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 structure of the MscL channel in membranes of varying thickness and curvature has been studied (Wang et al. 2018). Temperature-sensitive mutants have revealed aspects of the thermodynamic stability of the MscL structure (Owada et al. 2019).

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). Water may act as a 'lubricant' (softener) during TM1 helix elongation that may play a role in gating (Bavi et al. 2016).

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).



This family belongs to the .

 

References:

Ajouz, B., C. Berrier, A. Garrigues, M. Besnard, and A. Ghazi. (1998). Release of thioredoxin via the mechanosensitive channel MscL during osmotic downshock of Escherichia coli cells. J. Biol. Chem. 273: 26670-26674.

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.

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.

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.

Bavi, N., O. Bavi, M. Vossoughi, R. Naghdabadi, A.P. Hill, B. Martinac, and Y. Jamali. (2016). Nanomechanical properties of MscL alpha helices: A steered molecular dynamics study. Channels (Austin) 0. [Epub: Ahead of Print]

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.

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.

Blount, P., S.I. Sukharev, P.C. Moe, M.J. Schroeder, H.R. Guy, and C. Kung. (1996a). Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli. EMBO J. 15: 4798-4805.

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.

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.

Chure, G., H.J. Lee, A. Rasmussen, and R. Phillips. (2018). Connecting the dots between mechanosensitive channel abundance, osmotic shock, and survival at single-cell resolution. J. Bacteriol. [Epub: Ahead of Print]

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.

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.

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.

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.

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.

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.

Katsuta, H., Y. Sawada, and M. Sokabe. (2018). Biophysical Mechanisms of Membrane-Thickness-Dependent MscL Gating: An All-Atom Molecular Dynamics Study. Langmuir. [Epub: Ahead of Print]

Kloda, A. and Martinac, B. (2002). Common evolutionary origins of mechanosensitive ion channels in archaea, bacteria and cell-walled eukarya. Archaea 1: 35-44.

Kung, C., B. Martinac, and S. Sukharev. (2010). Mechanosensitive channels in microbes. Annu. Rev. Microbiol. 64: 313-329.

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.

Liu, Z., C.S. Gandhi, and D.C. Rees. (2009). Structure of a tetrameric MscL in an expanded intermediate state. Nature 461: 120-124.

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.

Owada, N., M. Yoshida, K. Morita, and K. Yoshimura. (2019). Temperature-sensitive mutants of MscL mechanosensitive channel. J Biochem. [Epub: Ahead of Print]

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.

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.

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.

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.

Sukharev, S. (1999). Mechanosensitive channels in bacteria as membrane tension reporters. FASEB J. 13: 55-61.

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.

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.

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.

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.

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.

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.

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.

Wang, Z., J.M. Jumper, S. Wang, K.F. Freed, and T.R. Sosnick. (2018). A Membrane Burial Potential with H-Bonds and Applications to Curved Membranes and Fast Simulations. Biophys. J. 115: 1872-1884.

Yoshimura, K., J. Usukura, and M. Sokabe. (2008). Gating-associated conformational changes in the mechanosensitive channel MscL. Proc. Natl. Acad. Sci. USA 105: 4033-4038.

Examples:

TC#NameOrganismal TypeExample
1.A.22.1.1

Large mechanosensitive ion channel: MscL; catalyzes efflux of ions (slightly cation selective), osmolytes and small proteins. Protein-lipid interactions are important for gating, dependent on TMS tilting (Iscla et al., 2011b).  The carboxyl-terminal cytoplasmic helices assemble into a pentameric bundle that resembles cartilage oligomeric matrix protein, and these are required for the selective formation of the pentamer (Ando et al. 2015). Lysophospholipids can increase the size of particles that can be transported (Foo et al. 2015). 500 - 700 channels are needed for 80% survival, a number of channels similar to that found in cell (Chure et al. 2018). its activation threshold decreases with membrane thickness; the membrane-thickness-dependent MscL opening mainly arises from structural changes in MscL to match the altered membrane thickness by stretching (Katsuta et al. 2018).

Bacteria

MscL of E. coli (P0A742)

 
1.A.22.1.10

Osmotic adaptation channel that influences sporulation and secondary metabolite production, Sco3190 (MscL) (Wang et al. 2007).

Actinobacteria

Sco3190 of Streptomyces coelicolor

 
1.A.22.1.11

Large conductance mechanosensitive channel protein, MscL, of 101 aas and 2 TMSs. When the membrane is stretched, MscL responds to the increase of membrane tension and opens a nonselective pore to about 30 A wide, exhibiting a large unitary conductance of approximately 3 nS. The structures of this archaeal MscL, trapped in the closed and expanded intermediate states, has been solved (Li et al. 2015). The comparative analysis of these two new structures reveals significant conformational rearrangements in the different domains of MscL. The large changes observed in the tilt angles of the two transmembrane helices (TMS1 and TMS2) fit well with the helix-pivoting model. Meanwhile, the periplasmic loop region transforms from a folded structure, containing an omega-shaped loop and a short beta-hairpin, to an extended and partly disordered conformation during channel expansion. Moreover, a significant rotating and sliding of the N-terminal helix (N-helix) is coupled to the tilting movements of TMS1 and TMS2. The dynamic relationships between the N-helix and TMS1/TMS2 suggest that the N-helix serves as a membrane-anchored stopper that limits the tilts of TM1 and TM2 in the gating process (Li et al. 2015).

MscL of Methanosarcina acetivorans

 
1.A.22.1.12

MscL protein of 171 aas and 2 or 3 TMSs.

MscL of Chondrus crispus (Carrageen Irish moss) (Polymorpha crispa)

 
1.A.22.1.13

Putative large-conductance mechanosensitive channel of 101 aas and 2 TMSs.

MscL channel of Tetraselmis virus 1

 
1.A.22.1.14

MscL homologue of 101 aas and 2 TMSs.

MscL of Cafeteria roenbergensis virus

            BV-PW1].
 
1.A.22.1.2Large mechanosensitive ion channel (3-D structure known)Bacteria MscL of Mycobacterium tuberculosis (P0A5K8)
 
1.A.22.1.3MscL; catalyzes ion and osmolyte release following osmmotic downshiftBacteriaMscL (YwpC) of Bacillus subtilis
 
1.A.22.1.4

MscL (activated by arachidonate (Balleza et al., 2010), 45% identical to MscL of Bacillus subtilis (1.A.22.1.3)).

Proteobacteria

MscL of Rhizobium etli (Q2KCQ1)

 
1.A.22.1.5

The pentameric MscL channel (Iscla et al., 2011).  The high resolution structure of a proposed closed but expanded tetrameric intermediate state has been determined (Liu et al. 2009).

Bacteria

MscL of Staphylococcus aureus (P68805)

 
1.A.22.1.6

MscL; rescues cells form osmotic downshift (Bucarey et al., 2012).

Bacteria

MscL of Micromonospora aurantica  (D9T6D3)

 
1.A.22.1.7

Large-conductance mechanosensitive channel, MscL

Cyanobacteria

MscL of Synechococcus sp.

 
1.A.22.1.8

Large-conductance mechanosensitive channel, MscL

Bacteria

MscL of Leuconostoc citreum

 
1.A.22.1.9

Large-conductance mechanosensitive channel, MscL

Bacteria

MscL of Renibacterium salmoninarum

 
Examples:

TC#NameOrganismal TypeExample