European Biophysics Journal

, Volume 34, Issue 5, pp 403–412

The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity

  • I-Jung Tsai
  • Zhen-Wei Liu
  • John Rayment
  • Christel Norman
  • Allan McKinley
  • Boris Martinac


The periplasmic loop of MscL, the mechanosensitive channel of large conductance, acts as a spring resisting the opening of the channel. Recently, a high-throughput functional screening of a range of MscL structural mutants indicated that the substitution of residue glutamine (Q) 65 with arginine (R) or leucine (L) leads to a wild-type (WT)-like and a loss-of-function (LOF) phenotype, respectively (Maurer and Dougherty J. Biol. Chem. 278(23):21076–21082, 2003). We used electron paramagnetic resonance (EPR) spectroscopy, single-channel recording and in vivo experiments to investigate further the effect of R and L mutation of Q65 on the gating mechanism of MscL. Structural analysis of Q65R and Q65L was carried out by coupling the site-directed spin labeling (SDSL) with EPR spectroscopy. A SDSL cysteine mutant of the isoleucine 24 residue (I24C-SL) in the first transmembrane domain, TM1, of MscL served as a reporter residue in EPR experiments. This was due to its strong spin–spin interaction with the neighboring I24C-SL residues in the MscL channel pentamer (Perozo et al.Nature 418:942–948, 2002). The effects of bilayer incorporation of lysophosphatidylcholine on the MscL mutants were also investigated. Functional analysis was carried out using patch-clamp recordings from these mutants and WT MscL reconstituted into artificial liposomes. Although our data are largely in agreement with the high-throughput mutational analysis of Maurer and Dougherty, this study shows that Q65R and Q65L form functional channels and that these mutations lead to partial gain-of-function (GOF) and LOF mutation, respectively. Overall, our study confirms and advances the notion that the periplasmic loop plays a role in setting the channel mechanosensitivity.


Mechanosensitive channel Electron paramagnetic resonance Lysophosphatidylcholine Patch-clamp Osmoregulation 



Analysis of variance




Electron paramagnetic resonance


Gain of function


Glutathione S-transferase


N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid


Inner diameter






Loss of function






Potassium-regulated mechanosensitive channel


Mechanosensitive channel of large conductance


Mechanosensitive channel of small conductance


Outer diameter


Phosphate-buffered saline


Standard deviation


Site-directed spin labeling


First transmembrane domain


Second transmembrane domain


Specially designed polymethylpentene


Wild type


  1. Ajouz B, Berrier C, Besnard M, Martinac B, Ghazi A (2000) Contributions of the different extramembranous domains of the mechanosensitive channel MscL to its response to membrane tension. J Biol Chem 275:1015–1022Google Scholar
  2. Altenbach C, Greenhalgh DA, Khorfana HG, Hubbell WL (1994) A collision gradient method to determine the immersion depth of nitroxides in lipids bilayers: application to spin-labeled mutants of bacteriorhodopsin. Proc Natl Acad Sci USA 91:1667–1671Google Scholar
  3. Biswas R, Kühne C, Brudvig GW, Gopalan V (2001) Use of EPR spectroscopy to study macromolecular structure and function. Sci Prog 84(1):45–68Google Scholar
  4. Blount P, Sukharev SI, Schroeder MJ, Nagle SK, Kung C (1996) Single residue substitutions that change gating properties of a mechanosensitive channel in Escherichia coli. Proc Natl Acad Sci USA 93:11652–11657Google Scholar
  5. Blount P, Schroeder M, Kung C (1997) Mutations in a bacterial mechanosensitive channel change the cellular response to osmotic stress. J Biol Chem 272:32150–32157CrossRefGoogle Scholar
  6. Boedeker Plastics I Rexolite Cross-Linked Polystyrene Specification. (Accessed March 2000)
  7. Delcour AH, Martinac B, Alder J, Kung C (1989) Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels. Biophys J 56:631–636Google Scholar
  8. Gullingsrud J, Schulten K (2003) Gating of MscL studied by steered molecular dynamics. Biophys J 85:2087–2099Google Scholar
  9. Hamill O, Martinac B (2001) Molecular basis of mechanotransduction in living cells. Physiol Rev 81(2):685–740Google Scholar
  10. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100PubMedGoogle Scholar
  11. Häse CC, Dain AC, Le Martinac B (1995) Purification and functional reconstitution of the recombinant large mechanosensititve ion channel (MscL) of Escherichia coli. J Biol Chem 270:18329–18334CrossRefGoogle Scholar
  12. Häse CC, Minchin RF, Kloda A, Martinac B (1997) Cross-linking studies and membrane localization and assembly of radio-labelled large mechanosensitive ion channel (MscL) of E. coli: mutants with altered channel gating and pressure sensitivity. J Membr Biol 157:17–25Google Scholar
  13. Jawaorski M, Sienkiewicz A, Scholes C (1997) Double-stacked dielectric resonators for sensitive EPR measurements. J Magn Reson 124:87–96Google Scholar
  14. Levin G, Blount P (2004) Cysteine scanning of MscL transmembrane domains reveals residues critical for mechanosensitive channel gating. Biophys J 86:2862–2870Google Scholar
  15. Levina N, Totemeyer S, Stokes NR, Louis P, Jones MA, Booth IR (1999) Protection of E. coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J 18:1730–1737CrossRefGoogle Scholar
  16. Li Y, Wray R, Blount P (2004) Intragenic suppression of gain-of-function mutations in the Escherichia coli mechanosensitive channel, MscL. Mol Microbiol 53:485–495Google Scholar
  17. Lowry OH, Rosebbrough NJ, Lewis Farr A, Randal RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  18. Markin VS, Martinac B (1991) Mechanosensitive ion channels as reporters of bilayer expansion. A theoretical model. Biophys J 60:1120–1127Google Scholar
  19. Martinac B (2004) Mechanosensitive ion channels: molecules of mechanotransduction. J Cell Sci 117:2449–2460CrossRefGoogle Scholar
  20. Martinac B, Adler J, Kung C (1990) Mechanosensitive ion channels of E.coli activated by amphipaths. Nature 348:261–263Google Scholar
  21. Maurer JA, Dougherty DA (2003) Generation and evaluation of a large mutational library from the Escherichia coli mechanosensitive channel of large conductance, MscL. Implications for channel gating and evolutionary design. J Biol Chem 278(23):21076–21082Google Scholar
  22. Maurer JA, Elmore DE, Lester HA, Dougherty DA (2000) Comparing and contrasting E. coli andM. tuberculosis mechanosensitive channels (MscL). New gain function mutations in the loop region. J Biol Chem 275(18):13336–13342Google Scholar
  23. Moe PC, Levin G, Blount P (2000) Pursuing the roots of mechanosensation: a structure based genetic analysis of the M. tuberculosis MscL channel. Biophys J 78:A804Google Scholar
  24. Ou X, Blount P, Hoffman R, Kung C (1998) One face of transmembrane helix is crucial in mechanosensitive channel gating. Proc Natl Acad Sci USA 95:11471–11475Google Scholar
  25. Park KH, Berrier C, Martinac B, Ghazi A (2004) Purification and functional reconstitution of N-halves and C-halves of the MscL channel. Biophys J 86:2129–2136Google Scholar
  26. Perozo E, Cortes DM, Sompornpisut P, Kloda A, Martinac B (2002a). Open structure of MscL and the gating mechanism of mechanosensitive channels. Nature 418:942–948Google Scholar
  27. Perozo E, Kloda A, Cortes M, Martinac B (2002b) Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating. Nat Struct Biol 9:696–703Google Scholar
  28. RESOMICS (2000) Dielectric resonators. Cat. No. 095E-7. Murata Manufacturing Co., Ltd.Google Scholar
  29. San Diego Plastics I Rexolite. (Accessed March 2000)
  30. Sukharev SI, Martinac B, Arshavsky VY, Kung C (1993) Two types of mechanosensitive channels in the E. coli cell envelop solubilization and functional reconstitution. Biophys J 65:177–183Google Scholar
  31. Sukharev SI, Durell SR, Guy HR (2001) Structural models of the MscL gating mechanism. Biophys J 81:917–936Google Scholar
  32. Yoshimura K, Batiza A, Schroeder M, Blount P, Kung C (1999) Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity. Biophys J 77:1960–1972Google Scholar
  33. Yoshimura K, Batiza A, Kung C (2001) Chemically charging the pore constriction opens the mechanosensitive channel MscL. Biophys J 80:2198–2206Google Scholar

Copyright information

© EBSA 2005

Authors and Affiliations

  • I-Jung Tsai
    • 1
    • 2
  • Zhen-Wei Liu
    • 1
    • 3
  • John Rayment
    • 1
    • 2
  • Christel Norman
    • 1
  • Allan McKinley
    • 2
  • Boris Martinac
    • 1
    • 3
  1. 1.School of Medicine and PharmacologyUniversity of Western AustraliaNedlandsAustralia
  2. 2.School of Biomedical and Chemical SciencesUniversity of Western AustralianCrawleyAustralia
  3. 3.School of Biomedical SciencesUniversity of QueenslandBrisbaneAustralia

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