Bacterial
mechanosensitive
channels:
progress
towards
an
understanding
of
their
roles
in
cell
physiology
§
Ian
R
Booth
1
,
2
Bacterial
mechanosensitive
channels
sense
the
changes
in
lateral
tension
in
the
bilayer
of
the
cytoplasmic
membrane
generated
by
rapid
water
flow
into
the
cell.
Two
major
structural
families
are
found
widely
distributed
across
bacteria
and
archaea:
MscL
and
MscS.
Our
understanding
of
the
mechanisms
of
gating
has
advanced
rapidly
through
genetic
analysis,
structural
biology
and
electrophysiology.
It
is
only
recently
that
the
analysis
of
the
physiological
roles
of
the
channels
has
kept
pace
with
mechanistic
studies.
Recent
advances
have
increased
our
understanding
of
the
role
of
the
channels
in
preventing
structural
perturbation
during
osmotic
transitions
and
its
relationship
to
water
flow
across
the
membrane.
It
is
to
these
recent
developments
that
this
review
is
dedicated.
Addresses
1
School
of
Medical
Sciences,
University
of
Aberdeen,
Aberdeen,
AB25
2ZD,
United
Kingdom
2
Visiting
Associate
in
Chemistry,
California
Institute
of
Technology,
Pasadena,
CA
91125,
United
States
Corresponding
author:
Booth,
Ian
R
(
i.r.booth@abdn.ac.uk
)
Current
Opinion
in
Microbiology
2014,
18
:16
–
22
This
review
comes
from
a
themed
issue
on
Cell
regulation
Edited
by
Cecilia
Arraiano
and
Gregory
M
Cook
1369-5274/$
–
see
front
matter,
#
2014
The
Author.
Published
by
Elsevier
Ltd.
All
rights
reserved.
http://dx.doi.org/10.1016/j.mib.2014.01.005
Introduction
Mechanosensitive
(MS)
channels
sense
changes
in
the
tension
in
the
lipid
bilayer
of
the
cytoplasmic
membrane
[
1
].
Bacterial
channels
have
been
well-studied
in
a
range
of
organisms
[
2
]
and
they
are
considered
to
be
useful
models
for
mechanotransduction
in
higher
organisms
[
3
].
Mammalian
channels
are
frequently
ion-selective
and
thus
generate
specific
signals
that
are
integrated
by
the
neuronal
system
leading
to
an
altered
behaviour.
In
contrast,
bacterial
mechanosensitive
channels
are
generally
non-specific
in
terms
of
the
ions
and
molecules
that
pass
through
the
open
pore.
Their
transition
from
the
closed
to
the
open
state
creates
a
transient
pore
of
quite
large
dimensions,
minimally
6
A
̊
diameter
(the
size
of
a
hydrated
K
+
ion)
through
to
30
A
̊
diameter
for
MscL
[
2
].
Their
proposed
major
role
in
cell
physiology
is
well-
established,
namely
protection
of
the
physical
integrity
of
the
cell
during
transitions
from
high
osmolarity
to
low
[
4
].
One
of
the
most
important
questions
remaining
addresses
channel
abundance,
structural
diversity
and
plurality
in
bacterial
species.
This
short
article
will
review
the
timing
of
channel
gating
and
its
importance
for
the
roles
of
the
channels.
Osmoregulation
and
cytoplasmic
solute
concentrations
Bacterial
cells
accumulate
solutes
in
their
cytoplasm
well
beyond
the
concentrations
that
might
be
required
for
metabolism.
In
most
bacteria
there
is
a
preference
for
the
accumulation
of
potassium
and
glutamate
[
5
].
How-
ever,
diverse
metabolic
anions
accumulate
to
millimolar
levels,
such
that
the
cytoplasm
may
contain
as
much
as
200
mM
osmotically
active
anions
even
when
grown
at
moderately
low
osmolarity
(
240
mOsm)
[
6
].
This
would
generate
a
net
turgor
pressure
of
4
atm
(
40
mOsm
solute
1
atm
[
2
];
the
osmolarity
of
the
medium
is
equivalent
to
6
atm)
directed
outwards
from
the
cell
(
Figure
1
).
Movement
of
water
across
the
membrane
into
the
cytoplasm
generates
the
turgor
pressure
and
provides
the
expansion
space
required
for
growth
through
biosynthesis
of
new
polymers.
Measure-
ment
of
turgor
pressure
is
extremely
difficult
and
there
is
no
certainty
that
this
parameter
does
not
vary
with
either
growth
conditions
or
with
the
identity
of
the
organism.
A
net
outward
pressure
of
4
atm
in
E.
coli
cells
was
suggested
[
7
],
but
recent
experiments
have
questioned
this
[
8
].
When
subjected
to
hyperosmotic
stress,
Gram
negative
bacteria
exhibit
a
biphasic
strategy
to
counter
water
loss.
In
the
initial
phase
potassium
and
glutamate
pools
increase
and
subsequently
these
ions
may
be
replaced
by
compatible
solutes,
such
as
trehalose,
betaine
and
proline
[
5
].
A
cell
adapted
to
high
osmolarity
is
at
risk
when
transferred
to
low
osmolarity
due
to
the
osmotically
driven
water
flow
into
the
cytoplasm.
A
decrease
in
the
external
osmolarity
of
800
mOsm
(equivalent
of
transfer
from
growth
medium
containing
0.5
M
NaCl
into
growth
medium
alone
or
approximating
the
transfer
of
cells
from
sea
water
to
fresh
water)
could
raise
the
turgor
pressure
by
20
atm
[
2
].
The
actual
increase
experienced
by
the
cells
§
This
is
an
open-access
article
distributed
under
the
terms
of
the
Creative
Commons
Attribution-NonCommercial-No
Derivative
Works
License,
which
permits
non-commercial
use,
distribution,
and
repro-
duction
in
any
medium,
provided
the
original
author
and
source
are
credited.
Available
online
at
www.sciencedirect.com
ScienceDirect
Current
Opinion
in
Microbiology
2014,
18
:16
–
22
www.sciencedirect.com
depends
on
the
rate
of
water
penetration
into
the
cyto-
plasm,
the
elasticity
of
the
peptidoglycan
(PTG)
and
the
activity
of
mechanosensitive
channels.
The
centrality
of
water
in
life
Central
to
understanding
the
core
physiology
of
bacterial
MS
channels
is
an
appreciation
of
the
rapidity
of
water
fluxes
across
the
lipid
bilayer.
The
membrane
bilayer
is
highly
permeable
to
water
and
in
some
bacterial
species
this
natural
permeability
is
further
augmented
by
expres-
sion
of
aquaporins
[
9
].
In
response
to
hyperosmotic
shock
[
10,11
]
(J
Mika,
PhD
Thesis,
Groningen,
2012)
and
hypoosmotic
shock
[
12
]
the
cell
shrinks
or
expands,
respectively,
on
the
very
rapid
timescales
(30%
volume
change
in
<
1
s
is
typical).
A
bacterial
cell
of
10
15
L
contains
3
–
4
10
10
water
molecules.
Considering
an
E.
coli
cell
as
a
cylinder
(
2
m
m
length
and
1
m
m
diameter)
that
can
expand
along
its
length
but
not
readily
change
its
diameter
(at
least
over
the
very
short
timescales
associ-
ated
with
osmotically
driven
water
movements),
which
is
consistent
with
current
theories
of
peptidoglycan
struc-
ture
[
13
],
an
expansion
of
12%
[
14
]
would
require
4
–
5
10
9
water
molecules
to
cross
the
membrane,
which,
in
E.
coli
,
can
occur
in
100
ms
[
10,11
]
(J
Mika,
PhD
Thesis,
Groningen,
2012).
The
capacity
to
withstand
rapid
water
movements
is
dependent
upon
the
operation
of
MS
channels
and
on
the
strength
of
the
cell
wall.
Peptidoglycan,
which
gives
the
cell
its
physical
integrity
and
shape
[
13
],
is
a
dynamic,
semi-elastic
polymer
con-
structed
from
oligosaccharides
of
varying
lengths
(N-
acetylglucosamine
and
N-acetylmuramic
acid
pentapep-
tide
units;
NAG-NAM-p5)
crosslinked
by
short
peptides.
Sugar
chains
are
organised
principally
in
the
circumfer-
ential
direction,
while
the
peptides
are
oriented
in
the
long
direction
of
the
cell
[
13
,15,16,17
].
Peptidoglycan
is
not
a
continuous
structure;
the
sugar
chains
are
of
variable
length
(a
single
circumference
requiring
many
indepen-
dent
polysaccharide
chains)
and
the
peptide
crosslinking
is
incomplete
[
18
].
This
variation
creates
a
mesh
in
which
there
are
holes
(of
varying
sizes)
that
are
bounded
by
the
sugars
and
peptides
[
18
].
Growth
of
E.
coli
cells
is
largely
by
extension
in
the
long
direction
and
this
requires
the
peptidoglycan
be
a
highly
dynamic
structure;
increasing
the
length
of
the
cell
is
principally
achieved
by
breakage
of
the
peptide
bonds
and
the
insertion
of
new
wall
material
[
13
].
Although
the
peptidoglycan
is
a
unique
structure
between
the
cytoplasmic
and
the
outer
mem-
branes,
there
are
important
connections
to
both
mem-
branes
through
synthetic
complexes
and
lipoproteins,
Bacterial
mechanosensitive
channel
function:
physiology
Booth
17
Figure
1
outer membrane (OM)
cytoplasm
periplasm
Peptidoglycan (PTG)
cytoplasmic membrane
LPS
Divalent cations, e.g. Ca
2+
Cytoplasmic solutes ~400 mOsm
External solutes
~240 mOsm
Turgor ~10 atm
Medium
~6 atm
PTG + OM
~4 atm
Water flow
Current Opinion in Microbiology
The
generation
of
turgor
and
resistance
to
the
force.
In
E.
coli
cells
growing
in
a
medium
of
240
mOsm
(a
standard
minimal
medium
or
LB
containing
5
g/L
NaCl)
one
may
confidently
expect
to
find
200
m
M
cytoplasmic
anions
and
300
m
M
K
+
.
Approximately
100
m
M
of
the
K
+
matches
fixed
anions
and
is
thus
not
considered
for
the
calculation
of
the
outward
turgor
of
10
atm.
Given
the
medium
contributes
6
atm
the
net
turgor
pressure
is
4
atm.
MS
channels
will
gate
if
there
is
a
net
outward
pressure
of
0.1
atm
and
thus
the
cell
wall
and
outer
membrane,
between
them,
contribute
a
resistance
of
4
atm
to
maintain
MS
channels
closed.
There
are
at
least
two
contributions
to
the
strength
of
the
cell
wall
—
the
first,
already
described,
is
the
crosslinking
of
the
peptidoglycan
and
the
second
is
the
outer
membrane
that
can
provide
some
resistive
force
through
the
binding
together
of
the
lipopolysaccharide
chains
by
divalent
cations
[
46
].
www.sciencedirect.com
Current
Opinion
in
Microbiology
2014,
18
:16
–
22
respectively
[
13
].
Some
of
these
connections
are
transi-
ent,
but
others,
for
example,
lipoprotein
linkages
are
covalent
bonds
to
the
PTG
peptides.
The
dynamic
nature
of
the
peptidoglycan
renders
the
cell
susceptible
to
physical
disruption
by
rapid
water
flow
into
the
cyto-
plasm.
Expansion
of
the
cell,
some
considerations
The
effect
of
increased
water
influx
into
the
cytoplasm
is
conditional
on
the
pre-existing
state
of
the
peptidoglycan.
Measurements
have
been
made,
by
atomic
force
micro-
scopy,
of
the
expansion
that
isolated
PTG
sacculi
can
undergo
in
response
to
applied
force
[
14
];
a
12%
expan-
sion
was
measured
for
every
1
atm
of
pressure
applied.
If
the
cell
can
expand
under
the
inflow
of
water
there
will
be
no
net
increase
in
pressure
on
the
membrane
provided
that
the
bilayer
can
increase
its
surface
area
on
the
same
timescale
as
the
water
movements.
However,
the
mem-
brane
has
a
limited
expansive
capacity
due
to
a
lack
of
extensive
phospholipid
reserves
—
estimates
suggest
2
–
4%
expansion,
by
increasing
the
distance
between
head-
groups
of
the
phospholipids,
as
a
mechanical
upper
limit
[
19
].
Moreover,
rapidly
growing
cells
are
likely
to
have
their
PTG
sacculus
already
stretched
and
it
is
not
clear
whether
an
immediate
expansion
on
this
scale
is
feasible
without
imposing
considerable
strain
on
the
wall.
MS
channels,
when
assayed
in
membrane
patches,
gate
at
an
imposed
pressure
on
the
lipid
bilayer
(i.e.
shorn
of
the
cell
wall)
of
around
0.1
atm
(depending
on
the
specific
lipid
context
and
the
shape
of
the
membrane
patch)
[
20
–
22
].
Clearly,
therefore,
as
the
PTG
achieves
its
maximum
expanded
stable
state
a
further
increase
in
turgor
pressure
will
lead
to
gating
of
the
MS
channel
complement.
Sukharev
and
colleagues
[
12
]
measured
the
rapidity
of
the
initial
swelling
upon
imposition
of
a
hypoosmotic
shock,
using
the
change
in
the
refractive
index
of
cells.
They
observed
swelling
to
occur
30
–
50
ms
after
low-
ering
the
external
osmolarity,
followed
by
channel
gating
after
150
–
200
ms.
Previous
studies
to
detect
the
respon-
siveness
of
MscS
and
MscL
to
tension
changes
suggested
that
the
channels
gate
3
–
5
m
s
after
the
tension
reaches
the
activation
threshold
[
23
].
This
suggests
that
channel
gating
100
ms
after
swelling
is
not
due
to
intrinsic
lack
of
responsiveness
of
the
channels,
but
may
be
due
to
generation
of
the
gating
signal
from
a
combination
of
water
inflow,
cell
expansion
and
other,
as
yet
unknown,
modulations
of
the
cell
envelope.
An
approximate
time-
line
for
swelling,
adaptation
and
death
can
be
constructed
(
Figure
2
)
that
allows
one
to
appreciate
the
extreme
rapidity
of
the
onset
of
hypoosmotic
stress
and
the
speed
of
the
response
required.
Cell
death
follows
multiple
paths
Failure
of
mechanosensitive
channels
to
open
leads
to
cell
death
[
4,24,25,26
].
The
precise
fate
of
individual
cells
lacking
MS
channels
is
complex.
Cell
fate
is
deter-
mined
by
a
combination
of
known
and
unknown
parameters
that
vary
between
individuals.
For
each
cell
these
include
the
turgor
pressure,
the
number
of
channels
and
the
strength
of
the
cell
wall.
In
addition,
the
rate
of
change
of
the
osmolarity
is
itself
a
major
determinant
of
cell
fate
[
27
]
(see
below).
Colony
counts
have
been
the
preferred
method
to
investigate
the
fate
of
channel-less
mutants
and
such
studies
have
been
informative
in
defin-
ing
the
core
role
of
mechanosensitive
channels
[
4,24,25,26
,28
].
These
assays
reveal
that
a
small,
but
significant,
fraction
of
mutant
cells
survive
hypoosmotic
shock.
When
such
survivors
are
re-cultured
they
recapi-
tulate
the
original
pattern
of
survival,
that
is
the
majority
die,
but
a
few
survive.
Thus,
the
majority
of
survivors
have
not
acquired
protective
mutations,
but
rather
their
18
Cell
regulation
Figure
2
Time line for hypoosmotic shock
swelling
gating
Leaking and bursting
1 s
20 min
Current Opinion in Microbiology
Timeline
for
swelling,
adaptation
or
death.
This
figure
illustrates
that,
from
measurements
by
stopped
flow
and
microscopy,
swelling
was
observed
30
–
50
ms
after
imposition
of
a
severe
(
900
mOsm)
hypoosmotic
shock
[
12
].
Changes
in
refractive
index
consistent
with
channel
gating
suggested
that
this
occurs
between
150
and
200
ms
after
shock
[
12
].
Initial
cell
disruption
events
were
first
observed
200
–
1000
ms
after
downshock
[
33
]
but
lytic
events
continue
for
at
least
20
min
[
4
]
(SS
Black
et
al.
,
unpublished
data;
M
Bialecka-Fornal
et
al
.,
personal
communication).
Current
Opinion
in
Microbiology
2014,
18
:16
–
22
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