Francis Kevin R, Wei Ling, Yu Shan Ping
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, 29425, USA.
Neurochem Res. 2015 Apr;40(4):674-87. doi: 10.1007/s11064-015-1514-4. Epub 2015 Jan 11.
Voltage-gated Na(+) channel activity is vital for the proper function of excitable cells and has been indicated in nervous system development. Meanwhile, the Src family of non-receptor tyrosine kinases (SFKs) has been implicated in the regulation of Na(+) channel activity. The present investigation tests the hypothesis that Src family kinases influence neuronal differentiation via a chronic regulation of Na(+) channel functionality. In cultured mouse embryonic stem (ES) cells undergoing neural induction and terminal neuronal differentiation, SFKs showed distinct stage-specific expression patterns during the differentiation process. ES cell-derived neuronal cells expressed multiple voltage-gated Na(+) channel proteins (Nav) and underwent a gradual increase in Na(+) channel activity. While acute inhibition of SFKs using the Src family inhibitor PP2 suppressed the Na(+) current, chronic inhibition of SFKs during early neuronal differentiation of ES cells did not change Nav expression. However, a long-lasting block of SFK significantly altered electrophysiological properties of the Na(+) channels, shown as a right shift of the current-voltage relationship of the Na(+) channels, and reduced the amplitude of Na(+) currents recorded in drug-free solutions. Immunocytochemical staining of differentiated cells subjected to the chronic exposure of a SFK inhibitor, or the Na(+) channel blocker tetrodotoxin, showed no changes in the number of NeuN-positive cells; however, both treatments significantly hindered neurite outgrowth. These findings suggest that SFKs not only modulate the Na(+) channel activation acutely, but the tonic activity of SFKs is also critical for normal development of functional Na(+) channels and neuronal differentiation or maturation of ES cells.
电压门控钠通道活性对于可兴奋细胞的正常功能至关重要，并且在神经系统发育中也有体现。同时，非受体酪氨酸激酶Src家族（SFKs）与钠通道活性的调节有关。本研究检验了Src家族激酶通过对钠通道功能的长期调节来影响神经元分化的假说。在经历神经诱导和终末神经元分化的培养小鼠胚胎干细胞（ES细胞）中，SFKs在分化过程中呈现出明显的阶段特异性表达模式。ES细胞来源的神经元细胞表达多种电压门控钠通道蛋白（Nav），并且钠通道活性逐渐增加。虽然使用Src家族抑制剂PP2急性抑制SFKs可抑制钠电流，但在ES细胞早期神经元分化过程中慢性抑制SFKs并不会改变Nav的表达。然而，长期阻断SFKs会显著改变钠通道的电生理特性，表现为钠通道电流-电压关系的右移，并降低在无药物溶液中记录的钠电流幅度。对慢性暴露于SFK抑制剂或钠通道阻滞剂河豚毒素的分化细胞进行免疫细胞化学染色，结果显示NeuN阳性细胞数量没有变化；然而，这两种处理均显著阻碍了神经突的生长。这些发现表明，SFKs不仅能急性调节钠通道的激活，而且SFKs的持续性活性对于功能性钠通道的正常发育以及ES细胞的神经元分化或成熟也至关重要。
