HCN Channel Phosphorylation Sites Mapped by Mass Spectrometry in Human Epilepsy Patients and in an Animal Model of Temporal Lobe Epilepsy.
HCN Channel Phosphorylation Sites Mapped by Mass Spectrometry in Human Epilepsy Patients and in an Animal Model of Temporal Lobe Epilepsy.
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人癫痫患者和颞叶癫痫动物模型中HCN通道磷酸化位点的质谱图。
DOI：
10.1016/j.neuroscience.2021.01.038
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发表时间：
2021-04-15
期刊：
影响因子：
3.3
通讯作者：
Poolos NP
中科院分区：
文献类型：
--
作者：
Concepcion FA;Khan MN;Ju Wang JD;Wei AD;Ojemann JG;Ko AL;Shi Y;Eng JK;Ramirez JM;Poolos NP
由于超极化激活的环核苷酸门控 (HCN) 离子通道调节皮质和海马主要神经元的兴奋性，因此这些通道在脑损伤或癫痫发生后癫痫发展过程中发生的过度兴奋中发挥关键作用。在毛果芸香碱诱导的癫痫持续状态产生的癫痫大鼠中，HCN 通道活性通过两种主要机制下调：门控超极化转变和 HCN 通道介导的电流幅度降低，Ih。由于这些机制受到各种磷酸化信号通路的调节，我们假设在癫痫发生过程中各个 HCN 通道氨基酸残基（磷酸位点）发生磷酸化变化。我们收集了因毛果芸香碱诱导的癫痫持续状态而致癫痫的雄性 Sprague Dawley 大鼠和年龄匹配的幼稚对照的 CA1 海马组织。我们还纳入了含有癫痫发作区域（EZ）的切除人脑组织，以便与我们的慢性癫痫大鼠进行比较。通过免疫沉淀和胰蛋白酶凝胶消化富集 HCN1 和 HCN2 亚型后，通过质谱分析样品。我们从 HCN1 和 HCN2 通道中鉴定出许多磷酸位点，代表了对 HCN 通道内磷酸化位点的新颖调查。我们发现人类和大鼠之间的 HCN 通道磷酸位点具有高水平的同源性。我们还发现了一种新的 HCN1 通道磷酸位点 S791，其在慢性癫痫阶段的磷酸化显着增加。拟磷突变体 S791D 的异源表达复制了慢性癫痫大鼠神经元中观察到的 Ih 门控超极化转变。这些结果表明 HCN1 通道磷酸化在癫痫中发生改变，并且可能具有重要的致病作用。
Because hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels modulate the excitability of cortical and hippocampal principal neurons, these channels play a key role in the hyperexcitability that occurs during the development of epilepsy after a brain insult, or epileptogenesis. In epileptic rats generated by pilocarpine-induced status epilepticus, HCN channel activity is downregulated by two main mechanisms: a hyperpolarizing shift in gating and a decrease in amplitude of the current mediated by HCN channels, Ih. Because these mechanisms are modulated by various phosphorylation signaling pathways, we hypothesized that phosphorylation changes occur at individual HCN channel amino acid residues (phosphosites) during epileptogenesis. We collected CA1 hippocampal tissue from male Sprague Dawley rats made epileptic by pilocarpine-induced status epilepticus, and age-matched naïve controls. We also included resected human brain tissue containing epileptogenic zones (EZs) where seizures arise for comparison to our chronically epileptic rats. After enrichment for HCN1 and HCN2 isoforms by immunoprecipitation and trypsin in-gel digestion, the samples were analyzed by mass spectrometry. We identified numerous phosphosites from HCN1 and HCN2 channels, representing a novel survey of phosphorylation sites within HCN channels. We found high levels of HCN channel phosphosite homology between humans and rats. We also identified a novel HCN1 channel phosphosite S791, which underwent significantly increased phosphorylation during the chronic epilepsy stage. Heterologous expression of a phosphomimetic mutant, S791D, replicated a hyperpolarizing shift in Ih gating seen in neurons from chronically epileptic rats. These results show that HCN1 channel phosphorylation is altered in epilepsy and may be of pathogenic importance.
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影响因子：
5.4
作者：
Brennan GP;Baram TZ;Poolos NP
通讯作者：
Poolos NP
DOI：
10.1523/jneurosci.1290-10.2010
发表时间：
2010-05-12
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
--
作者：
Jung S;Bullis JB;Lau IH;Jones TD;Warner LN;Poolos NP
通讯作者：
Poolos NP
影响因子：
4.5
作者：
Bauer, CK;Falk, T;Schwarz, JR
通讯作者：
Schwarz, JR
影响因子：
3.4
作者：
Eng, Jimmy K.;Jahan, Tahmina A.;Hoopmann, Michael R.
通讯作者：
Hoopmann, Michael R.
影响因子：
5.3
作者：
Brewster, A;Bender, RA;Baram, TZ
通讯作者：
Baram, TZ