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Molecular characterization of the Na+/H+-antiporter NhaA from Salmonella Typhimurium.
[typhoid]
Na
+
/
H
+
antiporters
are
integral
membrane
proteins
that
are
present
in
almost
every
cell
and
in
every
kingdom
of
life
.
They
are
essential
for
the
regulation
of
intracellular
pH-value
,
Na
+
-
concentration
and
cell
volume
.
These
secondary
active
transporters
exchange
sodium
ions
against
protons
via
an
alternating
access
mechanism
,
which
is
not
understood
in
full
detail
.
Na
+
/
H
+
antiporters
show
distinct
species-
specific
transport
characteristics
and
regulatory
properties
that
correlate
with
respective
physiological
functions
.
Here
we
present
the
characterization
of
the
Na
+
/
H
+
antiporter
NhaA
from
Salmonella
enterica
serovar
Thyphimurium
LT
2
,
the
causing
agent
of
food-born
human
gastroenteritis
and
typhoid
like
infections
.
The
recombinant
antiporter
was
functional
in
vivo
and
in
vitro
.
Expression
of
its
gene
complemented
the
Na
+
-
sensitive
phenotype
of
an
E
.
coli
strain
that
lacks
the
main
Na
+
/
H
+
antiporters
.
Purified
to
homogeneity
,
the
antiporter
was
a
dimer
in
solution
as
accurately
determined
by
size-exclusion
chromatography
combined
with
multi-angle
laser-light
scattering
and
refractive
index
monitoring
.
The
purified
antiporter
was
fully
capable
of
electrogenic
Na
+
(
Li
+
)
/
H
+
-
antiport
when
reconstituted
in
proteoliposomes
and
assayed
by
solid-supported
membrane-based
electrophysiological
measurements
.
Transport
activity
was
inhibited
by
2
-
aminoperimidine
.
The
recorded
negative
currents
were
in
agreement
with
a
1
Na
+
(
Li
+
)
/
2
H
+
stoichiometry
.
Transport
activity
was
low
at
pH
7
and
up-regulation
above
this
pH
value
was
accompanied
by
a
nearly
10
-
fold
decrease
of
KmNa
(
16
mM
at
pH
8
.
5
)
supporting
a
competitive
substrate
binding
mechanism
.
K
+
does
not
affect
Na
+
affinity
or
transport
of
substrate
cations
,
indicating
that
selectivity
of
the
antiport
arises
from
the
substrate
binding
step
.
In
contrast
to
homologous
E
.
coli
NhaA
,
transport
activity
remains
high
at
pH
values
above
8
.
5
.
The
antiporter
from
S
.
Typhimurium
is
a
promising
candidate
for
combined
structural
and
functional
studies
to
contribute
to
the
elucidation
of
the
mechanism
of
pH-dependent
Na
+
/
H
+
antiporters
and
to
provide
insights
in
the
molecular
basis
of
species-
specific
growth
and
survival
strategies
.