Molecular Mechanisms of Epilepsy-Causing Mutations in IKM channels: anti-epileptic effect of Lipophilic compounds.

IKM 通道引起癫痫的分子机制：亲脂性化合物的抗癫痫作用。

• 批准号: 10318601
• 负责人: Rene Barro-Soria
• 金额: $ 33.58万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318601
• 关键词: Affect; Alternative Therapies; Antiepileptic Agents; Brain; CRISPR/Cas technology; Cessation of life; Cysteine; Data; Defect; Dependence; Development; Disease; Drug Design; Epilepsy; Functional disorder; Gene Delivery; Genes; Goals; Head; Homeostasis; Human; Induced Mutation; Induced pluripotent stem cell derived neurons; Inherited; Intellectual functioning disability; Intervention; Ion Channel; Lead; Length; Link; Maintenance; Maps; Measures; Molecular; Movement; Mutagenesis; Mutate; Mutation; Neurons; Pathogenesis; Patients; Persons; Polyunsaturated Fatty Acids; Potassium Channel; Proteins; Research; Seizures; Site; Technology; Testing; Variant; Viral; Work; Xenopus oocyte; base; early onset; functional restoration; genetic variant; genome editing; improved; induced pluripotent stem cell; lipophilicity; mutant; neuronal excitability; patch clamp; pi bond; programs; reduce symptoms; sensor; unnatural amino acids; voltage

Abnormal neuronal activity in the brain leads to epileptic seizures that, when repeated or prolonged, can cause neuronal damage resulting in delayed psychomotor development and intellectual disability. Most genetic variants associated with epilepsy are in genes encoding ion channels, including potassium channels that regulate neuronal excitability such as IKM channels. Inherited mutations in the IKM channel cause a wide spectrum of early-onset epileptic disorders. The long-term goal of this research program is to understand the mechanisms by which the wt IKM channel work, how epilepsy-causing mutations lead to dysfunction of IKM channels and to design drugs that correct IKM dysfunction. The objective of this application is to determine how epilepsy-inducing mutations in the IKM subunits KCNQ2 and KCNQ3 cause channel malfunction. Because polyunsaturated fatty acids (PUFAs) have been shown to alleviate the symptoms of intractable epileptic seizures, we will investigate the mechanisms by which these compounds reverse channel malfunction and therefore improve neuronal function. The overarching hypothesis is that that epilepsy-causing mutations in KCNQ channels affect voltage sensor movement and that PUFAs can restore normal voltage dependence of voltage sensor movement in mutated KCNQ channels. The rationale for the proposed research is that understanding the molecular basis by which different mutations in the IKM channel are linked to epilepsy will not only help explain epilepsy pathogenesis but also provide clues for intervention strategies. Guided by preliminary data, we will test our hypothesis by pursuing three specific aims: (1) determine the mechanisms by which epilepsy-causing mutations affect IKM channels function. (2) determine how PUFAs affect voltage sensor and gate movements of IKM channels bearing epilepsy-associated mutations and to identify which PUFA variants restores channel function, and (3) determine whether PUFAs reduce hyperexcitability on neurons bearing epilepsy-causing mutations in IKM channels. Under the first Aim, we will combine cysteine accessibility and VCF approaches to simultaneously measure voltage sensor movement and gate opening in the wt IKM and a set of epilepsy-associated mutants. This will allow us to determine how mutations affect the movement of the voltage sensor and the activation gate in KCNQ2 and KCNQ3 channels. We will also incorporate unnatural amino acids (UUAs) into mutated channels (UUAs mutagenesis) to further map the molecular determinants of channel dysfunction. Under Aim 2, we will test PUFA variants with different chain lengths, different acyl chains and different types of polar head groups to determine the molecular mechanism of PUFA’s effects on these mutations. Under the third Aim, we will test PUFA variants that can correct channel function and restore activity in iPSC-derived cortical neurons bearing epilepsy-associated mutations in KCNQ2 and/or KCNQ3. The proposed research is significant because the anticipated results will provide the mechanistic basis for how mutations cause IKM channel defects and will show proof-of-concept that PUFAs can act as antiepileptic drugs.

大脑中异常的神经元活动会导致癫痫发作，当反复发作或持续时间延长时，可能会导致癫痫发作 神经元损伤导致精神运动发育延迟和智力残疾。最具遗传性 与癫痫相关的变异存在于编码离子通道的基因中，包括钾通道 调节神经元兴奋性，如IKM通道。IKM渠道中的遗传突变导致广泛的 早发性癫痫障碍的谱系。这项研究计划的长期目标是了解 Wt IKM通道的工作机制，致痫突变如何导致IKM功能障碍 并设计纠正IKM功能障碍的药物。此应用程序的目标是确定如何 KCNQ2和KCNQ3亚基KCNQ2和KCNQ3的致痫突变可导致通道功能障碍。因为 多不饱和脂肪酸(PUFAs)已被证明可以缓解顽固性癫痫的症状 ，我们将研究这些化合物逆转通道故障和 从而改善神经功能。最重要的假设是，导致癫痫的突变在 KCNQ通道影响电压传感器的运动，PUFA可以恢复正常的电压依赖 电压传感器在突变的KCNQ通道中的运动。建议进行这项研究的理由是 了解IKM通道中不同突变与癫痫有关的分子基础 不仅有助于解释癫痫的发病机制，也为干预策略提供线索。指导原则 初步数据，我们将通过追求三个具体目标来验证我们的假设：(1)通过以下方式确定机制 哪些致痫突变会影响IKM通道的功能。(2)确定多不饱和脂肪酸对电压传感器的影响 携带癫痫相关突变的IKM通道的门和门运动以及识别哪种PUFA 变种恢复通道功能，以及(3)确定多不饱和脂肪酸是否降低神经元的过度兴奋性 在IKM通道中携带导致癫痫的突变。在第一个目标下，我们将结合半胱氨酸的可及性 和VCF方法，以同时测量wt IKM中的电压传感器移动和栅极开度 一组与癫痫相关的突变体。这将使我们能够确定突变是如何影响 KCNQ2和KCNQ3通道中的电压传感器和激活门。我们还将把非自然的 将氨基酸(UUA)导入突变通道(UUA突变)以进一步定位 渠道功能障碍。在目标2下，我们将测试具有不同链长、不同酰基链的多不饱和脂肪酸变体 以及不同类型的极性头部基团，以确定多不饱和脂肪酸对这些分子机制的影响 突变。在第三个目标下，我们将测试可以纠正通道功能和恢复活动的PUFA变体 在IPSC来源的皮质神经元中，携带癫痫相关的KCNQ2和/或KCNQ3突变。这个 拟议的研究具有重要意义，因为预期结果将为如何 突变会导致IKM通道缺陷，并将证明多不饱和脂肪酸可以作为抗癫痫药物。