James Thomas F, Nenov Miroslav N, Wildburger Norelle C, Lichti Cheryl F, Luisi Jonathan, Vergara Fernanda, Panova-Electronova Neli I, Nilsson Carol L, Rudra Jai S, Green Thomas A, Labate Demetrio, Laezza Fernanda
Department of Pharmacology & Toxicology, USA; Neuroscience Graduate Program, USA.
Department of Pharmacology & Toxicology, USA.
Biochim Biophys Acta. 2015 Apr;1850(4):832-44. doi: 10.1016/j.bbagen.2015.01.011. Epub 2015 Jan 20.
Phosphorylation plays an essential role in regulating voltage-gated sodium (Na(v)) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Na(v) channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Na(v) channels.
We used patch-clamp electrophysiology to record sodium currents from Na(v)1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues.
We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Na(v)1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Na(v)1.2-encoded currents. Neither mRNA nor total protein expression was changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Na(v)1.2 channel indicates that cell surface expression of CD4-Na(v)1.2 C-tail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3β phosphorylates T(1966) at the C-terminal tail of Na(v)1.2.
These findings provide evidence for a new mechanism by which GSK3 modulates Na(v) channel function via its C-terminal tail.
These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.
磷酸化在调节电压门控钠（Na(v)）通道和兴奋性方面起着至关重要的作用。然而，令人惊讶的是，已被鉴定为Na(v)通道调节剂的激酶数量有限。我们推测，糖原合酶激酶3（GSK3）作为一种与多种脑部疾病相关的关键激酶，可能直接调节神经元Na(v)通道。
我们采用膜片钳电生理学技术记录稳定表达于HEK-293细胞中的Na(v)1.2通道的钠电流。通过RT-PCR、蛋白质印迹法或共聚焦显微镜对mRNA和蛋白质水平进行定量分析，并利用体外磷酸化和质谱鉴定磷酸化残基。
我们发现，将细胞暴露于GSK3抑制剂XIII可显著增强Na(v)1.2的峰值电流密度，这一表型可通过用小干扰RNA（siRNA）沉默GSK3来重现。相反，GSK3β的过表达抑制了Na(v)1.2编码的电流。GSK3抑制后，mRNA和总蛋白表达均未改变。对表达Na(v)1.2通道胞内结构域的CD4嵌合构建体进行细胞表面标记表明，在药理学抑制GSK3后，CD4-Na(v)1.2 C末端的细胞表面表达上调，导致质膜上表面斑点增加。最后，结合体外磷酸化和高分辨率质谱，我们进一步证明GSK3β使Na(v)1.2 C末端尾部的T(1966)磷酸化。
这些发现为GSK3通过其C末端尾部调节Na(v)通道功能的新机制提供了证据。
这些发现为理解几种神经精神疾病中常见的信号功能障碍提供了基础知识。
