Src Tyrosine Kinases Regulate Neuronal Differentiation of Mouse Embryonic Stem Cells Via Modulation of Voltage-Gated Sodium Channel Activity

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 (Na_v) 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 Na_v 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.