Skip to main content
SpringerLink
Log in

Recombinant Production, Reconstruction in Lipid–Protein Nanodiscs, and Electron Microscopy of Full-Length α-Subunit of Human Potassium Channel Kv7.1

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

Voltage-gated potassium channel Kv7.1 plays an important role in the excitability of cardiac muscle. The α-subunit of Kv7.1 (KCNQ1) is the main structural element of this channel. Tetramerization of KCNQ1 in the membrane results in formation of an ion channel, which comprises a pore and four voltage-sensing domains. Mutations in the human KCNQ1 gene are one of the major causes of inherited arrhythmias, long QT syndrome in particular. The construct encoding full-length human KCNQ1 protein was synthesized in this work, and an expression system in the Pichia pastoris yeast cells was developed. The membrane fraction of the yeast cells containing the recombinant protein (rKCNQ1) was solubilized with CHAPS detergent. To better mimic the lipid environment of the channel, lipid–protein nanodiscs were formed using solu- bilized membrane fraction and MSP2N2 protein. The rKCNQ1/nanodisc and rKCNQ1/CHAPS samples were purified using the Rho1D4 tag introduced at the C-terminus of the protein. Protein samples were examined using transmission electron microscopy with negative staining. In both cases, homogeneous rKCNQ1 samples were observed based on image analysis. Statistical analysis of the images of individual protein particles solubilized in the detergent revealed the presence of a tetrameric structure confirming intact subunit assembly. A three-dimensional channel structure reconstructed at 2.5-nm resolution represents a compact density with diameter of the membrane part of ~9 nm and height ~11 nm. Analysis of the images of rKCNQ1 in nanodiscs revealed additional electron density corresponding to the lipid bilayer fragment and the MSP2N2 protein. These results indicate that the nanodiscs facilitate protein isolation, purification, and stabilization in solution and can be used for further structural studies of human Kv7.1.

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

Similar content being viewed by others

Abbreviations

CaM:

calmodulin

FSC:

Fourier shell correlation

Kv:

voltage-gated K+ channel

LPN:

lipid–protein nanodisc

MP:

membrane protein

MSA:

multidimensional statistical analysis

MSP:

membrane scaffold protein

PIP2 :

phosphatidylinositol-4,5-bisphosphate

rKCNQ1:

recombinant analog of α-subunit of human KCNQ1 channel Kv7.1

TCEP:

Tris(2-carboxyethyl)phosphine

TEM:

transmission electron microscopy

TM:

transmembrane

VSD:

voltage-sensitive domain

References

  1. Yellen, G. (2002) The voltage-gated potassium channels and their relatives, Nature, 419, 35–42.

    Article  PubMed  CAS  Google Scholar 

  2. Singer-Lahat, D., Chikvashvili, D., and Lotan, I. (2008) Direct interaction of endogenous Kv channels with syntax-in enhances exocytosis by neuroendocrine cells, PLoS One, 3, e1381.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. MacDonald, P. E., and Wheeler, M. B. (2003) Voltage-dependent K+ channels in pancreatic beta cells: role, regu-lation and potential as therapeutic targets, Diabetologia, 46, 1046–1062.

    Article  PubMed  CAS  Google Scholar 

  4. Pal, S., Hartnett, K. A., Nerbonne, J. M., Levitan, E. S., and Aizenman, E. (2003) Mediation of neuronal apoptosis by Kv2.1-encoded potassium channels, J. Neurosci., 23, 4798–4802.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Deutsch, C., and Chen, L. Q. (1993) Heterologous expres-sion of specific K+ channels in T lymphocytes: functional consequences for volume regulation, Proc. Natl. Acad. Sci. USA, 90, 10036–10040.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Camacho, J. (2006) Ether a go-go potassium channels and cancer, Cancer Lett., 233, 1–9.

    Article  PubMed  CAS  Google Scholar 

  7. Judge, S. I. V., and Bever, C. T. (2006) Potassium channel blockers in multiple sclerosis: neuronal Kv channels and effects of symptomatic treatment, Pharmacol. Ther., 111, 224–259.

    Article  PubMed  CAS  Google Scholar 

  8. Beekwilder, J. P., O’Leary, M. E., van den Broek, L. P., van Kempen, G. T. H., Ypey, D. L., and van den Berg, R. J. (2003) Kv1.1 channels of dorsal root ganglion neurons are inhibited by n-butyl-p-aminobenzoate, a promising anes-thetic for the treatment of chronic pain, J. Pharmacol. Exp. Ther., 304, 531–538.

    Article  PubMed  CAS  Google Scholar 

  9. Watanabe, H., Nagata, E., Kosakai, A., Nakamura, M., Yokoyama, M., Tanaka, K., and Sasai, H. (2000) Disruption of the epilepsy KCNQ2 gene results in neural hyperexcitability, J. Neurochem., 75, 28–33.

    Article  PubMed  CAS  Google Scholar 

  10. Tester, D. J., and Ackerman, M. J. (2014) Genetics of long QT syndrome, Methodist Debakey Cardiovasc. J., 10, 29–33.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Nakajo, K., Ulbrich, M. H., Kubo, Y., and Isacoff, E. Y. (2010) Stoichiometry of the KCNQ1–KCNE1 ion channel complex, Proc. Natl. Acad. Sci. USA, 107, 18862–18867.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Zaydman, M. A., Silva, J. R., Delaloye, K., Li, Y., Liang, H., Larsson, H. P., Shi, J., and Cui, J. (2013) Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening, Proc. Natl. Acad. Sci. USA, 110, 13180–13185.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Shamgar, L., Ma, L., Schmitt, N., Haitin, Y., Peretz, A., Wiener, R., Hirsch, J., Pongs, O., and Attali, B. (2006) Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations, Circ. Res., 98, 1055–1063.

    Article  PubMed  CAS  Google Scholar 

  14. Kosenko, A., and Hoshi, N. (2013) A change in configura-tion of the calmodulin–KCNQ channel complex underlies Ca2+-dependent modulation of KCNQ channel activity, PLoS One, 8, e82290.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Inanobe, A., Tsuzuki, C., and Kurachi, Y. (2015) An epithelial Ca2+-sensor protein is an alternative to calmod-ulin to compose functional KCNQ1 channels, Cell Physiol. Biochem., 36, 1847–1861.

    Article  PubMed  CAS  Google Scholar 

  16. Jensen, M. O., Jogini, V., Borhani, D. W., Leffler, A. E., Dror, R. O., and Shaw, D. E. (2012) Mechanism of voltage gating in potassium channels, Science, 336, 229–233.

    Article  PubMed  CAS  Google Scholar 

  17. Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B. T., and MacKinnon, R. (2003) X-ray structure of a volt-age-dependent K+ channel, Nature, 423, 33–41.

    Article  PubMed  CAS  Google Scholar 

  18. Long, S. B., Tao, X., Campbell, E. B., and MacKinnon, R. (2007) Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment, Nature, 450, 376–382.

    Article  PubMed  CAS  Google Scholar 

  19. McCusker, E. C., Bane, S. E., O’Malley, M. A., and Robinson, A. S. (2007) Heterologous GPCR expression: a bottleneck to obtaining crystal structures, Biotechnol. Prog., 23, 540–547.

    Article  PubMed  CAS  Google Scholar 

  20. Lee, S.-Y., Lee, A., Chen, J., and MacKinnon, R. (2005) Structure of the KvAP voltage-dependent K+ channel and its dependence on the lipid membrane, Proc. Natl. Acad. Sci. USA, 102, 15441–15446.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Whicher, J. R., and MacKinnon, R. (2016) Structure of the voltage-gated K+ channel Eag1 reveals an alternative volt-age sensing mechanism, Science, 353, 664–669.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Lee, C.-H., and MacKinnon, R. (2017) Structures of the human HCN1 hyperpolarization-activated channel, Cell, 168, 111–120.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Wang, W., and MacKinnon, R. (2017) Cryo-EM structure of the open human ether-a-go-go-related K+ channel hERG, Cell, 169, 422–430.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  25. Grinkova, Y. V., Denisov, I. G., and Sligar, S. G. (2010) Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers, Protein Eng. Des. Sel., 23, 843–848.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Yoshiura, C., Kofuku, Y., Ueda, T., Mase, Y., Yokogawa, M., Osawa, M., Terashima, Y., Matsushima, K., and Shimada, I. (2010) NMR analyses of the interaction between CCR5 and its ligand using functional reconstitu-tion of CCR5 in lipid bilayers, J. Am. Chem. Soc., 132, 6768–6777.

    Article  PubMed  CAS  Google Scholar 

  27. Shenkarev, Z. O., Lyukmanova, E. N., Solozhenkin, O. I., Gagnidze, I. E., Nekrasova, O. V., Chupin, V. V., Tagaev, A. A., Yakimenko, Z. A., Ovchinnikova, T. V., Kirpichnikov, M. P., and Arseniev, A. S. (2009) Lipid–protein nanodiscs: possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides, Biochemistry (Moscow), 74, 756–765.

    Article  CAS  Google Scholar 

  28. Ludtke, S. J. (2016) Single-particle refinement and vari-ability analysis in EMAN2.1, Methods Enzymol., 579, 159–189.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Van Heel, M., Portugal, R., Schatz, M., Orlova, E., and Verkleij, A. (eds.) (2009) Handbook on DVD 3D-EM in Life Sciences, 3D-EM Network of Excellence, European Commission, London.

    Google Scholar 

  30. Devaraneni, P. K., Devereaux, J. J., and Valiyaveetil, F. I. (2011) In vitro folding of KvAP, a voltage-gated K+ channel, Biochemistry, 50, 10442–10450.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Parcej, D. N., and Eckhardt-Strelau, L. (2003) Structural characterization of neuronal voltage-sensitive K+ channels heterologously expressed in Pichia pastoris, J. Mol. Biol., 333, 103–116.

    Article  PubMed  CAS  Google Scholar 

  32. Dhillon, M. S., Cockcroft, C. J., Munsey, T., Smith, K. J., Powell, A. J., Carter, P., Wrighton, D. C., Rong, H., Yusaf, S. P., and Sivaprasadarao, A. (2014) A functional Kv1.2-hERG chimeric channel expressed in Pichia pastoris, Sci. Rep., 4, 4201.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Molbaek, K., Scharff-Poulsen, P., Helix-Nielsen, C., Klaerke, D. A., and Pedersen, P. A. (2015) High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae, Microb. Cell Fact., 14, 15.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Dahimene, S., Alcolea, S., Naud, P., Jourdon, P., Escande, D., Brasseur, R., Thomas, A., Baro, I., and Merot, J. (2006) The N-terminal juxtamembranous domain of KCNQ1 is critical for channel surface expression: implica-tions in the Romano–Ward LQT1 syndrome, Circ. Res., 99, 1076–1083.

    Article  PubMed  CAS  Google Scholar 

  35. Oprian, D. D., Molday, R. S., Kaufman, R. J., and Khorana, H. G. (1987) Expression of a synthetic bovine rhodopsin gene in monkey kidney cells, Proc. Natl. Acad. Sci. USA, 84, 8874–8878.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Lorenz-Fonfria, V., Peralvarez-Marin, A., Padros, E., and Lazarova, T. (2011) Chap. 12. Solubilization, purification, and characterization of integral membrane proteins, in Production of Membrane Proteins: Strategies for Expression and Isolation (Robinson, A. S., ed.), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, pp. 317–360.

    Chapter  Google Scholar 

  37. Sokolova, O. S., Shaitan, K. V., Grizel’, A. V., Popinako, A. V., Karlova, M. G., and Kirpichnikov, M. P. (2012) Three-dimensional structure of human Kv10.2 ion channel studied by single particle electron microscopy and molecu-lar modeling, Russ. J. Bioorg. Chem., 38, 152–158.

    Article  CAS  Google Scholar 

  38. Rath, A., Glibowicka, M., Nadeau, V. G., Chen, G., and Deber, C. M. (2009) Detergent binding explains anomalous SDS-PAGE migration of membrane proteins, Proc. Natl. Acad. Sci. USA, 106, 1760–1765.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Frauenfeld, J., Gumbart, J., Sluis, E. O., van der Funes, S., Gartmann, M., Beatrix, B., Mielke, T., Berninghausen, O., Becker, T., Schulten, K., and Beckmann, R. (2011) Cryo-EM structure of the ribosome–SecYE complex in the membrane environment, Nat. Struct. Mol. Biol., 18, 614–621.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Gao, Y., Cao, E., Julius, D., and Cheng, Y. (2016) TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action, Nature, 534, 347–351.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Shen, P. S., Yang, X., DeCaen, P. G., Liu, X., Bulkley, D., Clapham, D. E., and Cao, E. (2016) The structure of the polycystic kidney disease channel PKD2 in lipid nanodiscs, Cell, 167, 763–773.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  42. Shenkarev, Z. O., Lyukmanova, E. N., Paramonov, A. S., Shingarova, L. N., Chupin, V. V., Kirpichnikov, M. P., Blommers, M. J., and Arseniev, A. S. (2010) Lipid–protein nanodiscs as reference medium in detergent screening for high-resolution NMR studies of integral membrane pro-teins, J. Am. Chem. Soc., 132, 5628–5629.

    Article  PubMed  CAS  Google Scholar 

  43. Van Heel, M. (1987) Angular reconstitution: a posteriori assignment of projection directions for 3D reconstruction, Ultramicroscopy, 21, 111–123.

    Article  PubMed  Google Scholar 

  44. Howard, R. J., Clark, K. A., Holton, J. M., and Minor, D. L. (2007) Structural insight into KCNQ Kv7 channel assembly and channelopathy, Neuron, 53, 663–675.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Merk, A., Bartesaghi, A., Banerjee, S., Falconieri, V., Rao, P., Davis, M. I., Pragani, R., Boxer, M. B., Earl, L. A., Milne, J. L. S., and Subramaniam, S. (2016) Breaking cryo-EM resolution barriers to facilitate drug discovery, Cell, 165, 1698–1707.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Shemyakin−Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Russia

    Z. O. Shenkarev, M. G. Karlova, D. S. Kulbatskii, M. P. Kirpichnikov & E. N. Lyukmanova

  2. Lomonosov Moscow State University, Faculty of Biology, 119991, Moscow, Russia

    M. G. Karlova, D. S. Kulbatskii, M. P. Kirpichnikov, E. N. Lyukmanova & O. S. Sokolova

Authors
  1. Z. O. Shenkarev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. G. Karlova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D. S. Kulbatskii
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. P. Kirpichnikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. N. Lyukmanova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. O. S. Sokolova
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Z. O. Shenkarev or O. S. Sokolova.

Additional information

Original Russian Text © Z. O. Shenkarev, M. G. Karlova, D. S. Kulbatskii, M. P. Kirpichnikov, E. N. Lyukmanova, O. S. Sokolova, 2018, published in Biokhimiya, 2018, Vol. 83, No. 5, pp. 735–748.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shenkarev, Z.O., Karlova, M.G., Kulbatskii, D.S. et al. Recombinant Production, Reconstruction in Lipid–Protein Nanodiscs, and Electron Microscopy of Full-Length α-Subunit of Human Potassium Channel Kv7.1. Biochemistry Moscow 83, 562–573 (2018). https://doi.org/10.1134/S0006297918050097

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297918050097

Keywords

Search

Navigation