An in vivo assay identifies changes in residue accessibility on mechanosensitive channel gating
MscL is a mechanosensitive channel of large conductance that functions as an "emergency release valve," allowing bacteria to survive acute hypoosmotic stress. Although Escherichia coli MscL is the best-studied mechanosensitive channel, structural rearrangements occurring during gating remain disputed. Introduction of a charged residue into the pore of MscL was shown to result in a reduced-viability phenotype. Here, we probe for residues in the transmembrane domains that are exposed to the aqueous environment in the presence and absence of hypoosmotic shock by reacting a charged sulfhydryl reagent with substituted cysteines. Subsequent analysis of cell viability allows for an assessment of residues exposed in the closed and opening states in vivo. The results suggest that the crystal structure of MscL derived from the Mycobacterium tuberculosis orthologue may reflect a nearly closed rather than fully closed state and support a clockwise rotation of the pore-forming first transmembrane domain on gating.
assay identifies changes in residue
accessibility on mechanosensitive channel gating
Jessica L. Bartlett, Gal Levin, and Paul Blount*
Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040
Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved May 28, 2004 (received for review March 23, 2004)
MscL is a mechanosensitive channel of large conductance that
functions as an ‘‘emergency release valve,’’ allowing bacteria to
survive acute hypoosmotic stress. Although Escherichia coli MscL is
the best-studied mechanosensitive channel, structural rearrange-
ments occurring during gating remain disputed. Introduction of a
charged residue into the pore of MscL was shown to result in a
reduced-viability phenotype. Here, we probe for residues in the
transmembrane domains that are exposed to the aqueous envi-
ronment in the presence and absence of hypoosmotic shock by
reacting a charged sulfhydryl reagent with substituted cysteines.
Subsequent analysis of cell viability allows for an assessment of
residues exposed in the closed and opening states in vivo. The
results suggest that the crystal structure of MscL derived from the
Mycobacterium tuberculosis orthologue may reﬂect a nearly closed
rather than fully closed state and support a clockwise rotation of
the pore-forming ﬁrst transmembrane domain on gating.
he ability to detect mechanical force is crucial for essentially
all life. One paradigm, which has been well studied at the
molecular and structural levels, is the ability of microbes to
detect membrane stretch invoked by osmotic environment.
Escherichia coli contains two mechanosensitive channels that
have been shown to be involved in osmotic regulation, MscL and
MscS (1). Homologues of these molecules are found in virtually
all microbes, including the Archea (2). The genes appear to
encode proteins that serve a redundant function in osmotic
adaptation because deletion of both mscS and mscL is required
to observe a phenotype (1). The resulting double-null strain is
osmotically fragile, showing reduced viability on acute hypotonic
shock (osmotic downshock).
To date, MscL is the best-studied mechanosensitive channel.
Many mutations that effect a gain-of-function (GOF) pheno-
type, in which the cells show slowed growth or decreased
viability, have been isolated and studied (3–6). The pivotal point
came when a crystal structure of an orthologous channel from
Mycobacterium tuberculosis was resolved to 3.5 Å (7). The
channel was found to be a homopentamer. However, the study
could not determine whether the solved structure reflects a
closed or ‘‘nearly closed’’ state of the channel (the channel
constricted to a 4-Å-diameter pore), nor could it accurately
predict the structural changes that occur on channel gating.
What was clear from the crystal structure was that the first
transmembrane domain (TM1) lines the lumen and that to
obtain the predicted open-pore size of ⬎30 Å in diameter (8),
large structural changes must occur. Subsequent studies have
predicted features of the channels’ open structure; one suggested
model is based on modeling, crosslinking, and disulfide-trapping
experiments (9, 10), and the other is based on EPR results (11).
Although agreement exists on general aspects of gating, such as
tilting of the transmembrane domains, several fundamental
differences exist, including the identity of specific residues likely
to line the open pore.
One approach that has been often used for identifying residues
within a channel pore is the substituted cysteine accessibility
method (SCAM) (12). In the SCAM, cysteine substitutions are
generated within the protein and sulfhydryl reagents are allowed
to react on gating. Hydrophilic, often charged, reagents are
typically used to assure that only residues that are accessible to
the aqueous environment are modified. If the residue is buried
within the closed channel but exposed on gating, then the
reagent will react with the cysteine only on channel opening. For
channels with small pore sizes, a decrease in conductance is often
observed on this modification.
Recently, Batiza et al. (13), studying E. coli MscL, adapted the
SCAM to test for accessibility in vivo. This group exploited
previous observations demonstrating that hydrophilic or charged
substitutions within TM1 often lead to decreased viability (4, 6).
This appears to be due to channel misgating because patch-
clamp analysis demonstrated that the channels gate at lower-
than-normal membrane tensions. In addition, reaction of
charged sulfhydryl reagents with a cysteine mutant, G22C,
demonstrated that these changes could be effected in situ,as
assayed by patch clamp (14). Batiza et al. (13) studied the
accessibility of a mutant with a single substitution, L19C, and
found a decreased viability on treatment of the charged sulfhy-
dryl reagent, [2-(trimethylammonium) ethyl]methanethiosul-
fonate bromide (MTSET), with intact cells. This decrease in
viability was observed only when the channel was gated by an
acute osmotic downshock, suggesting that the residue was only
exposed on gating.
We recently generated a mutant library in which every amino
acid in the transmembrane domains had been sequentially
replaced with cysteine. Because wild-type E. coli MscL contains
no cysteines, each substituted cysteine is unique within the
subunit. Every mutant in the library was assayed for its ability to
confer phenotypic changes, and many were characterized elec-
trophysiologically (5). Although not as severe as reported (3–6),
GOF-effecting cysteine substitutions were noted in the middle of
TM1, near the proposed channel constriction point and toward
the periplasmic and cytoplasmic regions of the second trans-
membrane domain (TM2). Here, we use this characterized
library for a modified in vivo SCAM. The ability to drastically
decrease viability on residue modification allows us the unique
ability to resolve aspects of the structure of MscL in different
conformational states while it resides in a living cell. Although
our results are consistent with the overall predictions of the
crystal structure, they suggest modifications needed to define the
fully closed state of the channel. Our results also provide strong
support for one of the contested models for structural changes
that occur during channel gating.
Materials and Methods
Strains. The cysteine mutant library was generated as described
(5). The wild-type E. coli mscL and cysteine substituted mscL
mutants were inserted into pB10b (17), and expression was
induced by using isopropyl
-D-thiogalactoside. The E. coli
This paper was submitted directly (Track II) to the PNAS ofﬁce.
Abbreviations: GOF, gain-of-function; TM1, ﬁrst transmembrane domain; TM2, second
transmembrane domain; SCAM, substituted cysteine accessibility method; LOF, loss-of-
function; MTSET, [2-(trimethylammonium) ethyl]methanethiosulfonate bromide.
*To whom correspondence should be addressed. E-mail: paul.blount@utsouthwestern.
© 2004 by The National Academy of Sciences of the USA
July 6, 2004
FRAG-1 (18) derivative strain, MJF455 ⌬mscL::Cm, ⌬mscS (1),
was used as host. MTSET was obtained from Toronto Research
Chemicals (North York, ON, Canada).
Functional Assay. Recently streaked colonies were grown
overnight at 37°C in 1 ml of citrate-phosphate-defined medium
(per liter: 8.57 g of Na
, 1.34 g of citric
acid, 1.0 g of NH
, 0.001 g of thiamine, 0.1 g of Mg
0.002 g of (NH
O) plus 1 mM ampicillin. The
fresh overnight culture was diluted 1:20 in 2 ml of this defined
medium and grown for l h. The culture was then diluted to an
of 0.05 in 2 ml of the same medium supplemented with 0.5
M NaCl. The cultures were then grown to an OD
at which stage expression was induced for1hwith1mM
-D-thiogalactoside. The induced cultures were diluted 1:20 into
citrate-phosphate medium containing (i) 0.5 M NaCl as control;
(ii) no additives for osmotic downshock; (iii) 0.5 M NaCl and 1
mM MTSET for MTSET alone; or (iv) 1 mM MTSET for
MTSET and osmotic downshock. Cells were grown with shaking
at 37°C for 15 min, and then six consecutive 1:10 serial dilutions
were made in medium containing either no salt (for the osmotic
downshock conditions) or 0.5 M NaCl (for the mock-shock
conditions). These diluted cultures were plated in duplicate and
grown overnight at 37°C on LB-ampicillin agar plates. The
colony-forming units were counted and averaged per experi-
ment; no statistically significant differences in colony-forming
units for the mock-shock control were observed between wild
type and any mutant (all were ⬇1.2 ⫻ 10
per OD unit). All data are presented as a percentage of the
Functional Assay Was Used to Scan the Transmembrane
Domains for Residue Positions.
The in vivo SCAM was used to
identify the amino acids of MscL that are exposed to the aqueous
environment on channel gating. We used a transmembrane-
domain cysteine library to assay all residues within TM1 and
TM2 (1). The mutated proteins were expressed in an mscL
double-null strain (MJF455). In the primary screen, the
library was exposed simultaneously to both the positively
charged sulfhydryl reagent MTSET and acute osmotic down-
shock. The treated cells were then plated and viability scored. As
seen in Fig. 1, several of the cysteine mutants demonstrated a
significant decrease in viability on treatment. Mutants exhibiting
⬍60% viability were targeted for further study.
All mutants were initially tested after 1 h of
thiogalactoside induction. However, consistent with a previous
study (5), at induced expression levels V23C conferred a GOF
phenotype that led to a viability too low to measure. Therefore,
V23C was assayed in the absence of induced expression. Previous
studies had demonstrated that without induction the vector
allows the expression of two to six channels per cell (15). As
Fig. 1. Viability of cysteine mutants challenged by osmotic downshock in the presence of MTSET. Cysteine mutants within the ﬁrst (Upper, TM1) and the second
(Lower, TM2) transmembrane domains were tested. Stars indicate channels that were tested at uninduced levels, WT and V23. Mutants showing ⬍60% viability
(ﬁlled bars) were targeted for further study. All experiments were performed in duplicate with three to eight independent experiments; SEM for each is shown.
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0402040101 Bartlett et al.
shown in Fig. 1, even at such low expression levels, the viability
ratio of treated to untreated V23A-containing cells was quite low
for this mutant, supporting it as a candidate for further study.
Mutants Targeted by the Primary Screen Fall into Four Major Cate-
At this point we sought to exclude mutants that form a
nonfunctional channel, or are loss-of-function (LOF), and thus
are not suitable reporters of residue accessibility on gating.
Toward this end, we assayed all the mutants indicated by the
initial screen for their viability subsequent to the osmotic
downshock in the absence of MTSET treatment. The double-null
MJF455 strain used in this study, lacking both mscS and mscL,
normally shows decreased viability on acute osmotic downshock.
Expression of a functional MscL channel rescues this osmotic-
lysis phenotype, even at uninduced expression levels [see wild
type (Fig. 1) and V23C (Table 1)], which we know to be only a
few channels per cell (15). We assayed all of the mutants for their
viability subsequent to the osmotic downshock in the absence of
MTSET treatment. As seen in Table 1, group I, seven of the 19
candidates identified in the primary screen were determined to
be LOF by their inability to rescue this osmotic-lysis phenotype.
The remaining 12 candidates identified in the primary screen
showed decreased viability only in the presence of MTSET. To
determine whether MTSET was reacting with the channel when
in the closed state, we tested viability in the absence of osmotic
downshock. Four candidates demonstrated decreased viability
on MTSET treatment alone (Table 1, group II). In contrast,
seven of the candidates showed decreased viability that de-
pended on both MTSET and osmotic downshock (Table 1,
). Four of these demonstrated some decrease
in viability when treated with MTSET alone (group III
was assigned to a fourth category, group IV, because it showed
the interesting property that MTSET actually increased viability
on osmotic downshock.
The modified SCAM used here allowed us to identify MscL
transmembrane residues exposed in different conformational
states while the channel is within its native environment of a
living cell. Sulfhydryl reagents, including methanethiosulfonate
compounds, have been used in the past to probe the pore of
several other channels (12). However, the pore of the MscL
channel is much larger than those other channels and is esti-
mated to be ⬇30 Å (8). Therefore, it seems unlikely that MTSET
would block the passage of ions, as is observed for other
channels. Instead, the assay depends on the assumption that the
addition of a large hydrophilic group onto the residue will cause
the channel to gate more easily. Precedent exists: previous
studies found that hydrophilic substitutions within the TM1
domain, which lines the pore (7), leads to channels that gate at
lower membrane tension, thus resulting in a GOF phenotype (3,
4, 6). These substitutions appear to result in channels that more
easily go through transitions between closed and open states (6,
16). The assumption appears to be valid given the finding that at
least one substituted residue per
-helical turn of the pore-
forming region of TM1 was found to lead to MTSET-dependent
decreases in viability (Fig. 2).
Although the primary screen used here does not distinguish
between mutated residues that become exposed on channel
gating and those conferring constitutive LOF phenotypes, it is a
simple matter to distinguish among these categories on subse-
quent analysis. In the previous study in which MTSET was used
to identify a residue exposed on gating, the authors used several
mscL-null host strains (13). Here, we have specifically used an
mscL and mscS double-null strain, MJF455 (1). This use has the
advantage of avoiding false-negative results; if a channel is
functionally compromised and is significantly less sensitive than
normal, then the MscS channel cannot shunt the turgor forces
induced by the osmotic downshock. However, because the
double-null strain is osmotically fragile, this approach will also
identify nonfunctional or LOF mutants. A decreased viability
could also be due to MTSET binding in the closed state and lead
to a nonfunctional channel that induces lysis on osmotic shock.
However, group III
shows a phenotype independent of shock,
and mutations in group III
gave consistent results when per-
formed in PB104 (4, 17), a MscS-containing strain (13, and data
not shown). The cysteine library used here has been character-
ized previously; although the vast majority of mutants were
shown to be functional, a few conferred LOF phenotypes due to
misfunctioning channels, as opposed to heterologous or de-
creased expression levels (5). Although the osmotic downshock
procedure was different between the original study and the
present one, a strong agreement exists between the two; most of
the mutants we identify as LOF here (group I) are consistent
with the previous study. A few mutants, specifically Q80C, F85C,
and A89C, were categorized as LOF in the previous study but
were not recognized here, presumably because of differing
experimental conditions; none of these mutants were shown to
react with MTSET. In addition, we categorize G76C and I92C
as LOF; these mutants were not assayed in the previous study
because of their GOF phenotype. These mutants may not truly
Table 1. Categorization of mutants as a result of the
Strain Osmotic shock MTSET MTSET ⫹ osmotic shock
Empty vector 4.6 ⫾ 0.2 122 ⫾ 1 3.3 ⫾ 0.1
WT MscL 93 ⫾ 196⫾ 185⫾ 1
G14C 16 ⴞ 1 122 ⫾ 324⫾ 2
D18C 26 ⴞ 9 113 ⫾ 10 18 ⫾ 6
K31C 29 ⴞ 3 116 ⫾ 935⫾ 1
L36C 33 ⴞ 4 87 ⫾ 411⫾ 1
G76C 40 ⴞ 2 96 ⫾ 825⫾ 5
I92C 56 ⴞ 4 111 ⫾ 541⫾ 2
F93C 47 ⴞ 2 107 ⫾ 449⫾ 3
G26C 13 ⫾ 2 <0.2 ⱕ0.2
G30C 79 ⫾ 4 <0.2 25 ⫾ 3
S34C 89 ⫾ 15 <0.2 ⱕ0.2
A38C 85 ⫾ 3 61 ⴞ 4 55 ⫾ 1
G22C 80 ⫾ 2 48 ⴞ 6 0.60 ⴞ 0.08
V23C* 83 ⫾ 4 45 ⴞ 712ⴞ 1
A27C 94 ⫾ 6 54 ⴞ 243ⴞ 2
V37C 95 ⫾ 3 56 ⴞ 328ⴞ 4
L19C 115 ⫾ 19 129 ⫾ 13 14 ⴞ 6
I24C 87 ⫾ 292⫾ 2 22 ⴞ 4
M42C 78 ⫾ 864⫾ 9 3.3 ⴞ 0.6
R13C 6 ⫾ 3 107 ⫾ 357⫾ 10
Shown is the percent survival ⫾ SEM under the different conditions as-
sayed. All experiments were performed in duplicate with three to eight
independent experiments. The ﬁrst column indicates the ability of the cells to
survive osmotic downshock alone, the second indicates their ability to survive
MTSET alone, and the third indicates their ability to survive both MTSET and
osmotic downshock. All V23 data, indicated by *, is from uninduced cultures
due to low viability when induced. Boldface indicates the condition in which
the largest and statistically signiﬁcant decreases in viability are observed.
Differences between osmotic shock and MTSET are statistically signiﬁcant (P ⬍
0.05 as determined by Student’s t test) for groups I, II, III
, and IV. Differences
between MTSET and MTSET ⫹ osmotic shock are statistically signiﬁcant for
groups I, III
, and IV and for G30C. Differences between osmotic shock and
MTSET ⫹ osmotic shock are statistically signiﬁcant for groups II, III
, and IV,
and for L36C.
Bartlett et al. PNAS
July 6, 2004
be nonfunctional, but the compromised viability in the LOF
assay may in fact be due to the combined stresses of the GOF
phenotype (misgating or leaking channels) combined with os-
We found that R13C appears to be sensitive to osmotic
downshock alone but is saved by the addition of MTSET (Table
1, group IV). The R13C substitution has been shown to confer
a GOF phenotype (5). As with G76C and I92C (discussed
above), the LOF phenotype may in fact be due to the combined
stresses of the GOF phenotype and osmotic downshock. A likely
interpretation is that introduction of the positively charged
MTSET restores channel function, and thus viability, because
the positive charge at this position is reestablished.
The results presented here give us an image of the vestibule
portion of the pore. Several residues reacted with MTSET
independent of osmotic downshock and elicited a loss of viability
phenotype (group II). These residues reside within the more
periplasmic portion of TM1 (Fig. 2). Although we cannot
Fig. 2. Relative position of reactive TM1 residues. An idealized helical wheel (Left) and helical net (Right) are shown. Residues have been colored according
to the condition under which decreased viability was seen. Blue indicates that the residues respond to MTSET alone; pink indicates that the residues respond
to MTSET alone but show an increased response on osmotic downshock; red indicates that the residues require both MTSET and osmotic downshock to show
decreased viability. On the right is the helical net representation of TM1. Enclosed residues react with MTSET; those within the shaded region are residues in the
gate that show increased accessibility to MTSET when the channel is gated by osmotic downshock.
Fig. 3. The location of reactive residues within current models for the closed MscL channel. The structure of the closed or nearly closed M. tuberculosis MscL
channel derived from x-ray crystallography is shown. Pictured are proﬁles of the complex (Left) viewed from the side (Top) and periplasm (Bottom), single
subunits (Middle), and a close-up of the TM1 domain (Right) . The approximate boundary of the membrane is indicated with green horizontal lines. Residues
in TM1 that react with MTSET are shown in CPK format colored according to the condition in which a decreased viability was seen. Blue indicates that the residues
respond to MTSET alone; pink indicates that the residues respond to MTSET alone but show an increased response on osmotic downshock; red indicates that
residues require both MTSET and osmotic downshock to show decreased viability. Note that in the model the blue-labeled residues are not totally facing the
lumen; a slight counterclockwise rotation of TM1 would be required.
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0402040101 Bartlett et al.
completely rule out the possibility that the cysteine substitution
influences the structure for a given mutated MscL, it is impres-
sive that when modeled onto the crystal structure of the M.
tuberculosis MscL channel (7), the residues appear to line one
face of the helix (Fig. 3). Note, however, that they do not directly
face the lumen but are rotated several degrees clockwise as
viewed from the periplasm. Although when the MscL channel
was originally crystallized, the authors noted that it appeared to
be in a closed or ‘‘nearly closed’’ state (7), it has more recently
become dogma that the crystal structure reflects the closed
conformation. Recently, a proposal was made that the current
structural models, including the crystal structure, do indeed
reflect a ‘‘nearly closed’’ state and that a slight counterclockwise
rotation of TM1 is necessary to achieve the native closed state
(5). The evidence underlying this proposal was the formation of
a disulfide bridge for a single cysteine mutant, G26C. Here, we
find four residues that appear to be exposed in the closed state
that support this hypothesis (Fig. 3). In sum, the findings are
consistent with the clockwise direction of rotation for TM1
during gating (counterclockwise for closure) suggested by data
obtained from site-directed spin-labeling and EPR spectroscopy
(11) but not consistent with the countermodel for gating that
proposes a slight rotation in the opposite direction (9, 10).
Several residues showed changes in MTSET accessibility on
osmotic shock. G30C showed an apparent decrease in accessi-
bility on shock (note the difference between MTSET ⫹ shock vs.
MTSET-alone values), suggesting that the residue is buried on
channel opening. Some mutants required both exposure to
MTSET and osmotic downshock for the largest decrease in
viability (groups III
), suggesting that these residues are
inaccessible in the closed state but become exposed on gating
(Fig. 2). In a previous study, V23C and V37C were identified as
conferring a strong GOF phenotype when expressed (5). Hence,
the small but measurable accessibility of MTSET to these
residues in the absence of osmotic downshock may simply be that
the reagent can react with channels that are misgating in vivo.
Similarly, G26 confers a GOF phenotype and may actually
require gating for reactivity. Hence, for members of group III
and even for selected members of group II, we may be under-
estimating the requirement of channel gating for MTSET reac-
A careful examination of residues categorized within groups
can help to predict a profile for the open-pore and
transition-state structures. Consistent with the hypothesis that
little if any of TM2 lines the pore (10), we found only TM1
mutants within these groups. Two of these TM1 residues reside
close to the periplasm, V37 and M42. Presumably these residues
are obscured by the periplasmic loop structure and are revealed
on gating. Exposure of more cytoplasmic residues may occur in
either of two conditions: either the residue is buried within the
protein and exposed as the channel opens, or the residue resides
within the cytoplasmic compartment and becomes accessible
when the channel allows the reagent to flow into the cell. L19C,
which is the most cytoplasmic residue identified within these
categories, has previously been shown by patch clamp to not be
accessible to MTSET when it was placed within the bath of an
excised patch (i.e., the cytoplasmic side). A similar electrophys-
iological study with G22C found that MTSET has some access
to this residue when applied to the periplasmic (pipette) but not
the cytoplasmic (bath) side of a patch (14). Because V23 and I24
are more periplasmic in location, it seems likely that they would
also not be accessible from the cytoplasmic side (Fig. 2). This
interpretation is consistent with the crystal structure, which
suggests these residues are buried within a tightly packed bundle
of TM1 helices surrounded by TM2s.
Perhaps the most significant finding is that I24 is exposed on
gating. This residue is tangential to the constriction point of the
closed pore. The crystal structure suggests that the constriction
point of the closed pore is V23 (V21 in M. tuberculosis) (7); but,
as discussed above, the disulfide bridging of G26C suggested that
this residue may be the constriction point in the fully closed state
(5). The latter interpretation is more consistent with the data
presented here (Figs. 2 and 3). Regardless, exposure of I24 to the
pore lumen would require a significant clockwise rotation of
TM1. Here again, the data support the clockwise rotation of
TM1 on channel gating as proposed (11). In the alternative
model proposed by Sukharev et al. (10), F29, not I24, is predicted
to line the open pore. Because F29 is on the opposite face of the
helix as I24 (Fig. 2), the data presented here strongly suggest that
the Sukharev et al. model is incorrect in its predictions of residues
lining the open pore. Hence, by using an in vivo SCAM, we have
been able to identify residues lining the lumen of the pore in
different conformational states of the molecule, support modi-
fications of the closed-state models of the channel, and provide
support for a clockwise rotation of the pore-forming first trans-
membrane domain on gating.
We thank Jennifer Trosky for her help in developing this assay and Dr.
Paul Moe for a critical reading of the manuscript. This work was
supported by National Institutes of Health Grants GM61028 and
DK60818, Welch Foundation Grant I-1420, and Air Force Office of
Scientific Review Grant F49620-01-1-0503.
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