Epilepsy and dendritic excitability

 描述（由申请人提供）：超极化激活的环核苷酸门控（HCN）通道是电压门控离子通道，可调节参与癫痫发病机制的几个脑区（包括海马、新皮质和丘脑）的兴奋性。积累的证据表明，下调Ih，HCN通道产生的电流，导致神经元的过度兴奋，和HCN 1通道，主要的皮质和海马亚型的基因缺失，加快获得性癫痫模型的癫痫发生率。最近的证据表明，HCN 1突变是一些严重癫痫和发育迟缓儿童早期癫痫性脑病的基础。因此，HCN 1通道病发生在人类遗传性癫痫和获得性癫痫的动物模型中。 在我们实验室最近的工作中，由化学惊厥剂诱导的癫痫持续状态（SE）诱导的癫痫与SE后1小时内开始的HCN 1通道表达丧失相关，并持续到慢性癫痫。HCN 1通道急性内化从海马锥体树突的表面膜后SE的第一个小时内，以及发病前几天后的HCN 1 mRNA表达的转录下调。我们现在第一次表明，HCN 1通道表面表达是由蛋白激酶C（PKC）活性以双向方式控制的，PKC对HCN 1的磷酸化增加导致HCN 1表面表达减少。使用质谱技术的新的初步数据显示，HCN 1通道在静止时磷酸化程度最低，但在S867和S868残基处磷酸化程度增加，这两个残基在PKC激活条件下也在幼稚组织中磷酸化。这些数据表明，HCN 1通道功能障碍在癫痫发生的最早表现是PKC介导的表面表达的损失和随之而来的损失HCN 1介导的电流（Ih）。 我们在R 01更新申请中的目标是进一步了解癫痫发生过程中HCN 1通道功能障碍的机制。我们建议进一步研究磷酸化信号转导机制，潜在的急性变化后，在HCN 1膜贩运SE;确定这些早期事件是否有助于或启动慢性HCN 1通道病;并表征人类HCN 1突变对HCN 1通道生物物理特性的影响。为此，我们将使用最先进的技术，包括：质谱;突变HCN 1通道表达的病毒转导体内; CA 1海马锥体神经元树突中的细胞电生理学;以及分析一组800名不明原因癫痫性脑病儿童中HCN 1突变的新数据集。

 DESCRIPTION (provided by applicant): Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that modulate excitability in several brain regions involved in the pathogenesis of epilepsy, including the hippocampus, neocortex, and thalamus. Accumulated evidence shows that downregulation of Ih, the current generated by HCN channels, causes neuronal hyperexcitability, and that genetic deletion of HCN1 channels, the main cortical and hippocampal subtype, accelerates the rate of epileptogenesis in acquired epilepsy models. More recent evidence shows that mutations in HCN1 underlie early life epileptic encephalopathy in some children with severe epilepsy and developmental delay. Thus HCN1 channelopathy occurs in both human genetic epilepsy and animal models of acquired epilepsy. In recent work from our laboratory, epilepsy induced by chemoconvulsant-induced status epilepticus (SE) was associated with loss of HCN1 channel expression that began within 1 hour post-SE, and persisted into chronic epilepsy. HCN1 channels were acutely internalized from the surface membrane of hippocampal pyramidal dendrites within the first hour following SE, well before the onset several days later of transcriptional downregulation of HCN1 mRNA expression. We now show for the first time that HCN1 channel surface expression is governed in a bidirectional fashion by protein kinase C (PKC) activity, with increased phosphorylation of HCN1 by PKC leading to decreased HCN1 surface expression. New preliminary data using mass spectrometry techniques show that HCN1 channels are minimally phosphorylated at rest, but undergo increased phosphorylation at S867 and S868 residues, which are also phosphorylated in naive tissue under conditions of PKC activation. These data suggest that the earliest manifestation of HCN1 channels dysfunction during epileptogenesis is PKC-mediated loss of surface expression and concomitant loss of HCN1-mediated current (Ih). Our goal in this R01 renewal application is to further understand the mechanisms of HCN1 channel dysfunction during the development of epilepsy. We propose to further study the phosphorylation signaling mechanisms underlying acute changes in HCN1 membrane trafficking after SE; determine whether these early events contribute to or initiate chronic HCN1 channelopathy; and characterize the effects of human HCN1 mutations on HCN1 channel biophysical properties. To do so, we will use state-of-the-art techniques including: mass spectrometry; viral transduction of mutant HCN1 channel expression in vivo; cellular electrophysiology in the dendrites of CA1 hippocampal pyramidal neurons; and analysis of a novel dataset of HCN1 mutations in a cohort of 800 children affected with epileptic encephalopathy of unknown cause.