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:

Localization of PIP2 activation gate in inward rectifier K+ channels

Abstract

Ion channels respond to changes in transmembrane voltage or ligand concentration by opening or closing an activation gate. In voltage-gated K+ channels, this gate has been localized to an intracellular bundle crossing. Here we examined whether this bundle crossing, or the more internal cytoplasmic pore, acts as a gate for PIP2 activation of inward rectifier K+ (Kir) channels expressed in Xenopus laevis oocytes. We studied the open/closed state-dependence of the accessibility of intracellular cationic modifiers to a position (residue Ile176 in the TM2 helix of Kir2.1) more external to the bundle crossing. Cd2+ blocked I176C mutant channels much more weakly in the closed state than in the open state, but Ag+ and sulfhydryl-specific methanethiosulfonate reagents modified the channels with similar rates in both states. These results suggest that the TM2 helices undergo conformation changes upon PIP2 binding/unbinding, but neither they nor the cytoplasmic pore close fully to form a physical gate for K+ conduction.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Homology model of the Kir2.1 pore based on a sequence alignment with KcsA.
Figure 2: State-dependence of Cd2+ block of I176C channels.
Figure 3: State-dependence of Ag+ modification of A184C channels.
Figure 4: State-dependence of Ag+ modification of I176C channels.
Figure 5: Apparent second-order rate constant for Ag+ modification of TM2 cysteines.
Figure 6: Ag+ modification of TM1 cysteines.
Figure 7: Apparent second-order rate constant for MTSEA and MTSET modification of I176C.
Figure 8: Representative single-channel currents of I176C in an inside-out patch.

Similar content being viewed by others

References

  1. Doyle, D.A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998).

    Article  CAS  Google Scholar 

  2. Jiang, Y. et al. The open pore conformation of potassium channels. Nature 417, 523–526 (2002).

    Article  CAS  Google Scholar 

  3. Liu, Y., Jurman, M.E. & Yellen, G. Gated access to the pore of a voltage-dependent K+ channel. Neuron 19, 175–184 (1997).

    Article  Google Scholar 

  4. Flynn, G.E. & Zagotta, W.N. Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels. Neuron 30, 689–698 (2001).

    Article  CAS  Google Scholar 

  5. del Camino, D. & Yellen, G. Tight steric closure at the intracellular activation gate of a voltage- gated K+ channel. Neuron 32, 649–656 (2001).

    Article  CAS  Google Scholar 

  6. Bruening-Wright, A., Schumacher, M.A., Adelman, J.P. & Maylie, J. Localization of the activation gate for small conductance Ca2+-activated channels. J. Neurosci. 22, 6499–6506 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Lu, T., Zhu, Y-G. & Yang, J. Cytoplasmic amino and carboxyl domains form a wide internal vestibule in an inwardly rectifying K+ channel. Proc. Natl. Acad. Sci. USA 96, 9926–9931 (1999).

    Article  CAS  Google Scholar 

  9. Nishida, M. & MacKinnon, R. Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 Å resolution. Cell 111, 957–965 (2002).

    Article  CAS  Google Scholar 

  10. Matsuda, H., Saigusa, A. & Irisawa, H. Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+. Nature 325, 156–158 (1987).

    Article  CAS  Google Scholar 

  11. Vandenberg, C.A. Inward rectification of a potassium channel in cardiac ventricular cells depends on internal magnesium ions. Proc. Natl. Acad. Sci. USA 84, 2560–2564 (1987).

    Article  CAS  Google Scholar 

  12. Lopatin, A.N., Makhina, E.N. & Nichols, C.G. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature 372, 366–369 (1994).

    Article  CAS  Google Scholar 

  13. Ficker, E., Taglialatela, M., Wible, B.A., Henley, C.M. & Brown, A.M. Spermine and spermidine as gating molecules for inward rectifier K+ channels. Science 266, 1068–1072 (1994).

    Article  CAS  Google Scholar 

  14. Fakler, B. et al. Strong voltage-dependent inward rectification of inward rectifier K+ channels is caused by intracellular spermine. Cell 80, 149–154 (1995).

    Article  CAS  Google Scholar 

  15. Hilgemann, D.W. & Ball, R. Regulation of cardiac Na+, Ca2+ exchange and KATP potassium channels by PIP2 . Science 273, 956–959 (1996).

    Article  CAS  Google Scholar 

  16. Huang, C-L., Feng, S. & Hilgemann, D.W. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gβγ . Nature 391, 803–806 (1997).

    Article  Google Scholar 

  17. Sui, J.L., Petit Jacques, P. & Logothetis, D.E. Activation of the atrial KACh channel by the betagamma subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidyl-inositol phosphates. Proc. Natl. Acad. Sci. USA 95, 1307–1312 (1999).

    Article  Google Scholar 

  18. Zhang, H., He, C., Yan, X., Mirshahi, T. & Logothetis, D.E. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions. Nat. Cell Biol. 1,183–188 (1999).

    Article  CAS  Google Scholar 

  19. Shyng, S.L., Cukras, C.A., Harwood, J. & Nichols, C.G. Structural determinants of PIP2 regulation of inward rectifier KATP channels. J. Gen. Physiol. 116, 599–608 (2000).

    Article  CAS  Google Scholar 

  20. Lopes, C., Zhang, H., Rohacs, T., Yang, J. & Logothetis, D.E. Alterations in conserved PIP2 and Kir channels interactions underlie channelopathies. Neuron 34, 933–944 (2002).

    Article  CAS  Google Scholar 

  21. Kobrinsky, E., Mirshahi, T., Zhang, H., Jin, T. & Logothetis, D.E. Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization. Nat. Cell Biol. 2, 507–514 (2000).

    Article  CAS  Google Scholar 

  22. Lu, T., Nguyen, B., Zhang, X-M. & Yang, J. Architecture of a K+ channel inner pore revealed by stoichiometric covalent modification. Neuron 22, 571–580 (1999).

    Article  CAS  Google Scholar 

  23. Akabas, M.H., Stauffer, D.A., Xu, M. & Karlin, A. Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science 258, 307–310 (1992).

    Article  CAS  Google Scholar 

  24. Karlin, A. & Akabas, M.H. Substituted-cysteine accessibility method. Methods Enzymol. 293, 123–145 (1998).

    Article  CAS  Google Scholar 

  25. Jin, T. et al. The βγ subunits of G proteins gate a K+ channel by pivoted bending of a transmembrane segment. Mol. Cell 10, 469–481 (2002).

    Article  CAS  Google Scholar 

  26. Loussouarn, G., Phillips, L.R., Masia, R., Rose, T. & Nichols C.G. Flexibility of the Kir6.2 inward rectifier K+ channel pore. Proc. Natl. Acad. Sci. USA 98, 4227–4232 (2001).

    Article  CAS  Google Scholar 

  27. Holmgren, M., Liu, Y., Xu, Y. & Yellen, G. On the use of thiol-modifying agents to determine channel topology. Neuropharmacology 35, 797–804 (1996).

    Article  CAS  Google Scholar 

  28. Perozo, E., Cortes, D.M. & Cuello, L.G. Structural rearrangements underlying K+-channel activation gating. Science 285, 73–78 (1999).

    Article  CAS  Google Scholar 

  29. Liu, Y.S., Sompornpisut, P. & Perozo E. Structure of the KcsA channel intracellular gate in the open state. Nat. Struct. Biol. 8, 883–887 (2001).

    Article  CAS  Google Scholar 

  30. Proks, P., Antcliff, J.F. & Ashcroft, F.M. The ligand-sensitive gate of a potassium channel lies close to the selectivity filter. EMBO Rep. 4, 70–75 (2003).

    Article  CAS  Google Scholar 

  31. Phillips, L.R., Enkvetchakul, D & Nichols, C. Gating dependence of inner pore access in inward rectifier K+ channels. Neuron 37, 953–962 (2003).

    Article  Google Scholar 

  32. Lopez-Barneo, J., Hoshi, T., Heinemann, S.H. & Aldrich, R.W. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels 1, 61–71 (1993).

    CAS  PubMed  Google Scholar 

  33. Yellen, G., Sodickson, D., Chen, T.-Y. & Jurman, M.E. An engineered cysteine in the external mouth of a K+ channel allows inactivation to be modulated by metal binding. Biophys. J. 66, 1068–1075 (1994).

    Article  CAS  Google Scholar 

  34. Liu, Y., Jurman, M.E. & Yellen, G. Dynamic rearrangement of the outer mouth of a K+ channel during gating. Neuron 16, 859–867 (1996).

    Article  CAS  Google Scholar 

  35. Kiss, L., LoTurco, J. & Korn, S.J. Contribution of the selectivity filter to in activation in potassium channels. Biophys. J. 76, 253–263 (1999).

    Article  CAS  Google Scholar 

  36. Immke, D., Wood, M., Kiss, L. & Korn, S.J. Potassium-dependent changes in the conformation of the Kv2.1 potassium channel pore. J. Gen. Physiol. 113, 819–836 (1999).

    Article  CAS  Google Scholar 

  37. Chapman, M.L., VanDongen, H.M.A. & VanDongen, A.M.J. Activation-dependent subconductance levels in the drk1 K+ channel suggest a subunit basis for ion permeation and gating. Biophys. J. 72, 708–719 (1997).

    Article  CAS  Google Scholar 

  38. Zheng, J. & Sigworth, F.J. Selectivity changes during activation of mutant shaker potassium channels. J. Gen. Physiol. 110, 101–117 (1997).

    Article  CAS  Google Scholar 

  39. Zheng, J., & Sigworth, F.J. Intermediate conductances during deactivation of heteromultimeric shaker potassium channels. J. Gen. Physiol. 112, 457–474 (1998).

    Article  CAS  Google Scholar 

  40. Zhou, M., Morais-Cabral, J.H., Mann, S. & MacKinnon, R. Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature 411, 657–661 (2001).

    Article  CAS  Google Scholar 

  41. Lu, T., Wu, L., Xiao, J. & Yang, J. Permeant ion-dependent changes in gating of Kir2.1 inward rectifier potassium channels. J. Gen. Physiol. 118, 509–521 (2001).

    Article  CAS  Google Scholar 

  42. So, I., Ashmole, I., Davies, N.W., Sutcliffe, M.J. & Stanfield, P.R. The K+ channel signature sequence of murine Kir2.1: mutations that affect microscopic gating but not ionic selectivity. J. Physiol. 531, 37–50 (2001).

    Article  CAS  Google Scholar 

  43. Proks, P., Antcliff, J.F. & Ashcroft, F.M. Mutations within the P-loop of Kir6.2 modulate the intraburst kinetics of the ATP-sensitive potassium channel. J. Gen. Physiol. 118, 341–353 (2001).

    Article  CAS  Google Scholar 

  44. Lu, T. et al. Probing ion permeation and gating in a K+ channel with backbone mutations in the selectivity filter. Nat. Neurosci. 4, 239–246 (2001).

    Article  CAS  Google Scholar 

  45. Trapp, S., Proks, P., Tucker, S.J. & Ashcroft, F.M. Molecular analysis of KATP channel gating and implications for channel inhibition by ATP. J. Gen. Physiol. 112, 333–350 (1998).

    Article  CAS  Google Scholar 

  46. Enkvetchakul, D., Loussouarn, G., Makhina, E., Shyng, S.-L. & Nichols, C.G. The kinetic and physical basis of KATP channel gating: toward a unified molecular understanding. Biophys. J. 78, 2334–2348 (2000).

    Article  CAS  Google Scholar 

  47. Yakubovich, D., Pastushenko, V., Bitler, A., Dessauer, C.W. & Dascal, N. Slow modal gating of single G protein-activated K+ channels expressed in Xenopus oocytes. J. Physiol. 524, 737–755 (2000).

    Article  CAS  Google Scholar 

  48. Choe, H., Palmer, L.G. & Sackin, H. Structural determinants of gating in inward-rectifier K+ channels. Biophys. J. 76, 1988–2003 (1999).

    Article  CAS  Google Scholar 

  49. Sadja, R., Smadja, K., Alagem, N. & Reuveny E. Coupling Gβγ-dependent activation to channel opening via pore elements in inwardly rectifying potassium channels. Neuron 29, 669–680 (2001).

    Article  CAS  Google Scholar 

  50. Yi, B.A., Lin, Y.F., Jan, Y.N. & Jan L.Y. Yeast screen for constitutively active mutant G protein-activated potassium channels. Neuron 29, 657–667 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Cui (Case Western), G. Zhu (Columbia) and members of our laboratory for discussion and reading the manuscript. This work was supported by a grant to J.Y. from the National Institute of Neurological Disorders and Stroke (NS45383). J.Y. is a recipient of the McKnight Scholar Award and the Scholar Research Programme of the EJLB Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jian Yang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xiao, J., Zhen, Xg. & Yang, J. Localization of PIP2 activation gate in inward rectifier K+ channels. Nat Neurosci 6, 811–818 (2003). https://doi.org/10.1038/nn1090

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn1090

This article is cited by

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