Forces from Lipids

A key observation on MscL and MscS (above) is that these mechanosensitive channels receive their gating forces from the lipid bilayer.  Purified MscL or MscS protein has been reassembled into artificial liposomes of defined phospholipids.  Patches sampled from such liposomes contain channels with their mechanosensitivity intact.  Since there are no other proteins in the patches to pass on the forces, these channels must receive their gating forces from the lipid bilayer (1). (Fig. 1)

Fig 1.  Force from the lipid bilayer gates the MscL channels.  (Top) Purified MscL protein is reconstituted into multilamellar liposomes after replacing the detergent with lipids.  Induced liposome blisters can be sampled with a patach-clamp pipette.  A suction applied to the pipette (broad open arrow) creates tension (small filled arrows) in the membrane patch and activates MscL channel.  (Bottom)  The increase in the number of channel openings in a patch (shown as unitary-conductance steps at the marked levels) is evident when the suction applied to such a patch of lipid bilayer increases from 30 to 40 mm Hg.  Since the patch has only purified MscL and lipids, the force that open these MscL must come from the lipid bilayer.  From (7).

The current model addresses the forces within the lipid bilayer itself.  The ordering the lipids at the membrane-water interface generates a large tension in each monolayer at the level of the phospholipid’s neck (2).  The force profile in the interior of the bilayer has been calculated (Fig. 2a).  Any material submerged in the bilayer, including channel proteins, is at equilibrium with these forces.

Fig 2.  The intrinsic forces in the lipid bilayer, and how applied forces can open MS channels. (a) The intrinsic force profile plotted as its direction and magnitude along the depth of the bilayer (left), and a cartoon of a channel protein in section (right), showing how the sharp tension (narrow arrows) near the lipid necks balanced by more diffused pressure nearby (broad arrows) is exerted on the channel-lipid interface (red).  (b).  The forces at the crucial channel-lipid interface (red) will change when the bilayer (green) is stretched or bent (left), or when the channel is displaced from the bilayer through a tether like an elevator (right).  It is also possible that the tether, through ancillary proteins, pulls on the lipids surrounding the channel (not shown).  In all cases, changes in the force profile at the interface (red) can become the ultimate trigger for the channel’s conformational change.  From (7).

External pressure can thin and deform the bilayer changing its internal force profile.  The mismatch at the channel-lipid interface can be the energetic drive for the channel protein to reach a new conformation, e.g. the open state.   In molecular dynamics simulation experiments, Schulten et al (2003) showed that MscL steered by forces at the levels lipid necks indeed open (3) in a manner similar to that predicted by subunit cross-linking and computational modeling (4). (Fig. 3).

Fig 3.  Opening E. coli MscL.   Helical segments (S1, S2, S3) and transmembrane helices (M1, M2) in one MscL subunit, as deduced from sequence and other analyses (left).  Side (upper center) and top (lower center) views of the closed channel backbone structure of MscL, by analogy to the crystal structure of the M. tuberculosis MscL homolog.  In a molecular dynamics simulation forces from the lipid are directed as indicated by the yellow arrows.  The open structure deduced from both modeling and experimentation (right).  The opening is huge (about 30 Angstrom in diameter): befitting its ability to release solutes indiscriminately.  The work to increase the area under tension constitutes the free energy difference that partitions the open and closed states. From (7).

Chemically unrelated amphipathic molecules preferentially added to one monolayer cause the bilayer to bend and thereby alter the internal force profile therein.  Such amphipaths can open MscS (5) and MscL (6).  The structure of MscL opened with amphipaths, as deduced from EPR spectroscopy by Perozo et al. (6), agrees with open structure predicted by other methods (4) in the main. 

The concept of mechanosensitive channels being gated by forces inherent in the lipid bilayer is in variant with the intuition that such channels in eukaryotes are pulled by cytoskeletons or extracellular “strings”.  Careful examination of existing evidence questions this intuition (7).  In hair cell, the gating spring may pull the channel away from the bilayer (Fig. 2b) or pull on the surrounding bilayer itself.

See (7, 8) for reviews.

In the 80s, our first patch-clamp survey of the yeast plasma membrane revealed a 36- picoSiemens stretch-activated conductance (9).   Inadvertently, our recent research showed that this channel is likely to be operated by membrane lipids.  Osmotic downshock induces a Ca2+ influx, presumably through this channel.  We examined this Ca2+ influx in each of the 4,906 yeast gene deletants and found several over-responders that seem to have sensitized channels.  Of all the possibilities (general metabolism, ion distribution, cytoskeleton, cell wall, membrane receptors etc.), most of the over-responders turned out to have deleted of proteins functioning in the biogenesis of phospholipids, sphingoliids, or ergosterol.  This unbiased examination of the yeast genome supports the force-from-lipid principle is applicable to eukaryotic as well as prokaryotic channels.

1.	Sukharev et al. (1997) Annu. Rev. Physiol. 59: 633.
2.	Cantor (1997) J. Physic. Chem.101: 1723.
3.	Gullingsrud & Schulten (2003) Biophys. J. 85: 2087.
4.	Sukharev et al. (2001) Nature 409: 720.
5.	Martinac et al. (1990) Nature 348: 261-263.
6.	Perozo et al. (2002) Nature 418: 94213.
7.	Kung (2005) Nature 436: 647.
8.	Anishkin & Kung (2005) Cur. Opin. Neurobiol. 15: 397.
9.	Gustin et al. (1988) Science 242: 762.
10.	Loukin et al. (2007) FASEB J. 8: 1813.

 

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