Molecular basis for ligand activation of the human KCNQ2 channel


The voltage-gated potassium channel KCNQ2 is responsible for M-current in neurons and is an important drug target to treat epilepsy, pain and several other diseases related to neuronal hyper-excitability. A list of synthetic compounds have been developed to directly activate KCNQ2, yet our knowledge of their activation mechanism is limited, due to lack of high-resolution structures. Here, we report cryo-electron microscopy (cryo-EM) structures of the human KCNQ2 determined in apo state and in complex with two activators, ztz240 or retigabine, which activate KCNQ2 through different mechanisms. The activator-bound structures, along with electrophysiology analysis, reveal that ztz240 binds at the voltage-sensing domain and directly stabilizes it at the activated state, whereas retigabine binds at the pore domain and activates the channel by an allosteric modulation. By accurately defining ligand-binding sites, these KCNQ2 structures not only reveal different ligand recognition and activation mechanisms, but also provide a structural basis for drug optimization and design.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: ztz240 and RTG activation on KCNQ2.
Fig. 2: The apo-state structure of KCNQ2.
Fig. 3: The ztz240-bound structure of KCNQ2 (KCNQ2-Z).
Fig. 4: The RTG-bound structure of KCNQ2 (KCNQ2-R).
Fig. 5: The structure of KCNQ2-CaM complex.

Data availability

Structure coordinates and cryo-EM density maps have been deposited in the protein data bank under accession numbers 7CR0 and EMD-30443 for apo KCNQ2, 7CR3 and EMD-30446 for apo KCNQ2-CaM, 7CR1 and EMD-30444 for ztz240-bound KCNQ2-Z, 7CR4 and EMD-30447 for ztz240-bound KCNQ2-CaM-Z, 7CR2 and EMD-30445 for RTG-bound KCNQ2-R, 7CR7 and EMD-30448 for RTG-bound KCNQ2-CaM-R.


  1. 1.

    Wang, H.-S. et al. KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science 282, 1890–1893 (1998).

    CAS  PubMed  Google Scholar 

  2. 2.

    Schroeder, B. C., Kubisch, C., Stein, V. & Jentsch, T. J. Moderate loss of function of cyclic-AMP-modulated KCNQ2/KCNQ3 K+ channels causes epilepsy. Nature 396, 687–690 (1998).

    CAS  PubMed  Google Scholar 

  3. 3.

    Singh, N. A. et al. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat. Genet. 18, 25–29 (1998).

    CAS  PubMed  Google Scholar 

  4. 4.

    Charlier, C. et al. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat. Genet. 18, 53–55 (1998).

    CAS  PubMed  Google Scholar 

  5. 5.

    Kato, M. et al. Clinical spectrum of early onset epileptic encephalopathies caused by KCNQ2 mutation. Epilepsia 54, 1282–1287 (2013).

    CAS  PubMed  Google Scholar 

  6. 6.

    Orhan, G. et al. Dominant‐negative effects of KCNQ2 mutations are associated with epileptic encephalopathy. Ann. Neurol. 75, 382–394 (2014).

    CAS  PubMed  Google Scholar 

  7. 7.

    Weckhuysen, S. et al. KCNQ2 encephalopathy: emerging phenotype of a neonatal epileptic encephalopathy. Ann. Neurol. 71, 15–25 (2012).

    CAS  PubMed  Google Scholar 

  8. 8.

    Wulff, H., Castle, N. A. & Pardo, L. A. Voltage-gated potassium channels as therapeutic targets. Nat. Rev. Drug Discov. 8, 982–1001 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Jentsch, T. J. Neuronal KCNQ potassium channels: physislogy and role in disease. Nat. Rev. Neurosci. 1, 21–30 (2000).

    CAS  PubMed  Google Scholar 

  10. 10.

    Soldovieri, M. V., Miceli, F. & Taglialatela, M. Driving with no brakes: molecular pathophysiology of Kv7 potassium channels. Physiology 26, 365–376 (2011).

    CAS  PubMed  Google Scholar 

  11. 11.

    Stott, J. B., Jepps, T. A. & Greenwood, I. A. KV7 potassium channels: a new therapeutic target in smooth muscle disorders. Drug Discov. Today 19, 413–424 (2014).

    CAS  PubMed  Google Scholar 

  12. 12.

    Zheng, Q. et al. Suppression of KCNQ/M (Kv7) potassium channels in dorsal root ganglion neurons contributes to the development of bone cancer pain in a rat model. Pain 154, 434–448 (2013).

    CAS  PubMed  Google Scholar 

  13. 13.

    Zhang, F. et al. Suppression of KCNQ/M potassium channel in dorsal root ganglia neurons contributes to the development of osteoarthritic pain. Pharmacology 103, 257–262 (2019).

    CAS  PubMed  Google Scholar 

  14. 14.

    Yu, T. et al. KCNQ2/3/5 channels in dorsal root ganglion neurons can be therapeutic targets of neuropathic pain in diabetic rats. Mol. Pain. 14, 1–15 (2018).

    CAS  Google Scholar 

  15. 15.

    Blackburn-Munro, G. & Jensen, B. S. The anticonvulsant retigabine attenuates nociceptive behaviours in rat models of persistent and neuropathic pain. Eur. J. Pharmacol. 460, 109–116 (2003).

    CAS  PubMed  Google Scholar 

  16. 16.

    Munro, G. & Dalby-Brown, W. Kv7 (KCNQ) channel modulators and neuropathic pain. J. Med. Chem. 50, 2576–2582 (2007).

    CAS  PubMed  Google Scholar 

  17. 17.

    Szelenyi, I. Flupirtine, a re-discovered drug, revisited. Inflamm. Res. 62, 251–258 (2013).

    CAS  PubMed  Google Scholar 

  18. 18.

    Wen, H. & Levitan, I. B. Calmodulin is an auxiliary subunit of KCNQ2/3 potassium channels. J. Neurosci. 22, 7991–8001 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Bernardo-Seisdedos, G. et al. Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7. 2 K+ channel. Proc. Natl. Acad. Sci. USA 115, 2395–2400 (2018).

    PubMed  Google Scholar 

  20. 20.

    Etxeberria, A. et al. Calmodulin regulates the trafficking of KCNQ2 potassium channels. FASEB J. 22, 1135–1143 (2008).

    CAS  PubMed  Google Scholar 

  21. 21.

    Zhang, Q. et al. Dynamic PIP2 interactions with voltage sensor elements contribute to KCNQ2 channel gating. Proc. Natl. Acad. Sci. USA 110, 20093–20098 (2013).

    CAS  PubMed  Google Scholar 

  22. 22.

    Wickenden, A. D., Yu, W., Zou, A., Jegla, T. & Wagoner, P. K. Retigabine, a novel anti-convulsant, enhances activation of KCNQ2/Q3 potassium channels. Mol. Pharmacol. 58, 591–600 (2000).

    CAS  PubMed  Google Scholar 

  23. 23.

    Main, M. J. et al. Modulation of KCNQ2/3 potassium channels by the novel anticonvulsant retigabine. Mol. Pharmacol. 58, 253–262 (2000).

    CAS  PubMed  Google Scholar 

  24. 24.

    Stafstrom, C. E., Grippon, S. & Kirkpatrick, P. Ezogabine (retigabine). Nat. Rev. Drug Discov. 10, 729–730 (2011).

    CAS  PubMed  Google Scholar 

  25. 25.

    Friedel, H. A. & Fitton, A. Flupirtine: a review of its pharmacological properties, and therapeutic efficacy in pain states. Drugs 45, 548–569 (1993).

    CAS  PubMed  Google Scholar 

  26. 26.

    Wickenden, A. D. et al. N-(6-chloro-pyridin-3-yl)-3, 4-difluoro-benzamide (ICA-27243): a novel, selective KCNQ2/Q3 potassium channel activator. Mol. Pharmacol. 73, 977–986 (2008).

    CAS  PubMed  Google Scholar 

  27. 27.

    Padilla, K., Wickenden, A. D., Gerlach, A. C. & McCormack, K. The KCNQ2/3 selective channel opener ICA-27243 binds to a novel voltage-sensor domain site. Neurosci. Lett. 465, 138–142 (2009).

    CAS  PubMed  Google Scholar 

  28. 28.

    Daniluk, J., Cooper, J. A., Stender, M. & Kowalczyk, A. Survey of physicians' understanding of specific risks associated with retigabine. Drugs Real World Outcomes 3, 155–163 (2016).

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Groseclose, M. R. & Castellino, S. An investigation into retigabine (ezogabine) associated dyspigmentation in rat eyes by MALDI imaging mass spectrometry. Chem. Res. Toxicol. 32, 294–303 (2019).

    CAS  PubMed  Google Scholar 

  30. 30.

    Sun, J. & MacKinnon, R. Cryo-EM structure of a KCNQ1/CaM complex reveals insights into congenital long QT syndrome. Cell 169, 1042–1050 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Sun, J. & MacKinnon, R. Structural basis of human KCNQ1 modulation and gating. Cell 180, 340–347 (2020).

    CAS  PubMed  Google Scholar 

  32. 32.

    Gao, Z. et al. Isoform-specific prolongation of Kv7 (KCNQ) potassium channel opening mediated by new molecular determinants for drug-channel interactions. J. Biol. Chem. 285, 28322–28332 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Strulovich, R., Tobelaim, W. S., Attali, B. & Hirsch, J. A. Structural insights into the M-channel proximal C-terminus/calmodulin complex. Biochemistry 55, 5353–5365 (2016).

    CAS  PubMed  Google Scholar 

  34. 34.

    Tao, X., Lee, A., Limapichat, W., Dougherty, D. A. & Mackinnon, R. A gating charge transfer center in voltage sensors. Science 328, 67–73 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Li, P. et al. The gating charge pathway of an epilepsy-associated potassium channel accommodates chemical ligands. Cell Res. 23, 1106–1118 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Schenzer, A. et al. Molecular determinants of KCNQ (Kv7) K+ channel sensitivity to the anticonvulsant retigabine. J. Neurosci. 25, 5051–5060 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Wuttke, T. V., Seebohm, G., Bail, S., Maljevic, S. & Lerche, H. The new anticonvulsant retigabine favors voltage-dependent opening of the Kv7.2 (KCNQ2) channel by binding to its activation gate. Mol. Pharmacol. 67, 1009–1017 (2005).

    CAS  PubMed  Google Scholar 

  38. 38.

    Lange, W. et al. Refinement of the binding site and mode of action of the anticonvulsant Retigabine on KCNQ K+ channels. Mol. Pharmacol. 75, 272–280 (2009).

    CAS  PubMed  Google Scholar 

  39. 39.

    Kim, R. Y. et al. Atomic basis for therapeutic activation of neuronal potassium channels. Nat. Commun. 6, 8116 (2015).

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Gamper, N., Li, Y. & Shapiro, M. S. Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin. Mol. Biol. Cell 16, 3538–3551 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Peretz, A. et al. Targeting the voltage sensor of Kv7.2 voltage-gated K+ channels with a new gating-modifier. Proc. Natl. Acad. Sci. USA 107, 15637–15642 (2010).

    CAS  PubMed  Google Scholar 

  42. 42.

    Ottosson, N. E. et al. A drug pocket at the lipid bilayer–potassium channel interface. Sci. Adv. 3, e1701099 (2017).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Ahuja, S. et al. Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist. Science 350, aac5464 (2015).

    Google Scholar 

  44. 44.

    Xu, H. et al. Structural basis of Nav1. 7 inhibition by a gating-modifier spider toxin. Cell 176, 702–715 (2019).

    CAS  PubMed  Google Scholar 

  45. 45.

    Shen, H., Liu, D., Wu, K., Lei, J. & Yan, N. Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins. Science 363, 1303–1308 (2019).

    CAS  Google Scholar 

  46. 46.

    Clairfeuille, T. et al. Structural basis of α-scorpion toxin action on Nav channels. Science 363, eaav8573 (2019).

    CAS  PubMed  Google Scholar 

  47. 47.

    Hong, L., Kim, I. H. & Tombola, F. Molecular determinants of Hv1 proton channel inhibition by guanidine derivatives. Proc. Natl. Acad. Sci. USA 111, 9971–9976 (2014).

    CAS  PubMed  Google Scholar 

  48. 48.

    Wang, A. W., Yang, R. & Kurata, H. T. Sequence determinants of subtype‐specific actions of KCNQ channel openers. J. Physiol. 595, 663–676 (2017).

    CAS  PubMed  Google Scholar 

  49. 49.

    Diver, M. M., Cheng, Y. & Julius, D. Structural insights into TRPM8 inhibition and desensitization. Science 365, 1434–1440 (2019).

    CAS  PubMed  Google Scholar 

  50. 50.

    Song, K. et al. Structural basis for human TRPC5 channel inhibition by two distinct inhibitors. bioRxiv (2020).

  51. 51.

    Zhao, Y. et al. Molecular basis for ligand modulation of a mammalian voltage-gated Ca2+ channel. Cell 177, 1495–1506 (2019).

    CAS  PubMed  Google Scholar 

  52. 52.

    Li, Y., Gamper, N., Hilgemann, D. W. & Shapiro, M. S. Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate. J. Neurosci. 25, 9825–9835 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Kumar, M. et al. Synthesis and evaluation of potent KCNQ2/3-specific channel activators. Mol. Pharmacol. 89, 667–677 (2016).

    CAS  PubMed  Google Scholar 

  54. 54.

    Shi, S. et al. Molecular mechanisms and structural basis of retigabine analogues in regulating KCNQ2 channel. J. Memb. Biol. 253, 167–181 (2020).

    CAS  Google Scholar 

  55. 55.

    Wang, L., Qiao, G., Hu, H., Gao, Z. & Nan, F. Discovery of novel retigabine derivatives as potent KCNQ4 and KCNQ5 channel agonists with improved specificity. ACS Med. Chem. Lett. 10, 27–33 (2019).

    CAS  PubMed  Google Scholar 

  56. 56.

    Morales-Perez, C. L., Noviello, C. M. & Hibbs, R. E. Manipulation of subunit stoichiometry in heteromeric membrane proteins. Structure 24, 797–805 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  63. 63.

    Schrodinger, L. The PyMOL molecular graphics system. Version 1.8 (2015).

    Google Scholar 

  64. 64.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Sigworth, F. J. The variance of sodium current fluctuations at the node of Ranvier. J. Physiol. 307, 97–129 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Yang, F., Cui, Y., Wang, K. & Zheng, J. Thermosensitive TRP channel pore turret is part of the temperature activation pathway. Proc. Natl. Acad. Sci. USA 107, 7083–7088 (2010).

    CAS  PubMed  Google Scholar 

Download references


Single-particle cryo-EM data were collected at Center of Cryo-Electron Microscopy at Zhejiang University. We are grateful to Dr. Xing Zhang, Shenghai Chang, and Xiaokang Zhang for their support in facility access and data acquisition. We thank Dr. Fan Yang and Lizhen Xu for their assistance with the noise analysis of the electrophysiological data. We thank the support of ECNU Multifunctional Platform for Innovation (001). This work was supported in part by the Ministry of Science and Technology of China (2018YFA0508100 to J.G. and Q.Z., and 2016YFA0500404 to S.Y.), the National Natural Science Foundation of China (31870724 to J.G., 31800699 to Q.Z., 31525001 and 31430019 to S.Y.), the Fundamental Research Funds for the Central Universities (to J.G. and H.Y.), the “Personalized Medicines-Molecular Signature-based Drug Discovery and Development”, the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12040220 to H.Y.), the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (2018ZX09711002 to Q.Z.), and the “XingFuZhiHua” funding of ECNU (44300-19311-542500/006 to H.Y.).

Author information




J.G., H.Y. and Q.Z. conceived and designed this project. X.L., J.W., D.Lai, D.Lv, S.Y. and J.G. prepared the samples, and performed data acquisition, image processing and structure determination; Q.Z., P.G., J.F., L.M. and H.Y. did electrophysiological recording. All authors participated in the data analysis and manuscript preparation.

Corresponding authors

Correspondence to Huaiyu Yang or Jiangtao Guo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, X., Zhang, Q., Guo, P. et al. Molecular basis for ligand activation of the human KCNQ2 channel. Cell Res (2020).

Download citation