of 11
Cell
Calcium
57
(2015)
140–150
Contents
lists
available
at
ScienceDirect
Cell
Calcium
jou
rn
al
hom
epage:
www.elsevier.com/locate/ceca
Invited
review
The
evolution
of
bacterial
mechanosensitive
channels
Ian
R.
Booth
a
,
b
,
,
Samantha
Miller
a
,
Axel
Müller
c
,
Laura
Lehtovirta-Morley
d
a
School
of
Medical
Sciences,
Institute
of
Medical
Sciences,
University
of
Aberdeen,
Foresterhill,
Aberdeen
AB25
2ZD,
UK
b
Division
of
Biology
and
Biological
Engineering,
California
Institute
of
Technology,
1200
E.
California
Blvd.,
Pasadena,
CA
91125,
USA
c
Division
of
Chemistry
and
Chemical
Engineering,
Broad
Institute,
California
Institute
of
Technology,
1200
E.
California
Blvd.,
Pasadena,
CA
91125,
USA
d
Institute
of
Biological
and
Environmental
Sciences,
Cruikshank
Building,
University
of
Aberdeen,
St
Machar
Drive,
Aberdeen
AB24
3UU,
UK
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
30
November
2014
Received
in
revised
form
16
December
2014
Accepted
17
December
2014
Available
online
25
December
2014
Keywords:
Mechanosensitive
channels
MscS
MscL
Gene
synteny
Potassium
channels
a
b
s
t
r
a
c
t
Mechanosensitive
channels
are
ubiquitous
and
highly
studied.
However,
the
evolution
of
the
bacterial
channels
remains
enigmatic.
It
can
be
argued
that
mechanosensitivity
might
be
a
feature
of
all
membrane
proteins
with
some
becoming
progressively
less
sensitive
to
membrane
tension
over
the
course
of
evo-
lution.
Bacteria
and
archaea
exhibit
two
main
classes
of
channels,
MscS
and
MscL.
Present
day
channels
suggest
that
the
evolution
of
MscL
may
be
highly
constrained,
whereas
MscS
has
undergone
elaboration
via
gene
fusion
(and
potentially
gene
fission)
events
to
generate
a
diversity
of
channel
structures.
Some
of
these
channel
variants
are
constrained
to
a
small
number
of
genera
or
species.
Some
are
only
found
in
higher
organisms.
Only
exceptionally
have
these
diverse
channels
been
investigated
in
any
detail.
In
this
review
we
consider
both
the
processes
that
might
have
led
to
the
evolved
complexity
but
also
some
of
the
methods
exploiting
the
explosion
of
genome
sequences
to
understand
(and/or
track)
their
distribution.
The
role
of
MscS-related
channels
in
calcium-mediated
cell
biology
events
is
considered.
©
2015
The
Authors.
Published
by
Elsevier
Ltd.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(
http://creativecommons.org/licenses/by-nc-nd/4.0/
).
1.
Introduction
Mechanosensitive
(MS)
channels
are
ubiquitous
and
belong
to
many
different
protein
families
[1–5]
,
which
in
itself
may
be
indica-
tive
of
multiple
evolutionary
origins.
These
channels
differ
widely
in
their
structural
complexity,
potential
functions
and
sensitivity
to
mechanical
forces.
Indeed
it
has
been
suggested
that
all
channel
proteins
have
the
potential
to
be
mechanosensitive
in
their
gat-
ing
[6]
.
Consequently
one
view
would
be
that
evolution
may
have
selected
for
the
loss
of
this
property
rather
than
mechanosensi-
tivity
being
selectively
acquired.
This
proposition
would
require
significant
constraints
on
the
primary
sequences
in
the
initial
chan-
nel
families
to
avoid
residues
that
lock
channels
in
an
inactive
state
[7,8]
,
followed
by
acquisition
of,
and
selection
for,
proteins
of
diminished
mechanosensitivity
during
the
time
course
of
evo-
lution.
Discussion
of
such
intrinsic
mechanosensitivity
has
focused
both
on
channels
that
exhibit
a
low
threshold
for
activation
[6]
and
on
those
with
higher
thresholds
for
example
the
well-characterised
bacterial
MS
channels,
MscS
and
MscL
[9–11]
and
the
eukaryotic
Corresponding
author
at:
School
of
Medical
Sciences,
Institute
of
Medical
Sci-
ences,
University
of
Aberdeen,
Foresterhill,
Aberdeen
AB25
2ZD,
UK.
E-mail
addresses:
i.r.booth@abdn.ac.uk
(I.R.
Booth),
sam.miller@abdn.ac.uk
(S.
Miller),
axel@caltech.edu
(A.
Müller),
l.lehtovirta@abdn.ac.uk
(L.
Lehtovirta-
Morley).
TRAAK
family
[4,12–14]
.
The
importance
of
the
specific
interac-
tions
of
mechanosensitive
channels
with
lipids
for
their
gating
has
become
a
dominant
theme
in
recent
years
and
there
is
a
criti-
cal
interplay
between
structure
and
mechanism
[13,15,16]
.
This
review
seeks
to
speculate
on
the
evolutionary
paths
to
current
bacterial
mechanosensitive
channels,
particularly
the
MscS
fam-
ily
[16,17]
.
This
structurally
diverse
family
is
built
around
a
core
mechanosensitive
channel
domain
that
has
become
decorated
with
additional
domains
often
of
unknown
function.
However,
the
EF-
hand
MscS
channels
provide
a
clear
example
of
elaborations
that
are
understood.
The
potential
role
of
channels
in
Ca
2+
signalling
is
also
considered
in
the
context
of
the
creation,
by
mechanosensi-
tive
channels,
of
a
non-specific
spore
across
the
lipid
bilayer
that
might
serve
as
a
conduit
for
cation
entry
(both
Ca
2+
and
Na
+
)
[18–21]
.
1.1.
In
the
beginning
There
are
few
certainties
around
the
evolution
of
life
from
the
primordial
cells
to
current
day
cell
complexity
[22–24]
.
However,
the
absence
of
a
rigid
cell
wall,
even
one
that
is
dynamic,
like
bacte-
rial
peptidoglycan,
seems
almost
a
certainty.
Equally,
given
the
pre-
dominance
of
phosphate-based
metabolism
throughout
life,
Ca
2+
exclusion
may
have
been
an
essential
prerequisite
for
evolution.
A
semipermeable
membrane
that
set
the
boundary
for
the
cytoplasm
would
have
surrounded
primordial
cells.
Such
cells
could
have
had
http://dx.doi.org/10.1016/j.ceca.2014.12.011
0143-4160/©
2015
The
Authors.
Published
by
Elsevier
Ltd.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(
http://creativecommons.org/licenses/by-nc-nd/4.0/
).
I.R.
Booth
et
al.
/
Cell
Calcium
57
(2015)
140–150
141
Water,
ions,
polar
solutes
Prot
oce
ll-1:
fay ac
id bil
ayer
me
mb
r
ane
mode
r
at
e
ly
p
ermeable
to
polar
s
olu
tes
Δp≈0
Water
Prot
oce
ll-2:
Phospholi
pid
bil
ayer –
primi
ve
chann
e
ls
cre
at
epa
ths
for
sel
ec
ve
perme
a
on
of
s
olu
tes
and ions.
X
+
N
-
S
Δp≈0
Wat
er
Prot
oce
ll-3:
Phospholi
pid
bil
ayer
ion pu
mps a
ll
ow
gr
adie
nts
to
be
est
ablish
ed
for
s
olu
tes
and ions
.
Δp
>>0
{ATP
,N
A
DH
}
{ATP
,N
A
DH
}
{AT
P,NADH}
X
+
N
-
S
Pumps
K
+
Na
+
Cl
-
S
;
{
ADP
,
Pi,
NAD}
R
eplaceme
nt
of
fay
acid bil
ayers
with phospholipids
or
e
ther lipid
s
Evo
lu

on of
c
om
plex
mem
brane
prote
ins
Fig.
1.
Evolution
of
membrane
processes.
Potential
events
in
the
early
evolution
of
membrane
processes.
The
model
envisages
three
initial
stages
of
complexity.
Protocell-1:
has
a
permeable
fatty
acid
bilayer
membrane
[23,25]
;
Protocell-2:
the
emergence
of
phospholipid
bilayers
and
the
necessary,
simple,
channels
envisaged
as
homo-oligomers
of
small
peptides
[112]
.
Protocell-3:
the
evolution
of
more
complex
membrane
proteins
enabling
the
generation
and
utilisation
of
ion
gradients.
In
Protocell-1
metabolism
generates
NAD(P)H
required
for
reductive
anabolism
and
ATP
by
substrate
level
phosphorylation.
The
intrinsic
leakiness
of
the
membrane
means
that
there
is
no
substantial
protonmotive
force
(

p
)
but
there
is
likely
to
be
a
Donnan
Potential
due
to
the
presence
of
fixed
anions
in
the
cytoplasm
on
DNA
and
RNA.
Protocell-2,
the
formation
of
a
phospholipid
bilayer
restricts
transmembrane
ion
(X
+
and
N
)
and
solute
(S)
movement
to
protein-lined
pathways,
such
as
simple
channels
constructed
from
homo-oligomers
of
single
peptides.
In
the
absence
of
ion
pumps

p
remains
close
to
zero
and
chemical
gradients
drive
solute
influx
to
equilibrium.
Protocell-3,
elaboration
of
membrane
proteins
through
gene
duplication,
fusion
and
divergence
leading,
ultimately,
to
energy
transducing
complexes
that
can
either
generate
or
utilize
a
protonmotive
force
(

p

0).
The
pathways
of
gene
divergence
allow
transporter
and
channels
proteins
to
acquire
specificity
and
channels
to
acquire
gating.
bilayers
constructed
from
two
leaflets
of
fatty
acids
[23,25]
rather
than
the
current
structure
derived
from
phospholipids
or
ether
lipids
(
Fig.
1
).
Fatty
acid
membranes
have
the
advantages
that
they
self-assemble
in
a
concentration-dependent
manner,
which
gives
an
early
basis
for
growth,
and
they
have
a
higher
permeability
to
polar
solutes
than
is
the
case
for
phospholipids.
Their
great
disad-
vantage
is
that
assembly
and
stability
of
the
bilayer
is
limited
to
a
small
set
of
potential
environmental
conditions
and
such
a
bilayer
would
certainly
have
constrained
the
range
of
pH
value
in
which
such
protocells
could
have
been
stable
[25]
.
Thus
a
major
driver
for
the
evolution
of
phospholipids
and
ether
lipids
would
have
been
the
greater
stability
of
the
bilayers,
allowing
cells
to
expand
into
a
wider
range
of
niches.
Simultaneously
the
lower
permeability
of
the
phospholipid
bilayer
would
have
required
mechanisms
for
solute
transfer.
One
scenario
is
that
the
first
channels
arose
to
facili-
tate
increased
selective
ion
permeation
to
counter
the
permeability
barrier
imposed
during
the
transition
from
fatty
acid
membranes
to
phospholipid
bilayers.
Simple
homo-oligomers
of
single
span
transmembrane
peptides,
like
Alamethicin,
or
the
slightly
more
complex
gramicidins
[26]
,
would
allow
selective
ion
movement
(
Fig.
1
).
Such
channels
as
these
can
exclude
Ca
2+
whilst
facilitating
the
movement
of
other
ions
e.g.
K
+
and
Na
+
.
Present
day
toxins,
e.g.
Helicobacter
VacA,
may
mimic
the
original
channels,
and
structural
remnants
may
exist
in
mechanosensitive
channels,
such
MscS.
In
the
latter
the
pore
is
in
essence
created
by
a
heptamer
of
transmem-
brane
helices
(TM3a)
anchored
in
the
bilayer
by
TM3b
[16,27]
.
The
VacA
toxin
creates
anionic-selective
pores
via
a
hexameric
assem-
bly
of
a
single
transmembrane
helix
that
is
organised
like
the
pore
helices
of
MscS
[28]
.
Subsequent
events,
such
as
those
described
below,
would
have
led
to
the
elaboration
of
complex
transporters
and
their
necessary
energy
coupling
(
Fig.
1
).
1.2.
The
problems
of
osmosis
Water
flows
relatively
freely
through
membranes,
even
in
the
absence
of
aquaporins
[29]
.
All
cells
have
a
problem
with
water
flow
during
metabolite
transformations
that
must
occur
in
the
cyto-
plasm
during
growth.
For
example,
a
simple
act,
such
as
initiating
metabolism
of
a
sugar,
generates
multiple
metabolites
[30]
lead-
ing
to
an
increase
in
cytoplasmic
osmolarity
and
a
driving
force
for
water
entry.
The
lytic
tension
of
Escherichia
coli
membranes
equates
to
around
250
mmHg
applied
pressure
across
the
mem-
brane
and
this
can
be
achieved
by
a
simple
rise
in
the
concentrations
of
cytoplasmic
metabolites
of
around
10
mM
[3]
.
In
addition,
extant
metabolism
predominantly
generates
anions
[30]
that
must
be
accompanied
by
either
protons
generated
from
metabolism
or
other
cations,
which
today
are
principally
potassium
or
sodium
ions,
though
some
contribution
from
magnesium
and
organic
poly-
cations
(polyamines)
are
also
evident
in
today’s
cytoplasm
[30]
.
The
movements
of
K
+
and
Na
+
in
exchange
for
protons
would
be
required
to
stabilise
the
cytoplasmic
pH
[31]
,
so
channel
functions
might
initially
have
also
evolved
for
this
purpose.
Inward
move-
ments
of
K
+
,
and
possibly
Na
+
,
are
now
mediated
by
channel-like
proteins
(Trk,
Kdp,
Ktr,
MthK
and
NaChBac)
[32–38]
although
some
of
these
proteins
may
have
evolved
different
modalities
(e.g.
Kdp;
142
I.R.
Booth
et
al.
/
Cell
Calcium
57
(2015)
140–150
[39]
).
In
contrast,
ion
efflux
is
usually
via
antiports
that
exchange
the
cytoplasmic
cations
for
external
protons
[40]
(see
below).
1.3.
Calcium
in
cells
Cells
almost
always
have
mechanisms
to
exclude
Ca
2+
since
high
concentrations
of
this
cation
in
the
cytoplasm
are
inimical,
due
to
the
insolubility
of
its
phosphate
salt
at
the
mildly
alkaline
pH
found
in
the
majority
of
bacterial
cells
[31]
.
Maintenance
of
low
Ca
2+
,
plus
other
properties
of
this
cation,
has
led
to
its
almost
unique
role
in
signalling
[26]
.
Thus,
during
ion
ingress
through
trans-
porters
and
channels,
organisms
exhibit
strong
selectivity
against
Ca
2+
despite
its
similar
ionic
radius
to
Na
+
and
also
have
active
antiport
mechanisms
to
exclude
this
divalent
ion
from
the
cyto-
plasm
[37,38,41–43]
.
Bacterial
Ca
2+
pools
of
around
90
nM
have
been
measured
in
cells
incubated
with
mM
external
Ca
2+
[42]
.
Selective
exclusion
of
Ca
2+
must
have
arisen
early
in
evolution,
given
that
it
is
desirable
not
to
allow
its
penetration
into
the
cyto-
plasm.
Present
day
mechanosensitive
channels
create
relatively
large,
transient,
non-selective
pores
in
the
membrane
and
equilibrate
ion
gradients,
if
only
for
several
minutes
following
their
gating
[44]
.
During
hypoosmotic
shock
in
the
presence
of
high
external
con-
centrations
of
Ca
2+
,
this
ion
can
penetrate
to
the
cytoplasm
through
an
open
MscS
or
MscL
pore
[18]
.
This
may
be
combined
with
the
exquisitely
sensitive
Ca
2+
-sensing
systems
to
effect
changes
in
gene
expression
[18,45,46]
.
There
is
a
subtle
difference
between
the
pro-
jected
early
evolutionary
events
to
exclude
Ca
2+
and
the
current
day
inadvertent
flooding
of
the
cytoplasm
with
this
ion
by
the
forma-
tion
of
a
pore
by
a
mechanosensitive
channel
or
a
toxin.
Control
is
the
essence
of
this
distinction
and
can
be
seen
in
yeast
[19,20]
and
in
Bacillus
[21]
to
effect
specific
physiological
events
(see
Section
4
).
1.4.
Mechanosensitive
channels
and
metabolism
Retention
of
mechanosensitivity
in
primordial
ion-specific
channels
might
have
originally
had
the
purpose
of
allowing
con-
trol
over
of
cation
uptake
(particularly
potassium),
limiting
influx
to
that
required
to
compensate
for
metabolically-generated
anions.
For
this
reason,
inhibition
of
channel
activity
by
bilayer
tension
(in
distinction
to
activation,
as
in
MscS,
TRAAK
and
MscL)
may
have
been
a
more
critical
evolutionary
event.
Once
metabolism
reaches
a
steady
state,
the
cytoplasmic
osmolarity
would
be
stable
but
the
cell
might
have
a
slight
net
outwardly
directed
turgor.
Intrinsi-
cally
the
volume
expansion
associated
with
metabolism
must
be
within
the
stability
limits
of
the
bilayer.
Ion
transport
functions
that
might
raise
the
turgor
pressure
to
unsustainable
levels
need
to
be
controlled.
The
lytic
threshold
in
naked
protoplasts,
as
measured
by
patch
clamp
technology,
is
quite
high
[3,9]
.
Thus
primordial
wall-less
cells
would
be
subject
to
small
routine
fluctuations
in
membrane
tension
as
a
result
of
transitions
in
metabolism.
The
major
bacterial
mechanosensitive
channels,
MscS
and
MscL,
exhibit
high
gating
thresholds
[9,11]
compared
with
known
K
+
channels
[6]
and
thus
the
high
threshold
may
only
have
been
selected
for
once
cells
evolved
a
cell
wall.
One
can
also
argue
that
the
high
gating
thresholds
prevent
inappropriate
gating
of
the
channels
since
the
latter
has
been
shown
to
be
a
lethal
event
[47]
.
The
extant
organisms
that
most
resemble
the
primordial
cells
are
probably
the
Mycoplasmas
and
bacterial
L-forms.
The
latter
are
generated
by
digestion
of
the
existing
cell
wall
to
release
the
protoplast
and,
while
they
are
intrinsically
very
interesting,
their
mechanosensitivity
should
principally
reflect
that
of
their
bacterial
progenitors
[48]
.
Intriguingly,
genetic
analysis
of
the
requirements
for
growth
of
Bacillus
L-forms
identified
enzymes
that
mediate
the
synthesis
of
branched
chain
lipids.
These
enzymes
were
critical
for
membrane
fluidity
and
cell
division,
but
not
for
cell
expansion,
which
was
unaffected
by
the
genetic
lesion
[49–51]
.
In
contrast
to
L-forms,
Mycoplasmas
have
existed
for
a
very
long
time
as
inde-
pendent
wall-less
bacteria
[48]
.
Mycoplasmas
are
stable
dividing
cells
that
exist
without
a
bacterial
cell
wall
but
incorporate
choles-
terol
to
facilitate
growth
[48]
.
Cholesterol
is
known
to
stabilise
membranes
by
stiffening
the
lipid
bilayer
[52]
.
Mycoplasmas
are
believed
to
have
evolved
by
degenerate
evolution
from
Lactobacilli
and
Clostridia.
These
two
genera
have
‘normal’
complements
of
mechanosensitive
channels
(i.e.
both
MscL
and
MscS-related
chan-
nels)
and
most
Mycoplasmas
reflect
their
origins
in
this
respect,
having
retained
mechanosensitive
channels
during
their
evolu-
tion.
Critically
the
retention
of
mechanosensitive
channels
over
the
600,000
years
of
severe
reductive
genome
complexity
in
the
Mycoplasma
genome
[48]
,
speaks
volumes
for
their
function
in
stabilizing
even
wall-less
cells.
2.
Current
roles
for
channels
in
cell
physiology
A
great
diversity
of
ion
channels,
both
selective
and
non-
selective,
have
been
found
in
bacterial
genomes
and
some
of
these
have
been
characterised
biochemically,
often
after
heterologous
expression
in
E.
coli
.
The
two
most
highly
characterised
families
are
the
K
channels
and
the
mechanosensitive
channels,
although
sig-
nificant
analysis
of
gating
of
the
Na
+
channel
has
also
been
reported
[37,38,53,54]
.
2.1.
Potassium
channels
Potassium
channels
are
the
major
route
for
K
+
accumulation
by
bacterial
cells.
In
higher
organisms
the
roles
of
K
+
channels
in
cell
physiology
are
more
varied
and
complex,
but
in
bacteria
and
some
yeasts
their
core
function
is
to
provide
a
regulated
pore
for
potassium
movements
across
the
membrane.
In
bacteria
the
chan-
nels
are
thought
to
be
one
half
of
the
solution
needed
to
maintain
a
potassium
pool
that
is
primarily
set
by
the
external
osmolarity
[55]
.
Thus,
both
influx
and
efflux
must
be
managed
and
while
the
former,
with
its
role
for
ion
channels,
has
been
moderately
well-
characterised,
the
efflux
routes
are
poorly
understood.
Potassium
efflux
systems
that
play
an
important
role
in
protecting
cells
against
toxic
insults
are
almost
ubiquitous
[56,57]
.
They
share
common
regulatory
structures
with
uptake
channels,
but
they
respond
to
specific
and
different
signals
[58]
.
The
debate
on
their
channel-like
functions
is
unresolved.
For
the
most
part
bacterial
potassium
channels
exist
either
as
complex,
multimeric,
assemblies
of
at
least
two
proteins
the
pore
domain
and
the
regulatory
KTN
(RCK)
domain
(TrkAH,
KtrAB
are
examples;
[32,36]
)
or
the
same
modules
as
a
single
polypep-
tides
bearing
two
domains
(MthK,
GsuK,
CglK;
[33,34,59,60]
).
In
some
cases
translation
sites
internal
to
the
structural
gene
result
in
the
expression
of
extra
regulatory
(KTN)
domains
that
are
crit-
ical
to
function
[33]
.
The
K
+
channel
pore
can
be
formed
from
either
four
identical
subunits
or
from
a
single
subunit
that
contains
four
similar
domains
the
consequence
is
that
the
symmetrical
pore
in
the
former
is
replaced
by
an
asymmetrical
structure
in
the
latter
(compare
MthK
with
TrkHA;
[33,36]
).
Variation
in
gene
struc-
ture,
and
thereby
the
organisation
of
protein
complexes
to
form
functional
channels,
indicates
multiple
evolutionary
pathways
to
similar
functional
solutions.
It
seems
highly
likely
that
the
TRAAK
and
related
mechanosensitive
channels
are
evolutionary
deriva-
tives
of
the
conventional
potassium
channels
that
drive
electrical
signalling
and
ion
accumulation
in
bacteria
[4,5,13]
.
Similar
com-
plexity
in
the
extant
structures
for
the
bacterial
multi-subunit
sugar
phosphotransferases
also
indicates
a
range
of
solutions
that
can
be
accommodated
by
the
functional
requirements
of
the
system