Program in Medical Neuroscience, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Department of Biology, School of Science, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202.
J Biol Chem. 2020 May 1;295(18):6151-6164. doi: 10.1074/jbc.RA119.012423. Epub 2020 Mar 11.
-Palmitoylation is a reversible post-translational lipid modification that dynamically regulates protein functions. Voltage-gated sodium channels are subjected to -palmitoylation and exhibit altered functions in different -palmitoylation states. Our aim was to investigate whether and how -palmitoylation regulates Nav1.6 channel function and to identify -palmitoylation sites that can potentially be pharmacologically targeted. Acyl-biotin exchange assay showed that Nav1.6 is modified by -palmitoylation in the mouse brain and in a Nav1.6 stable HEK 293 cell line. Using whole-cell voltage clamp, we discovered that enhancing -palmitoylation with palmitic acid increases Nav1.6 current, whereas blocking -palmitoylation with 2-bromopalmitate reduces Nav1.6 current and shifts the steady-state inactivation in the hyperpolarizing direction. Three -palmitoylation sites (Cys, Cys, and Cys) were identified. These sites differentially modulate distinct Nav1.6 properties. Interestingly, Cys is exclusive to Nav1.6 among all Nav isoforms and is evolutionally conserved in Nav1.6 among most species. Cys-palmitoylation regulates current amplitude uniquely in Nav1.6. Furthermore, we showed that eliminating -palmitoylation at specific sites alters Nav1.6-mediated excitability in dorsal root ganglion neurons. Therefore, our study reveals -palmitoylation as a potential isoform-specific mechanism to modulate Nav activity and neuronal excitability in physiological and diseased conditions.
棕榈酰化是一种可动态调节蛋白质功能的可逆翻译后脂质修饰。电压门控钠离子通道受到棕榈酰化的影响，并在不同的棕榈酰化状态下表现出功能改变。我们的目的是研究棕榈酰化是否以及如何调节 Nav1.6 通道功能，并确定潜在的可药物靶向的棕榈酰化位点。酰基辅酶 A 交换测定显示，Nav1.6 在小鼠大脑和 Nav1.6 稳定的 HEK293 细胞系中被棕榈酰化修饰。通过全细胞膜片钳技术，我们发现用棕榈酸增强棕榈酰化会增加 Nav1.6 电流，而用 2-溴棕榈酸阻断棕榈酰化会减少 Nav1.6 电流并使稳态失活向超极化方向移动。鉴定出三个棕榈酰化位点（Cys、Cys 和 Cys）。这些位点差异调节不同的 Nav1.6 特性。有趣的是，Cys 在所有 Nav 亚型中是 Nav1.6 所特有的，并且在大多数物种的 Nav1.6 中是进化保守的。Cys 棕榈酰化在 Nav1.6 中独特地调节电流幅度。此外，我们还表明，在特定位点消除棕榈酰化会改变背根神经节神经元中 Nav1.6 介导的兴奋性。因此，我们的研究揭示了棕榈酰化作为一种潜在的亚型特异性机制，可调节生理和病理条件下 Nav 活性和神经元兴奋性。
