The MscL of E. coli is perhaps the best understood of these mechanosensors.
Mechanically gated MscL activity was first observed in patch clamp experiments con-
ducted on giant E. colispheroplasts, and subsequent purification of MscL and reconsti-
tution in artificial lipid bilayers demonstrated that the channel retained its characteristic
mechanosensitive conductance (~3 nS) and gating kinetics even in a cell-free environ-
ment (Sukharev et al. 1993; Sukharev et al. 1994). These findings convincingly estab-
lished that changes in membrane tension alone can be sufficient to activate a
mechanosensitive ion channel.
Sequence analysis revealed that the E. coli(Eco-)MscL encodes a 136 amino acid
membrane protein with cytoplasmic N- and C-terminal regions and two -helical trans-
membrane domains (designated TM1 and TM2) connected by a periplasmic loop (Blount
et al. 1996). Determination of the crystal structure of a homolog of Eco-MscL, the MscL
ofMyobacterium tuberculosis(Chang et al. 1998), showed that five MscL subunits form
a homopentamer, with TM1 domains forming the funnel-shaped permeation pathway and
TM2 helices interacting with the lipid bilayer and surrounding the central barrel of TM1
domains (Figure 5.4a). On the cytoplasmic side of the channel, the five C-terminal he-
lices assemble into a bundle not required for gating but presumed to act as a size-
exclusion filter (Anishkin et al. 2003). Though unresolved in the crystal structure, the
five amphiphilic N-terminal (S1) domains were predicted to organize as -helices, inter-
acting to form another bundle situated between the channel pore and the C-terminal
“hanging basket” (Sukharev et al. 2001b; Sukharev et al. 2001a).
How do these different channel domains contribute to the mechanical gating of MscL?
According to a current model based on computer simulations and experiments using cys-
teine substitutions to stabilize specific domain interactions, the channel is activated by
the sequential tension-induced opening of two gates (Sukharev et al. 2001b). Upon
stretch of the membrane, the channel pore increases in diameter from ~0.2 nm to about
3.5 nm (Doyle 2004). This dramatic change in channel conformation is thought to occur
as the transmembrane domains TM1 and TM2 undergo significant tilting, thereby swing-
ing away from the central channel axis. This iris-like expansion is considered the initial
gating event because it would open the hydrophobic constriction at the narrow end of the
funnel-shaped TM1 pore acting as a barrier to ion permeation. However, removal of this
constriction is not sufficient to elicit full conductance of the channel. In fact, the initial
conformational change only seems to draw the channel into a low subconducting state
(Anishkin et al. 2005) (Figure 5.4a and Color Section). Further extension is required to
activate the channel completely. The transition to the fully open state is thought to depend
on the disruption of the N-terminal bundle connected to the TM1 domains via linker re-
gions. Only when the TM barrel fully expands at close to lytic tensions is force transmit-
ted via the linkers to this bundle, pulling it apart and thereby opening the second gate to
release huge amounts of ions and small solutes (Sukharev et al. 2001b).
5.3.2. Gating through Tethers: The Mechanoreceptor for Gentle Touch in
Caenorhabditis elegans
The nematode C. elegansresponds to gentle touch (as little as 10-μN force) with an
avoidance reaction, moving backward when the stimulus is applied to the head and mov-