Baumgartner Timothy John, Dvorak Nolan Michael, Goode Nana Aboadwe, Haghighijoo Zahra, Marosi Mate, Singh Jully, Singh Aditya Kumar, Laezza Fernanda
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, USA.
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, USA.
J Biol Chem. 2025 Feb;301(2):108162. doi: 10.1016/j.jbc.2025.108162. Epub 2025 Jan 8.
Voltage-gated Na+ (Na) channels are the primary determinants of the action potential in excitable cells. Na channels rely on a wide and diverse array of intracellular protein-protein interactions (PPIs) to achieve their full function. Glycogen synthase kinase 3β (GSK3β) has been previously identified as a modulator of Na1.6-encoded currents and neuronal excitability through PPI formation with Na1.6 and phosphorylation of its C-terminal domain (CTD). Here, we hypothesized that GSK3β functions as a scaffold in a regulatory PPI complex with the Na1.6 CTD. Mutagenesis screening using the split-luciferase complementation assay indicated that the axin-binding domain (ABD) of GSK3β (262-299) is necessary for complex formation between the Na1.6 CTD and GSK3β, and that residues within this domain are drivers of GSK3β-mediated regulation of the channel. Overexpression of an ABD-GFP fusion construct in human embryonic kidney 293 cells stably expressing Na1.6 significantly reduced Na1.6 nanocluster density compared with GFP alone. In addition, overexpression of the ABD-GFP fusion construct ablates GSK3β-mediated potentiation of Na1.6-encoded currents and alters channel kinetics. Finally, in vivo AAV-mediated overexpression of the ABD-GFP construct in the CA1 hippocampal region induced a reduction in maximal action potential firing and an increase in action potential current threshold in a manner resembling previously reported effects of GSK3β silencing in neurons. Taken together, these results not only suggest that GSK3β-mediated regulation of Na1.6 extends beyond transient phosphorylation but also implicates the ABD as a critical regulatory domain that facilitates GSK3β's functional effects on Na1.6 and neuronal excitability.
电压门控钠（Na）通道是可兴奋细胞动作电位的主要决定因素。钠通道依靠广泛多样的细胞内蛋白质-蛋白质相互作用（PPI）来实现其完整功能。糖原合酶激酶3β（GSK3β）先前已被确定为通过与Na1.6形成PPI并使其C末端结构域（CTD）磷酸化来调节Na1.6编码电流和神经元兴奋性的调节剂。在此，我们假设GSK3β在与Na1.6 CTD的调节性PPI复合物中起支架作用。使用分裂荧光素酶互补测定法进行的诱变筛选表明，GSK3β（262 - 299）的轴蛋白结合结构域（ABD）是Na1.6 CTD与GSK3β之间形成复合物所必需的，并且该结构域内的残基是GSK3β介导的通道调节的驱动因素。与单独的GFP相比，在稳定表达Na1.6的人胚肾293细胞中过表达ABD - GFP融合构建体可显著降低Na1.6纳米簇密度。此外，ABD - GFP融合构建体的过表达消除了GSK3β介导的Na1.6编码电流的增强作用，并改变了通道动力学。最后，在体内通过AAV介导在CA1海马区过表达ABD - GFP构建体，以类似于先前报道的神经元中GSK3β沉默效应的方式，诱导最大动作电位发放减少和动作电位电流阈值增加。综上所述，这些结果不仅表明GSK3β介导的对Na1.6的调节不仅限于瞬时磷酸化，还暗示ABD是促进GSK3β对Na1.6和神经元兴奋性产生功能影响的关键调节结构域。
