Program in Medical Neuroscience, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
Cells. 2022 Jul 4;11(13):2108. doi: 10.3390/cells11132108.
Aberrant Nav1.6 activity can induce hyperexcitability associated with epilepsy. Gain-of-function mutations in the gene encoding Nav1.6 are linked to epilepsy development; however, the molecular mechanisms mediating these changes are remarkably heterogeneous and may involve post-translational regulation of Nav1.6. Because calcium/calmodulin-dependent protein kinase II (CaMKII) is a powerful modulator of Nav1.6 channels, we investigated whether CaMKII modulates disease-linked Nav1.6 mutants. Whole-cell voltage clamp recordings in ND7/23 cells show that CaMKII inhibition of the epilepsy-related mutation R850Q largely recapitulates the effects previously observed for WT Nav1.6. We also characterized a rare missense variant, R639C, located within a regulatory hotspot for CaMKII modulation of Nav1.6. Prediction software algorithms and electrophysiological recordings revealed gain-of-function effects for R639C mutant channel activity, including increased sodium currents and hyperpolarized activation compared to WT Nav1.6. Importantly, the R639C mutation ablates CaMKII phosphorylation at a key regulatory site, T642, and, in contrast to WT and R850Q channels, displays a distinct response to CaMKII inhibition. Computational simulations demonstrate that modeled neurons harboring the R639C or R850Q mutations are hyperexcitable, and simulating the effects of CaMKII inhibition on Nav1.6 activity in modeled neurons differentially reduced hyperexcitability. Acute CaMKII inhibition may represent a promising mechanism to attenuate gain-of-function effects produced by Nav1.6 mutations.
Nav1.6 活性异常可引起与癫痫相关的过度兴奋。编码 Nav1.6 的基因中的功能获得性突变与癫痫的发展有关；然而，介导这些变化的分子机制非常复杂，可能涉及 Nav1.6 的翻译后调节。由于钙/钙调蛋白依赖性蛋白激酶 II（CaMKII）是 Nav1.6 通道的强大调节剂，我们研究了 CaMKII 是否调节与疾病相关的 Nav1.6 突变体。ND7/23 细胞中的全细胞膜片钳记录显示，CaMKII 对癫痫相关突变 R850Q 的抑制作用在很大程度上再现了先前观察到的 WT Nav1.6 的作用。我们还对位于 CaMKII 调节 Nav1.6 的调节热点内的罕见错义变体 R639C 进行了表征。预测软件算法和电生理记录显示，R639C 突变通道的功能具有功能获得效应，包括与 WT Nav1.6 相比，钠电流增加和超极化激活。重要的是，R639C 突变使 Nav1.6 中 CaMKII 磷酸化的关键调节位点 T642 失活，与 WT 和 R850Q 通道不同，它对 CaMKII 抑制的反应也不同。计算模拟表明，携带 R639C 或 R850Q 突变的模拟神经元过度兴奋，模拟 CaMKII 抑制对 Nav1.6 活性的影响可降低神经元的过度兴奋。急性 CaMKII 抑制可能是减轻 Nav1.6 突变引起的功能获得效应的有前途的机制。
