Cell Type-Specific Roles of the Na-Activated K Current in KCNT1-Related Epilepsy

编码Na+激活的K+通道的KCNT1基因的变异导致了几个严重的 儿童癫痫障碍，很难治疗。此前，致病的 当在非神经细胞中检测时，KCNT1变体被证明增加了通道电流， 从而引出了一个基本问题，即如何增加通常与 抑制神经元兴奋性会导致以过度、同步为特征的疾病 神经元活动。我们已经用同源致病人建立了两个小鼠模型 有频繁癫痫发作的KCNT1变种。在这两种模型中，皮质抑制神经元(CIN) 在膜兴奋性和动作电位产生方面表现出强烈的损害，而 兴奋性神经元(CEN)则不起作用。这些数据表明，KCNT1的功能增益(GOF) 突变通过损害CINS的功能而导致儿童癫痫，而KCNT1通道 在调节神经元兴奋性方面发挥特定细胞类型的作用。这项提案将考验这些 通过(1)确定KCNT1阻断和GOF如何影响CIN膜兴奋性的假说 亚型，(2)使用新开发的KCNT1选择性抑制剂来测量电流和 CIN亚型中的致病变异是如何改变它的，以及(3)测试它是如何受损的 CINs亚群的膜兴奋性影响其在体内的活性，及其相互关系 癫痫发作或大脑皮层过度兴奋。确定这些脆弱的细胞类型，潜在的 机制，以及它们功能失调的体内效应将使我们能够将细胞缺陷与 癫痫样活动和癫痫发作。这一结果将促进我们对这一规定的理解 CIN的兴奋性、KCNT1介导的电流的生理作用与疾病 KCNT1 GOF在体内癫痫模型中的作用机制。这将缩小知识差距。 离子通道变异体的生物物理效应与由此导致的细胞功能障碍之间的关系 网络，并有可能改善针对重症患者的精确治疗 儿童期癫痫。

Variants in the KCNT1 gene, which encodes a Na+-activated K+ channel, cause several severe childhood epilepsy disorders that are largely refractory to treatment. Previously, pathogenic KCNT1 variants were shown to increase channel current when assayed in non-neuronal cells, leading to the fundamental question of how increasing a current that is usually associated with dampening neuronal excitability leads to disorders characterized by excessive, synchronous neuronal activity. We have created two mouse models with orthologous pathogenic human KCNT1 variants that have frequent seizures. In both models, cortical inhibitory neurons (cINs) show strong impairments in membrane excitability and action potential generation, whereas excitatory neurons (cENs) do not. These data suggest that KCNT1 gain-of-function (GOF) variants cause childhood epilepsy by impairing the function of cINs, and that KCNT1 channels play cell-type-specific roles in regulating neuronal excitability. This proposal will test these hypotheses by (1) determining how KCNT1 block and GOF affect membrane excitability of cIN subtypes, (2) using newly-developed KCNT1-selective inhibitors to measure the current and how it is altered by disease-causing variants in cIN subtypes, and (3) testing how impaired membrane excitability in subpopulations of cINs affects their activity in vivo, and how this relates to seizures or cortical hyperexcitability. Determining these vulnerable cell types, the underlying mechanisms, and the in vivo effects of their dysfunction will allow us to relate cellular deficits to epileptiform activity and seizures. The results will advance our understanding of the regulation of cIN excitability, the physiological roles of the KCNT1-mediated current, and the disease mechanisms of KCNT1 GOF in an in vivo model of epilepsy. This will narrow the knowledge gap between the biophysical effects of ion channel variants and the resulting dysfunction of networks, and has the potential to improve the targeting of precision therapies for severe childhood epilepsies.