Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Hayashi T, Huganir RL (2004) Tyrosine phosphorylation and regulation of the AMPA receptor by SRC family tyrosine kinases. J Neurosci 24:6152–6160

    Article  CAS  PubMed  Google Scholar 

  2. Hilborn MD, Vaillancourt RR, Rane SG (1998) Growth factor receptor tyrosine kinases acutely regulate neuronal sodium channels through the src signaling pathway. J Neurosci 18:590–600

    CAS  PubMed  Google Scholar 

  3. Salter MW (1998) Src, N-methyl-d-aspartate (NMDA) receptors, and synaptic plasticity. Biochem Pharmacol 56:789–798

    Article  CAS  PubMed  Google Scholar 

  4. Knoll B, Drescher U (2004) Src family kinases are involved in EphA receptor-mediated retinal axon guidance. J Neurosci 24:6248–6257

    Article  PubMed  Google Scholar 

  5. Toutant M, Studler JM, Burgaya F, Costa A, Ezan P, Gelman M, Girault JA (2000) Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms. Biochem J 348(Pt 1):119–128

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Osterhout DJ, Wolven A, Wolf RM, Resh MD, Chao MV (1999) Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase. J Cell Biol 145:1209–1218

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Theus MH, Wei L, Francis K, Yu SP (2006) Critical roles of Src family tyrosine kinases in excitatory neuronal differentiation of cultured embryonic stem cells. Exp Cell Res 312:3096–3107

    Article  CAS  PubMed  Google Scholar 

  8. Meyn MA 3rd, Schreiner SJ, Dumitrescu TP, Nau GJ, Smithgall TE (2005) SRC family kinase activity is required for murine embryonic stem cell growth and differentiation. Mol Pharmacol 68:1320–1330

    Article  CAS  PubMed  Google Scholar 

  9. Hille B (1992) Ionic channels of excitable membranes. Sinauer Associates Inc, Sunderland

    Google Scholar 

  10. Westenbroek RE, Merrick DK, Catterall WA (1989) Differential subcellular localization of the RI and RII Na+ channel subtypes in central neurons. Neuron 3:695–704

    Article  CAS  PubMed  Google Scholar 

  11. Westenbroek RE, Noebels JL, Catterall WA (1992) Elevated expression of type II Na+ channels in hypomyelinated axons of shiverer mouse brain. J Neurosci 12:2259–2267

    CAS  PubMed  Google Scholar 

  12. Goldin AL, Barchi RL, Caldwell JH, Hofmann F, Howe JR, Hunter JC, Kallen RG, Mandel G, Meisler MH, Netter YB, Noda M, Tamkun MM, Waxman SG, Wood JN, Catterall WA (2000) Nomenclature of voltage-gated sodium channels. Neuron 28:365–368

    Article  CAS  PubMed  Google Scholar 

  13. Burgess DL, Kohrman DC, Galt J, Plummer NW, Jones JM, Spear B, Meisler MH (1995) Mutation of a new sodium channel gene, Scn8a, in the mouse mutant ‘motor endplate disease’. Nat Genet 10:461–465

    Article  CAS  PubMed  Google Scholar 

  14. Felts PA, Yokoyama S, Dib-Hajj S, Black JA, Waxman SG (1997) Sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1): different expression patterns in developing rat nervous system. Brain Res Mol Brain Res 45:71–82

    Article  CAS  PubMed  Google Scholar 

  15. Wiesel TN (1999) Early explorations of the development and plasticity of the visual cortex: a personal view. J Neurobiol 41:7–9

    Article  CAS  PubMed  Google Scholar 

  16. Xu X, Shrager P (2005) Dependence of axon initial segment formation on Na+ channel expression. J Neurosci Res 79:428–441

    Article  CAS  PubMed  Google Scholar 

  17. Zhang LI, Poo MM (2001) Electrical activity and development of neural circuits. Nat Neurosci 4(Suppl):1207–1214

    Article  CAS  PubMed  Google Scholar 

  18. Pineda RH, Svoboda KR, Wright MA, Taylor AD, Novak AE, Gamse JT, Eisen JS, Ribera AB (2006) Knockdown of Nav1.6a Na+ channels affects zebrafish motoneuron development. Development 133:3827–3836

    Article  CAS  PubMed  Google Scholar 

  19. Bergey GK, Fitzgerald SC, Schrier BK, Nelson PG (1981) Neuronal maturation in mammalian cell culture is dependent on spontaneous electrical activity. Brain Res 207:49–58

    Article  CAS  PubMed  Google Scholar 

  20. Ahern CA, Zhang JF, Wookalis MJ, Horn R (2005) Modulation of the cardiac sodium channel NaV1.5 by Fyn, a Src family tyrosine kinase. Circ Res 96:991–998

    Article  CAS  PubMed  Google Scholar 

  21. Ahn M, Beacham D, Westenbroek RE, Scheuer T, Catterall WA (2007) Regulation of Na(v)1.2 channels by brain-derived neurotrophic factor, TrkB, and associated Fyn kinase. J Neurosci 27:11533–11542

    Article  CAS  PubMed  Google Scholar 

  22. Beacham D, Ahn M, Catterall WA, Scheuer T (2007) Sites and molecular mechanisms of modulation of Na(v)1.2 channels by Fyn tyrosine kinase. J Neurosci 27:11543–11551

    Article  CAS  PubMed  Google Scholar 

  23. Lu WY, Xiong ZG, Lei S, Orser BA, Dudek E, Browning MD, MacDonald JF (1999) G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors. Nat Neurosci 2:331–338

    Article  CAS  PubMed  Google Scholar 

  24. Holmes TC, Fadool DA, Ren R, Levitan IB (1996) Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain. Science 274:2089–2091

    Article  CAS  PubMed  Google Scholar 

  25. MacFarlane SN, Sontheimer H (2000) Modulation of Kv1.5 currents by Src tyrosine phosphorylation: potential role in the differentiation of astrocytes. J Neurosci 20:5245–5253

    CAS  PubMed  Google Scholar 

  26. Wischmeyer E, Doring F, Karschin A (1998) Acute suppression of inwardly rectifying Kir2.1 channels by direct tyrosine kinase phosphorylation. J Biol Chem 273:34063–34068

    Article  CAS  PubMed  Google Scholar 

  27. Bain G, Gottlieb DI (1998) Neural cells derived by in vitro differentiation of P19 and embryonic stem cells. Perspect Dev Neurobiol 5:175–178

    CAS  PubMed  Google Scholar 

  28. Bain G, Mansergh FC, Wride MA, Hance JE, Isogawa A, Rancourt SL, Ray WJ, Yoshimura Y, Tsuzuki T, Gottlieb DI, Rancourt DE (2000) ES cell neural differentiation reveals a substantial number of novel ESTs. Funct Integr Genomics 1:127–139

    Article  CAS  PubMed  Google Scholar 

  29. Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI (1995) Embryonic stem cells express neuronal properties in vitro. Dev Biol 168:342–357

    Article  CAS  PubMed  Google Scholar 

  30. Blake RA, Broome MA, Liu X, Wu J, Gishizky M, Sun L, Courtneidge SA (2000) SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling. Mol Cell Biol 20:9018–9027

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Tatton L, Morley GM, Chopra R, Khwaja A (2003) The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases. J Biol Chem 278:4847–4853

    Article  CAS  PubMed  Google Scholar 

  32. Cui L, Jiang J, Wei L, Zhou X, Fraser JL, Snider BJ, Yu SP (2008) Transplantation of embryonic stem cells improves nerve repair and functional recovery after severe sciatic nerve axotomy in rats. Stem Cells 26:1356–1365

    Article  CAS  PubMed  Google Scholar 

  33. Wei L, Cui L, Snider BJ, Rivkin M, Yu SS, Lee CS, Adams LD, Gottlieb DI, Johnson EM Jr, Yu SP, Choi DW (2005) Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia. Neurobiol Dis 19:183–193

    Article  CAS  PubMed  Google Scholar 

  34. Cooper JA, Gould KL, Cartwright CA, Hunter T (1986) Tyr527 is phosphorylated in pp60c-src: implications for regulation. Science 231:1431–1434

    Article  CAS  PubMed  Google Scholar 

  35. Piwnica-Worms H, Saunders KB, Roberts TM, Smith AE, Cheng SH (1987) Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell 49:75–82

    Article  CAS  PubMed  Google Scholar 

  36. Yung LY, Tso PH, Wu EH, Yu JC, Ip NY, Wong YH (2008) Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins. Cell Signal 20:1538–1544

    Article  CAS  PubMed  Google Scholar 

  37. Urbano FJ, Buno W (2000) Neurotrophin regulation of sodium and calcium channels in human neuroblastoma cells. Neuroscience 96:439–443

    Article  CAS  PubMed  Google Scholar 

  38. Tucker BA, Rahimtula M, Mearow KM (2008) Src and FAK are key early signalling intermediates required for neurite growth in NGF-responsive adult DRG neurons. Cell Signal 20:241–257

    Article  CAS  PubMed  Google Scholar 

  39. Traxler P, Bold G, Frei J, Lang M, Lydon N, Mett H, Buchdunger E, Meyer T, Mueller M, Furet P (1997) Use of a pharmacophore model for the design of EGF-R tyrosine kinase inhibitors: 4-(phenylamino)pyrazolo[3,4-d]pyrimidines. J Med Chem 40:3601–3616

    Article  CAS  PubMed  Google Scholar 

  40. Schmidt JW, Brugge JS, Nelson WJ (1992) pp60src tyrosine kinase modulates P19 embryonal carcinoma cell fate by inhibiting neuronal but not epithelial differentiation. J Cell Biol 116:1019–1033

    Article  CAS  PubMed  Google Scholar 

  41. Beggs HE, Soriano P, Maness PF (1994) NCAM-dependent neurite outgrowth is inhibited in neurons from Fyn-minus mice. J Cell Biol 127:825–833

    Article  CAS  PubMed  Google Scholar 

  42. Ignelzi MA Jr, Miller DR, Soriano P, Maness PF (1994) Impaired neurite outgrowth of src-minus cerebellar neurons on the cell adhesion molecule L1. Neuron 12:873–884

    Article  CAS  PubMed  Google Scholar 

  43. Chen C, Bharucha V, Chen Y, Westenbroek RE, Brown A, Malhotra JD, Jones D, Avery C, Gillespie PJ 3rd, Kazen-Gillespie KA, Kazarinova-Noyes K, Shrager P, Saunders TL, Macdonald RL, Ransom BR, Scheuer T, Catterall WA, Isom LL (2002) Reduced sodium channel density, altered voltage dependence of inactivation, and increased susceptibility to seizures in mice lacking sodium channel beta 2-subunits. Proc Natl Acad Sci USA 99:17072–17077

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Brackenbury WJ, Djamgoz MB (2006) Activity-dependent regulation of voltage-gated Na+ channel expression in Mat-LyLu rat prostate cancer cell line. J Physiol 573:343–356

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Feng S, Pflueger M, Lin SX, Groveman BR, Su J, Yu XM (2012) Regulation of voltage-gated sodium current by endogenous Src family kinases in cochlear spiral ganglion neurons in culture. Pflugers Arch 463:571–584

    Article  CAS  PubMed  Google Scholar 

  46. Wang Y, Wagner MB, Kumar R, Cheng J, Joyner RW (2003) Inhibition of fast sodium current in rabbit ventricular myocytes by protein tyrosine kinase inhibitors. Pflugers Arch 446:485–491

    Article  CAS  PubMed  Google Scholar 

  47. Maier SK, Westenbroek RE, Schenkman KA, Feigl EO, Scheuer T, Catterall WA (2002) An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart. Proc Natl Acad Sci USA 99:4073–4078

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Xie H, Ziskind-Conhaim L (1995) Blocking Ca(2+)-dependent synaptic release delays motoneuron differentiation in the rat spinal cord. J Neurosci 15:5900–5911

    CAS  PubMed  Google Scholar 

  49. Borodinsky LN, Coso OA, Fiszman ML (2002) Contribution of Ca2 + calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells. J Neurochem 80:1062–1070

    Article  CAS  PubMed  Google Scholar 

  50. D’Ascenzo M, Piacentini R, Casalbore P, Budoni M, Pallini R, Azzena GB, Grassi C (2006) Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation. Eur J Neurosci 23:935–944

    Article  PubMed  Google Scholar 

  51. Carey MB, Matsumoto SG (1999) Spontaneous calcium transients are required for neuronal differentiation of murine neural crest. Dev Biol 215:298–313

    Article  CAS  PubMed  Google Scholar 

  52. Moran D (1991) Voltage-dependent -L-type Ca2 + channels participate in regulating neural crest migration and differentiation. Am J Anat 192:14–22

    Article  CAS  PubMed  Google Scholar 

  53. Cantrell AR, Tibbs VC, Westenbroek RE, Scheuer T, Catterall WA (1999) Dopaminergic modulation of voltage-gated Na+ current in rat hippocampal neurons requires anchoring of cAMP-dependent protein kinase. J Neurosci 19:RC21

    CAS  PubMed  Google Scholar 

  54. Cantrell AR, Tibbs VC, Yu FH, Murphy BJ, Sharp EM, Qu Y, Catterall WA, Scheuer T (2002) Molecular mechanism of convergent regulation of brain Na(+) channels by protein kinase C and protein kinase A anchored to AKAP-15. Mol Cell Neurosci 21:63–80

    Article  CAS  PubMed  Google Scholar 

  55. Morgan PJ, Ortinau S, Frahm J, Kruger N, Rolfs A, Frech MJ (2009) Protection of neurons derived from human neural progenitor cells by veratridine. NeuroReport 20:1225–1229

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by NIH Grants NS0458710 (SPY), NS057255 (SPY), NS058710 (LW), and the American Heart Association Established Investigator Award (LW) and the Grant-in-Aid Grant 12GRNT12060222 (SPY).

Author information

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, 29425, USA

    Kevin R. Francis

  2. Department of Anesthesiology, Emory University School of Medicine, 101 Woodruff Circle, Suite 620, Atlanta, GA, 30322, USA

    Ling Wei & Shan Ping Yu

  3. Department of Neurology, Emory University, Atlanta, GA, 30322, USA

    Ling Wei

Authors
  1. Kevin R. Francis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ling Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shan Ping Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shan Ping Yu.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Francis, K.R., Wei, L. & Yu, S.P. Src Tyrosine Kinases Regulate Neuronal Differentiation of Mouse Embryonic Stem Cells Via Modulation of Voltage-Gated Sodium Channel Activity. Neurochem Res 40, 674–687 (2015). https://doi.org/10.1007/s11064-015-1514-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-015-1514-4

Keywords

Search

Navigation