Tan Xiao-Feng, Fernández-Mariño Ana I, Li Yan, Chang Tsg-Hui, Swartz Kenton J
Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
Department of Physiology and Biophysics, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA.
Nature. 2025 Aug 6. doi: 10.1038/s41586-025-09339-7.
Action potentials are generated by opening of voltage-activated sodium (Na) and potassium (K) channels, which can rapidly inactivate to shape the nerve impulse and contribute to synaptic facilitation and short-term memory. The mechanism of fast inactivation was proposed to involve an intracellular domain that blocks the internal pore in both Na and K channels; however, recent studies in Na and K channels support a mechanism in which the internal pore closes during inactivation. Here we investigate the mechanism of fast inactivation in the Shaker K channel using cryo-electron microscopy, mass spectrometry and electrophysiology. We resolved structures of a fully inactivated state in which the non-polar end of the N terminus plugs the internal pore in an extended conformation. The N-terminal methionine is deleted, leaving an alanine that is acetylated and interacts with a pore-lining isoleucine residue where RNA editing regulates fast inactivation. Opening of the internal activation gate is required for fast inactivation because it enables the plug domain to block the pore and repositions gate residues to interact with and stabilize that domain. We also show that external K destabilizes the inactivated state by altering the conformation of the ion selectivity filter rather than by electrostatic repulsion. These findings establish the mechanism of fast inactivation in K channels, revealing how it is regulated by RNA editing and N-terminal acetylation, and providing a framework for understanding related mechanisms in other voltage-activated channels.
动作电位由电压激活的钠（Na）通道和钾（K）通道的开放产生，这些通道可迅速失活以塑造神经冲动，并有助于突触易化和短期记忆。快速失活的机制被认为涉及一个细胞内结构域，该结构域可阻断Na通道和K通道的内部孔道；然而，最近对Na通道和K通道的研究支持一种机制，即在失活过程中内部孔道会关闭。在这里，我们使用冷冻电子显微镜、质谱和电生理学研究了Shaker K通道中快速失活的机制。我们解析了完全失活状态的结构，其中N端的非极性末端以伸展构象堵塞内部孔道。N端的甲硫氨酸被删除，留下一个被乙酰化的丙氨酸，它与一个孔道内衬异亮氨酸残基相互作用，RNA编辑在此调节快速失活。快速失活需要内部激活门的开放，因为它使堵塞结构域能够阻断孔道，并重新定位门控残基以与该结构域相互作用并使其稳定。我们还表明，外部K通过改变离子选择性过滤器的构象而不是通过静电排斥来使失活状态不稳定。这些发现确立了K通道中快速失活的机制，揭示了它是如何由RNA编辑和N端乙酰化调节的，并为理解其他电压激活通道中的相关机制提供了一个框架。
