated to determine the primary mechanism responsible for the seizure-induced dephosphorylating of connexin 43 and downstream effects on gap-junction coupling, potassium currents, and glutamate transporter currents prior to and after the onset of spontaneous seizures. Chronic video-EEG monitoring and freeze-fracture replica immunogold labeling (FRIL) will be used to determine whether seizure-induced alterations in phosphorylated connexin 43 membrane expression and potential increases in seizure frequency associated with phosphorylation-mediated hemi channel opening can be prevented with pharmacological or transgenic methods. The proposal will provide valuable translational information about how phosphatase inhibitors or kinase agonists may be exploited to prevent the progression of epileptogenesis and achieve and disease-modification.

描述（由申请人提供）：毛果石诱导的状态癫痫病（SE）诱导反应性神经胶质病并以时间依赖的方式激活海马星形胶质细胞中的钙信号传导（1，2）。基本研究表明，在SE后48小时，在SE后30天，连接蛋白磷酸化显着降低。该数据与先前的研究结合在一起，表明可以通过从星形胶质细胞中释放谷氨酸来引发阵发性去极化移位3，这表明通过在海马星形胶质细胞中脱磷酸化结膜43通过去磷酸化增加了间隙 - 连接半通道的开放概率可能有助于粘液性生成的进展。该提案的主要目标是表征丝氨酸/苏氨酸激酶和磷酸酶在癫痫发作引起的连接蛋白43的去磷酸化中的作用，具体是，以评估状态癫痫症的假说急性激活磷酸酶（并可能在特定时间点降低渗透速率），从而使磷酸化酶降低，从而在特定的时间点降低，从而在散孔中降低了磷酸化酶，从而在渗透速率中降低了磷酸化酶，从而使磷酸化酶降低了磷酸化酶。连接43个含有HEMI通道的开放概率和星形胶质细胞间隙 - 在海马中癫痫发生期间的间隙 - 连接耦合。分子方法将用于表征磷酸酶和激酶活化的时间过程。 SE之后的多个时间点，在海马切片中的激酶和磷酸酶活性之间的平衡变化时，将对间隙结耦合的变化进行定量测量。激酶和磷酸酶活性将在药理学和遗传上进行调节，以确定负责癫痫发作诱导的连接蛋白43的去磷酸化的主要机制，以及对间隙结构偶联，钾电流和谷氨酸转运蛋白和谷氨酸转运蛋白的影响和下游影响。慢性视频EEG监测和冻结折叠复制品免疫金标记（FRIL）将用于确定癫痫发作诱导的磷酸化连接蛋白43膜表达的变化是否可以通过药理或转基因方法来预防与磷酸化介导的Hemi通道相关的癫痫发作频率的增加。该提案将提供有关如何利用磷酸酶抑制剂或激酶激动剂的宝贵翻译信息，以防止癫痫生成和实现和疾病调整的进展。