First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel antiseizure strategies. Impaired GABAergic inhibition is key to the development of such benzodiazepine-resistant seizures, as well as the pathophysiology of status epilepticus (SE). It is emerging that reduced or impaired neuronal K+/Cl– cotransporter 2 (KCC2) activity contributes to deficits in γ-aminobutyric acid (GABA)-mediated inhibition and increased seizure vulnerability. The with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signaling pathway inhibits neuronal KCC2 via KCC2-T1007 phosphorylation. A selective WNK kinase inhibitor, WNK463, was recently synthesized by Novartis. Exploiting WNK463, we test the hypothesis that pharmacological WNK inhibition will enhance KCC2 activity, increase the efficacy of GABAergic inhibition, and thereby limit seizure activity in animal models. Immunoprecipitation and Western blot analysis were used to examine WNK463’s effects on KCC2-T1007 phosphorylation, in vitro and in vivo. A thallium (Tl+) uptake assay was used in human embryonic kidney (HEK-293) cells expressing KCC2 to test WNK463’s effects on KCC2-mediated Tl+ transport. Gramicidin-perforated- and whole-cell patch-clamp recordings in cortical rat neurons were used to examine WNK463’s effects on KCC2-mediated Cl– transport. In mouse brain slices (entorhinal cortex), field recordings were utilized to examine WNK463’s effects on 4-aminopyridine-induced seizure activity. Last, WNK463 was directly deliver to the mouse hippocampus in vivo and tested in a kainic acid model of diazepam-resistant SE. WNK463 significantly reduces KCC2-T1007 phosphorylation in vitro and in vivo (mice). In human embryonic kidney 293 (HEK-293) cells expressing KCC2, WNK463 greatly enhanced the rates Tl+ transport. However, the drug did not enhance Tl+ transport in cells expressing a KCC2-phospho null T1007 mutant. In cultured rat neurons, WNK463 rapidly reduced intracellular Cl– and consequently hyperpolarized the Cl– reversal potential (EGABA). In mature neurons that were artificially loaded with 30 mM Cl–, WNK463 significantly enhanced KCC2-mediated Cl– export and hyperpolarized EGABA. In a 4-aminopyridine model of acute seizures, WNK463 reduced the frequency and number of seizure-like events (SLEs). Finally, in an in vivo kainic acid (KA) model of diazepam-resistant SE, WNK463 slowed the onset and reduced the severity of KA-induced status epilepticus. Last, WNK463 prevented the development of pharmaco-resistance to diazepam in drug-treated mice. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. Our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant SE.

中文翻译：

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抑制无赖氨酸激酶可增强 K+/Cl- 协同转运蛋白 2 活性并限制癫痫持续状态

在大约 30% 的癫痫患者中，一线苯二氮卓类药物治疗未能终止癫痫发作，这凸显了对新型抗癫痫发作策略的需求。受损的 GABA 能抑制是此类苯二氮卓类药物耐药性癫痫发作以及癫痫持续状态 (SE) 的病理生理学发展的关键。神经元 K+/Cl- 协同转运蛋白 2 (KCC2) 活性降低或受损会导致 γ-氨基丁酸 (GABA) 介导的抑制作用不足和癫痫易感性增加。无赖氨酸激酶 (WNK)-STE20/SPS1 相关的脯氨酸/富含丙氨酸 (SPAK) 激酶信号通路通过 KCC2-T1007 磷酸化抑制神经元 KCC2。诺华公司最近合成了一种选择性 WNK 激酶抑制剂 WNK463。利用 WNK463，我们检验了药理学 WNK 抑制将增强 KCC2 活性的假设，增加 GABA 能抑制的功效，从而限制动物模型中的癫痫发作活动。免疫沉淀和蛋白质印迹分析用于检查 WNK463 在体外和体内对 KCC2-T1007 磷酸化的影响。在表达 KCC2 的人胚肾 (HEK-293) 细胞中使用铊 (Tl+) 摄取测定，以测试 WNK463 对 KCC2 介导的 Tl+ 转运的影响。使用皮质大鼠神经元中的短杆菌肽穿孔和全细胞膜片钳记录来检查 WNK463 对 KCC2 介导的 Cl- 转运的影响。在小鼠脑切片（内嗅皮层）中，利用现场记录来检查 WNK463 对 4-氨基吡啶诱导的癫痫发作活动的影响。最后，WNK463 在体内直接递送至小鼠海马体，并在耐地西泮 SE 的红藻氨酸模型中进行了测试。WNK463 在体外和体内（小鼠）显着降低 KCC2-T1007 磷酸化。在表达 KCC2 的人胚肾 293 (HEK-293) 细胞中，WNK463 大大提高了 Tl+ 转运速率。然而，该药物并未增强表达 KCC2-phospho 无效 T1007 突变体的细胞中的 Tl+ 转运。在培养的大鼠神经元中，WNK463 迅速降低细胞内 Cl-，从而使 Cl- 反转电位 (EGABA) 超极化。在人工加载 30 mM Cl– 的成熟神经元中，WNK463 显着增强了 KCC2 介导的 Cl– 输出和超极化 EGABA。在急性癫痫发作的 4-氨基吡啶模型中，WNK463 降低了癫痫样事件 (SLE) 的频率和数量。最后，在地西泮耐药 SE 的体内红藻氨酸 (KA) 模型中，WNK463 减缓了 KA 诱导的癫痫持续状态的发作并降低了严重程度。最后的，WNK463 可防止药物治疗小鼠对地西泮产生耐药性。我们的研究结果表明，急性 WNK463 治疗增强了神经元中的 KCC2 活性，并限制了两种成熟的癫痫发作和癫痫模型中的癫痫发作负担。我们的工作表明，增加 KCC2 活性的药物可能是缓解地西泮耐药性 SE 的有用辅助疗法。