Deficits in KCC2 activity and the pathophysiology of Status Epilepticus

描述（由申请人提供）：电中性K+/Cl-共转运蛋白2（KCC 2）允许神经元维持低细胞内Cl-浓度，这是快速生长的必要先决条件。 由A型γ-氨基丁酸（GABAAR）介导的突触抑制。与此一致，KCC 2活性的缺陷导致癫痫发作，并且被认为是癫痫持续状态（SE）病理学的中心。SE是最具破坏性的癫痫形式，在美国每年有42，000人死亡，还有数十万严重脑损伤病例。SE随着时间的推移变得不那么容易处理，导致耐药性癫痫发作的发展，导致死亡率和发病率增加。在美国，SE每年的成本为48亿美元。与其在调节神经元Cl-稳态中的重要作用一致，在难治性癫痫患者和SE动物模型中观察到KCC 2活性的缺陷。因此，了解SE导致KCC 2失活的机制具有明确的临床意义。KCC 2功能通过该蛋白质的C-末端细胞内结构域内的关键调节残基的磷酸化受到正和负调节。具体地，丝氨酸940（S940）通过蛋白激酶C的磷酸化增强KCC 2活性，而相邻的苏氨酸残基906和1007通过无赖氨酸激酶（WNK）的磷酸化降低转运蛋白活性（Lee等人，2007; 2011; Riehart，2009）。因此，在SE过程中可能导致KCC 2失活的一种机制是这些关键调控残基磷酸化的修饰。为了解决这个问题，我们已经利用了针对S940和T906的磷酸特异性抗体。此外，我们已经创造了小鼠，其中这些关键调节残基的磷酸化通过突变为丙氨酸而被阻止。最后，我们使用了WNK 3缺陷的小鼠，WNK 3是在成年大脑中表达的主要同种型。使用这些新型试剂的初步研究使我们能够制定一个总体假设，将在这里进行测试;“SE期间神经元活性的持续升高导致S940的去磷酸化，但T906/1007的磷酸化增强，导致KCC 2快速抑制的事件，直接导致SE病理生理学的GABA能抑制功效降低”。我们的研究将集中于以下具体目标。具体目标1。为了检验KCC 2磷酸化缺陷有助于SE特异性目的2的发展和致死性的假设。为了检验S940 A小鼠在SE特异性目标3期间表现出增强的T906磷酸化和KCC 2活性的选择性缺陷的假设。为了检验减少KCC 2的WNK依赖性磷酸化可以防止SE发展的假设。总的来说，这些实验将为SE的病理生理学提供关键的机制见解，并可能有助于开发新的治疗方法，以限制这种破坏性疾病的影响。

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.