Deficits in KCC2 activity and the pathophysiology of Status Epilepticus

电子中和钾/氯共转运体2(KCC2)使神经元维持较低的细胞内Cl2浓度， A型γ-氨基丁酸介导快速突触抑制的必要条件 (GABAAR)与此一致，KCC2活性缺陷会导致癫痫发作，并被认为是 癫痫持续状态(SE)病理学。自发性癫痫是最具破坏性的癫痫形式，占4.2万 美国每年的死亡人数，以及数十万例严重的脑损伤病例。Se变少了 随着时间的推移易于处理，导致出现抗药性癫痫，从而增加死亡率和 发病率。在美国，SE每年的成本为48亿美元。与其在以下方面的基本作用一致 在顽固性癫痫患者中，存在调节神经元氯稳态的KCC2活性缺陷，以及 在SE的动物模型中。因此，了解SE导致KCC2失活的机制 具有明显的临床意义。KCC2函数通过正向和负向调制 该蛋白的C末端胞内结构域内的关键调节残基的磷酸化。具体来说， 蛋白激酶C对丝氨酸940(S940)的磷酸化增强了KCC2的活性，而对KCC2的磷酸化 邻近的苏氨酸残基906和1007通过非赖氨酸激酶(WNKS)降低转运蛋白活性 (Lee等人，2007年；2011年；Riehart，2009年)。因此，可能导致KCC2失活的一种机制是 Se是这些关键调控残基的磷酸化修饰。为了解决这个问题，我们有 利用针对S940和T906的磷酸特异性抗体。此外，我们还创造了老鼠，在其中 这些关键调控残基的磷酸化已通过突变为丙氨酸而被阻止。最后，我们 利用了WNK3缺乏的小鼠，WNK3是WNK在成人大脑中表达的主要亚型。初步 使用这些新试剂的研究使我们能够形成一个将被检验的总体假说 在此，“SE期间神经元活性的持续升高导致S940去磷酸化，但是 增强T906/1007的磷酸化，导致KCC2快速抑制的事件，减少 GABA能抑制的疗效直接有助于SE的病理生理学“。我们的研究将 重点关注以下具体目标。 具体目标1.检验KCC2磷酸化缺陷导致 自发性硬化症的发展和致命性 特定目的2.检验S940A小鼠表现出增强的T906磷酸化和 SE期间KCC2活性的选择性亏损 具体目的3.验证减少WNK依赖的KCC2磷酸化的假说 阻止自发性硬化症的发展。 总的来说，这些实验将为SE的病理生理学提供关键的机械学见解，并可能 帮助开发新的治疗方法，以限制这种破坏性疾病的影响。

The electroneutral K+/Cl- co-transporter 2 (KCC2) allows neurons to maintain low intracellular Clconcentrations, an essential prerequisite for fast synaptic inhibition mediated by type A γ-aminobutyric acid (GABAAR). Consistent with this, deficits in KCC2 activity lead to seizures and are believed to be central to the pathology of Status Epilepticus (SE). SE is the most devastating form of epilepsy, and accounts for 42,000 deaths per year in the US, and hundreds of thousand more cases of severe brain damage. SE becomes less tractable with time, leading to the development of drug resistant seizures, resulting in increased mortality and morbidity. SE is associated with a cost of $4.8 billion per year in the US. Consistent with its essential role in regulating neuronal Cl- homeostasis, deficits in KCC2 activity are seen in patients with intractable epilepsy, and in animal models of SE. Therefore, understanding the mechanisms by which SE leads to inactivation of KCC2 is of clear clinical significance. KCC2 function is subject to both positive and negative modulation via phosphorylation of key regulatory residues within the C-terminal intracellular domain of this protein. Specifically, phosphorylation of serine 940 (S940) by protein kinase C enhances KCC2 activity, while phosphorylation of the adjacent threonine residues 906 and 1007 by with-no-lysine kinases (WNKs) decreases transporter activity (Lee et al., 2007; 2011; Riehart, 2009). Thus, one mechanism that may contribute to KCC2 inactivation during SE is modifications in the phosphorylation of these key regulatory residues. To address this issue, we have utilized phospho-specific antibodies against S940 and T906. In addition, we have created mice in which the phosphorylation of these key regulatory residues has been prevented via mutation to alanines. Finally, we have made use of mice deficient in WNK3, the principle WNK isoform expressed in the adult brain. Preliminary studies using these novel reagents have allowed us to formulate an overarching hypothesis that will be tested here; “Persistent elevations in neuronal activity during SE lead to dephosphorylation of S940, but enhanced phosphorylation of T906/1007, events that lead to rapid inhibition of KCC2, reductions in the efficacy of GABAergic inhibition that directly contribute to the pathophysiology of SE”. Our studies will focus on the following specific aims. Specific Aim 1. To test the hypothesis that deficits in KCC2 phosphorylation contribute to the development and lethality of SE Specific Aim 2. To test the hypothesis that S940A mice exhibit enhanced T906 phosphorylation and a selective deficit in KCC2 activity during SE Specific Aim 3. To test the hypothesis that reducing WNK dependent phosphorylation of KCC2 prevents the development of SE. Collectively these experiments will provide key mechanistic insights into the pathophysiology of SE, and may aid the development of novel therapeutics to limit the impact of this devastating disorder.