小胶质细胞BK通道调控颞叶癫痫发生的分子机制及其靶标评估

The activation of microglia has been proved to be one of the most important pathological changes in epileptogenesis. Our previous study found that the expression of microglia BK channels (large-conductance calcium-activated potassium channels) were significantly increased in the lesion of temporal lobe epilepsy and the hippocampus of kainic acid model. However, the function of microgilal BK channels was still unclear. Our preliminary data showed that microgilal BK channels could not only positively cooperate with Orai1/CRACs, one of the key components for microglia activation, but also up-regulate the expression of IL-1β and TNF-1α. Based on the results above, it is speculated that the abnormal activation of microglial BK channels is closely involved in the pathogenesis of epilepsy. The project aims to investigate the following subjects by using BK knockout mice, electroencephalogram (EEG) recording, brain slice patch clamping, living cell imaging and optogenetics: 1. the changes in animal behavior, cell morphology and EEG induced by the abnormal expression of microglial BK channels in temporal lobe epilepsy model; 2. the molecular mechanism of neuronal excitability facilitated by BK channels activation; 3. the pharmacological effects of specific BK channel blocker martentoxin on temporal lobe epilepsy as well as the structure information of the interaction between microglial BK channel and martentoxin. The project will not only enrich the molecular etiology of epileptogenesis mediated by BK channels, but also provide a potential molecular target for treating temporal lobe epilepsy.

小胶质细胞的活化被证明是癫痫发生的重要病理改变之一。本申项前期研究发现小胶质细胞BK通道（大电导钙激活钾通道）在颞叶癫痫灶和海人酸模型的海马区表达增多，其具体功能仍不明确。预实验结果表明BK通道可正相调控小胶质细胞活化的关键分子Orai1/CRAC，上调炎症因子IL-1β和TNF-1α的表达。由此提示，小胶质细胞BK通道的异常激活紧密参与了癫痫发生的病理过程。本申项拟利用BK通道基因敲除小鼠结合在体脑电、脑片膜片钳、活细胞成像、光遗传学及单细胞测序等手段，探讨：1、小胶质细胞BK通道异常表达对颞叶癫痫的在体行为、细胞形态与脑电的改变；2、小胶质细胞BK通道的激活易化神经元兴奋性的分子机制；3、特异性BK通道阻断剂martentoxin对颞叶癫痫的整体调制效应及其与小胶质细胞BK通道动态互作的分子结构信息。丰富由BK通道介导癫痫发生的分子病因，为临床应对颞叶癫痫的药物干预提供可能的分子靶标。

小胶质细胞的活化被证明是癫痫发生的重要病理改变之一。本申项前期研究发现小胶质细胞BK通道（大电导钙激活钾通道）在颞叶癫痫灶和海人酸模型的海马区表达增多，其具体功能仍不明确。本项目综合运用了细胞分子生物学、电生理学、药理学等多学科交叉方法，探讨了小胶质细胞BK通道异常表达对颞叶癫痫的在体行为、其调节神经元兴奋性的分子机制、特异性BK通道阻断剂martentoxin（MarTX）对颞叶癫痫的整体调制效应。.本项目初步研究成果揭示了小胶质细胞BK通道在颞叶癫痫灶和海人酸模型的海马区表达增多；我们发现了一个全新的KCNMA1基因（编码BK通道α亚基）突变位点，并鉴定了3个BK通道的突变体，结果显示E155Q、R458T、E884K突变体电流密度减小，I-V曲线朝着去极化方向偏移，更慢激活，β4亚基使得E155Q的激活时间常数（τ）变大，激活速率减小；BK基因敲除小鼠存在自发性癫痫、运动和认知障碍表型，并表现出焦虑样行为；BK通道除了影响神经元动作电位的复极化，可正相调控小胶质细胞活化的关键分子Orai1/CRAC，调节小胶质细胞溶酶体TRPML1的钙释放功能，抑制细胞自噬，介导炎症与癫痫的发生；MarTX对颞叶癫痫的调制效应优于MarTX突变体，并能有效保护神经元和调节神经元的兴奋性。.本项目结果丰富由BK通道介导癫痫发生的分子病因，为临床应对颞叶癫痫的药物干预提供可能的分子靶标。