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1.A.22 The Large Conductance Mechanosensitive Ion Channel (MscL) Family

MscL of E. coli has been extensively characterized, and limited functional studies have been performed on some of its homologues (Häse et al., 1995; Sukharev et al., 1996, 1999; Sukharev et al., 1999, 2001). The MscL protein of E. coli is 136 amino acyl residues in length and spans the membrane twice as & alpha;-helices (Blount et al., 1996a,b). It forms a homopentameric channel with ten transmembrane spanners (Blount et al., 1996a,b; Sukharev et al., 1999). The channel transports ions fairly nonspecifically with slight selectivity for cations over anions (Sukharev et al., 1994). Mechanosensitivity has been demonstrated for several MscL homologues using patch-clamp methodology (Blount et al., 1996a,b; Blount et al., 1997; Sukharev et al., 1996). It has been shown to release proteins such as thioredoxin during osmotic downshift (Ajouz et al., 1998). Expression of the E. coli mscL gene has been shown to protect Vibrio alginolyticus and Bacillus subtilis from cell lysis during osmotic downshift (Nakamaru et al., 1999; Hoffmann et al., 2008). The levels of both MscL and MscS channels in Bacillus subtilis are high during exponential phase growth, very low in stationary phase and non-detectable in spores (Wahome et al., 2009). Bacterial mechanosensitive channels, MscL and MscS, reflect an intimate coupling of protein conformation with the mechanics of the surrounding membrane. The membrane serves as an adaptable sensor that responds to an input of applied force and converts it into an output signal. The cell can exploit this information in a number of ways: ensuring cellular viability in the presence of osmotic stress and perhaps also serving as a signal transducer for membrane tension (Haswell et al., 2011).  MscL is gated by changes in bilayer deformation and by the membrane potential (Andersson et al. 2008).

The three-dimensional structure of the M. tuberculosis MscL has been solved to 3.5 Å resolution, and the crystal structure has been shown to reflect that in the intact cell membrane (Chang et al., 1998; Perozo et al., 2001). This structure provided the basis for a model that explains gate opening and closing in response to membrane tension. Tension is proposed to expand the 10 TMS/5 subunit transmembrane barrel via the linker between the two TMSs [S1 (N-terminal) and M1 (C-terminal)]. S1 segments form a bundle when the channel is closed, and cross-linking between S1 segments prevents opening. S1 and M1 interact in the open channel, and cross-linking S1 to M1 impedes channel closing. The opening of MscL is accompanied by the disassociation of a carboxl-terminal protrusion and pore formation (Yoshimura et al., 2008). Phylogenetic, structural and functional analysis have been presented by Pivetti et al. (2003). How these channels may respond to change in the mechanical environment the lipid bilayer provides is discussed by Kung et al. (2010).  Channel opening uses a helix-tilt mechanism and opens to a 2.8 nm diameter pore (Wang et al. 2014).

Price et al. (2011) have demonstrated in vitro synthesis and oligomerization of the mechanosensitive channel, MscL, into functional ion channels. They showed that insertion requires YidC (2.A.9.3.1) but subsequent oligomerization to the functional pentamer occurs spontaneously.  MscL acts as an 'emergency relief valve', protecting bacteria from lysis upon acute osmotic down-shock. MscL is reversibly and directly gated by changes in membrane tension. In the open state, MscL forms a non- selective 3 nS conductance channel which gates at tensions close to the lytic limit of the bacterial membrane. An earlier crystal structure at 3.5 A resolution of a pentameric MscL from Mycobacterium tuberculosis represented a closed-state or non-conducting conformation. MscL has a complex gating behaviour; it exhibits several intermediates between the closed and open states, including one putative non-conductive expanded state and at least three sub-conducting states.  Liu et al. 2009 presented the crystal structure of a carboxy-terminal truncation mutant (Delta95-120) of MscL from Staphylococcus aureus (SaMscL(CDelta26)) at 3.8 A resolution.  SaMscL(CDelta26) forms a tetrameric channel with both transmembrane helices tilted away from the membrane normal at angles close to that inferred for the open state, probably corresponding to a non-conductive but partially expanded intermediate state (Liu et al. 2009).

The generalized transport reactions are:

(a) proteins (in) → proteins (out)

(b) ions (out) ions (in)

(c) osmolytes (in) osmolytes (out).

References associated with 1.A.22 family:

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Andersson, M., G. Okeyo, D. Wilson, H. Keizer, P. Moe, P. Blount, D. Fine, A. Dodabalapur, and R.S. Duran. (2008). Voltage-induced gating of the mechanosensitive MscL ion channel reconstituted in a tethered lipid bilayer membrane. Biosens Bioelectron 23: 919-923. 17996439
Ando C., Liu N. and Yoshimura K. (2015). A cytoplasmic helix is required for pentamer formation of the Escherichia coli MscL mechanosensitive channel. J Biochem. 158(2):109-14. 25697390
Balleza, D., F. Gómez-Lagunas, and C. Quinto. (2010). Cloning and functional expression of an MscL ortholog from Rhizobium etli: characterization of a mechanosensitive channel. J. Membr. Biol. 234: 13-27. 20177670
Blount, P., M.J. Schroeder, and C. Kung. (1997). Mutations in a bacterial mechanosensitive channel change the cellular response to osmotic stress. J. Biol. Chem. 272: 32150-32157. 9405414
Blount, P., S.I. Sukharev, M.J. Schroeder, S.K. Nangle, and C. Kung. (1996b). Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli. Proc. Natl. Acad. Sci. USA 93: 11652-11657. 8876191
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Bucarey, S.A., K. Penn, L. Paul, W. Fenical, and P.R. Jensen. (2012). Genetic Complementation of the Obligate Marine Actinobacterium Salinispora tropica with the Large Mechanosensitive Channel Gene mscL Rescues Cells from Osmotic Downshock. Appl. Environ. Microbiol. 78: 4175-4182. 22492446
Chang, G., R.H. Spencer, A.T. Lee, M.T. Barclay, and D.C. Rees. (1998). Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science 282: 2220-2226. 9856938
Foo A., Battle AR., Chi G., Hankamer B., Landsberg MJ. and Martinac B. (2015). Inducible release of particulates from liposomes using the mechanosensitive channel of large conductance and L-alpha-lysophosphatidylcholine. Eur Biophys J. 44(7):521-30. 26143502
Hase, C.C., A.C. Le Dain, and B. Martinac. (1995). Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli. J. Biol. Chem. 270: 18329-18334. 7543101
Haswell, E.S., R. Phillips, and D.C. Rees. (2011). Mechanosensitive channels: what can they do and how do they do it? Structure 19: 1356-1369. 22000509
Hoffmann, T., C. Boiangiu, S. Moses, and E. Bremer. (2008). Responses of Bacillus subtilis to hypotonic challenges: physiological contributions of mechanosensitive channels to cellular survival. Appl. Environ. Microbiol. 74: 2454-2460. 18310427
Iscla, I., R. Wray, and P. Blount. (2011). An in vivo screen reveals protein-lipid interactions crucial for gating a mechanosensitive channel. FASEB J. 25: 694-702. 21068398
Iscla, I., R. Wray, and P. Blount. (2011). The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo. Protein. Sci. 20: 1638-1642. 21739498
Kloda, A. and Martinac, B. (2002). Common evolutionary origins of mechanosensitive ion channels in archaea, bacteria and cell-walled eukarya. Archaea 1: 35-44. 15803657
Kung, C., B. Martinac, and S. Sukharev. (2010). Mechanosensitive channels in microbes. Annu. Rev. Microbiol. 64: 313-329. 20825352
Li J., Guo J., Ou X., Zhang M., Li Y. and Liu Z. (2015). Mechanical coupling of the multiple structural elements of the large-conductance mechanosensitive channel during expansion. Proc Natl Acad Sci U S A. 112(34):10726-31. 26261325
Liu, Z., C.S. Gandhi, and D.C. Rees. (2009). Structure of a tetrameric MscL in an expanded intermediate state. Nature 461: 120-124. 19701184
Nakamaru, Y., Y. Takahashi, T. Unemoto, and T. Nakamura. (1999). Mechanosensitive channel functions to alleviate the cell lysis of marine bacterium, Vibrio alginolyticus, by osmotic downshock. FEBS Lett. 444: 170-172. 10050752
Perozo, E., A. Kloda, D.M. Cortes, and B. Martinac. (2001). Site-directed spin-labeling analysis of reconstituted Mscl in the closed state. J Gen Physiol 118: 193-206. 11479346
Pivetti, C.D., M.-R. Yen, S. Miller, W. Busch, Y.-H. Tseng, I.R. Booth, and M.H. Saier, Jr. (2003). Two families of mechanosensitive channel proteins. Microbiol. Mol. Biol. Rev. 67: 66-85. 12626684
Price, C.E., A. Kocer, S. Kol, J.P. van der Berg, and A.J. Driessen. (2011). In vitro synthesis and oligomerization of the mechanosensitive channel of large conductance, MscL, into a functional ion channel. FEBS Lett. 585: 249-254. 21134371
Saier, M.H., Jr., B.H. Eng, S. Fard, J. Garg, D.A. Haggerty, W.J. Hutchinson, D.L. Jack, E.C. Lai, H.J. Liu, D.P. Nusinew, A.M. Omar, S.S. Pao, I.T. Paulsen, J.A. Quan, M. Sliwinski, T.-T. Tseng, S. Wachi, and G.B. Young. (1999). Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim. Biophys. Acta 1422: 1-56. 10082980
Sukharev, S. (1999). Mechanosensitive channels in bacteria as membrane tension reporters. FASEB J. 13: 55-61. 10352145
Sukharev, S., M. Betanzos, C.S. Chiang, and H.R. Guy. (2001). The gating mechanism of the large mechanosensitive channel MscL. Nature 409: 720-724. 11217861
Sukharev, S., M.J. Schroeder, and D.R. McCaslin. (1999). Stoichiometry of the large conductance bacterial mechanosensitive channel of E. coli. A biochemical study. J. Memb. Biol. 171: 183-193. 10501827
Sukharev, S.I., P. Blount, B. Martinac, F.R. Blattner, and C. Kung. (1994). A large-conductance mechanosensitive channel in E. coliencoded by mscL alone. Nature 368: 265-268. 7511799
Sukharev, S.I., P. Blount, B. Martinac, H.R. Guy, and C. Kung. (1996). MscL: a mechanosensitive channel in Escherichia coli. In: Organellar Ion Channels and Transporters, The Rockefeller University Press, pp. 133-141. 8809939
Wahome, P.G., A.E. Cowan, B. Setlow, and P. Setlow. (2009). Levels and localization of mechanosensitive channel proteins in Bacillus subtilis. Arch. Microbiol. 191: 403-414. 19252899
Wang, C.X., H.X. Ge, X.P. Hou, and Y.Q. Li. (2007). Roles of larger conductance mechanosensitive channels (MscL) in sporulation and Act secretion in Streptomyces coelicolor. J Basic Microbiol 47: 518-524. 18072238
Wang, Y., Y. Liu, H.A. Deberg, T. Nomura, M.T. Hoffman, P.R. Rohde, K. Schulten, B. Martinac, and P.R. Selvin. (2014). Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel. Elife 3: e01834. 24550255
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