Prolonged inhibition of CaMKII results in hyperexcitability by increasing NaV1.2 expression and its interaction with CaM

Dysfunction of calcium/calmodulin (CaM)-dependent kinase II (CaMKII) has been involved in hyperexcitability-related disorders including epilepsy. However, the exact mechanism by which CaMKII inhibits neuronal excitability remains to be elucidated. In the present study, we found that seizure-like events recorded by EEG in both wild-type and genetic epilepsy model rats were markedly augmented in response to the prolonged inhibition of CaMKII, indicating that the inactivation of CaMKII enhanced the neuronal excitability. Electrophysiological recording showed that CaMKII inhibition induced hyperexcitability of cultured hippocampal neurons and potentiation of neuronal activity in induced pluripotent stem cell (iPSC)-derived cortical neurons. We further revealed that CaMKII inhibition enhanced the persistent slow inactivating sodium current (I_NaP) of hippocampal neurons, which was attributed to increased neuronal expression of the voltage-gated sodium channel Na_V1.2. Our findings suggest that Na_V1.2 expression was transcriptionally upregulated by the negative regulator nuclear receptor subfamily 4 group A member 2 (<em>NR4A2</em>). Furthermore, CaMKII inhibition induced interaction between Na_V1.2 and CaM through immunoprecipitation assay. Intriguingly, a peptide that antagonized the binding of CaM to the IQ domain of Na_V1.2 prevented neuronal hyperexcitability induced by CaMKII inhibition <em>in vivo</em> and <em>in vitro</em>. Altogether, we unveil that prolonged CaMKII inhibition leads to hyperexcitability through increasing the expression of Na_V1.2 and its association with CaM. Thus, our study uncovers a novel signaling mechanism by which CaMKII maintains to appropriate neuronal excitability.钙/钙调蛋白（calcium/calmodulin, CaM）依赖性蛋白激酶II（CaMKII）功能异常，已被证实参与癫痫等神经元过度兴奋性相关疾病的发病过程。然而，CaMKII抑制神经元兴奋性的确切分子机制仍有待阐明。本研究发现，在野生型及遗传性癫痫模型大鼠的脑电图（electroencephalogram, EEG）记录中，痫样放电事件在延长CaMKII抑制后显著增强，这表明CaMKII失活会提升神经元兴奋性。电生理记录结果显示，CaMKII抑制可诱导培养的海马神经元出现兴奋性亢进，并增强诱导多能干细胞（induced pluripotent stem cell, iPSC）来源的皮层神经元的神经元活动。本研究进一步揭示，CaMKII抑制会增强海马神经元的持续性缓慢失活钠电流（persistent slow inactivating sodium current, I_NaP），该效应源于电压门控钠通道Na_V1.2的神经元表达水平升高。我们的研究结果表明，Na_V1.2的表达会被其负调控因子核受体亚家族4A组2成员（nuclear receptor subfamily 4 group A member 2, NR4A2）在转录水平上调。此外，通过免疫沉淀实验证实，CaMKII抑制会促进Na_V1.2与CaM的结合互作。值得注意的是，一种能够拮抗CaM与Na_V1.2的IQ结构域结合的多肽，可在体内（in vivo）和体外（in vitro）阻断CaMKII抑制诱导的神经元兴奋性亢进。综上，本研究阐明了延长CaMKII抑制会通过上调Na_V1.2的表达及其与CaM的结合，引发神经元兴奋性亢进。因此，本研究揭示了CaMKII维持神经元兴奋性处于适宜水平的全新信号通路机制。