Nav1.6 is the primary voltage-gated sodium channel isoform expressed in mature axon initial segments and nodes, making it critical for initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may have profound effects on input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism regulating ion channel function. Because Nav1.6 and the multifunctional Ca2+/CaM-dependent protein kinase II (CaMKII) are independently linked to excitability disorders, we sought to investigate modulation of Nav1.6 function by CaMKII signaling. We show that inhibition of CaMKII, a Ser/Thr protein kinase associated with excitability, synaptic plasticity, and excitability disorders, with the CaMKII-specific peptide inhibitor CN21 reduces transient and persistent currents in Nav1.6-expressing Purkinje neurons by 87%. Using whole-cell voltage clamp of Nav1.6, we show that CaMKII inhibition in ND7/23 and HEK293 cells significantly reduces transient and persistent currents by 72% and produces a 5.8-mV depolarizing shift in the voltage dependence of activation. Immobilized peptide arrays and nanoflow LC-electrospray ionization/MS of Nav1.6 reveal potential sites of CaMKII phosphorylation, specifically Ser-561 and Ser-641/Thr-642 within the first intracellular loop of the channel. Using site-directed mutagenesis to test multiple potential sites of phosphorylation, we show that Ala substitutions of Ser-561 and Ser-641/Thr-642 recapitulate the depolarizing shift in activation and reduction in current density. Computational simulations to model effects of CaMKII inhibition on Nav1.6 function demonstrate dramatic reductions in spontaneous and evoked action potentials in a Purkinje cell model, suggesting that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate neuronal excitability.

中文翻译：

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CaMKII 增强电压门控钠通道 Nav1.6 活性和神经元兴奋性。

Nav1.6 是在成熟轴突初始段和节点中表达的主要电压门控钠通道同种型，使其对神经元冲动的启动和传播至关重要。因此，Nav1.6 调制和功能障碍可能对正常和病理条件下神经元的输入输出特性产生深远影响。磷酸化是一种强大且可逆的调节离子通道功能的机制。由于 Nav1.6 和多功能 Ca2+/CaM 依赖性蛋白激酶 II (CaMKII) 与兴奋性障碍独立相关，我们试图通过 CaMKII 信号传导研究 Nav1.6 功能的调节。我们表明抑制 CaMKII，一种与兴奋性、突触可塑性和兴奋性障碍相关的 Ser/Thr 蛋白激酶，使用 CaMKII 特异性肽抑制剂 CN21 将表达 Nav1.6 的浦肯野神经元中的瞬时和持续电流减少 87%。使用 Nav1.6 的全细胞电压钳，我们表明 ND7/23 和 HEK293 细胞中的 CaMKII 抑制显着减少了 72% 的瞬态和持续电流，并在激活的电压依赖性中产生 5.8-mV 的去极化偏移。Nav1.6 的固定肽阵列和纳流 LC-电喷雾电离/MS 揭示了 CaMKII 磷酸化的潜在位点，特别是通道的第一个细胞内环内的 Ser-561 和 Ser-641/Thr-642。使用定点诱变来测试磷酸化的多个潜在位点，我们表明 Ser-561 和 Ser-641/Thr-642 的 Ala 替换概括了激活和电流密度降低的去极化转变。