Abstract
The bacterial mechanosensitive channels MscS and MscL are gated by an increase in membrane tension when the bacterium experiences hypoosmotic shock. It has been well established that membrane lipids modulate the mechanosensitivity and gating behavior of these channels. The focus of this study is a negatively charged phospholipid, cardiolipin, which has been shown to localize at curved regions of the bacterial cell, including the poles and the septum, and to have a strong preference for binding to membrane proteins. Here we characterize the effect of cardiolipin on MscS, the mechanosensitive channel of small conductance, using patch-clamp electrophysiology. We compare the gating kinetics and mechanosensitivity of the channel in both azolectin and mixtures of pure lipids DOPE/DOPC liposomes with and without cardiolipin. In azolectin liposomes, the addition of 10 % cardiolipin abolishes hysteresis of MscS, but MscL remains largely unaffected, indicating that cardiolipin may stabilize the closed state of MscS. On the other hand, mixtures of DOPE/DOPC abolish the hysteresis gating of MscS even in the absence of cardiolipin, and the addition of cardiolipin increases the opening and closing thresholds of both MscS and MscL. In addition, we show that MscS gates more frequently when cardiolipin is present in both the azolectin and pure lipid systems; this dose-dependent effect ultimately destabilizes the open state of MscS and we consider the functional implications of this cardiolipin effect in the bacterial osmotic response. Our results show that cardiolipin modulates the mechanosensitivity and gating characteristics of MscS, indicating its important role in the physiology of bacterial cells.
Similar content being viewed by others
References
Battle AR, Petrov E, Pal P, Martinac B (2009) Rapid and improved reconstitution of bacterial mechanosensitive ion channel proteins MscS and MscL into liposomes using a modified sucrose method. FEBS Lett 583:407–412
Bavi N, Nakayama Y, Bavi O, Cox CD, Qin QH, Martinac B (2014) Biophysical implications of lipid bilayer rheometry for mechanosensitive channels. Proc Natl Acad Sci USA 111:13864–13869
Berrier C, Besnard M, Ajouz B, Coulombe A, Ghazi A (1996) Multiple mechanosensitive ion channels from E. coli, activated at different thresholds of applied pressure. J Membr Biol 151:175–187
Booth IR, Blount P (2012) The MscS and MscL families of mechanosensitive channels act as microbial emergency release valves. J Bacteriol 194:4802–4809
Cantor RS (1999) The influence of membrane lateral pressures on simple geometric models of protein conformational equilibria. Chem Phys Lipids 101(1):45–56
Cox CD, Wann KT, Martinac B (2014) Selectivity mechanisms in MscS-like channels: from structure to function. Channels (Austin) 8:5–12
Delcour AH, Martinac B, Adler J, Kung C (1989) Modified reconstitution method used in patch-clamp studies of E. coli ion channels. Biophys J 56(3):631–636
Errington J (2003) Dynamic proteins and a cytoskeleton in bacteria. Nat Cell Biol 5:175–178
Häse CC, Le Dain AC, Martinac B (1995) Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of E. coli. J Biol Chem 270:18329–18334
Hurst AC, Gottlieb PA, Martinac B (2009) Concentration dependent effect of GsMTx4 on mechanosensitive channels of small conductance in E. coli spheroplasts. Eur Biophys J 38:415–425
Kamaraju K, Gottlieb PA, Sachs F, Sukharev S (2010) Effects of GsMTx4 on bacterial mechanosensitive channels in inside-out patches from giant spheroplasts. Biophys J 99(9):2870–2878
Laganowsky A, Reading E, Allison TM, Ulmschneider MB, Degiacomi MT, Baldwin AJ, Robinson CV (2014) Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510:172–175
Laloux G, Jacobs-Wagner C (2014) How do bacteria localize proteins to the cell pole? J Cell Sci 127:11–19
Levina N, Tötemeye S, Stokes NR, Louis P, Jones IA, Booth IR (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):1730–1737
Lewis RN, McElhaney RN (2009) The physicochemical properties of cardiolipin bilayers and cardiolipin-containing lipid membranes. Biochim Biophys Acta 1788(10):2069–2079
Macdonald AG, Martinac B (2005) Effect of high hydrostatic pressure on the bacterial mechanosensitive channel MscS. Eur Biophys J 34(5):434–441
Marom M, Safonov R, Amram S, Avneon Y, Nachliel E, Gutman M, Zohary K, Azem A, Tsfadia Y (2009) Interaction of the Tim44 C-terminal domain with negatively charged phospholipids. Biochemistry 48:11185–11195
Marsh D (2007) Lateral pressure profile, spontaneous curvature frustration, and the incorporation and conformation of proteins in membranes. Biophys J 93(11):3884–3899
Martinac B (2007) 3.5 billion years of mechanosensory transduction: structure and function of mechanosensitive channels in prokaryotes. Mechanosensitive ion channels, part A. Curr Top Membr 58:25–57
Martinac B (2011) Bacterial mechanosensitive channels as a paradigm for mechanosensory transduction. Cell Physiol Biochem 28(6):1051–1060
Martinac B, Nomura T, Chi G, Petrov E, Rohde PR, Battle AR, Foo A, Constantine M, Rothnagel R, Carne S, Deplazes E, Cornell B, Cranfield CG, Hankamer B, Landsberg MJ (2014) Bacterial mechanosensitive channels: models for studying mechanosensory transduction. Antioxid Redox Signal 20:952–969
Neidhardt FC et al (1996) E. coli and Salmonella typhimurium: cellular and molecular biology, 2nd edn. ASM Press, Washington, DC
Nomura T, Cranfield CG, Deplazes E, Owen DM, Macmillan A, Battle AR, Constantine M, Sokabe M, Martinac B (2012) Differential effects of lipids and lyso-lipids on the mechanosensitivity of the mechanosensitive channels MscL and MscS. Proc Natl Acad Sci USA 109:8770–8775
Oliver PM, Crooks JA, Leidl M, Yoon EJ, Saghatelian A, Weibel DB (2014) Localization of anionic phospholipids in E. coli cells. J Bacteriol 196:3386–3398
Paradies G, Paradies V, Ruggiero FM, Petrosillo G (2014) Cardiolipin and mitochondrial function in health and disease. Antioxid Redox Signal 20:1925–1953
Perozo E, Kloda A, Cortes DM, Martinac B (2001) Site-directed spin-labeling analysis of reconstituted MscL in the closed state. J Gen Physiol 118:193–206
Perozo E, Kloda A, Cortes DM, Martinac B (2002) Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating. Nat Struct Biol 9:696–703
Renner LD, Weibel DB (2011) Cardiolipin microdomains localize to negatively curved regions of E. coli membranes. Proc Natl Acad Sci USA 108:6264–6269
Romantsov T, Stalker L, Culham DE, Wood JM (2008) Cardiolipin controls the osmotic stress response and the subcellular location of transporter ProP in E. coli. J Biol Chem 283(18):12314–12323
Romantsov T, Guan Z, Wood JM (2009) Cardiolipin and the osmotic stress responses of bacteria. Biochim Biophys Acta 1788(10):2092–2100
Romantsov T, Battle AR, Hendel JL, Martinac B, Wood JM (2010) Protein localization in E. coli cells: comparison of the cytoplasmic membrane proteins ProP, LacY, ProW, AqpZ, MscS, and MscL. J Bacteriol 192:912–924
Stokes NR, Murray HD, Subramaniam C, Gourse RL, Louis P, Bartlett W, Miller S, Booth IR (2003) A role for mechanosensitive channels in survival of stationary phase: regulation of channel expression by RpoS. PNAS 100(26):15959–15964
Sukharev S (2002) Purification of the small mechanosensitive channel of E. coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes. Biophys J 83:290–298
Sukharev SI, Sigurdson WJ, Kung C, Sachs F (1999) Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL. J Gen Physiol 113:525–540
Vasquez V, Cortes DM, Furukawa H, Perozo E (2007) An optimized purification and reconstitution method for the MscS channel: strategies for spectroscopical analysis. Biochemistry 46:6766–6773
Vasquez V, Sotomayor M, Cordero-Morales J, Schulten K, Perozo E (2008) A structural mechanism for MscS gating in lipid bilayers. Science 321:1210–1214
Weber H, Polen T, Heuveling J, Wendisch VF, Hengge R (2005) Genome-wide analysis of the general stress response network in E. coli: sigmaS-dependent genes, promoters, and sigma factor selectivity. J Bacteriol 187(5):1591–1603 (for complete set of data including MscL, MscS and ybhO, refer to the website: http://www.bcp.fu-berlin.de/en/biologie/arbeitsgruppen/mikrobiologie/ag_hengge/scidata/index.html). Accessed 28 Mar 2015
Weiss C, Oppliger W, Vergeres G, Demel R, Jeno P, Horst M, de Kruijff B, Schatz G, Azem A (1999) Domain structure and lipid interaction of recombinant yeast Tim44. Proc Natl Acad Sci USA 96:8890–8894
Yoshimura K, Nomura T, Sokabe M (2004) Loss-of-function mutations at the rim of the funnel of mechanosensitive channel MscL. Biophys J 86(4):2113–2120
Zhong DL, Blount P (2013) Phosphatidylinositol is crucial for the mechanosensitivity of mycobacterium tuberculosis MscL. Biochemistry 52:5415–5420
Acknowledgments
We thank the Griffith Health Institute, Griffith University for a New Researcher Grant award to ARB, the Japanese Society for Promotion of Science (JSPS) for a fellowship to YN, and the National Health and Medical Research Council of Australia for the Principal Research Fellowship APP1044628 and Grant APP104780 to BM.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Special issue: Biophysics of mechanotransduction.
Rights and permissions
About this article
Cite this article
Ridone, P., Nakayama, Y., Martinac, B. et al. Patch clamp characterization of the effect of cardiolipin on MscS of E. coli . Eur Biophys J 44, 567–576 (2015). https://doi.org/10.1007/s00249-015-1020-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-015-1020-2