(Received for publication, April 12, 1995; and in revised form, November 15, 1995)
From the
The action of aldosterone to increase apical membrane
permeability in responsive epithelia is thought to be due to activation
of sodium channels. This channel is regulated, in part, by G-proteins,
but it is not known if this mechanism is regulated by aldosterone. We
report that aldosterone stimulates the expression of the 41-kDa
subunit of the heterotrimeric GTP-binding proteins
in A-6 cells. Both mRNA and the total amount of this protein are
increased by aldosterone. The G-protein is palmitoylated in response to
the steroid, and the newly synthesized subunit is found to co-localize
with the sodium channel. Aldosterone stimulation of sodium transport is
significantly inhibited by inhibition of palmitoylation. These results
suggest that aldosterone regulates sodium channel activity in epithelia
through stimulation of the expression and post-translational targeting
of a channel regulatory G-protein subunit.
Aldosterone increases epithelial sodium reabsorption in part by
activating Na channels already present in the apical
membrane(1, 2) . The mechanism of steroid-induced
activation of pre-existing channels is not known. Na
channels of the type regulated by aldosterone have been shown in
patch clamp studies to be gated by heterotrimeric
G-proteins(3, 4) , similar to other ion
channels(5, 6) , and the
subunit of
the heterotrimeric G-proteins is known to be topographically localized
with the channel(7) . A number of G-protein
subunits have
been shown to be post-translationally acylated, and these modifications
promote membrane targeting and
attachment(8, 9, 10) . Although G-proteins
are transcriptionally regulated in epithelial cells under conditions of
growth and differentiation(11, 12) , it is not known
if they are synthesized, acylated, or targeted during stimulation of
Na
transport by steroids. We examined the possibility
that aldosterone enhances association of the
G-protein with the Na
channel by directing its
synthesis and post-translational covalent lipid modification.
To determine if G-protein content was increased by
aldosterone, A6 cells were labeled with
[S]methionine in the presence and absence of 0.1
µM aldosterone. Whole cell lysates were first incubated
with an antibody directed against the common GTP binding site of
G-proteins (GA/1), and the immunoprecipitated proteins were subjected
to SDS-PAGE and autoradiography. Densitometry revealed that aldosterone
enhanced metabolic labeling of a 41-45-kDa GTP-binding protein
3-fold compared to control (Fig. 1a). Other GTP-binding
proteins were also labeled. To identify the protein labeled at 41 kDa,
cell lysates were then subjected to immunoprecipitation with an
affinity-purified antibody (EC/2) specific for G
(DuPont NEN)(7, 16) , directed against the
carboxyl-terminal decapeptide of
, analyzed by
SDS-PAGE and fluorography (Fig. 1b). Aldosterone
enhanced metabolic labeling of G
by 3-4-fold
compared to controls. Next, whole cell lysates were subjected to
Western blot analysis with EC/2 and visualized using
peroxidase-conjugated second antibodies and chemiluminescence
conjugates. Aldosterone increased the amount of the 41-kDa
G-protein 2-3-fold by densitometry (Fig. 1c).
Figure 1:
Effect of
aldosterone on G protein content. a and b, A6 cells were labeled with
[
S]methionine (100 µCi/ml) for 6 h followed
by either 1
10
M aldosterone (A) or diluent (C) for an additional 4 h. Whole cell
lysates were prepared as described under ``Experimental
Procedures.'' a, 200 µg of whole cell lysate protein
was subjected to immunoprecipitation with G-protein antibody raised to
the amino acid sequence (GTSNNSGKSTIVKQMK) part of the GTP binding
domain, GA/1 (DuPont NEN). b, or with antibody to the
carboxyl-terminal decapeptide (KNNLKECGLY) of G
,
derived from clone EC/2 (DuPont NEN). SDS-PAGE and fluorography were
performed as described under ``Experimental Procedures.''
Shown are representative fluorograms of 4 experiments. Molecular mass
marker positions were determined from Coomassie Blue-stained gels and
are noted on the left of the figure. Fluorography was carried
out for 2 weeks at -80 °C. c, A6 cells were treated
with 1
10
M aldosterone or diluent
for 18 h, and whole cell lysates were protein-matched, subjected to
SDS-PAGE, and transferred to nitrocellulose for Western blotting.
Membranes were probed with EC/2 antibody and goat anti-rabbit second
antibody, and visualization was accomplished using an ECL kit
(Amersham), an enhanced chemiluminescence system and fluorography.
Shown is a representative fluorogram of 4 experiments. Molecular mass
marker positions are noted on the left.
To determine whether the increased
expression of G was due to an increase in mRNA
levels, A6 cells were serum-depleted overnight, then exposed to 0.1
µM aldosterone for 12-16 h, poly(A
)
mRNA was isolated, and Northern blot analysis was performed. The mRNA
was probed with a specific G
probe and a probe for
-actin, a housekeeping gene (Fig. 2). Densitometry revealed
that aldosterone induced a 1.6-2-fold specific increase in
G
message. These results indicate that aldosterone
may increase Na
transport via an increase in G-protein
mRNA expression.
Figure 2:
Northern blot hybridization of A6 mRNA for
G and
-actin. C, control; A,
aldosterone-treated. 5 µg of mRNA was electrophoresed through 1%
formaldehyde-agarose gels. A specific G
oligonucleotide probe was synthesized to examine if the mRNA for
the
subunit of the Na
channel
complex was regulated by aldosterone. The blot was probed with a
specific 5`-
P-labeled
oligonucleotide
probe (CCTGGCAGCTTCCCAAA) and exposed at -80 °C for 48 h to
Kodak X-Omat film. This probe does not recognized G
or G
mRNA (data not shown). The autoradiograph
for
-actin mRNA was obtained by stripping the G
probe and rehybridizing with the second probe to detect actin for
quantification. To control for variability, the G
mRNA density values were normalized to the value for the
-actin hybridization within each sample. This is a representative
autoradiograph of 3 experiments. Three 150-mm filters of confluent A6
cells were used for mRNA isolation under each condition for each
experiment.
Experiments were designed to determine whether the
newly synthesized G-protein becomes associated with the sodium channel.
A6 cells were metabolically labeled with
[S]methionine in the presence and absence of
10
M aldosterone. Whole cell lysates were
protein-matched and incubated with polyclonal antisera raised against a
highly purified preparation of sodium channel isolated from bovine
renal papilla (17) or preimmune rabbit serum using the same
conditions which had previously described the association of G
with the sodium channel complex(7) . Immunoprecipitated
proteins were subjected to SDS-PAGE under reducing conditions and
autoradiographed. Fig. 3a demonstrates that aldosterone
enhances the co-localization of the labeled 41-45-kDa
G-protein with the sodium channel
(2.5-2.6-fold increase by densitometry). Consistent with previous
observations(33) , most other channel subunits are not
metabolically labeled with [
S]methionine over
the time course employed here. In order to ensure that aldosterone
selectively increases the expression of this subunit of the channel
complex, the experiment was repeated with an extended period of
metabolic labeling with [
S]methionine prior to
aldosterone exposure and immunoprecipitation with the sodium channel
antibody performed as described above. Fig. 3b demonstrates that channel subunits are metabolically labeled at
50, 95, 70, 55, and 41-45 kDa. Only the 41-45-kDa subunit
is significantly enhanced in labeling by aldosterone. This finding is
consistent with previous electrophysiological and biochemical evidence
that aldosterone acts primarily by activating pre-existing
channels(1, 2) . To determine whether
post-translational modifications with lipids target the induced
G-protein, we examined the effects of aldosterone on palmitoylation and
myristoylation of G
. A6 cells were labeled with
[
C]palmitate in the presence of 1 µM aldosterone or diluent. Cells were homogenized and crude membrane
fraction was isolated by centrifugation at 100,000
g for 1 h and subjected to SDS-PAGE. As shown in Fig. 4a, aldosterone stimulated palmitoylation of
several membrane proteins including a 41-45-kDa protein. There
were also enhanced labeling of a broad band around 30 kDa, although
palmitoylation of a smaller molecular mass protein at 18 kDa was not
enhanced by aldosterone. This pattern of palmitoylation of membrane
proteins was similar with both the 4- and 18-h exposure to aldosterone.
In order to determine whether the 41-45-kDa palmitoylated protein
was in fact a G-protein associated with the channel, we undertook
immunoprecipitation of cellular proteins with both G-protein and
Na
channel antibodies following incubation with
isotopically labeled acyl groups in the presence or absence of
aldosterone. Whole cell lysates from A6 cells metabolically labeled
with [
C]palmitate in the presence of 0.1
µM aldosterone or diluent were protein-matched and
immunoprecipitated with with GA/1 G-protein antibody (Fig. 4b). Treatment with aldosterone resulted in a
2-fold enhancement of labeling of the 41-kDa G-protein. Several other
G-proteins also appear to be palmitoylated in response to aldosterone.
Similar experiments were performed with
[
H]myristate but failed to show incorporation of
the isotopically labeled lipid into the 41-kDa G-protein in either the
presence or absence of aldosterone, although other proteins were
clearly labeled (data not shown). In A6 cells, it appears that the
41-kDa G-protein is palmitoylated but not myristoylated. A6 cells were
next metabolically labeled with [
C]palmitate in
the presence and absence of aldosterone, and whole cell lysates were
subjected to immunoprecipitation with the Na
channel
antibody previously used to localize the G-protein to the
channel(7) . When protein-matched samples were
immunoprecipitated with this antibody, aldosterone specifically
increased palmitoylation of a 41-kDa protein (2-3-fold by
densitometry) (Fig. 4c).
Figure 3:
Sodium channel localization of
G. a, A6 cells were labeled with
[
S]methionine in the presence of 10
M aldosterone or diluent as described in Fig. 1.
200 µg of whole cell lysate was subjected to immunoprecipitation
with the sodium channel antibody. Immunoprecipitated proteins were
resolved on 15% polyacrylamide gels under reducing conditions. Proteins
were visualized by fluorography. Molecular mass marker positions are
shown on the left. A, aldosterone-treated. C, control. n = 4 for each (4-8-week
exposure) experiment. In aldosterone-treated cells only, the 41-kDa
band is metabolically labeled and co-localized with the sodium channel
complex. b, aldosterone selectively increases the expression
the 41-45-kDa G
subunit relative to the other
channel subunits. A6 cells were labeled with
[
S]methionine for 24 h and then exposed to 10
M aldosterone or diluent for an additional
4 h. 200 µg of whole cell lysate was then subjected to
immunoprecipitation with the sodium channel antibody or preimmune
rabbit serum. Immunoprecipitated proteins were resolved on 5-15%
SDS-PAGE gels and visualized by autoradiography. Shown is a
representative experiment of 4 experiments. A,
immunoprecipitate from aldosterone-treated cells. C,
immunoprecipitate from control cells. The last lane on the right labeled(-) represents whole cell lysates from metabolically
labeled cells treated with aldosterone which were immunoprecipitated
with preimmune rabbit serum. Numbers shown to the left demonstrate the molecular weight of the resolved channel subunits
as determined from migration of molecular mass standards. Aldosterone
increases the metabolic labeling only of the 41-45-kDa subunit of
the sodium channel (average 2.5 times by
densitometry).
Figure 4:
a, short (4-h) or long term (18-h)
Aldosterone exposure stimulates the palmitoylation of membrane
proteins. A6 cells were labeled with
[C]palmitate for 18 h in the presence of
10
M aldosterone for either 4 h or 18 h.
Control cells were labeled with [
C]palmitate for
18 h in the presence of diluent. Cells were homogenized, and a crude
membrane fraction was isolated by centrifugation at 100,000
g for 1 h. Samples were protein-matched and subjected to
electrophoresis on 15% SDS-PAGE gels and subjected to autoradiography.
Shown is a representative of 4 separate experiments. Migration of
molecular mass markers is shown on the left. C,
control; A, aldosterone-treated cell: A4, 4 h of
aldosterone; A18, 18 h of aldosterone. b,
G
is palmitoylated in response to aldosterone. A6
cells were labeled with [
C]palmitate in the
presence of 10
M aldosterone or diluent.
200 µg of whole cell lysates were subjected to immunoprecipitation
with GA/1 G-protein antibody, and immunoprecipitated proteins were
resolved using SDS-PAGE and fluorography. A,
aldosterone-treated; C, control. Migration of molecular mass
markers are indicated on the left. A 41-kDa G-protein is
palmitoylated in response to aldosterone. Shown is a representative
fluorograph of 4 separate experiments. c, sodium channel
associated G
is palmitoylated in aldosterone-treated
cells. A6 cells were labeled with [
C]palmitate
in the presence or absence of 10
M aldosterone. 200 µg of whole cell lysate was subjected to
immunoprecipitation with sodium channel antibody. Immunoprecipitated
proteins were resolved under reducing conditions on 15% polyacrylamide
gels as in Fig. 3and exposed at -80 °C for 10 weeks.
Shown is a representative fluorogram of 3 separate
experiments.
The physiological relevance
of this observation was examined using an inhibitor of palmitoylation,
2-fluoropalmitic acid(18) . 2-Fluoropalmitic acid (Biomol,
Plymouth Meeting, PA) had no effect on basal sodium transport over
short time courses at the concentration of 37.5 µM (Fig. 5), but markedly inhibited aldosterone-induced
stimulation of sodium transport. In order to demonstrate that the
action of this inhibitor might in fact be related to inhibition of
G-protein palmitoylation, cells were metabolically labeled
with[C]palmitate in the presence and absence of
aldosterone and 37.5 µM 2-fluoropalmitic acid.
Protein-matched whole cell lysates were subjected to
immunoprecipitation with GA/1 and subjected to SDS-PAGE and
autoradiography. Fig. 6demonstrates that aldosterone stimulates
palmitoylation of several G-proteins including a band at 41 kDa and
another around 30 kDa, and that labeling of these proteins with the
acyl group is markedly inhibited by 2-fluoropalmitic acid.
Figure 5:
Effect of 2-fluoropalmitic acid on
aldosterone-stimulated sodium transport. A6 cells were grown on
Millipore filters (4.2 cm) to confluence and used when they
exhibited stable resistance. Basal short circuit current (I
) was measured. Then, 10
M aldosterone or diluent with or without
2-fluoropalmitic acid (37.5 µM) in amphibian media was
added, and I
was measured at 1, 2, and 3 h. Data
are reported as means ± S.E. n = 8 for each
group. Triangles, aldosterone; squares,
2-fluoropalmitic acid + aldosterone; circles,
2-fluoropalmitic acid. *, p < 0.05 (aldosterone versus 2-fluoropalmitic acid + aldosterone). Data analysis was
performed using one-way analysis of variance on NCSF statistical
software (Hintze, Kaysville, UT).
Figure 6:
2-Fluoropalmitic acid inhibits
aldosterone-stimulated palmitoylation of G. A6 cells
were labeled with [
C]palmitate with
10
M aldosterone or diluent with or without
37.5 µM 2-fluoropalmitic acid overnight. 200 µg of
whole cell lysate protein was subjected to immunoprecipitation with
GA/1 antibody, and immunoprecipitated proteins were resolved by
SDS-PAGE and exposed for 2 weeks at -80 °C. Migration of
molecular mass markers is shown on the left. A,
aldosterone-treated; C, control; AF, aldosterone- and
2-fluoropalmitic acid-treated cells; F, 2-fluoropalmitic acid.
Shown is a representative fluorograph of 3
experiments.
The 41-kDa G subunit has been shown
previously to increase the open time of the apical sodium channel in
excised apical membrane patches from A6 cells (3) and to be a
component of the 700-kDa sodium channel complex(7) . Our
results demonstrate that aldosterone stimulates the expression of mRNA
for this subunit and results in an increase in the total amount of this
protein, which becomes associated with the sodium channel complex.
Since this G-protein is thought to have a gating effect on the sodium
channel, it seems reasonable to propose that increased expression and
localization of this subunit may be one mechanism whereby aldosterone
activates quiescent channels already present at or near the apical
membrane(1) . It also seems likely that aldosterone may direct
a mechanism that localizes this G-protein to a site adjacent to the
channel.
Several types of post-translational modifications have been
described that are associated with membrane targeting or attachment of
G-proteins. A number of G-protein subunits have been shown to be
post-translationally acylated with either palmitate or myristate at
sites near their amino termini, and these modifications promote
membrane attachment(8, 9, 10) . Smaller
molecular weight G-proteins are targeted to membranes by a sequence of
events involving highly conserved carboxyl-terminal cysteine residues.
This pathway involves first prenylation of a cysteine residue, cleavage
of the terminal amino acids, and subsequent carboxylmethylation and/or
acylation (20, 21, 22) . Several previous
observations suggest that these targeting pathways might be involved in
aldosterone action. First, aldosterone stimulates acylation-deacylation
reactions(23) , and inhibition of these reactions blocks both
the transport response and the localization of aldosterone-induced
proteins to membranes(24) . Second, aldosterone stimulates
carboxylmethylation reactions which result in increased channel
activation(25, 26, 27) . The results
presented here indicate that aldosterone stimulates the palmitoylation,
but not myristoylation, of the 41-kDa G
protein.
These studies have also shown that aldosterone induces increased
metabolic labeling and palmitoylation of several small GTP-binding
proteins most notably at 30 and 18 kDa ( Fig. 1and Fig. 6and (32) ). The identity of these G-proteins and
their relation to aldosterone stimulation of sodium transport is not
known. Inhibitor studies suggest that palmitoylation of one or more
G-proteins may be required for the full expression of the early
transport response to aldosterone. As G
is the only
G-protein currently known to be associated with a channel regulatory
function, the simplest hypothesis to explain our results is a model
whereby aldosterone stimulates localization of the G-protein by
lipidation reactions.
The specific site of post-translational
lipidations of the G-protein subunit have not been identified in these
studies. It seems unlikely that this represents the pathway of
carboxyl-terminal lipidation and carboxylmethylation described for the
targeting of small molecular weight GTP-binding proteins for several
reasons. First, palmitoylation of the subunit of G-proteins has
been described primarily as an amino-terminal
modification(8, 9) . Second, although
carboxylmethylation has been implicated in the action of aldosterone on
the sodium channel(25, 26, 27) , recent
studies with purified channel complex indicate that the 95-kDa subunit
is carboxylmethylated rather than the 41-kDa subunit(28) .
Carboxylmethylation of this 95-kDa subunit results in rapid activation
of the channel(28) . While these in vitro studies do
not necessarily rule out carboxylmethylation of the 41-kDa subunit in
intact cells, they are consistent with the recent observations of
Sariban-Sohraby et al.(29) , which suggest that
aldosterone stimulates the methylation of a 90-95-kDa membrane
protein in A6 cells.
Taken together, the observations that aldosterone stimulates localization of a 41-kDa subunit of the channel and carboxylmethylation of a 90-95-kDa subunit(30, 31) , suggest the possibility that there may be more than one action at the channel. Such a suggestion has previously been made by Asher and Garty (30) on the basis of vesicle studies. They described an early stimulation of transport by aldosterone that was not stable to vesicle preparation and a prolonged effect that was stable to vesicle preparation. Carboxylmethylation reactions of membrane-bound proteins are known to be reversible reactions due to the presence of methylesterases in the membrane(31) . We speculate that aldosterone may activate sodium channels already residing in the apical membrane, both through an early, reversible carboxylmethylation and a later, more stable association of a gating G-protein.