Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones

Abstract

Ion channels formed by the TRP (transient receptor potential) superfamily of proteins act as sensors for temperature, osmolarity, mechanical stress and taste1,2. The growth cones of developing axons are responsible for sensing extracellular guidance factors, many of which trigger Ca2+ influx at the growth cone3,4; however, the identity of the ion channels involved remains to be clarified. Here, we report that TRP-like channel activity exists in the growth cones of cultured Xenopus neurons and can be modulated by exposure to netrin-1 and brain-derived neurotrophic factor, two chemoattractants for axon guidance. Whole-cell recording from growth cones showed that netrin-1 induced a membrane depolarization, part of which remained after all major voltage-dependent channels were blocked. Furthermore, the membrane depolarization was sensitive to blockers of TRP channels. Pharmacological blockade of putative TRP currents or downregulation of Xenopus TRP-1 (xTRPC1) expression with a specific morpholino oligonucleotide abolished the growth-cone turning and Ca2+ elevation induced by a netrin-1 gradient. Thus, TRPC currents reflect early events in the growth cone's detection of some extracellular guidance signals, resulting in membrane depolarization and cytoplasmic Ca2+ elevation that mediates the turning of growth cones.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Membrane depolarization and putative TRPC currents induced by netrin-1 and BDNF.
Figure 2: Growth-cone turning induced by netrin-1 or BDNF, and the effect of SKF-96365.
Figure 3: Effects of xTRPC1 MO on TRPC currents and growth-cone turning induced by netrin-1.
Figure 4: xTRPC1 mediates netrin-1-induced Ca2+ elevation in the growth cone.
Figure 5: xTRPC1 mediates netrin-1-induced membrane depolarization.

References

  1. 1

    Clapham, D. E. TRP channels as cellular sensors. Nature 426, 517–524 (2003)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Montell, C., Birnbaumer, L. & Flockerzi, V. The TRP channels, a remarkably functional family. Cell 108, 595–598 (2002)

    CAS  Article  Google Scholar 

  3. 3

    Hong, K., Nishiyama, M., Henley, J., Tessier-Lavigne, M. & Poo, M. Calcium signalling in the guidance of nerve growth by netrin-1. Nature 403, 93–98 (2000)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Henley, J. & Poo, M. M. Calcium mediates bidirectional growth cone turning induced by myelin-associated glycoprotein. Neuron 44, 909–916 (2004)

    CAS  Article  Google Scholar 

  5. 5

    Henley, J. & Poo, M. M. Guiding neuronal growth cones using Ca2+ signals. Trends Cell Biol. 14, 320–330 (2004)

    CAS  Article  Google Scholar 

  6. 6

    Nishiyama, M. et al. Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning. Nature 423, 990–995 (2003)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Ming, G. L. et al. cAMP-dependent growth cone guidance by netrin-1. Neuron 19, 1225–1235 (1997)

    MathSciNet  CAS  Article  Google Scholar 

  8. 8

    Kim, S. J. et al. Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1. Nature 426, 285–291 (2003)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Li, H. S., Xu, X. Z. & Montell, C. Activation of a TRPC3-dependent cation current through the neurotrophin BDNF. Neuron 24, 261–273 (1999)

    CAS  Article  Google Scholar 

  10. 10

    Zhu, X., Jiang, M. & Birnbaumer, L. Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry. J. Biol. Chem. 273, 133–142 (1998)

    CAS  Article  Google Scholar 

  11. 11

    Schwarz, G., Droogmans, G. & Nilius, B. Multiple effects of SKF 96365 on ionic currents and intracellular calcium in human endothelial cells. Cell Calcium 15, 45–54 (1994)

    CAS  Article  Google Scholar 

  12. 12

    Greka, A., Navarro, B., Oancea, E., Duggan, A. & Clapham, D. E. TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nature Neurosci. 6, 837–845 (2003)

    CAS  Article  Google Scholar 

  13. 13

    Song, H. J., Ming, G. L. & Poo, M. M. cAMP-induced switching in turning direction of nerve growth cones. Nature 388, 275–279 (1997)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Jung, S. et al. Lanthanides potentiate TRPC5 currents by an action at extracellular sites close to the pore mouth. J. Biol. Chem. 278, 3562–3571 (2003)

    CAS  Article  Google Scholar 

  15. 15

    Inoue, R. et al. The transient receptor potential protein homologue TRP6 is the essential component of vascular alpha(1)-adrenoceptor-activated Ca(2 + )-permeable cation channel. Circ. Res. 88, 325–332 (2001)

    CAS  Article  Google Scholar 

  16. 16

    Gomez, T. M., Robles, E., Poo, M. & Spitzer, N. C. Filopodial calcium transients promote substrate-dependent growth cone turning. Science 291, 1983–1987 (2001)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Li, Y. et al. Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor. Nature doi:10.1038/nature03477 (this issue)

  18. 18

    Bobanovic, L. K. et al. Molecular cloning and immunolocalization of a novel vertebrate trp homologue from Xenopus . Biochem. J. 340, 593–599 (1999)

    CAS  Article  Google Scholar 

  19. 19

    Brereton, H. M., Harland, M. L., Auld, A. M. & Barritt, G. J. Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes. Mol. Cell. Biochem. 214, 63–74 (2000)

    CAS  Article  Google Scholar 

  20. 20

    Lintschinger, B. et al. Coassembly of Trp1 and Trp3 proteins generates diacylglycerol- and Ca2+-sensitive cation channels. J. Biol. Chem. 275, 27799–27805 (2000)

    CAS  PubMed  Google Scholar 

  21. 21

    Strubing, C., Krapivinsky, G., Krapivinsky, L. & Clapham, D. E. TRPC1 and TRPC5 form a novel cation channel in mammalian brain. Neuron 29, 645–655 (2001)

    CAS  Article  Google Scholar 

  22. 22

    Strubing, C., Krapivinsky, G., Krapivinsky, L. & Clapham, D. E. Formation of novel TRPC channels by complex subunit interactions in embryonic brain. J. Biol. Chem. 278, 39014–39019 (2003)

    Article  Google Scholar 

  23. 23

    Xu, X. Z., Li, H. S., Guggino, W. B. & Montell, C. Coassembly of TRP and TRPL produces a distinct store-operated conductance. Cell 89, 1155–1164 (1997)

    CAS  Article  Google Scholar 

  24. 24

    Amiri, H., Schultz, G. & Schaefer, M. FRET-based analysis of TRPC subunit stoichiometry. Cell Calcium 33, 463–470 (2003)

    CAS  Article  Google Scholar 

  25. 25

    Schaefer, M., Plant, T. D., Stresow, N., Albrecht, N. & Schultz, G. Functional differences between TRPC4 splice variants. J. Biol. Chem. 277, 3752–3759 (2002)

    CAS  Article  Google Scholar 

  26. 26

    Goel, M., Sinkins, W. G. & Schilling, W. P. Selective association of TRPC channel subunits in rat brain synaptosomes. J. Biol. Chem. 277, 48303–48310 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Hofmann, T., Schaefer, M., Schultz, G. & Gudermann, T. Subunit composition of mammalian transient receptor potential channels in living cells. Proc. Natl Acad. Sci. USA 99, 7461–7466 (2002)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Lohof, A. M. et al. Asymmetric modulation of cytosolic cAMP activity induces growth cone turning. J. Neurosci. 12, 1253–1261 (1992)

    CAS  Article  Google Scholar 

  29. 29

    Gu, X. & Spitzer, N. C. Low-threshold Ca2+ current and its role in spontaneous elevations of intracellular Ca2+ in developing Xenopus neurons. J. Neurosci. 13, 4936–4948 (1993)

    CAS  Article  Google Scholar 

  30. 30

    Schweitz, H. et al. Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons. Proc. Natl Acad. Sci. USA 91, 878–882 (1994)

    ADS  CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank G. J. Barritt for the gift of xTRP-1 antibody. This work was supported by a grant from NIH and a NSF predoctoral fellowship (to G.X.W.)

Author information

Affiliations

Authors

Corresponding author

Correspondence to Mu-ming Poo.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure S1

Comparison of neurite growth in Xenopus spinal neurons with or without loading of xTRPC1 morpholino oligo (MO). (JPG 24 kb)

Supplementary Figure S2

Western blot of xTRPC1 on Xenopus spinal cord extracts. (JPG 24 kb)

Supplementary Figure S3

Voltage-dependent channels are not affected by the blockade of TRPC1. (JPG 37 kb)

Supplementary Figure Legends

Legends to accompany the above Supplementary Figures. (DOC 23 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wang, G., Poo, M. Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones. Nature 434, 898–904 (2005). https://doi.org/10.1038/nature03478

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.