Skip to main content
SpringerLink
Log in

Electrophysiology on Channel-Forming Proteins in Artificial Lipid Bilayers: Next-Generation Instrumentation for Multiple Recordings in Parallel

  • Protocol
  • First Online:
Patch Clamp Electrophysiology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2188))

Abstract

Artificial lipid bilayers have been used for several decades to study channel-forming pores and ion channels in membranes. Until recently, the classical two-chamber setups have been primarily used for studying the biophysical properties of pore forming proteins. Within the last 10 years, instruments for automated lipid bilayer measurements have been developed and are now commercially available. This chapter focuses on protein purification and reconstitution of channel-forming proteins into lipid bilayers using a classic setup and on the commercially available systems, the Orbit mini and Orbit 16.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Miller C (1986) Ion channel reconstitution, 1st edn. Springer, New York, NY

    Book  Google Scholar 

  2. Mueller P, Rudin DO (1963) Induced excitability in reconstituted cell membrane structure. J Theor Biol 4:268–280. https://doi.org/10.1016/0022-5193(63)90006-7

    Article  CAS  Google Scholar 

  3. Mueller P, Rudin DO (1967) Action potential phenomena in experimental bimolecular lipid membranes. Nature 213:603–604. https://doi.org/10.1038/213603a0

    Article  CAS  Google Scholar 

  4. Mueller P, Rudin DO (1967) Development of K+-Na+ discrimination in experimental bimolecular lipid membranes by macrocyclic antibiotics. Biochem Biophys Res Commun 26:398–404. https://doi.org/10.1016/0006-291x(67)90559-1

    Article  CAS  Google Scholar 

  5. Bean RC, Shepherd WC, Chan H, Eichner J (1969) Discrete conductance fluctuations in lipid bilayer protein membranes. J Gen Physiol 53:741–757

    Article  CAS  Google Scholar 

  6. Ehrenstein G, Lecar H, Nossal R (1970) The nature of the negative resistance in bimolecular lipid membranes containing excitability-inducing material. J Gen Physiol 55:119–133. https://doi.org/10.1085/jgp.55.1.119

    Article  CAS  Google Scholar 

  7. Hladky SB, Haydon DA (1970) Discreteness of conductance change in bimolecular lipid membranes in the presence of certain antibiotics. Nature 225:451–453. https://doi.org/10.1038/225451a0

    Article  CAS  Google Scholar 

  8. Gordon LG, Haydon DA (1972) The unit conductance channel of alamethicin. Biochim Biophys Acta Biomembr 255:1014–1018. https://doi.org/10.1016/0005-2736(72)90415-4

    Article  CAS  Google Scholar 

  9. Eisenberg M, Hall JE, Mead C (1973) The nature of the voltage-dependent conductance induced by alamethicin in black lipid membranes. J Membr Biol 14:143–176

    Article  CAS  Google Scholar 

  10. Behrends JC (2012) Evolution of the ion channel concept: the historical perspective. Chem Rev 112:6218–6226. https://doi.org/10.1021/cr300349g

    Article  CAS  Google Scholar 

  11. Hille B (2001) Ion channels of excitable membranes, 3rd edn. Sinauer Associates, Inc, Sunderland, MA

    Google Scholar 

  12. Sakmann B, Neher E (1995) Single-channel recording. Spinger, New York, NY

    Book  Google Scholar 

  13. White SH (1986) The physical nature of planar bilayer membranes. In: Miller C (ed) Ion channel reconst., 1st edn. Springer, Boston, MA, pp 3–35

    Chapter  Google Scholar 

  14. Finkelstein A (1974) Bilayers: formation, measurements, and incorporation of components. Methods Enzymol 32:489–501. https://doi.org/10.1016/0076-6879(74)32049-6

    Article  CAS  Google Scholar 

  15. Ehrlich BE (1992) Planar lipid bilayers on patch pipettes: bilayer formation and ion channel incorporation. Methods Enzymol 207:463–470. https://doi.org/10.1016/0076-6879(92)07033-k

    Article  CAS  Google Scholar 

  16. Miller C (1983) First steps in the reconstruction of ionic channel functions in model membranes. In: Barker JL, McKelvy JF (eds) Curr. methods cell. neurobiol. Wiley, New York, NY, pp 1–37

    Google Scholar 

  17. Williams AJ (1994) An introduction to the methods available for ion channel reconstitution. In: Ogden D (ed) Microelectrode Tech Plymouth work. Handbook. Company of Biologists Limited, Cambridge, UK, pp 79–99

    Google Scholar 

  18. Mittermeier L, Demirkhanyan L, Stadlbauer B et al (2019) TRPM7 is the central gatekeeper of intestinal mineral absorption essential for postnatal survival. Proc Natl Acad Sci U S A 116:4706–4715. https://doi.org/10.1073/pnas.1810633116

    Article  CAS  Google Scholar 

  19. Baaken G, Sondermann M, Schlemmer C et al (2008) Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents. Lab Chip 8:938–944. https://doi.org/10.1039/b800431e

    Article  CAS  Google Scholar 

  20. Baaken G, Ankri N, Schuler A-K et al (2011) Nanopore-based single-molecule mass spectrometry on a lipid membrane microarray. ACS Nano 5:8080–8088. https://doi.org/10.1021/nn202670z

    Article  CAS  Google Scholar 

  21. Baaken G, Halimeh I, Bacri L et al (2015) High-resolution size-discrimination of single nonionic synthetic polymers with a highly charged biological nanopore. ACS Nano 9:6443–6449. https://doi.org/10.1021/acsnano.5b02096

    Article  CAS  Google Scholar 

  22. del Rio Martinez JM, Zaitseva E, Petersen S et al (2015) Automated formation of lipid membrane microarrays for ionic single-molecule sensing with protein nanopores. Small 11:119–125. https://doi.org/10.1002/smll.201402016

    Article  CAS  Google Scholar 

  23. Warner. https://www.warneronline.com/classic-bilayer-cups-chambers. Accessed 23 July 2019

  24. Elements. https://elements-ic.com/blmkit-page/. Accessed 23 July 2019

  25. Tanford C (1980) The hydrophobic effect: formation of micelles and biological membranes. Wiley Interscience, New York, NY

    Google Scholar 

  26. White SH, Petersen DC, Simon S, Yafuso M (1976) Formation of planar bilayer membranes from lipid monolayers. A critique. Biophys J 16:481–489. https://doi.org/10.1016/S0006-3495(76)85703-7

    Article  CAS  Google Scholar 

  27. Niles WD, Levis RA, Cohen FS (1988) Planar bilayer membranes made from phospholipid monolayers form by a thinning process. Biophys J 53:327–335. https://doi.org/10.1016/S0006-3495(88)83110-2

    Article  CAS  Google Scholar 

  28. He Y, Wang K, Yan N (2014) The recombinant expression systems for structure determination of eukaryotic membrane proteins. Protein Cell 5:658–672. https://doi.org/10.1007/s13238-014-0086-4

    Article  CAS  Google Scholar 

  29. Popot J-L (2014) Folding membrane proteins in vitro: a table and some comments. Arch Biochem Biophys 564:314–326. https://doi.org/10.1016/j.abb.2014.06.029

    Article  CAS  Google Scholar 

  30. Schwarzer TS, Hermann M, Krishnan S et al (2017) Preparative refolding of small monomeric outer membrane proteins. Protein Expr Purif 132:171–181. https://doi.org/10.1016/j.pep.2017.01.012

    Article  CAS  Google Scholar 

  31. Jensen HM, Eng T, Chubukov V et al (2017) Improving membrane protein expression and function using genomic edits. Sci Rep 7:13030. https://doi.org/10.1038/s41598-017-12901-7

    Article  CAS  Google Scholar 

  32. Ishchenko A, Abola EE, Cherezov V (2017) Crystallization of membrane proteins: an overview. In: Wlodawer A, Dauter Z, Jaskolski M (eds) Protein crystallogr, Methods mol. Biol, vol 1607. Humana, New York, NY, pp 117–141

    Chapter  Google Scholar 

  33. Kubicek J, Block H, Maertens B et al (2014) Expression and purification of membrane proteins. Methods Enzymol 541:117–140. https://doi.org/10.1016/B978-0-12-420119-4.00010-0

    Article  CAS  Google Scholar 

  34. Niederweis M, Ehrt S, Heinz C et al (1999) Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis. Mol Microbiol 33:933–945. https://doi.org/10.1046/j.1365-2958.1999.01472.x

    Article  CAS  Google Scholar 

  35. Niederweis M (2003) Mycobacterial porins - new channel proteins in unique outer membranes. Mol Microbiol 49:1167–1177. https://doi.org/10.1046/j.1365-2958.2003.03662.x

    Article  CAS  Google Scholar 

  36. Faller M, Niederweis M, Schulz GE (2004) The structure of a mycobacterial outer-membrane channel. Science 303:1189–1192. https://doi.org/10.1126/science.1094114

    Article  CAS  Google Scholar 

  37. Derrington IM, Butler TZ, Collins MD et al (2010) Nanopore DNA sequencing with MspA. Proc Natl Acad Sci U S A 107:16060–16065. https://doi.org/10.1073/pnas.1001831107

    Article  Google Scholar 

  38. Manrao EA, Derrington IM, Laszlo AH et al (2012) Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat Biotechnol 30:349–353. https://doi.org/10.1038/nbt.2171

    Article  CAS  Google Scholar 

  39. Rues R-B, Henrich E, Boland C et al (2016) Cell-free production of membrane proteins in Escherichia coli lysates for functional and structural studies. In: Mus-Veteau I (ed) Heterologous expr. membr. proteins, Methods Mol. Biol, vol 1432. Humana, New York, NY, pp 1–21

    Chapter  Google Scholar 

  40. He W, Felderman M, Evans AC et al (2017) Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development. J Biol Chem 292:15121–15132. https://doi.org/10.1074/jbc.M117.784561

    Article  CAS  Google Scholar 

  41. Kovácsová G, Gustavsson E, Wang J et al (2015) Cell-free expression of a functional pore-only sodium channel. Protein Expr Purif 111:42–47. https://doi.org/10.1016/j.pep.2015.03.002

    Article  CAS  Google Scholar 

  42. Focke PJ, Hein C, Hoffmann B et al (2016) Combining in vitro folding with cell free protein synthesis for membrane protein expression. Biochemistry 55:4212–4219. https://doi.org/10.1021/acs.biochem.6b00488

    Article  CAS  Google Scholar 

  43. Renauld S, Cortes S, Bersch B et al (2017) Functional reconstitution of cell-free synthesized purified Kv channels. Biochim Biophys Acta Biomembr 1859:2373–2380. https://doi.org/10.1016/j.bbamem.2017.09.002

    Article  CAS  Google Scholar 

  44. Winterstein L-M, Kukovetz K, Rauh O et al (2018) Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers. J Gen Physiol 150:jgp.201711904. https://doi.org/10.1085/jgp.201711904

    Article  CAS  Google Scholar 

  45. Henrich E, Peetz O, Hein C et al (2017) Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology. Elife 6:e20954. https://doi.org/10.7554/eLife.20954

    Article  Google Scholar 

  46. Foshag D, Henrich E, Hiller E et al (2018) The E. coli S30 lysate proteome: a prototype for cell-free protein production. New Biotechnol 40:245–260. https://doi.org/10.1016/j.nbt.2017.09.005

    Article  CAS  Google Scholar 

  47. Rues R-B, Gräwe A, Henrich E, Bernhard F (2017) Membrane protein production in E. coli lysates in presence of preassembled nanodiscs. In: Burgess-Brown N (ed) Heterologous gene expr. E. coli, Methods Mol. Biol, vol 1586. Humana, New York, NY, pp 291–312

    Chapter  Google Scholar 

  48. Banerjee S, Nimigean CM (2011) Non-vesicular transfer of membrane proteins from nanoparticles to lipid bilayers. J Gen Physiol 137:217–223. https://doi.org/10.1085/jgp.201010558

    Article  CAS  Google Scholar 

  49. Patriarchi T, Shen A, He W et al (2018) Nanodelivery of a functional membrane receptor to manipulate cellular phenotype. Sci Rep 8:3556. https://doi.org/10.1038/s41598-018-21863-3

    Article  CAS  Google Scholar 

  50. Reiter R, Zaitseva E, Baaken G et al. (2019) Activity of the Gramicidin A Ion Channel in a Lipid Membrane with Switchable Physical Properties. Langmuir 35:14959–14966. https://doi.org/10.1021/acs.langmuir.9b02752

  51. Stockbridge RB, Tsai M-F (2015) Lipid reconstitution and recording of recombinant ion channels. Methods Enzymol 556:385–404. https://doi.org/10.1016/bs.mie.2014.12.028

    Article  CAS  Google Scholar 

  52. Knol J, Sjollema K, Poolman B (1998) Detergent-mediated reconstitution of membrane proteins. Biochemistry 37:16410–16415. https://doi.org/10.1021/bi981596u

    Article  CAS  Google Scholar 

  53. Woodbury DJ (1999) Nystatin/ergosterol method for reconstituting ion channels into planar lipid bilayers. Methods Enzymol 294:319–339. https://doi.org/10.1016/S0076-6879(99)94020-X

    Article  CAS  Google Scholar 

  54. de Planque M, de Planque M, Mendes G et al (2006) Controlled delivery of membrane proteins to artificial lipid bilayers by nystatin-ergosterol modulated vesicle fusion. IEE Proc Nanobiotechnol 153:21–30. https://doi.org/10.1049/ip-nbt:20050039

    Article  CAS  Google Scholar 

  55. Sakmann B, Neher E (2009) Single-channel recording, 2nd edn. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-1229-9

    Book  Google Scholar 

  56. Ogden D (1994) Microelectrode techniques: the Plymouth workshop handbook, 2nd edn. Company of Biologists Limited, Cambridge, UK

    Google Scholar 

  57. Shermann-Gold R (2012) The Axon guide. https://mdc.custhelp.com/euf/assets/content/AxonGuide3rdedition.pdf. Accessed 23 July 2019

  58. Forstater JH, Briggs K, Robertson JWF et al (2016) MOSAIC: a modular single-molecule analysis interface for decoding multistate nanopore data. Anal Chem 88:11900–11907. https://doi.org/10.1021/acs.analchem.6b03725

    Article  CAS  Google Scholar 

  59. MOSAIC. https://pages.nist.gov/mosaic/. Accessed 23 July 2019

  60. Wyllie DJ, Behe P, Colquhoun D (1998) Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors [published erratum appears in J Physiol (Lond) 1998 Nov 1;512(Pt 3):939]. J Physiol 510:1–18

    Article  CAS  Google Scholar 

  61. Lape R, Colquhoun D, Sivilotti LG (2008) On the nature of partial agonism in the nicotinic receptor superfamily. Nature 454:722–727. https://doi.org/10.1038/nature07139

    Article  CAS  Google Scholar 

  62. Colquhoun D, Hawkes AG (2009) The principles of the stochastic interpretation of ion-channel mechanisms. In: Sakmann B, Neher E (eds) Single channel rec., 2nd edn. Springer Science+Business Media LLC, New York, NY, pp 397–482

    Google Scholar 

  63. Colquhoun D, Hawkes AG (1994) The interpretation of single channel recordings. In: Ogden DC (ed) Microelectrode tech. Plymouth work. Handbook., 2nd edn. Company of Biologists Limited, Cambridge, UK, pp 141–188

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Ionera Technologies GmbH, Freiburg, Germany

    Ekaterina Zaitseva, Mordjane Boukhet & Gerhard Baaken

  2. Nanion Technologies GmbH, Munich, Germany

    Alison Obergrussberger, Conrad Weichbrodt & Niels Fertig

  3. Institute of Biophysical Chemistry & Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Frankfurt am Main, Germany

    Frank Bernhard & Christopher Hein

  4. Laboratory for Membrane Physiology and Technology, Department of Physiology, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Jan C. Behrends

Authors
  1. Ekaterina Zaitseva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alison Obergrussberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Conrad Weichbrodt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mordjane Boukhet
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Frank Bernhard
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christopher Hein
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gerhard Baaken
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Niels Fertig
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jan C. Behrends
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alison Obergrussberger .

Editor information

Editors and Affiliations

  1. University of Reading, Reading, UK

    Mark Dallas

  2. Sophion Bioscience A/S, Ballerup, Denmark

    Damian Bell

1 Electronic Supplementary Material

The air bubble lipid bilayer formation technique on the Orbit mini. (MP4 56896 kb)

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Zaitseva, E. et al. (2021). Electrophysiology on Channel-Forming Proteins in Artificial Lipid Bilayers: Next-Generation Instrumentation for Multiple Recordings in Parallel. In: Dallas, M., Bell, D. (eds) Patch Clamp Electrophysiology. Methods in Molecular Biology, vol 2188. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0818-0_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0818-0_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0817-3

  • Online ISBN: 978-1-0716-0818-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation