CaM和CaMKII共同调控电压门控性钠通道在癫痫发病机制中的作用及相关多肽的治疗策略

Voltage-gated sodium channel (VGSC) is one of the fundamental research standpoints in pathogenesis of epilepsy and antiepileptic drug therapy, and to explore the modulation of VGSC expression and function in epilepsy is of great scientific value and significance. We previously found that calmodulin (CaM) increased channel activity of VGSC in epilepsy, and calcium/calmodulin-dependent protein kinase II (CaMKII) was abnormally expressed in spontaneously epileptic rats (SER)and low magnesium model of epilepsy. So far, the modulation of CaM, CaMKII and the interaction between CaM and CaMKII on VGSC in epilepsy is still unclear, which is the key to reveal neuronal membrane excitability mechanisms in epilepsy. This study proposes a hypothesis that CaM and CaMKII can co-modulate VGSC in epilepsy by interaction and promotion. We would like to apply patch-clamp technique and molecular biology to clarify the modulation of lobe-specific function of CaM by N-lobe and C-lobe, CaMKII in the mediation of phosphorylation effects, and the interaction between CaM and CaMKII on VGSC channel expression, localization, binding and electrophysiological features in epilepsy.In addition, the antiepileptic effect of CaMKII polypeptides is also to be evaluated. The study is to further reveal the pathogenesis of epilepsy, and to provide a more valuable theoretical and practical basis for screening VGSC subtypes, CaM and CaMKII drug targets.

电压门控性钠通道（VGSC）是研究癫痫发病机制与抗痫药物治疗的根本立足点，探讨癫痫中VGSC表达与功能的调控具有重大的科学价值与意义。我们前期应用自发性癫痫大鼠（SER）和无镁癫痫细胞模型发现钙调蛋白（CaM）可增加癫痫VGSC活性；钙调蛋白依赖性蛋白激酶II（CaMKII）在模型中表达异常。目前癫痫中CaM 和CaMKII调节VGSC的作用关系仍不清楚，而这正是揭示癫痫神经元兴奋性产生的关键所在。本课题提出CaM与CaMKII互相影响与促进，共同调控癫痫钠通道的假说，拟采用膜片钳和分子生物学等技术阐明CaM N环和C环介导的结构特异性、CaMKII介导的磷酸化作用以及CaM与CaMKII的相互作用对癫痫钠通道的表达、定位、结合和电生理功能调控作用与机制，评估相关多肽的抗痫策略，旨在揭示癫痫发病机制，为筛选钠通道特异性亚型、CaM和CaMKII相关药物靶点提供更有价值的理论与实践依据。

钙调蛋白（CaM）与钙调蛋白依赖性蛋白激酶II（CaMKII）对癫痫电压门控性钠通道（VGSC）表达与功能的调控作用仍不清楚，而这正是揭示癫痫神经细胞膜兴奋性产生的关键机制。本项目我们应用膜片钳和分子生物学技术率先证实CaM与CaMKII共同调节癫痫VGSC活性，主要结论如下：⑴抑制CaMKII活性可诱导海马神经元中增加的持续型慢失活钠电流（INaP）和过度兴奋。此外，在CaMKII抑制后，应用EEG 发现Wistar和震颤大鼠出现癫痫样发作次数增加，提示CaMKII抑制导致超兴奋与INaP增加相关，表明CaMKII可能是癫痫的潜在治疗靶点。⑵应用膜片钳技术发现VGSC通道活性在低镁状态下对Ca2+/CaM的调节更加敏感。当使用CaM12和CaM34（在N或C-环中具有失活的钙离子结合位点的CaM突变体）时，低镁诱导神经元中VGSCs敏感性增加的特性仍部分存在，但使用CaM1234（四个钙离子结合位点均失活）时，低镁诱导神经元中VGSCs敏感度增加的特性消失，提示CaM N/C-环上需要有功能的钙离子结合位点是CaM调节VGSC的关键环节。⑶应用pull-down 技术检测CaM及其钙结合位点突变体（CaM12，CaM34和CaM1234）和截短蛋白（N-环和C-环）与NaV1.1 IQ的结合，发现Ca2+/CaM与NaV1.1 IQ具有浓度依赖性结合。ApoCaM（无Ca2+形式钙调蛋白）与NaV1.1 IQ的结合优先于Ca2+/CaM。另外，CaM的C-域是参与apoCaM与NaV1.1 IQ结合的主要结构域，而CaM的N-环是Ca2+/CaM与NaV1.1 IQ结合的主要结构域。而且，由CaMKII介导的磷酸化作用使Ca2+/CaM与NaV1.1 IQ的结合增加。（4）神经肽Y的mRNA水平在震颤大鼠海马和颞叶皮质中表达上调，且通过其受体激活CaM/CaMKII信号通路发挥抗痫作用。本研究提出Ca2+/CaM/CaMKII通路调节VGSC活性新机制，证明CaMKII通过磷酸化钠通道进而促进CaM结合VGSC，进一步引起通道持续钠电流和通道活性的增加，最终导致癫痫的产生和发展。本研究进一步揭示癫痫发病机制，为筛选VGSC、CaM/ CaMKII相关多肽药物靶点提供很有价值的实验依据。