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 α-helices (Blount et al., 1996a,b). It forms a homopentameric channel with ten TMSs (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). Mutational loss of the hydrophobic interaction between membrane lipids and the periplasmic rim of the channel's funnel impairs the function of MscL, presumably by blocking channel opening (Yoshimura et al. 2004). Cyclodextrins can be used for structural and functional studies of mechanosensitive channels (Zhang et al. 2021). Structural elements in water and ion permeation through an MscL have been identified using molecular dynamics simulation (Naeini et al. 2022). Crea et al. 2022 used an azobenzene-derived lipid analogue to optically activate MscL. Such an approach allows photoactivation and control of cellular processes as complex as gravitropism and turgor sensing in plants, contractility of the heart, and sensing pain, hearing, and touch in animals.

Each subunit of an MscL consists of two transmembrane segments TMS1 and TMS2 connected by a periplasmic loop. The closed pore is lined by five TMS1 helices. The two halves of the protein were cloned, and the first half conferred channel activity by itself while the second half conferred mechanosensitivity when exposed to the first half (Park et al. 2004). 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).  Membrane tension is not a mediator of long-range intracellular signaling, but local variations in tension mediate distinct processes in sub-cellular domains (Shi et al. 2018). Allosteric activation of MscL channels is triggered by lipid-mediated modification of mechanosensitive nano-pockets. Single-channel recordings have revealed a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension (Kapsalis et al. 2019). Synergistic modes of regulation by lipid molecules in membrane tension-activated mechanosensitive MscL channels have been decribed and discussed (Wang et al. 2021). Hybrid-supported lipid bilayers (HSLBs) contain phospholipids and diblock copolymers. Manzer et al. 2021 used cell-free expression of MscL to assemble it with HSLB by either cotranslational integration of the protein into hybrid vesicles, followed by fusion of these proteoliposomes, or by preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB.

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 MscL channel functions as a last-ditch emergency release valve, discharging cytoplasmic solutes upon decreases in the osmotic pressure.  Opening this large gated pore allows passage of  molecules up to 30 Å in diameter (Immadisetty et al. 2022). MscL undergoes large conformational changes and  contains structural/functional themes that recur in higher organisms and help elucidate how other, structurally more complex, channels function. These features of MscL include (i) the ability to directly sense and respond to biophysical changes in the membrane, (ii) an alpha helix ('slide helix') or series of charges ('knot in a rope') at the cytoplasmic membrane boundary to guide transmembrane movements, and (iii) important subunit interfaces that, when disrupted, appear to cause the channel to gate inappropriately (Immadisetty et al. 2022). MscL may have medical applications: the modality of the MscL channel can be changed, suggesting its use as a triggered nanovalve in nanodevices, including those for drug targeting. The antibiotic streptomycin opens MscL and uses it as one of the primary paths to the cytoplasm.

Assembling transmembrane proteins on organic electronic materials is a promising approach to couple biological functions to electrical readouts. A biosensing device produced in such a way would enable both the monitoring and regulation of physiological processes and the development of new analytical tools to identify drug targets and new protein functionalities. Transmembrane proteins can be interfaced with bioelectronics through supported lipid bilayers (SLBs). Manzer et al. 2023 demonstrated that cell-free expression systems allow for the one-step integration of MscL into SLBs assembled on an organic conducting polymer, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS).  MscL adopts the correct orientation, remains mobile in the SLB, and is active on the polyelectrolyte surface using optical and electrical readouts (Manzer et al. 2023).

The generalized transport reactions are:

(a) proteins (in) → proteins (out)

(b) ions (out) ions (in)

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