Deficits in KCC2 activity and the pathophysiology of Status Epilepticus

描述(申请人提供)：电子中和钾/氯-共转运体2(KCC2)允许神经元维持低的细胞内氯浓度，这是FAST的基本先决条件 A型γ-氨基丁酸(GABA)介导的突触抑制。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)的核心病理。SE是最具破坏性的癫痫形式，在美国每年造成4.2万人死亡，数十万人严重脑损伤。随着时间的推移，SE变得不那么容易处理，导致耐药癫痫的发生，导致死亡率和发病率的增加。在美国，SE每年的成本为48亿美元。在顽固性癫痫患者和SE动物模型中可以看到KCC2活性的缺陷，这与其在调节神经元氯稳态方面的基本作用一致。因此，了解SE导致KCC2失活的机制具有明显的临床意义。KCC2的功能受到正向和负向的调节，通过该蛋白C末端胞内结构域中的关键调节残基的磷酸化来实现。具体地说，蛋白激酶C对丝氨酸940(S940)的磷酸化增强了KCC2的活性，而无赖氨酸激酶(WNKS)对相邻的苏氨酸残基906和1007的磷酸化降低了转运蛋白的活性(Lee等人，2007；2011；Riehart，2009)。因此，在SE过程中可能导致KCC2失活的一个机制是这些关键调节残基的磷酸化修饰。为了解决这个问题，我们利用了针对S940和T906的磷酸特异性抗体。此外，我们还创造了小鼠，在小鼠中，这些关键调控残基的磷酸化通过突变为丙氨酸而被阻止。最后，我们利用了缺乏WNK3的小鼠，WNK3是WNK在成人大脑中表达的主要亚型。使用这些新试剂的初步研究使我们能够形成一个总体假说，并将在这里进行测试：“在SE期间神经元活动的持续升高导致S940的去磷酸化，但T906/1007的磷酸化增强，导致KCC2的快速抑制，GABA能抑制的有效性降低，这直接参与了SE的病理生理学”。我们的研究将集中在以下具体目标上。特异性目的1.验证KCC2磷酸化缺陷与SE特异性目的蛋白的发生和致死性有关的假说2.检验S940A小鼠在SE特异性目的蛋白过程中T906磷酸化增强和KCC2活性选择性缺陷的假说3.验证减少WNK依赖性KCC2磷酸化以阻止SE发生的假说。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，并可能有助于开发新的治疗方法来限制这种毁灭性疾病的影响。

DESCRIPTION (provided by applicant): The electroneutral K+/Cl- co-transporter 2 (KCC2) allows neurons to maintain low intracellular Cl- concentrations, 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 thousands 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.