Patch Clamp Electrophysiology for the Study of Bacterial Ion Channels in Giant Spheroplasts of E. coli

  • Boris MartinacEmail author
  • Paul R. Rohde
  • Charles G. Cranfield
  • Takeshi Nomura
Part of the Methods in Molecular Biology book series (MIMB, volume 966)


Ion channel studies have been focused on ion channels from animal and human cells over many years. Based on the knowledge acquired, predominantly over the last 20 years, a large diversity of ion channels exists in cellular membranes of prokaryotes as well. Paradoxically, most of what is known about the structure of eukaryotic ion channels is based on the structure of bacterial channels. This is largely due to the suitability of bacterial cells for functional and structural studies of biological macromolecules in a laboratory environment. Development of the “giant spheroplast” preparation from E. coli cells was instrumental for functional studies of ion channels in the bacterial cell membrane. Here we describe detailed protocols used for the preparation of giant spheroplasts as well as protocols used for the patch-clamp recording of native or heterologously expressed ion channels in E. coli spheroplast membrane.

Key words

Bacteria Archaea Cephalexin Lysozyme Mechanosensitive channels K+ channels Patch clamp Laplace’s law 



We wish to thank Maryrose Constantine as well as Drs. Anna Kloda, Evgeny Petrov, and Andrew Battle for critical reading and constructive comments on a draft of the manuscript. This work was supported by the Australian Research Council, the National Health and Medical Research Council of Australia, and Yamada Science Foundation.


  1. 1.
    Kubalski A, Martinac B (eds) (2005) Bacterial ion channels and their eukaryotic homologues. ASM, Washington, DCGoogle Scholar
  2. 2.
    Hamill OP et al (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391(2):85–100PubMedCrossRefGoogle Scholar
  3. 3.
    Ruthe HJ, Adler J (1985) Fusion of bacterial spheroplasts by electric fields. Biochim Biophys Acta 819(1):105–113PubMedCrossRefGoogle Scholar
  4. 4.
    Martinac B et al (1987) Pressure-sensitive ion channel in Escherichia coli. Proc Natl Acad Sci USA 84(8):2297–2301PubMedCrossRefGoogle Scholar
  5. 5.
    Martinac B, Saimi Y, Kung C (2008) Ion channels in microbes. Physiol Rev 88(4):1449–1490PubMedCrossRefGoogle Scholar
  6. 6.
    Blount P et al (1999) Mechanosensitive channels of bacteria. Methods Enzymol 294:458–482PubMedCrossRefGoogle Scholar
  7. 7.
    Martinac B (2011) Bacterial mechanosensitive channels as a paradigm for mechanosensory transduction. Cell Physiol Biochem 28(6):1051–1060PubMedCrossRefGoogle Scholar
  8. 8.
    Hamill OP, Martinac B (2001) Molecular basis of mechanotransduction in living cells. Physiol Rev 81(2):685–740PubMedGoogle Scholar
  9. 9.
    Nguyen T et al (2005) The effects of parabens on the mechanosensitive channels of E. coli. Eur Biophys J 34(5):389–395PubMedCrossRefGoogle Scholar
  10. 10.
    Levina N et al (1999) Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J 18(7):1730–1737PubMedCrossRefGoogle Scholar
  11. 11.
    Sukharev SI et al (1993) Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution. Biophys J 65(1):177–183PubMedCrossRefGoogle Scholar
  12. 12.
    Sukharev SI, Blount P, Martinac B, Kung C (1994) Functional reconstitution as an assay for biochemical isolation of channel proteins: application to the molecular identification of a bacterial mechanosensitive channel. Methods 6:51–59CrossRefGoogle Scholar
  13. 13.
    Häse CC, Le Dain AC, Martinac B (1995) Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli. J Biol Chem 270(31):18329–18334PubMedCrossRefGoogle Scholar
  14. 14.
    Edwards MD et al (2005) Pivotal role of the glycine-rich TM3 helix in gating the MscS mechanosensitive channel. Nat Struct Mol Biol 12(2):113–119PubMedCrossRefGoogle Scholar
  15. 15.
    Kloda A, Martinac B (2001) Structural and functional differences between two homologous mechanosensitive channels of Methanococcus jannaschii. EMBO J 20(8):1888–1896PubMedCrossRefGoogle Scholar
  16. 16.
    Schumann U, Edwards MD, Rasmussen T, Bartlett W, van West P, Booth IR (2010) YbdG in Escherichia coli is a threshold-setting mechanosensitive channel with MscM activity. Proc Natl Acad Sci USA 107:12664–12669PubMedCrossRefGoogle Scholar
  17. 17.
    Blount P et al (1996) Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli. Proc Natl Acad Sci USA 93(21):11652–11657PubMedCrossRefGoogle Scholar
  18. 18.
    Saimi Y, Loukin SH, Zhou X-L, Martinac B, Kung C (1999) Ion channels in microbes. Methods Enzymol 294:507–524PubMedCrossRefGoogle Scholar
  19. 19.
    Cortes DM, Cuello LG, Perozo E (2001) Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating. J Gen Physiol 117(2):165–180PubMedCrossRefGoogle Scholar
  20. 20.
    Cordero-Morales JF, Cuello LG, Perozo E (2006) Voltage-dependent gating at the KcsA selectivity filter. Nat Struct Mol Biol 13(4):319–322PubMedCrossRefGoogle Scholar
  21. 21.
    Blount P, Sukharev SI, Moe PC, Martinac B, Kung C (1999) Mechanosensitive channels in bacteria. Methods Enzymol 294:458–482PubMedCrossRefGoogle Scholar
  22. 22.
    Santos JS et al (2006) Molecular template for a voltage sensor in a novel K+ channel. I. Identification and functional characterization of KvLm, a voltage-gated K+ channel from Listeria monocytogenes. J Gen Physiol 128(3):283–292PubMedCrossRefGoogle Scholar
  23. 23.
    Vásquez V et al (2008) A structural mechanism for MscS gating in lipid bilayers. Science 321(5893):1210–1214PubMedCrossRefGoogle Scholar
  24. 24.
    Sukharev SI, Martinac B, Arshavsky VY, Kung C (1993) Two types of mechanosensitive channels in the E. coli cell envelope: solubilization and functional reconstitution. Biophys J 65:177–183PubMedCrossRefGoogle Scholar
  25. 25.
    Moe PC, Blount P, Kung C (1998) Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation. Mol Microbiol 28(3):583–592PubMedCrossRefGoogle Scholar
  26. 26.
    Börngen K et al (2010) The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation. Biochim Biophys Acta 1798(11):2141–2149PubMedCrossRefGoogle Scholar
  27. 27.
    Kloda A, Martinac B (2001) Molecular identification of a mechanosensitive channel in archaea. Biophys J 80(1):229–240PubMedCrossRefGoogle Scholar
  28. 28.
    Petrov E, Palanivelu D, Rohde PR, Constantine M, Minor D, Martinac B (2011) Patch-clamp characterization of the MscS-like mechanosensitive channel from Silicibacter pomeroyi. Biophysical Society Meeting Abstracts. Biophys J Suppl 100(3):281a, Abstract, 1532-PosCrossRefGoogle Scholar
  29. 29.
    Li Y et al (2002) Ionic regulation of MscK, a mechanosensitive channel from Escherichia coli. EMBO J 21(20):5323–5330PubMedCrossRefGoogle Scholar
  30. 30.
    Kuo MM et al (2008) The desensitization gating of the MthK K+ channel is governed by its cytoplasmic amino terminus. PLoS Biol 6(10):e223PubMedCrossRefGoogle Scholar
  31. 31.
    Sakmann B, Neher E (1995) Single-channel recording. Plenum, New YorkCrossRefGoogle Scholar
  32. 32.
    Sokabe M, Sachs F, Jing ZQ (1991) Quantitative video microscopy of patch clamped membranes stress, strain, capacitance, and stretch channel activation. Biophys J 59(3):722–728PubMedCrossRefGoogle Scholar
  33. 33.
    Yoshimura K et al (1999) Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity. Biophys J 77(4):1960–1972PubMedCrossRefGoogle Scholar
  34. 34.
    Haswell ES, Phillips R, Rees DC (2011) Mechanosensitive channels: what can they do and how do they do it? Structure 19(10):1356–1369PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Boris Martinac
    • 1
    • 2
    Email author
  • Paul R. Rohde
    • 1
  • Charles G. Cranfield
    • 1
  • Takeshi Nomura
    • 1
  1. 1.Victor Chang Cardiac Research InstituteDarlinghurstAustralia
  2. 2.St. Vincent’s Clinical SchoolUniversity of New South WalesSydneyAustralia

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