Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Electrochemical coupling in the voltage-dependent phosphatase Ci-VSP

Nature Chemical Biology volume 6, pages 369–375 (2010)Cite this article

Subjects

A Corrigendum to this article was published on 17 September 2010

This article has been updated

Abstract

In the voltage-sensing phosphatase Ci-VSP, a voltage-sensing domain (VSD) controls a lipid phosphatase domain (PD). The mechanism by which the domains are allosterically coupled is not well understood. Using an in vivo assay, we found that the interdomain linker that connects the VSD to the PD is essential for coupling the full-length protein. Biochemical assays showed that the linker is also needed for activity in the isolated PD. We also identified a late step of VSD motion in the full-length protein that depends on the linker. Notably, we found that this VSD motion requires PI(4,5)P2, a substrate of Ci-VSP. These results suggest that the voltage-driven motion of the VSD turns the enzyme on by rearranging the linker into an activated conformation, and that this activated conformation is stabilized by PI(4,5)P2. We propose that Ci-VSP activity is self-limited because its decrease of PI(4,5)P2 levels decouples the VSD from the enzyme.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Linker and catalytic site mutants reduce or abolish activity.
Figure 2: Mutations of PD catalytic site alter VSD motion.
Figure 3: Linker charge neutralization eliminates effect of catalytic site mutations on VSD motion.
Figure 4: Linker mutations alter late component of VSD motion.
Figure 5: PI(4,5)P2 depletion alters VSD motion in a manner that is dependent on the VSD-PD linker.
Figure 6: Model of linker regulation and coupling in Ci-VSP.

Similar content being viewed by others

Change history

  • 17 September 2010

    After publication, the authors found second-site mutations within two of the Ci-VSP constructs. The R253Q G214C and R254Q G214C constructs have been remade without the additional mutations and the data recollected. The data originally presented in Figure 1, Supplementary Figures 5 and 6 and Supplementary Table 1 have been updated to reflect the minor changes in the magnitude of the constructs’ effects. The changes do not affect the conclusions of the study. The changes have been made in the HTML and PDF versions of the article.

References

  1. Murata, Y., Iwasaki, H., Sasaki, M., Inaba, K. & Okamura, Y. Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature 435, 1239–1243 (2005).

    Article  CAS  Google Scholar 

  2. Okamura, Y., Murata, Y. & Iwasaki, H. Voltage-sensing phosphatase: actions and potentials. J. Physiol. (Lond.) 587, 513–520 (2009).

    Article  CAS  Google Scholar 

  3. Worby, C.A. & Dixon, J.E. Phosphoinositide phosphatases: emerging roles as voltage sensors? Mol. Interv. 5, 274–277 (2005).

    Article  CAS  Google Scholar 

  4. Lu, Z., Klem, A.M. & Ramu, Y. Ion conduction pore is conserved among potassium channels. Nature 413, 809–813 (2001).

    Article  CAS  Google Scholar 

  5. Lu, Z., Klem, A.M. & Ramu, Y. Coupling between voltage sensors and activation gate in voltage-gated K+ channels. J. Gen. Physiol. 120, 663–676 (2002).

    Article  CAS  Google Scholar 

  6. Long, S.B., Campbell, E.B. & Mackinnon, R. Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309, 897–903 (2005).

    Article  CAS  Google Scholar 

  7. Murata, Y. & Okamura, Y. Depolarization activates the phosphoinositide phosphatase Ci-VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2. J. Physiol. (Lond.) 583, 875–889 (2007).

    Article  CAS  Google Scholar 

  8. Iwasaki, H. et al. A voltage-sensing phosphatase, Ci-VSP, which shares sequence identity with PTEN, dephosphorylates phosphatidylinositol 4,5-bisphosphate. Proc. Natl. Acad. Sci. USA 105, 7970–7975 (2008).

    Article  CAS  Google Scholar 

  9. Halaszovich, C.R., Schreiber, D.N. & Oliver, D. Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5′-phosphatase. J. Biol. Chem. 284, 2106–2113 (2009).

    Article  CAS  Google Scholar 

  10. Maehama, T., Taylor, G.S. & Dixon, J.E. PTEN and myotubularin: novel phosphoinositide phosphatases. Annu. Rev. Biochem. 70, 247–279 (2001).

    Article  CAS  Google Scholar 

  11. Iijima, M., Huang, Y.E., Luo, H.R., Vazquez, F. & Devreotes, P.N. Novel mechanism of PTEN regulation by its phosphatidylinositol 4,5-bisphosphate binding motif is critical for chemotaxis. J. Biol. Chem. 279, 16606–16613 (2004).

    Article  CAS  Google Scholar 

  12. Walker, S.M., Leslie, N.R., Perera, N.M., Batty, I.H. & Downes, C.P. The tumour-suppressor function of PTEN requires an N-terminal lipid-binding motif. Biochem. J. 379, 301–307 (2004).

    Article  CAS  Google Scholar 

  13. Vazquez, F. et al. Tumor suppressor PTEN acts through dynamic interaction with the plasma membrane. Proc. Natl. Acad. Sci. USA 103, 3633–3638 (2006).

    Article  CAS  Google Scholar 

  14. Rahdar, M. et al. A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN. Proc. Natl. Acad. Sci. USA 106, 480–485 (2009).

    Article  CAS  Google Scholar 

  15. Villalba-Galea, C.A., Miceli, F., Taglialatela, M. & Bezanilla, F. Coupling between the voltage-sensing and phosphatase domains of Ci-VSP. J. Gen. Physiol. 134, 5–14 (2009).

    Article  CAS  Google Scholar 

  16. Mannuzzu, L.M., Moronne, M.M. & Isacoff, E.Y. Direct physical measure of conformational rearrangement underlying potassium channel gating. Science 271, 213–216 (1996).

    Article  CAS  Google Scholar 

  17. Kohout, S.C., Ulbrich, M.H., Bell, S.C. & Isacoff, E.Y. Subunit organization and functional transitions in Ci-VSP. Nat. Struct. Mol. Biol. 15, 106–108 (2008).

    Article  CAS  Google Scholar 

  18. Hossain, M.I. et al. Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatases. J. Biol. Chem. 283, 18248–18259 (2008).

    Article  CAS  Google Scholar 

  19. Villalba-Galea, C.A., Sandtner, W., Starace, D.M. & Bezanilla, F. S4-based voltage sensors have three major conformations. Proc. Natl. Acad. Sci. USA 105, 17600–17607 (2008).

    Article  CAS  Google Scholar 

  20. Steck, P.A. et al. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet. 15, 356–362 (1997).

    Article  CAS  Google Scholar 

  21. Duerr, E.M. et al. PTEN mutations in gliomas and glioneuronal tumors. Oncogene 16, 2259–2264 (1998).

    Article  CAS  Google Scholar 

  22. Gronbaek, K., Zeuthen, J., Guldberg, P., Ralfkiaer, E. & Hou-Jensen, K. Alterations of the MMAC1/PTEN gene in lymphoid malignancies. Blood 91, 4388–4390 (1998).

    CAS  PubMed  Google Scholar 

  23. Downes, C.P. et al. Acute regulation of the tumour suppressor phosphatase, PTEN, by anionic lipids and reactive oxygen species. Biochem. Soc. Trans. 32, 338–342 (2004).

    Article  CAS  Google Scholar 

  24. Xiao, Y. et al. PTEN catalysis of phospholipid dephosphorylation reaction follows a two-step mechanism in which the conserved aspartate-92 does not function as the general acid–mechanistic analysis of a familial Cowden disease-associated PTEN mutation. Cell. Signal. 19, 1434–1445 (2007).

    Article  CAS  Google Scholar 

  25. Flint, A.J., Tiganis, T., Barford, D. & Tonks, N.K. Development of “substrate-trapping” mutants to identify physiological substrates of protein tyrosine phosphatases. Proc. Natl. Acad. Sci. USA 94, 1680–1685 (1997).

    Article  CAS  Google Scholar 

  26. Julius, D., MacDermott, A.B., Axel, R. & Jessell, T.M. Molecular characterization of a functional cDNA encoding the serotonin 1c receptor. Science 241, 558–564 (1988).

    Article  CAS  Google Scholar 

  27. Suh, B.C., Inoue, T., Meyer, T. & Hille, B. Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science 314, 1454–1457 (2006).

    Article  CAS  Google Scholar 

  28. Varnai, P., Thyagarajan, B., Rohacs, T. & Balla, T. Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells. J. Cell Biol. 175, 377–382 (2006).

    Article  CAS  Google Scholar 

  29. Lukacs, V. et al. Dual regulation of TRPV1 by phosphoinositides. J. Neurosci. 27, 7070–7080 (2007).

    Article  CAS  Google Scholar 

  30. Campbell, R.B., Liu, F. & Ross, A.H. Allosteric activation of PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate. J. Biol. Chem. 278, 33617–33620 (2003).

    Article  CAS  Google Scholar 

  31. Redfern, R.E. et al. PTEN phosphatase selectively binds phosphoinositides and undergoes structural changes. Biochemistry 47, 2162–2171 (2008).

    Article  CAS  Google Scholar 

  32. Borah, H.N., Boruah, R.C. & Sandhu, J.S. Microwave-induced one-pot synthesis of N-carboxyalkyl maleimides and phthalimides. J. Chem. Res. (S) 272–273 (1998).

Download references

Acknowledgements

This work was supported by grants R01NS035549 (E.Y.I.), U24NS57631 (E.Y.I.) and R01DC007664 (D.L.M.) from the US National Institutes of Health and by an American Heart Association Established Investigator Award (D.L.M.). We thank Y. Okamura (Japanese National Institute for Physiological Sciences) for kindly providing the Ci-VSP cDNA, T. Meyer (Stanford University) for kindly providing the LDR and CFInp cDNA, E. Reuveny (Weizmann Institute of Science) for providing the IRK1 construct, S. Nakanishi (Osaka Bioscience Institute) for providing the mGluR1α receptor, J. Groves and P. Nair (University of California, Berkeley) for access to and instructions on use of the lipid extruder, and H. Janovjak, K. Nakajo, E. Peled and F. Tombola for helpful discussions.

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

    Susy C Kohout & Ehud Y Isacoff

  2. Chemical Biology Graduate Program, University of California, Berkeley, California, USA

    Sarah C Bell & Ehud Y Isacoff

  3. Cardiovascular Research Institute, University of California, San Francisco, California, USA

    Lijun Liu, Qiang Xu & Daniel L Minor Jr

  4. Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA

    Daniel L Minor Jr

  5. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA

    Daniel L Minor Jr

  6. California Institute for Quantitative Biosciences, University of California, San Francisco, California, USA

    Daniel L Minor Jr

  7. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Daniel L Minor Jr & Ehud Y Isacoff

  8. Helen Wills Neuroscience Institute, University of California, Berkeley, California, USA

    Ehud Y Isacoff

  9. Material Science Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Ehud Y Isacoff

Authors
  1. Susy C Kohout
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah C Bell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lijun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qiang Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daniel L Minor Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ehud Y Isacoff
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.C.K. designed, conducted and analyzed the VCF experiments and the confocal experiment, conducted and analyzed the malachite green assay and the CD spectroscopy, and wrote the paper. S.C.B. designed, conducted and analyzed the in vivo activity assay and edited the paper. L.L. expressed and purified the cytosolic domain proteins and edited the paper. Q.X. helped design the malachite green assay and expressed and purified the cytosolic domain proteins. D.L.M. designed and initiated the CD spectroscopy and edited the paper. E.Y.I. designed the VCF experiments and the in vivo activity assay and wrote the paper.

Corresponding author

Correspondence to Ehud Y Isacoff.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Tables 1–3, Supplementary Methods and Supplementary Results (PDF 237 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kohout, S., Bell, S., Liu, L. et al. Electrochemical coupling in the voltage-dependent phosphatase Ci-VSP. Nat Chem Biol 6, 369–375 (2010). https://doi.org/10.1038/nchembio.349

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.349

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing