Deficits in KCC2 activity and the pathophysiology of Status Epilepticus

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， A型γ-氨基丁酸介导的快速突触抑制的必要前提 （加巴）。与此一致，KCC 2活性的缺陷导致癫痫发作，并且被认为是癫痫发作的核心。 癫痫持续状态（SE）的病理学。SE是最具破坏性的癫痫形式， 死亡人数，以及数十万例严重脑损伤病例。SE变得更少 随着时间的推移，导致耐药性癫痫发作的发展，导致死亡率增加， 发病率。在美国，SE每年的成本为48亿美元。与其在以下方面的重要作用相一致： 调节神经元Cl-稳态，在难治性癫痫患者中观察到KCC 2活性的缺陷， 在SE的动物模型中。因此，了解SE导致KCC 2失活的机制， 具有明显的临床意义。KCC 2功能受到正调制和负调制， 该蛋白质的C-末端胞内结构域内的关键调节残基的磷酸化。具体地说， 丝氨酸940（S940）被蛋白激酶C磷酸化增强KCC 2活性，而丝氨酸940（S940）被蛋白激酶C磷酸化增强KCC 2活性。 邻近的苏氨酸残基906和1007通过无赖氨酸激酶（WNK）降低转运蛋白活性 (Lee例如，2007; 2011; Riehart，2009）。因此，一种可能导致KCC 2失活的机制是， SE是这些关键调节残基磷酸化的修饰。为了解决这个问题，我们 利用针对S940和T906的磷酸化特异性抗体。此外，我们还创造了小鼠， 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 利用WNK 3缺陷的小鼠，WNK 3是在成年大脑中表达的主要同种型。初步 使用这些新试剂的研究使我们能够制定一个总体假设， “SE期间神经元活性的持续升高导致S940的去磷酸化，但 T906/1007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能抑制的功效”。我们的研究将 着重于以下具体目标。 具体目标1。为了验证KCC 2磷酸化缺陷导致细胞凋亡的假设， SE的发生和致死 具体目标2。为了验证S940 A小鼠表现出增强的T906磷酸化和T906磷酸化的假设， SE期间KCC 2活性的选择性缺陷 具体目标3。为了检验减少KCC 2的WNK依赖性磷酸化 阻止了SE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制见解， 有助于开发新的治疗方法，以限制这种毁灭性疾病的影响。

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.