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: 10543145
• 负责人: Rene Barro-Soria
• 金额: $ 31.71万
• 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/10543145
• 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; 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; 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; structural determinants; 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，当反复或延长时， 神经元损伤导致延迟的精神发育和智力残疾。大多数遗传 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通道中的遗传突变导致广泛的 早发性癫痫疾病的范围。这项研究计划的长期目标是了解 野生型IKM通道的工作机制，癫痫突变如何导致IKM功能障碍 并设计纠正IKM功能障碍的药物。本申请的目的是确定如何 IKM亚基KCNQ 2和KCNQ 3中的癫痫诱导突变导致通道功能障碍。因为 多不饱和脂肪酸（PUFA）已被证明可以缓解顽固性癫痫的症状， 癫痫发作，我们将研究这些化合物逆转通道功能障碍的机制， 从而改善神经元功能。最重要的假设是， KCNQ通道影响电压传感器运动，PUFA可以恢复正常的电压依赖性， 突变的KCNQ通道中的电压传感器移动。拟议研究的理由是， 了解IKM通道中不同突变与癫痫相关的分子基础， 不仅有助于解释癫痫发病机制，而且为干预策略提供线索。指导 初步的数据，我们将测试我们的假设，追求三个具体目标：（1）确定机制， 哪些致痫突变影响IKM通道功能。(2)确定PUFA如何影响电压传感器 以及携带癫痫相关突变的IKM通道的门控运动，并确定哪些PUFA 变体恢复通道功能，以及（3）确定PUFA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，我们将结合联合收割机半胱氨酸可及性 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， 一组癫痫相关的突变体这将使我们能够确定突变如何影响细胞的运动。 电压传感器和KCNQ 2和KCNQ 3通道中的激活门。我们还将结合非自然的 氨基酸（UUAs）突变的通道（UUAs诱变），以进一步映射的分子决定簇 通道功能障碍在目标2下，我们将测试具有不同链长、不同酰基链的PUFA变体， 和不同类型的极性头部基团，以确定PUFA对这些影响的分子机制， 突变。在第三个目标下，我们将测试可以纠正通道功能并恢复活性的PUFA变体 在iPSC衍生的皮质神经元中，KCNQ 2和/或KCNQ 3中携带癫痫相关突变。的 拟议的研究是重要的，因为预期的结果将提供机制的基础， 突变导致IKM通道缺陷，并将证明PUFA可以作为抗癫痫药物的概念。