Sensing and Responding to Membrane Tension: The Bacterial MscL
Channel as a Model System
Irene Iscla and Paul Blount*
Department of Physiology, UT Southwestern Medical Center at Dallas, Dallas, Texas
tion; cardiovascular regulation; kidney function; and osmoregulation. Many channels from an assortment of families are now
candidates for eukaryotic mechanosensors and proprioception, as well as cardiovascular regulation, kidney function, and osmo-
regulation. Bacteria also possess two families of mechanosensitive channels, termed MscL and MscS, that function as osmotic
emergency release valves. Of the two channels, MscL is the most conserved, most streamlined in structure, and largest in
conductance at 3.6 nS with a pore diameter in excess of 30 A˚; hence, the structural changes required for gating are exaggerated
and perhaps more easily defined. Because of these properties, as well as its tractable nature, MscL represents a excellent model
for studying how a channel can sense and respond to biophysical changes of a lipid bilayer. Many of the properties of the MscL
channel, such as the sensitivity to amphipaths, a helix that runs along the membrane surface and is connected to the pore via
a glycine, a twisting and turning of the transmembrane domains upon gating, and the dynamic changes in membrane interac-
tions, may be common to other candidate mechanosensors. Here we review many of these properties and discuss their struc-
tural and functional implications.
Mechanosensors are important for many life functions, including the senses of touch, balance, and propriocep-
Many membrane proteins appear to sense mechanical
forces. Some channels appear to be membrane-tension-
gated and are often referred to as mechanosensitive (MS)
channels, whereas other channels, specifically the ligand-
and even voltage-gated channels, have been found to be
modulated by membrane tension (1). Voltage and MS
channels may even have a common evolutionary origin
(2). Results from studies of amphipaths suggest that many
of the characterized MS proteins, including microbial
sensors and pumps, sense the biophysical properties of the
membrane, specifically the tension within it (3–6). Given
that protein-lipid interactions are of prime importance for
these families of channels, surprisingly little is known about
the dynamics of protein-lipid interactions upon membrane
protein activation or channel gating. However, one mole-
cule, the bacterial MS channel of large conductance
(MscL), is now evolving as a paradigm of the dynamics of
protein-lipid interactions upon channel gating.
Two crystal structures have been obtained for MscL. The
first shows a homopentameric structure that appears to be
a closed or nearly closed state of the Mycobacterium
tuberculosis MscL (Tb-MscL) channel. Several lines of
evidence suggest that this structure may reflect a channel
that is close to but does not completely reflect the in vivo
closed state (7). The second structure shows a C-terminal-
truncated Staphylococcus aureus channel (Sa-MscL) with
a homotetrameric oligomerization rather than a pentameric
one as expected (8). Although the structure was originally
thought to reflect an intermediate-gating state, it did not fit
well with some expectations for structural changes that
should occur. Specifically, several lines of evidence sug-
gested that a corkscrew motion of the first transmembrane
domain (TMD1) occurs early in the gating process (7,9),
but this was not seen in this putative gating intermediate.
Indeed, in subsequent studies, no significant amount of
Sa-MscL tetramer was observed in vivo (9,10). The reorga-
nization of the pentamer into a tetrameric complex appears
to be a detergent-dependent process. Specifically, the deter-
gent LDAO, which was used in the crystallization process,
appears to induce this oligomeric shift, whereas the deter-
gents Triton X-100 and C5E8 do not (9). Interestingly, the
oligomeric rearrangement appears to be reversible (9).
Although there are some indications that the truncation of
the protein may have played a role in amplifying this oligo-
meric reorganization (11), the observation that even the
truncated Sa-MscL protein oligomerizes into pentamers
in vivo strongly supports the notion that tetramerization of
this channel is largely a detergent-dependent phenomenon
(10). Perhaps MscL, as a mechanosensor, is more sensitive
to changes in its environment because of its intimate
connection to the lipid bilayer. This observation of deter-
gent-dependent reorganization of the protein not only serves
as a cautionary tale for interpreting crystallographic results,
it may also underscore the importance of using a normal
lipid-bilayer environment, or detergents that better mimic
this milieu, to determine physiological structural elements.
Because it appears to be closer to a physiological state,
we show the nearly closed structure derived from the Tb-
MscL (Fig. 1, top). The protein starts with an amphipathic
a-helix that appears to lie along the membrane. A glycine
hinge is seen at position 12 (14 in Escherichia coli), leading
to TMD1, which as can be seen from above forms the pore
Submitted April 17, 2012, and accepted for publication June 12, 2012.
Editor: Eduardo Perozo.
? 2012 by the Biophysical Society
Biophysical Journal Volume 103 July 2012 169–174 169
constriction site, followed by a periplasmic loop region, and
then the second transmembrane domain (TMD2), which is
in contact with the lipid bilayer. The protein ends with a
pentameric a-helix bundle towhich each of the five subunits
contributes, and a linker connects TMD2 with this helical
bundle. Because of its tractability, almost all functional
studies have focused on the E. coli MscL (Eco-MscL). In
orchestration with another channel family, MscS, the
MscL channel functions as a biological emergency release
valve that opens upon acute decreases in the osmotic envi-
ronment to protect the cell from lysis (12). Upon stimula-
tion, the channel opens a massive pore that has been
predicted by molecular sieving experiments to exceed
30 A˚(13). The channel is predicted to decrease its thickness
within the membrane and to open like the iris of an old-
fashioned camera, with the first TMD largely forming the
pore (14,15). A model for the type of rearrangement that
occurs among the TMDs for the Eco-MscL, as determined
by EPR and other methods (15), is shown in the bottom
panel of Fig. 1. Early mutagenesis work with Eco-MscL
suggested that residues along both the periplasmic and cyto-
plasmic aqueous/lipid boundaries are important for normal
function (16,17); however, the periplasmic region of the
pore forms a vestibule, whereas the cytoplasmic region
better defines the pore constriction. Indeed, as discussed
below, recent evidence centers more on the cytoplasmic sub-
domains and the importance of the dynamics of protein-lipid
interactions in this region.
In this review, we first concentrate on what we refer to as
lipid-protein interactions (specifically, the important and
not-so-important properties of the lipid membrane) and
discuss what the channel may actually be sensing. We assess
whether the channel senses the pressure across the mem-
brane or the tension within it. We then discuss whether
hydrophobic mismatch or changes in the lateral pressure
profile of the membrane have a vital role in transducing
the energy of membrane tension to the protein to induce
conformational changes. Next, we address the issue of
whether specific lipid headgroups interact with the protein
and modify channel activity. Finally, we discuss what is
known about protein-lipid interactions in different regions
of the protein, including recent studies on domains within
the cytoplasmic aqueous-lipid interface.
Pressure across the membrane or the tension
within it, hydrophobic mismatch, or changes
in the lateral pressure profile: what is actually
Perhaps the first issue to address when discussing what is
actually sensed is whether the channel senses pressure
across a membrane (analogously to a trashcan lid that opens
upon pressure within the can) or the tension lateral to and
within the membrane. By La Place’s law, tension (T) within
a membrane is proportional to the pressure across it (P) and
the radius of curvature (r) of the matrix in the following rela-
tionship: T ¼ 1/2 P $ r. The pressure can be measured by
a pressure transducer, and with good optics one can observe
the radius of curvature of a patched membrane and thus
calculate the tension. Moe and Blount (18) studied several
membrane patches with quite different radius-of-curvature
values, and found that although they yielded different pres-
sure profiles for Eco-MscL gating, when tension was calcu-
lated and plotted, they all gated at the same calculated
tension. Hence, it appears that pressure across the mem-
brane plays no detectable role in MscL channel gating,
and membrane tension is by far the primary stimulus.
We are left, then, with one or more of the biophysical
properties of the membrane contributing to the sensed stim-
ulus. The first property to consider is membrane thinning.
The best estimates are that a normal, naked biological
membrane can compress by only a few percent, depending
on its composition (19). It seems unlikely that this would
be enough to induce large protein shifts in conformation.
On the other hand, when a protein is within a membrane,
w e i vc ims a l p i repwe i ve d i s
M. tuberculosis and a model for the structure of the TMDs of E. coli
MscL in the open structure. The top panels show the structure of the nearly
closed Tb-MscL as resolved in the crystal structure. The relatively simple
topology of each MscL subunit can be observed in the side view (left),
with a single subunit highlighted in red for clarity. The approximate
position of the lipid membrane is indicated by the horizontal blue lines.
The N-terminus forms a helix that lies along the membrane and connects
with TMD1. TMD1 crosses the membrane lining the pore of the channel,
as can be clearly observed in the periplasmic view (right). TMD1 is con-
nected to TMD2 by a periplasmic loop. TMD2 surrounds the TMD1 helix
and is in contact with the membrane. Finally, a cytoplasmic loop connects
TMD2with a cytoplasmic helixthat forms a bundle at the C-terminalend of
the channel. The bottom panels show similar side (left) and periplasmic
(right) views of a theoretical model based on EPR and other experimental
data (15) for what the TMDs might look like in the open structure. Note that
the N- and C-terminal domains and periplasmic loop are not shown; the
TMDs are connected with a straight line for orientation purposes.
Structure of the homopentameric MS channel MscL from
Biophysical Journal 103(2) 169–174
170Iscla and Blount
it can influence the membrane thickness around it through
hydrophobic mismatch (20). This suggests a potential inter-
active dynamics between membrane thickness and protein
structure, i.e., each can influence the other. Indeed, when
a Tb-MscL F80W channel was studied by quenching of
Trp fluorescence by brominated phospholipids in lipids of
different lengths, the results showed that lipid-binding
constants changed by only a factor of 1.5 in the chain length
range from C12 to C24, which is much less than expected
from theories of hydrophobic mismatch in which the protein
is treated as a rigid body (21). This suggests that MscL
distorts to match changes in bilayer thickness. Consistent
with this proposed protein-lipid interplay, as discussed
above and shown in Fig. 1 (bottom), the Eco-MscL protein
is thought to thin in the plane of the membrane upon gating;
thus, it is undoubtedly changing the thickness of the
membrane surrounding it. To directly determine whether
membrane thickness plays a role in Eco-MscL channel
gating, Perozo et al. (6) reconstituted the protein into lipids
of varying carbonyl chain length. Thinning of the membrane
did not lead to channels that gated spontaneously, but did
make them easier to gate, suggesting it may modulate gating
but was not the primary stimulus. On the other hand, the
same study demonstrated that the amphipath lysophosphati-
dylcholine (LPC), which would intercalate into the mem-
brane asymmetrically, gated the channel. Thus, it appears
that distortion of the membrane’s lateral pressure profile is
a stimulus for gating. These data are consistent with several
other mechanosensors that appear to be activated by such
amphipaths (1). However, it is important to note that
changes within the lateral pressure profile, rather than the
curving of the membrane per se, are the stimulus for
MscL gating (18).
Are negatively charged lipids required for normal
For several membrane proteins, including osmotically regu-
lated transporters (22), negatively charged lipids appear to
play a vital role in their function. For MscL, the data are
complicated. One of the standard assays for MscL activity
in a reconstituted system is the flux of calcein from lipo-
somes. The calcein dye is quenched when at high concentra-
tion in the liposomes, and only gives signal when fluxed.
One can activate the channel chemically using the amphi-
path LPC, or by inducing a charge in the pore by treating
an MscL cysteine mutant (usually Eco-MscL G22C) with
the charged sulfhydryl reagent MTSETþ. When incorpo-
rated into liposomes containing only the zwitterionic head-
group PC, Eco-MscL was shown to have a blunted calcein
flux response compared with liposomes containing nega-
tively charged lipids (23). This finding was interpreted to
mean that a significant number of channels are in an inactive
conformation when they are incorporated into membranes
devoid of anionic lipids. Indeed, basic calculations suggest
that the incorporation rate of active channels into liposomes,
even those containing anionic lipids, is only 0.7%. However,
previous findings obtained by patch-clamp assay showed
that when Eco-MscL is incorporated into pure PC lipids,
its function is indistinguishable from that observed when
it is incorporated into mixed lipid bilayers containing
anionic lipids (18). One of the major differences between
the flux and patch-clamp studies is the process used to incor-
porate the purified MscL into the lipid bilayers. MscL was
incorporated directly into the liposomes for the flux studies,
whereas a dehydration step, which allows the lipids to form
a multibilayer lattice, was used for the patch-clamp studies.
In the latter approach, liposomes, or blisters, were encour-
aged to form after rehydration by the addition of Mg2þ
(24). Could the differences in results then be due to the effi-
ciency of incorporation into the lipids, or even the clustering
of channels into a subpopulation of vesicles? The MscL
protein does appear to incorporate at reasonable levels in
the flux system (21); however, a direct and quantitative
comparison of incorporation into different lipids has not
been performed to date. Furthermore, although the observa-
tion that brominated lipids efficiently quench the fluores-
cence of a tryptophan MscL mutant (F93W) suggests that
MscL channels are not highly clustered (23), MscL channel
clustering has been proposed by another study (25). Regard-
less, it is clear that in one liposome reconstitution system,
fewer functional channels are incorporated when anionic
lipids are absent. The dehydration/rehydration approach
may bypass this by either forcing incorporation or selecting
for bilayers bearing functional channels in the blister forma-
tion process. In summary, although anionic lipids appear to
play a role in the incorporation of functional MscL channels
in one reconstituted system, in another system the patch-
clamp data demonstrate that anionic lipids are not required
for normal activity, and thus the hypothesis that anionic
lipids regulate MscL function in vivo remains speculative.
The N-terminal amphipathic a-helix: S1 stabilizer,
anchor domain, and slide helix
In the original M. tuberculosis crystal structure, the
N-terminal region of the protein (specifically, the first nine
amino acids) was not resolved (26). However, a reevaluation
of the structure resolved the placement of this region as an
amphipathic a-helix lying along the cytoplasmic membrane
(27). This region is highly conserved, suggesting its impor-
tance. In particular, there are two phenylalanines. According
to the E. coli register for amino acid location, the phenylal-
anines at positions 7 and 10 are 97% and 100% conserved,
respectively, in an alignment of 232 species; in the few
outliers, they are substituted only by leucines (28). The S1
domain motif of XXYYFYYFXX (with X being hydro-
phobic and Y polar amino acids) is conserved in 79%,
showing only small variations. This motif is even conserved
in species that have as many as 30 additional amino acids
Biophysical Journal 103(2) 169–174
N-terminal to this helix. In addition, early mutagenic and
deletion studies touted the importance of this region of the
protein for channel function. Even small deletions in this
region of three, eight, or 11 amino acids, or substitution of
nine amino acids of the S1 domain with a random sequence,
can lead to channels with either decreased sensitivity or total
loss of activity (29,30).
Iscla et al. (28) serially mutated every residue in the
Eco-MscL S1 helix to cysteine, and evaluated the proba-
bility of disulfide bridging between subunits. They found
that several of the mutant channel activities under variable
redox potentials were consistent with the crystal structure
showing this region running along the cytoplasmic mem-
brane. In evaluating the potential function of the S1 domain,
it may be important to note that all current models for the
gating of MscL predict a significant tilting of the TMD1
membrane upon opening (15,31–33), and several lines of
evidence also suggest a clockwise corkscrewing of TMD1
of almost 180?, as would be observed from the periplasm
(14,15,28,33,34). It therefore seems likely that the S1
domain helps to define the tilt and turns of TMD1 by main-
taining its interaction with the membrane, thus serving as an
anchor (Fig. 2 A). Interestingly, the two most conserved resi-
dues within the S1 domain, F7 and F10 (represented as red
hexagons in Fig. 2 A), have a high affinity for the lipid envi-
ronment. Given this functional role, the highly conserved
glycine at position 14 in E. coli would appear to be a hinge
between TMD1 and S1 domains (Fig. 2 A). This structure,
a helical membrane running along the cytoplasmic mem-
brane and connected to the pore domain via a flexible
glycine, is found in several other channels where its pertur-
bation can often have functional consequences. For
example, the bacterial inward rectifying Kþchannel KirBac
harbors a cytoplasmic a-helix running parallel to the mem-
brane (slide helix) (35), which was found to directly interact
at the cytoplasmic end of the putative pore-forming TMD6
domain of the MS yeast TRPY1 channel was shown to be
(37). This region could potentially form an amphipathic
a-helix, although the structure of this channel has not yet
been determined. A crystallographic structure of MscS,
another bacterial MS channel from an independent channel
family, also appears to contain an a-helix along the cyto-
plasmic/membrane surface just adjacent to the pore
(27,38). In similarity to MscL, current models of MscS
gating predict a tiltingof the pore domain (39), anda glycine
(Gly-113) is also present between these two domains, which
probably serves as a hinge (27,38). Finally, a recently solved
structure of the mammalian lipid-MS channel TRAAK
shows a cytoplasmic amphipathic helix after the first pore
domain. The ability of this inner helix to interact with both
the hydrophobic tails and acidic headgroups of the
nel’s mechanosensitivity (40). This is only a partial list;
several other examples of channels containing cytoplasmic
a-helix running parallel to the membrane also exist. Thus,
MscL may be using a common strategy among channels,
i.e., using a slide helix as an anchor domain to stabilize the
opening transmembrane pore within the bilayer.
Dynamics of the cytoplasmic end of TMD2
Early random mutagenesis studies not only indicated the
importance of the pore domain in channel function but
also revealed a handful of gain-of-function and loss-of-
function mutations lining the aqueous-lipid interface at the
cytoplasmic end of TMD2 (41,42). The cytoplasmic end
of the TMD2 has been defined experimentally. Probe acces-
sibility data and electron paramagnetic resonance (EPR)
studies determined a membrane span for Eco-MscL of 25
residues from His-74 to Leu-98 (43), and tryptophan fluo-
rescence spectra of Tb-MscL showed a transmembrane
span comparable to that observed by EPR (44).
Five amino acids after the predicted end of TMD2, there
is a cluster of charged residues (RKKEE) from residues
104–108 in Eco-MscL (Fig. 2 B). An early mutagenesis
study of Eco-MscL showed that a C-terminal deletion at
this point leads to nonfunctional channels (29), and another
study suggested that changing the composition of the region
can change the pH modulation of the channel (45). The
RKK within the sequence has also been suggested to
interact with anionic phospholipids, and mutation of these
residues to glutamines influences channel conformation
and function (46).
TMDs and the lipid membrane. (A) TMD1 and the N-terminal helix (S1)
are represented in the closed (left) and open (right) states. The position
of Gly-14 between the S1 and TMD1 domains is shown as a green sphere.
The conserved phenylalanine residues at positions 7 and 10 in the
S1 domain are shown as red hexagons. (B) A single MscL subunit is repre-
sented in closed (left) and open (right) states. TMD2, the cytoplasmic loop,
and the C-terminal helix are highlighted in a darker color. The positions of
residue N103 and the charged cluster (RKKEE) are shown as a green
hexagon and a red star, respectively.
Schematic representation of the interactions between MscL
Biophysical Journal 103(2) 169–174
172 Iscla and Blount
In a recent study, Iscla et al. (47) investigated the potential
dynamics of the region. They screened a library of indi-
vidual cysteine mutations on the cytoplasmic side of
TMD2 from residues L98C–E108C before and after post-
translational modification with hydrophobic MTS reagents.
Importantly, the screen involved an invivoassessment of the
viability of the cells expressing the modified protein,
precluding the need for any protein purification or in vitro
assays. The scan revealed a hot spot at position N103 in
which changes in polarity showed the greatest effects in
channel function with multiple reagents. Patch-clamp anal-
ysis, site-directed mutagenesis, and tryptophan fluorescence
measurements confirmed these findings. In summary, the
data strongly suggest that this region is critical for MscL
gating, and that N103 transiently enters the headgroup
region of the bilayer during the normal gating process, as
shown in Fig. 2 B. Interestingly, a series of charged amino
acids distal to a cytoplasmic transmembrane helix were
noted in other channel families predicted to have MS
channel members (see Kloda et al. (45) for a discussion),
suggesting that here again, MscL may be utilizing
conserved or analogous molecular mechanisms.
The bacterial MscL channel is the largest conducting gated
pore and functions as an osmotic emergency release valve.
By exploiting the exaggerated conformational changes
that occur in the MscL channel, as well as its tractable
nature, investigators have been able to use an assortment
of approaches including bacterial genetics involving in vivo
screens, biochemical screens, and electrophysiological
characterization in both native and synthetic membranes
to obtain a detailed assessment of the structure-function
relationships within this channel. It thus serves as a model
for how a protein can sense and respond to a mechanical
force. Many of the findings regarding how this channel func-
tions now appear to be common themes. We now know that
MscL senses the tension in the membrane—more specifi-
cally, changes within the lateral pressure profile (6,18).
MS currents from yeast (48) and chick skeletal muscle
(49) have also shown to respond to tension, suggesting
that this may be the stimulus sensed by eukaryotic mechano-
sensors as well. Indeed, when exposed to amphipaths that
intercalate into the membrane and thus cause stress within
the bilayer, many eukaryotic channels show lower thresh-
olds or spontaneous activity, suggesting that membrane
tension is a common stimulus for MS channel activation
(1). Furthermore, the observation that the channel can
function in a bilayer composed of the zwitterionic phospha-
tidylcholine, which is not synthesized by E. coli, demon-
strates that interactions with negatively charged lipids or
native lipid headgroups are not required for normal MS
channel activity (18). Both TMDs tilt within the membrane,
and TMD1 rotates in a corkscrew fashion clockwise, as
observed from the periplasm (14,15,33,34). The S1 amphi-
pathic helix that runs along the cytoplasmic membrane and
is attached to this gating pore domain via a flexible glycine
appears to serve as a stabilizer for the twists and turns this
pore-forming TMD1 must undergo, and such an anchor or
slide helix appears to be a common feature among many
channels (28). Finally, we now have evidence that at least
one region of the protein near the lipid-interacting TMD2
is extremely dynamic and embeds itself within the mem-
brane during the gating process. Future experiments will
show whether this feature is shared with other mechanosen-
sors. Undoubtedly, given its tractable nature, the MscL
channel will continue to reveal how it senses and responds
to membrane stretch. Investigators must now determine
whether these gating principles are also common among
mechanosensors and other channels.
P.B. was supported by grants I-1420 from the Welch Foundation,
NNH08ZTT003N NRA from NASA, RP100146 from the Cancer Preven-
tion and Research Institute of Texas, and AI080807 and GM061028
from the National Institutes of Health. I.I. was supported by grant
12SDG8740012 from the National American Heart Association.
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