E. coli MscL

We found the activities and later cloned the gene of the Mechanosensitive channel of Large conductance, MscL, of E. coli. Although mechanosensitive (MS) channels are found in many systems and play roles in the senses of touch, hearing, etc., MscL and MscS (below) are the few tangible models to study the biochemical and biophysical details of mechanosensation.

We first encountered MS channel activities in a patch-clamp survey of the surface of giant cells generated from bacteria  (1) (Fig. 1). These activities could be biochemically separated by gel filtration (2). Using reconstitution as an assay (patch clamping reconstituted fractions), the MscL protein was identified, and the corresponding gene cloned (3).

Fig. 1  Patch-clamping E. coli.  (left)  Procedures were perfected to convert E. coli into giant cells and then to round spheroplasts appropriate for patch-clamp experiments.  Activities of channels in the membrane patch drawn into the pipette electrode can be registered.  Calibration bar 10 μm.   (right) Suctions (monitored through a manometer, bottom trace) applied to the patch through the pipette activate two types of mechanosensitive channels:  MscS  and MscL.  Stepwise increase in current (upper trace) indicate the opening of individual channel molecules.  MscS is mechanosesnsitive channel of smaller conductance; MscL is of larger conductance.

Gain-of-function mutants with "loose-cannon" channel activities (4), site-directed mutations (5,6), and biochemical studies (7) led to a model of the MscL-channel subunit having two-transmembrane alpha helices (TM1 and TM2) (8).  The Rees laboratory (9) solved a crystal structure of the MscL homolog in Mycobacteium tuberculosis. The channel is a homopentamer, with the five TM1's coming together towards the cytoplasm to close the pore (Fig. 2).  The open structure has been deduced by a variety of studies by other laboratories.

Fig. 2  MscL structure.  (left) Side and (right) top view.  Ribbon diagrams of the backbone of MscL protein from M. tuberculosis (9.)  Each channel is a homopentamer.  Each subunit (differently colored) includes two transmembrane  alpha helices, TM1 and TM2.  The crystallized form is in a closed state, in which the five TM1s converge at the cytoplasmic end like a funnel to obstruct the pathway.

The Booth laboratory cloned the second MS-channel gene (mscS), (10), and showed that the mscL- mscS- double mutant bacteria lyse upon an osmotic downshock (Fig. 3). This work supports the previous hypothesis that these MS channels function as emergency valves. Upon downshocks, they open to jettison solutes, reducing the turgor and thereby preventing the bacteria from bursting (Fig. 3).

Fig. 3  Mechanosensitive channels function as emergency release valves in vivo.  (top).  E. coli cell in a normal environment (left) and in the rain (or upon dilution in the laboratory, right).  A bacterium (shown as a rod), having adjusted its cytoplasm to the relatively high osmolarity of the surrounding milieu (dark red, the red dots being solutes, not water), is confronted with a sudden dilution of its environment upon the onset of rain (light red).  Entry of water (not shown) through the lipid bilayer (water channel is not needed) swells the bacterium (now oval-shaped) and stretches open the mechanosensitive channels to jettison solutes (red puffs), enabling it to reach a new equilibrium and escaping osmolysis (and returns to being rod-shaped).  (bottom)  Genetically deleting the two major mechanosensitive channels (MscL and MscS) removes the emergency release valves.  Swelling of the double mutant cell causes osmolysis. See (13).

See (11, 12, 13) for reviews.

1. Martinac et al. (1987 ) PNAS84: 2297.
2. Sukharev et al. 1993)  Biophy. J. 65: 1.
3. Sukharev et al. (1994) Nature368: 265
4. Ou et al. (1998) PNAS95:11471.
5. Yoshimura et al. (1999) Biophy. J. 77: 144.
6. Yoshimura et al. (2001) Biophy. J. 80: 2198.
7. Blount et al., (1996) EMBO J. 15: 4798.
8. Sukharev et al. (1997) Annu. Rev. Physiol. 59: 633.
9. Chang et al. (1998) Science282: 2220.
10. Levina et al. (1998) EMBO J.18: 1730.
11. Sukharev and Corey (2004) Science STKE. re4.
12.  Anishkin and Kung (2005) Cur. Opin. Neurobiol. 15: 397.
13.  Kung (2005) Nature436: 647.

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