5
‑
HT
3
Receptor
MX Helix
Contributes
to Receptor
Function
James
Mocatta,
Susanne
M. Mesoy,
Dennis
A. Dougherty,
and Sarah
C. R. Lummis
*
Cite This:
ACS Chem.
Neurosci.
2022,
13, 2338−2345
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ABSTRACT:
5-HT
3
receptors
are
members
of
the
family
of
pentameric
ligand-gated
ion
channels.
Each
subunit
has
an
extracellular,
transmembrane,
and
intracellular
domain.
Only
part
of
the
intracellular
domain
structure
has
been
solved,
revealing
it
contains
two
α
-helical
segments;
one,
the
MA
helix,
is an
extension
of
M4,
while
the
other,
the
MX
helix,
is formed
from
residues
located
close
to
the
end
of
M3.
This
MX
helix
is
in
distinct
locations
in open
and
closed
receptor
structures,
suggesting
it may
play
a role
in
function.
Here,
we
explore
this
hypothesis
using
functional
responses
of Ala-substituted
mutant
receptors
expressed
in HEK293
cells.
The
data
show
altering
many
of the
MX
residues
results
in a small
decrease
in EC
50
(up
to
5-fold),
although
in one
(H232A)
this
is increased.
Radiolabeled
ligand
binding
on
selected
mutants
showed
no
change
in
binding
affinity,
indicating
an
effect
on
gating
and
not
binding.
In
addition,
five
mutations
(P316A,
V317A,
P318A,
D319A,
and
H323A)
initially
resulted
in
nonfunctional
receptors,
but
the
function
could
be
rescued
by
coexpression
with
a chaperone
protein,
suggesting
a likely
role
in
assembly
or
folding.
Examination
of
previously
obtained
MD
simulation
data
shows
that
the
extent
of
MX
encompassed
by
membrane
lipids
differs
considerably
in
the
open
and
closed
structures,
suggesting
that
lipid
−
protein
interactions
in
this
region
could
have
a major
effect
on
channel
opening
propensity.
We
conclude
that
the
MX
helix
can
modulate
the
function
of the
receptor
and
propose
that
its
interactions
with
membrane
lipids
play
a major
role
in this.
KEYWORDS:
Cys-loop
receptor,
binding
site, mutagenesis,
noncanonical
amino acid, nonsense
suppression
■
INTRODUCTION
Cys-loop
receptors
are
part
of the
pentameric
ligand-gated
ion
channel
(pLGIC)
superfamily,
proteins
that
are
critical
for
fast
synaptic
transmission
in
the
central
and
peripheral
nervous
systems
of
both
vertebrates
and
invertebrates
and
typified
by
the
nicotinic
acetylcholine
receptor
(nAChR).
1
They
can
be
either
homomeric
or
(more
usually)
heteromeric.
Each
of
the
five
subunits
possesses
a large
extracellular,
ligand-binding
domain
containing
the
eponymous
Cys-loop
(a
13
amino
acid
disulfide-bonded
peptide
loop),
a
channel-forming
trans-
membrane
domain,
consisting
of
four
membrane-spanning
segments,
M1
−
M4,
and
an
intracellular
domain.
The
intra-
cellular
domain
is the
least
well
understood,
despite
playing
roles
in
receptor
trafficking,
single-channel
conductance,
and
modulation.
The
structure
of
the
5-HT
3
A
homopentamer
has
been
solved
by
both
X-ray
crystallography
2
and
cryo-electron
microscopy
(cryo-EM).
3,4
These
data
reveal
a largely
β
-sheet
extracellular
domain
(ECD)
and
α
-helical
transmembrane
domain
(TMD),
as expected
for
a pLGIC.
Unusually,
however,
the
structures
also
reveal
some
details
of
the
intracellular
domain
(ICD),
a domain
that
is frequently
removed
prior
to
structural
examination;
these
data
suggest
that
the
ICD
is
mostly
disordered,
with
two
regions
of
α
-helix,
which
have
been
named
the
MA
and
MX
helices.
The
MA
helix
is
effectively
a cytoplasmic
extension
of M4,
while
the
MX
helix
is
close
to
the
end
of
M3
and
is
located
at
the
TMD/ICD
interface
adjacent
to
M4
(Figure
1).
As
bacterial
pLGICs
do
not
have
an
ICD
and
replacement
with
a short
peptide
in
vertebrate
receptors
does
not
ablate
function,
5
this
region
has
not
been
well
studied.
Yet,
it is
increasingly
becoming
apparent
that
the
ICD
can
have
major
effects,
e.g.,
binding
of
some
cytoplasmic
molecules
here
can
inhibit
ion
flux.
6
A comparison
of
putative
open
and
closed
(resting)
receptor
conformations
suggests
that
the
MX
helix
moves
significantly
during
channel
opening:
in
the
unbound
(apo)
state,
it lies
almost
parallel
to
the
plasma
membrane,
while
when
the
receptor
is open,
it is displaced
laterally
by
up
to
18
Å (Figure
1)
pulling
a post-M3
loop
outwards
from
the
central
axis
of
symmetry.
This
post-M3
loop
extends
away
from
the
M4
helix,
creating
lateral
portals
(dimensions
16.0
Å
Received:
June
10,
2022
Accepted:
July
6, 2022
Published:
July
22,
2022
Research
Article
pubs.acs.org/chemneuro
© 2022
The Authors.
Published
by
American
Chemical
Society
2338
https://doi.org/10.1021/acschemneuro.2c00339
ACS Chem.
Neurosci.
2022,
13, 2338
−
2345
×
11.4
Å)
that
would
allow
the
passage
of hydrated
Na
+
ions.
These
data
suggest
that
the
MX
helix
motion
is correlated
with
channel
opening
and
could
play
a role
in
modulating
signal
transduction.
A further
interesting
feature
of the
MX
helix
is that
it may
be
unique
to
the
cationic
pLGICs:
no
MX-like
helical
sections
have
yet
been
observed
in
glycine
and
GABA
A
receptor
structures,
and,
more
compelling,
an
alignment
of
this
region
reveals
some
conservation
of MX
between
the
cationic
5-HT
3
receptors
and
nACh
receptors
but
no
obvious
similar
region
in
anionic
pLGICs
(Figure
2).
We
therefore
decided
to
study
the
roles
of
the
individual
amino
acids
that
contribute
to the
MX
helix
using
Ala-scanning
mutagenesis,
and
to
use
these
data,
combined
with
structural
data,
to consider
if this
region
contributes
to receptor
function.
■
RESULTS
Functional
Characterization
of Mutant
and WT 5-HT
3
R.
To
examine
the
function
of wild-type
(WT)
and
mutant
5-
HT
3
A receptors,
we
transfected
them
into
HEK293
cells
and
probed
5-HT-elicited
responses
in
a
Flexstation
using
membrane
potential
sensitive
dye.
Concentration
−
response
curves
for
WT
receptors
revealed
a 5-HT
EC
50
of
0.3
μ
M
(pEC
50
= 6.5
±
0.04)
and
a Hill
coefficient
of
2.5
±
0.5,
consistent
with
previously
published
data.
7
Functional
Characterization
of Mutant
5-HT
3
R.
Five
mutants
did
not
respond
to
5-HT
following
expression
(P316A,
V317A,
P318A,
D319A,
and
H323A).
These
were
then
coexpressed
with
RIC-3,
a well-established
chaperone
of
the
5-HT
3
A receptor,
8
which
has
no
significant
effect
on
the
EC
50
of
WT
receptors.
All
of
the
nonfunctional
receptors
functioned
when
they
were
coexpressed
with
RIC-3;
thus,
our
data
suggest
that
Ala
substitution
of
these
residues
in
MX
is
deleterious
to
5-HT
3
R expression.
The
parameters
determined
for
mutant
receptors
(with
RIC-
3 for
those
that
were
initially
nonfunctional)
reveal
that
Ala
substitutions
resulted
in
small
changes
in
EC
50
s for
13/18
of
the
altered
receptors
(Table
1).
All
these
changes
decreased
EC
50
s except
for
H323A,
which
caused
an
increase
in
EC
50
(Figure
3).
Typical
Flexstation
traces
and
concentration
−
response
curves
for
this
mutant,
WT,
and
P318A
(a
mutant
that
causes
a decrease
in EC
50
) are
shown
in Figure
4.
Radiolabeled
Ligand
Binding.
EC
50
values
are
useful
measures
to
compare
the
effect
of a mutation
on
the
function
of
the
protein,
but
they
do
not
reveal
whether
the
mutation
affects
binding
or
gating.
One
method
to
explore
this
is to
perform
radiolabeled
ligand
binding
to examine
the
parameters
associated
with
the
binding
site.
Our
data
(Figure
5),
using
a
subset
of mutant
receptors,
revealed
that
the
binding
affinity
of
mutant
and
WT
receptors
was
not
significantly
different.
These
data
indicate
that
the
changes
in EC
50
we
observed
were
likely
due
to
changes
in gating.
Further
Characterization
of Residue
P318.
P318
is
located
at the
start
of
the
MX
helix
(Figure
6)
and
a P318A
substitution
was
typical
in
causing
a decrease
in
EC
50
; we
therefore
decided
to
further
explore
the
role
of
this
residue
using
an
expression
in
oocytes
with
both
canonical
and
noncanonical
amino
acids.
The
data
(Figure
6A,B,
Table
2)
show
that
the
small
decrease
in
EC
50
observed
with
P318A-
containing
receptors
in
our
HEK
cell
experiments
was
maintained
when
using
oocyte
expression,
and
a similar
small
decrease
was
seen
with
a P318V
substitution
but
not
Figure
1.
MX
helix
is in different
locations
in the
resting
(dark
green,
pdbid:
6BE1)
and
open
(light
green,
6DG8)
5-HT
3
R states.
This
indicates
MX
moves
upwards
and
outwards
relative
to
M4
when
the
receptor
transitions
from
the
resting
to
the
open
state.
Specific
residues
alter
positions
by
up
to
18
Å.
Figure
2.
Sequence
alignment
of
M3
and
MX
regions
of
anionic
and
cationic
pLGICs
shows
that
the
MX
helix
may
not
be
present
in
anionic
receptors.
ACS Chemical
Neuroscience
pubs.acs.org/chemneuro
Research
Article
https://doi.org/10.1021/acschemneuro.2c00339
ACS Chem.
Neurosci.
2022,
13, 2338
−
2345
2339
with
a P318G
mutation.
For
the
noncanonical
amino
acid
experiments,
EC
50
s were
similar
to
WT
for
those
substituted
with
cis
-4-fluoroproline
(CFP),
trans
-4-fluoroproline
(TFP),
and
α
-hydroxyvaline
(Vah),
but
again
we
observed
a decreased
EC
50
when
P318
was
substituted
with
pipecolic
acid
(Pip)
or
2-methylproline
(2MeP).
These
noncanonical
amino
acids
are
all
proline
analogues
that
can
probe
the
unusual
properties
of
proline;
these
properties
include
an
increased
propensity
for
a
cis
peptide
bond,
a decreased
hydrogen
bonding
capacity,
and
a kink
or bulge
in an
α
-helix.
CFP
and
TFP
are
close
analogues
of
Pro,
but
the
incorporation
of
a single
F in
a
cis
or
trans
configuration
results
in different
cis
peptide
bond
propensities
(TFP
= 12%,
CFP
= 28%
9
);
thus
our
data
(no
difference)
suggest
that
this
aspect
of
Pro
plays
no
role
here.
However,
a
different
shape
or
size
at position
318,
as
in Pip
(differs
from
Pro
in having
a larger
ring)
or
2MeP
(a
methyl
group),
results
in a similar
change
in EC
50
to
that
we
observed
with
Ala
and
Val.
In
contrast,
Vah
substitution
(generating
receptors
with
a
backbone
ester
in
place
of
an
amide)
suggests
that
the
decreased
hydrogen
bonding
capacity
allows
Vah
to maintain
a
WT-like
EC
50
. Overall,
these
data
are
not
incompatible
with
a
more
bulky
residue
at P318
being
deleterious
to
function,
and
our
speculative
hypothesis
is that
this
impairs
the
flexibility
of
this
region,
which
needs
to rapidly
shift
between
α
−
helical
(in
the
open
state,
Figure
6D)
and
non-
α
-helical
(in
the
closed
state,
Figure
6E).
In Silico
Studies.
To
explore
the
potential
for
lipid
interactions
with
MX,
we
examined
and
compared
the
800
ns
simulations
of
two
5-HT
3
R
structures
taken
from
Mem-
ProtMD.
10
The
currently
available
high-resolution
5-HT
3
R
structures
are
assigned
a range
of states
mainly
based
on
their
pore
diameters.
To
avoid
variations
due
to
different
methods
and
preparation
protocols,
we
selected
two
structures
from
a
single
group,
i.e.,
prepared
with
similar
equipment/protocols,
and
compared
a structure
classed
as
resting
(6BE1)
with
one
classed
as
open
(6DG8).
The
difference
in the
position
of MX
in the
6BE1
and
6DG8
structures
(Figure
1)
is maintained
even
at the
end
of the
800
ns
simulations
(Figure
7).
Panels
A
and
B show
how
MX
moves
from
below
the
membrane
to
completely
within
it:
in
the
6BE1
structure
(Figure
7A),
the
tip
of the
MX
is contacting
the
polar
headgroups
of the
lower
lipid
bilayer
and
the
rest
of
MX
is intracellular.
In
the
6DG8
structure
(Figure
7B),
MX
is
fully
embedded
within
the
membrane,
with
the
tip
even
reaching
some
upper
leaflet
lipids
and
the
bottom/hinge
area
now
contacting
the
intramembrane
sides
of
the
polar
headgroups.
Panels
C
and
D
show
the
positions
of
some
residues
we
identified
as affecting
EC
50
(Table
1, Figure
2)
and
how
their
environment
differs
between
the
open
and
closed
states.
D319,
H323,
and
L326
go
from
being
entirely
cytoplasmic
to
fully
embedded
in
the
membrane
�
D319
and
to
some
degree
H323
interacting
with
polar
headgroups
and
L326
thrust
into
the
hydrophobic
lipid
tails.
This
also
shows
the
major
rotation
that
the
distal
end
of
MX
undergoes,
with
L326,
R328,
and
W331
in
very
different
orientations
with
respect
to
M4
in
the
two
different
channel
states.
Unfortunately,
we
can
draw
no
conclusions
about
specific
lipid
interactions
under
physiological
conditions
from
these
simulations,
as
they
were
performed
in
phosphatidylcholine-
only
membranes.
Nevertheless,
the
images
show
that
a number
of
MX
residues
move
between
hydrophilic
and
hydrophobic
environments,
and
so
this
region
could
be
an
important
contributor
to
channel
opening/closing
dynamics.
■
DISCUSSION
The
aim
of this
study
was
to
determine
whether
the
MX
helix
residues
play
a role
in
the
5-HT
3
R
function
by
testing
the
function
and
expression
of receptors
when
each
individual
MX
residue
was
mutated
to
alanine.
The
data
indicate
that
five
of
the
residues
are
likely
involved
in
assembly
and/or
surface
expression
of
the
receptor,
as
a chaperone
is
required
for
Table
1. Functional
Parameters
for WT and Mutant
5-
HT
3
R
a
mutant
pEC
50
(M)
EC
50
(
μ
M)
n
H
n
WT
6.50
±
0.04
0.32
2.5
±
0.5
20
WT
6.52
±
0.06
0.30
2.0
±
0.2
8
P316A
7.18
±
0.06
b
0.07
1.7
±
0.3
4
V317A
7.06
±
0.05
b
0.09
2.0
±
0.4
8
P318A
6.93
±
0.06
b
0.12
1.4
±
0.3
8
D319A
7.04
±
0.04
b
0.09
2.4
±
0.5
4
W320A
6.73
±
0.02
0.19
3.6
±
0.4
4
L321A
6.76
±
0.06
0.18
1.6
±
0.2
4
R322A
6.73
±
0.04
0.19
1.8
±
0.2
4
H323A
6.16
±
0.05
b
0.69
2.4
±
0.5
4
L324A
6.80
±
0.04
0.16
3.3
±
0.8
4
V325A
6.84
±
0.03
b
0.14
2.5
±
0.3
4
L326A
7.06
±
0.03
b
0.09
3.2
±
0.5
4
D327A
6.96
±
0.03
b
0.11
4.4
±
1.2
4
R328A
6.83
±
0.07
b
0.15
1.7
±
0.4
4
I329A
6.88
±
0.06
b
0.13
2.5
±
0.8
4
W331A
6.96
±
0.05
b
0.11
2.3
±
0.5
4
I332A
6.84
±
0.04
b
0.14
3.2
±
1.0
4
L333A
6.78
±
0.05
0.17
2.8
±
0.8
4
C334A
6.98
±
0.04
b
0.11
3.4
±
0.8
4
a
Bold
= data
obtained
when
coexpressed
with
RIC-3.
p
=
−
log;
pEC
50
s are
used
as
they
are
normally
distributed
about
the
mean.
Data
= mean
±
SEM.
b
Significantly
different
to
WT,
ANOVA
with
Dunnett’s
multiple
comparison
test;
p
< 0.05.
Figure
3.
Plot
showing
many
mutants
result
in a decrease
of
EC
50
(
i.e.,
some
gain
of function)
with
one
showing
an
increase
( i.e.,
some
loss
of function).
Data
from
Table
1. Black
= significantly
different
to
WT,
ANOVA
with
Dunnett’s
multiple
comparison
test;
p
< 0.05.
ACS Chemical
Neuroscience
pubs.acs.org/chemneuro
Research
Article
https://doi.org/10.1021/acschemneuro.2c00339
ACS Chem.
Neurosci.
2022,
13, 2338
−
2345
2340
function.
In
addition,
a large
number
of
the
mutant
receptors
resulted
in
a small
decrease
in
EC
50
, indicating
a gain
of
function,
with
one
showing
an
increase
(Figure
3).
Radio-
labeled
binding
studies
showed
no
change
in the
ligand
affinity
at the
binding
site,
suggesting
that
the
mutations
resulted
in a
change
in gating.
Images
showing
the
position
of lipids
relative
to
MX
following
800
ns
MD
simulations
are
consistent
with
the
movement
of this
helix
into
the
membrane
when
the
pore
opens.
The
data,
which
are
discussed
in
more
detail
below,
suggest
that
residues
in this
α
-helix
can
contribute
to
receptor
function
and
may
be
critical
for
transducing
the
effects
of
changes
in the
ICD
(such
as when
a modulator
binds)
to
the
pore.
MX as a Region
Involved
in Receptor
Expression.
Previous
studies
in the
nAChR
have
shown
that
the
equivalent
region
in
these
receptors
regulates
receptor
assembly
and
trafficking.
11
Our
data
show
that
five
mutants,
which
did
not
initially
respond
to
5-HT
yet
did
function
when
coexpressed
with
RIC-3,
support
a similar
role
here.
RIC-3
is
a well-
established
chaperone
of
the
5-HT
3
A
receptor,
acting
via
interactions
with
the
MX
helix
to
facilitate
receptor
assembly.
6,8,24
Our
data
suggest
that
Ala
substitution
of certain
residues
in
MX
impedes
assembly
in
the
absence
of
RIC-3.
The
affected
residues
(Figure
8)
include
D319
and
H323,
which
are
charged
residues
that
face
away
from
M4,
and
we
speculate
that
these
residues
may
be
important
for
interaction
with
lipid
head
groups
to
guide
the
correct
insertion
of
this
region
of
the
receptor
into
the
bilayer.
Mutations
Resulting
in a Gain
of Function.
Most
of the
Ala-substituted
receptors
showed
small
decreases
in
EC
50
s,
which
the
radiolabeled
binding
data
suggest
are
not
due
to
a
change
in
binding
affinity,
and
thus
may
reflect
enhanced
gating
of
the
receptor.
This
is reminiscent
of
data
obtained
from
studies
of the
M4
region
of some
pLGICs,
e.g.,
in ELIC,
substitution
with
Ala
resulted
in
small
decreases
in
EC
50
s,
12
supporting
previous
data
that
suggested
that
this
helix
contributes
to
their
function.
13
−
16
In
the
case
of
ELIC,
it
was
proposed
that
closer
packing
of
M4
with
adjacent
transmembrane
helices
and/or
greater
conformational
flexi-
bility
is
advantageous.
Considering
these
as
potential
explanations
for
our
MX
data,
closer
helical
packing
is unlikely
as
there
is a limited
association
of
MX
with
adjacent
helices.
Greater
conformational
flexibility
is a possibility,
as
there
is a
change
not
only
in
the
location
but
also
in
the
length
of
the
helix
between
the
open
and
closed
states
of
the
receptor
(Figure
1).
A
further
explanation,
which
we
consider
more
likely,
is
a change
in
the
association
with
adjacent
lipids:
previously
performed
simulations
show
very
different
potential
MX
−
lipid
interactions
in
the
resting
and
open
states,
and
we
used
a snapshot
from
these
simulations
to
show
the
positions
of lipids
close
to MX
(Figure
7).
These
images
show
that
lipids
would
be
able
to
interact
with
all
the
residues
in
MX
in
the
open
state
but
only
some
in
the
closed
state,
i.e.,
lipid
interactions
could
change
significantly
during
gating.
This
could
explain
our
data,
as
any
changes
to
MX
residues
could
alter
their
lipid
interactions
and
hence
modify
the
stability
of
MX
in the
open
and/or
closed
state.
Other
routes
that
modify
MX,
such
as
a change
in
the
structure
of
the
loops
on
either
side
of
this
helix,
could
have
a similar
effect.
Indeed,
we
speculate
that
in vivo
the
MX
helix
may
be
the
route
by
which
modifications
to
the
M3-M4
loop
are
transduced
to
the
pore,
as
this
region
has
a number
of
motifs
including
phosphor-
ylation
sites
and
binding
sites
for
regulatory
and
cytoplasmic
signaling
molecules.
Figure
4.
Typical
fluorescent
responses
(F
in arbitrary
units,
AU)
of HEK293
cells
expressing
5-HT
3
A receptors
and
stimulated
at 20
s with
a range
of
concentrations
of
5-HT
(0.01
−
3
μ
M),
and
concentration
−
response
curves
derived
from
these
and
similar
data.
See
Table
1 for
parameters.
Figure
5.
Example
of
radioligand
binding
curves
from
HEK
cells
expressing
WT
or
mutant
receptors.
Parameters
derived
from
such
curves
are
similar:
WT:
K
d
= 0.25
±
0.1
nM,
B
max
= 1.1
±
0.4
pmol/
mg
protein;
P318A:
K
d
= 0.16
±
.
06
nM,
B
max
= 0.9
±
0.2
pmol/mg
protein;
H323A:
K
d
= 0.18
±
.
02
nM,
B
max
= 1.0
±
0.1
pmol/mg
protein
(data
= mean
±
SEM,
n
= 4).
ACS Chemical
Neuroscience
pubs.acs.org/chemneuro
Research
Article
https://doi.org/10.1021/acschemneuro.2c00339
ACS Chem.
Neurosci.
2022,
13, 2338
−
2345
2341