(Received for publication, September 20, 1995; and in revised form, December 5, 1995 )
From the
Agents known to increase cAMP levels in renal and intestinal
epithelia decrease sodium absorption by inhibiting NHE3, an isoform of
the Na/H
exchanger expressed at high
levels in apical membranes of these cells. In contrast, the ubiquitous,
housekeeping isoform of the exchanger (NHE1) is stimulated by cAMP in
some cell types. Optimal activity of NHE3 as well as NHE1 requires the
presence of ATP. To gain insight into the molecular mechanisms of ATP
dependence and cAMP regulation of NHE3, a series of mutations were
constructed by progressively truncating segments of the C-terminal
cytoplasmic domain of the transporter at amino acid positions 684, 638,
and 579 (named NHE3
684, NHE3
638, and NHE3
579). In
addition, chimeric antiporters were constructed with the N-terminal
transmembrane domain of NHE3 linked to the entire cytoplasmic region of
NHE1 (chimera NHE3/1) or vice versa (chimera NHE1/3). These
constructs were heterologously expressed in antiport-deficient Chinese
hamster ovary cells, and their activities were assessed by fluorimetric
measurements of intracellular pH and by radioisotope determinations of
Na
influx. Forskolin, which directly stimulates
adenylate cyclase, inhibited NHE3 as well as NHE1/3, but not NHE3/1,
suggesting that the cytoplasmic domain of NHE3 was sufficient to confer
sensitivity to inhibition by cAMP. Forskolin also inhibited the
truncated mutant NHE3
684 to an extent similar to that for wild
type NHE3. However, the inhibitory effect was greatly reduced in
NHE3
638 and more profound truncations (NHE3
579) obliterated
the effect of forskolin. These findings suggest that a region found
between amino acids 579 and 684 is essential for the cAMP response of
NHE3. In contrast, comparable ATP dependence was observed in all
exchanger constructs examined. These observations indicate that ATP
dependence is conferred by a region of the molecule in or adjacent to
the transmembrane domain, which is most conserved between isoforms. It
is concluded that different sites, and therefore different mechanisms,
underlie inhibition of NHE3 by cAMP and by depletion of ATP.
Na/H
exchange (NHE) (
)activity is present in virtually all mammalian cells and
catalyzes the electroneutral exchange of one sodium for one proton. The
exchange of Na
for H
, which is
characteristically sensitive to inhibition by amiloride and its
analogs(1, 2) , is driven by the concentration
gradients of these ions and does not require direct expenditure of
metabolic energy. Nevertheless, the presence of ATP is required for
optimal exchanger activity by a mechanism that is poorly
understood(3) .
NHE activity is thought to be essential for
pH homeostasis(4, 5) , transepithelial ion and water
transport(6) , and cell volume regulation (7) and may
also play a role in cell proliferation (5) and
adhesion(8, 9) . This functional versatility prompted
the search for variant forms of the transporter. To date, five
mammalian isoforms of the Na/H
exchanger (NHE1 to NHE5) have been
identified(10, 11, 12, 13, 14, 15) .
The isoforms share a similar hydropathy profile that indicates the
existence of two major structural domains: a predominantly hydrophobic
transmembranous N terminus and a more hydrophilic cytoplasmic C
terminus. The N-terminal domain, which is highly conserved among
isoforms, is predicted to span the membrane 10-12 times and is
believed responsible for catalyzing Na
and
H
exchange and conferring amiloride
sensitivity(16, 17) . The more variable C terminus is
thought to extend into the cytosol and to play a role in regulating the
activity and subcellular distribution of the exchangers(16) .
It contains potential sites for phosphorylation by protein kinases and,
in some isoforms, includes a segment with affinity for
calmodulin(10, 11, 18, 19, 20) .
NHE1, the ``housekeeping'' isoform, is present in nearly
all mammalian cells examined to date. In epithelial cells, it localizes
predominantly to the basolateral membrane. The other isoforms have a
more restricted tissue distribution. NHE2 to NHE4 are abundant in
epithelial cells of kidney, intestine, and stomach, whereas NHE5
resides primarily in brain, spleen, and testis. Of these, NHE3 has been
studied in most detail. It is restricted to the apical (brush border)
membranes of some epithelial cells of the renal and gastrointestinal
tracts, where it mediates Na reabsorption(21, 22, 23) . In these
tissues, the rate of Na
reabsorption is variable and
stringently controlled, primarily by agents that modulate the levels of
adenosine 3`,5`-cyclic monophosphate (cAMP). Because NHE3 is the
predominant exchanger in epithelial brush border membranes, this
isoform is postulated to be the direct target of the cAMP-dependent
protein kinase A (PKA)(24) . However, direct demonstration of
the regulation of NHE3 by PKA in native tissues is complicated by the
coexistence of multiple isoforms in different cell types and even in
different membranes of the same cell. These confounding factors can be
overcome by heterologous transfection of NHE3 into cells devoid of
endogenous exchanger activity. Using this approach, it has recently
been demonstrated that the activity of NHE3 is markedly depressed by
elevating intracellular cAMP using agents such as forskolin. (
)
The molecular basis of the regulatory effects of cAMP
on NHE3 is not clear. The cytosolic domain of this isoform contains
several consensus sites for phosphorylation by PKA(11) , some
of which may mediate the observed inhibition. On the other hand,
phosphorylation-independent regulation of other isoforms has been
described(7, 25) . In an attempt to better understand
the regulation of NHE3, a mutational analysis was conducted by
generating a series of progressive truncations of the regulatory
C-terminal domain that contains the putative phosphorylation sites. For
comparative purposes, the behavior of NHE1 was also examined since, in
osteoblasts, it has been reported to be activated by
cAMP(26) . Definition of the site(s) that confer NHE3-specific
sensitivity to cAMP was accomplished by creating chimeras of NHE1 and
NHE3. The functional behavior of these mutants was studied in isolation
by transfection into AP-1 cells, a Chinese hamster ovary line that
lacks endogenous Na/H
exchange(27) . These transfectants were also used to
analyze the structural basis of the paradoxical ATP dependence of the
exchanger.
Polyclonal antibodies to the NHE1 isoform
of the Na/H
exchanger were raised by
injecting rabbits with a fusion protein constructed with
-galactosidase of Escherichia coli, containing the last
157 residues (658-815) of the human NHE1. Polyclonal antibodies
to the NHE3 isoform were generated against a glutathione S-transferase fusion protein with residues 565-690 of
the rat NHE3. Antibodies were affinity-purified as
described(7, 28) .
Bicarbonate-free medium RPMI
1640 was buffered with 25 mM HEPES to pH 7.3 and adjusted to
290 ± 5 mosM. Phosphate-buffered saline consisted of
(in mM): 140 NaCl, 10 KCl, 8 sodium phosphate, 2 potassium
phosphate, pH 7.4. The isotonic Na-rich medium used in
the fluorimetric pH measurements contained (in mM): 140 NaCl,
3 KCl, 1 MgCl
, 10 glucose, 20 HEPES, pH 7.3. The isotonic
Na
-free medium contained the same salts, but NaCl was
substituted by N-methyl-D-glucamine. The isotonic
K
-rich medium had the same composition as
Na
-rich medium, except that NaCl was replaced by KCl.
In all cases the osmolarity was set to 290 ± 5 mosM with the major salt.
Figure 1:
Diagram of
Na/H
exchanger chimeras and deletion
mutants. A, diagram of the mammalian expression plasmids
containing the rat parental Na
/H
exchanger isoforms NHE1 and NHE3. Indicated in the figure are
relevant restriction endonuclease sites used in the construction of
chimeras of NHE1 and NHE3 and deletion mutations of NHE3. B,
linear profile and nomenclature of chimeras of NHE1 and NHE3 and
deletion mutations of NHE3. Details of their construction are provided
under ``Experimental
Procedures.''
Calibration of the fluorescence ratio versus pH was performed for each experiment by equilibrating the cells in
isotonic K-rich medium buffered to varying pH values
(between 7.45 and 5.85) in the presence of the
K
/H
ionophore nigericin (5
µM). Calibration curves were constructed by plotting the
extracellular pH, which is assumed to be identical to the internal pH,
against the corresponding fluorescence ratio(33) .
Cellular
buffering power was determined in the pH range studied by
pulsing cells with weak electrolytes, as described(34) .
H
efflux rates (mM/s) were calculated by
multiplying the rate of pH
recovery
(dpH
/dt) times the buffering capacity, at the
corresponding pH values. Na
/H
exchange rates were analyzed using the general allosteric model
described by the Hill equation shown in .
V is the H (equivalent) efflux at a
particular cytosolic H
concentration [S]; V
is the maximal H
efflux rate,
and n is the Hill coefficient, a minimal estimate of the
number of binding sites for H
. Though n was
greater than 1, the data were adequately fit using a single intrinsic
binding constant, K, suggesting that the affinities of the
individual sites were of the same order. Experimental results were fit
to the above model using a nonlinear regression data analysis program,
Enzfitter from Bios Corp.
Na/H
exchange was initially
assessed fluorimetrically in cells treated with or without forskolin,
an agent that stimulates adenylate cyclase and increases intracellular
cAMP. No attempt was made to compare the absolute rates of transport
between stable transfectants, which can reflect parameters other than
the length of the construct, such as the site(s) of genomic
integration, the number of incorporated cDNA copies/cell, and
differences in intracellular processing of mutated proteins.
Acid-loaded cells were exposed to Na
, and the rate of
recovery of pH
was used as a measure of exchanger activity.
It is noteworthy that in all cases the rate of alkalinization was
negligible in the absence of Na
. As shown in Fig. 2A, untreated cells transfected with full-length
NHE3 recovered readily from the acid load upon reintroduction of
Na
(solid circles). Consistent with
observations in renal proximal tubule cells(35, 36) ,
pretreatment with forskolin markedly depressed the activity of NHE3 (open circles). To more precisely evaluate the mechanism of
inhibition, the rate of Na
/H
exchange
was estimated at varying pH
. H
(equivalent) fluxes were calculated from the rate of change of
pH
(
pH/
t) and the buffering power,
determined independently throughout the pH range studied. As summarized
in Fig. 2B, the H
efflux rate in
control cells was highest at acidic pH
and decreased
thereafter approaching quiescence near pH 6.8. As described in isolated
renal brush borders (1) , the exchange process showed
cooperativity with respect to H
. The data were fit
adequately by the Hill equation (Fig. 2B, inset), yielding a coefficient of
3. Treatment with 10
µM forskolin reduced Na
/H
exchange activity at all pH
values tested.
Importantly, the pH
dependence of the exchanger was shifted
to more acidic levels by nearly 0.4 units. The Hill coefficient was not
markedly different when cAMP was elevated (
2.7), suggesting that
cooperativity with respect to H
persisted under these
conditions. Therefore, the effect of cAMP appears to be largely due to
a reduced affinity for intracellular H
. The effect of
cAMP on V
could not be properly assessed due to
insufficient data points below pH 6.0. Such data were not collected
because of concerns regarding potential cellular damage caused by
extremely acidic conditions. In this figure, the data are means
± S.E. of 27/54 (control/treated) individual determinations.
Figure 2:
Effect of forskolin on the activity of
full-length and C-terminally truncated NHE3: pH determinations. AP-1
cells transfected with full-length NHE3 (A and B),
NHE3684 (C and D), NHE3
638 (E and F) or NHE3
579 (G and H) were loaded with BCECF
in bicarbonate-free medium. The cells were acid-loaded with ammonium
and treated with (open symbols) or without (solid
symbols) 10 µM forskolin during the final 10 min of
incubation with the dye. Next, the pH
recovery
induced by addition of Na
was measured
fluorimetrically. Results in each panel are the mean ± S.E. of
at least 20 cells from three separate experiments. Left panels illustrate the recovery of pH
, while the right panels show the pH
dependence of
the rate of H
efflux, calculated from the mean
pH/
t and the buffering power, as described under
``Experimental Procedures.'' The insets show the
rate as a function of [H
]. Data points were
determined experimentally, while the line was fitted using the Hill
equation (see ``Experimental
Procedures'').
The inhibitory effect of forskolin on NHE3 was confirmed measuring
amiloride-sensitive Na uptake. As illustrated in Fig. 3A, activation of the adenylate cyclase reduced
influx by nearly 50% at pH
6.3, in good agreement with the
fluorescence determinations of pH
.
Figure 3:
Effects of forskolin and ATP depletion on
the activity of full-length and C-terminally truncated NHE3:
Na uptake determinations. AP-1 cells transfected with
either full-length NHE3, NHE3
684, NHE3
638 or NHE3
579
were grown to confluence on 24-well plates. A, the monolayers
were washed twice with Na
-medium (see
``Experimental Procedures'' for composition of media). The
cells were then preincubated in the same solution in the absence or
presence of 10 µM forskolin for 15 min at 37 °C. Next,
the medium was removed and the cells were preincubated for 3 min in
K
-nigericin solution to clamp the intracellular pH at
6.3 in the continued absence or presence of 10 µM forskolin. At the end of this equilibration period, the solutions
were aspirated and replaced with the same media supplemented with
NaCl (1 µCi/ml), 1 mM ouabain, with or
without 2 mM amiloride. Radioisotope uptake was terminated
after 10 min by aspiration and extensive washing with ice-cold
Na
-solution and the samples were processed as
described under ``Experimental Procedures.'' Background
Na
influx that was not inhibitable by 2
mM amiloride was subtracted from the total influx. Data are
expressed as the mean percentage of amiloride-inhibitable
Na
influx ± S.E. (n = 16) from four independent experiments. B, the
cell monolayers were incubated for 10 min in control or ATP-depleting
solutions at 37 °C. Na
/H
exchanger activity was then measured as described above. Data are
expressed as the mean percentage of amiloride-inhibitable
Na
influx ± S.E. (n = 8-12) from 2-3 independent experiments.
Significance of differences between control and experimental
measurements was calculated using two-tailed Student's t test and is indicated by an asterisk (p <
0.001).
The behavior of the
684 truncation was almost indistinguishable from that of the
full-length NHE3. As shown in Fig. 2(C and D), this mutant displayed similar recovery rates, a sharp
pH
dependence and cooperativity with a Hill coefficient of
2.6. Importantly, NHE3
684 was also susceptible to inhibition
by cAMP which, as in wild type NHE3, was manifested as an acidic shift
in the pH
sensitivity of exchange. The data are means
± S.E. of 34/27 (control/treated) individual determinations.
Na
uptake determinations also reflected
the inhibitory effect. The isotope influx rates were over 60% lower in
the forskolin-treated samples (Fig. 3A). Together,
these findings imply that the C-terminal 147 amino acids are not
required for the cAMP response.
Truncation at amino acid 638 had
little effect on the basal functional behavior of untreated cells, but
greatly reduced its responsiveness to cAMP. The pH sensitivity and
cooperativity of unstimulated NHE3638 were similar to those of the
full-length NHE3 (Fig. 2, E and F; Hill
coefficient
2). However, the inhibitory effect of forskolin was
only marginal in this mutant, with a small divergence noted primarily
at higher pH. In this figure the data are means ± S.E. of 35/27
(control/treated) individual determinations. The reduced effectiveness
of forskolin was also apparent when activity was assessed
radioisotopically (Fig. 3A).
An additional further
loss of responsiveness was noted in the NHE3579 truncated mutant.
The absolute recovery rates of these cells were low (note time scale in Fig. 2G), precluding detailed analysis at near neutral
pH
. Nevertheless, in the range where the measurements were
reliable elevation of cAMP did not inhibit exchange. In fact, a slight
stimulatory effect was recorded. The data are means ± S.E. of
37/31 (control/treated) individual determinations. The loss of
susceptibility to forskolin was also noted when
Na
uptake was measured (Fig. 3A). The influx rates with and without forskolin
were indistinguishable. In summary, the region encompassed by residues
579-684 appears to play a central role in mediating the
inhibitory action of cAMP.
Figure 4:
Effect of ATP depletion on the activity of
full-length and C-terminally truncated NHE3: pH determinations. AP-1
cells transfected with full-length NHE3 (A and B),
NHE3684 (C and D), NHE3
638 (E and F) or NHE3
579 (G and H) were loaded
with BCECF in bicarbonate-free medium. The cells were acid-loaded with
ammonium and treated with (open symbols) or without (solid
symbols) a glucose-free medium containing 5 mM deoxyglucose plus 1 µg/ml antimycin in order to deplete ATP
during the final 10 min of incubation with the dye. Next, the
pH
recovery induced by addition of Na
was measured fluorimetrically. Results in each panel are the mean
± S.E. of at least 20 cells from three separate experiments. Left panels illustrate the recovery of
pH
, while the right panels show the
pH
dependence of the rate of H
efflux, calculated from the mean
pH/
t and the buffering
power, as described under ``Experimental Procedures.'' The insets show the rate as a function of
[H
]. Data points were determined
experimentally, while the line was fitted using the Hill equation (see
``Experimental Procedures'').
The inhibitory effect
of ATP depletion was preserved in NHE3684 truncated mutants. The
data are means ± S.E. of 20/20 (control/treated) individual
determinations. Internally consistent results were obtained when
pH
recoveries were measured fluorimetrically (Fig. 4, C and D) and when Na
uptake was determined radioisotopically (Fig. 3B). Similarly, a profound inhibition of
H
extrusion (Fig. 4, E and H) (
)and Na
influx (Fig. 3B)
was noted when NHE3
638 and NHE3
579 transfectants were
subjected to the ATP depletion protocol. In these figures the data are
means ± S.E. of 61/59 and 47/29 (control/treated) individual
determinations, respectively. Thus, unlike the effect of cAMP, the
inhibition induced by metabolic depletion remains essentially
unaffected when most of the cytosolic tail of the exchanger has been
deleted.
The expression of the chimeras was probed using
antibodies directed to the cytosolic domains of either NHE1 or NHE3
(see ``Experimental Procedures''). As shown earlier,
anti-NHE1 antibodies recognized a wide band of approximately 110 kDa in
NHE1-transfected AP-1 cells (Fig. 5A). The width of
this band has been attributed to carbohydrate
heterogeneity(38) . The specificity of the antibody is
indicated by its failure to react with NHE3 or with the NHE1/3 chimera.
However, positive reactivity was detected in the NHE3/1 chimera,
supporting the presence of the cytosolic domain of NHE1. Significantly,
the immunoreactive band was sharper and had a molecular mass of 80
kDa, similar to that of NHE3, which unlike NHE1 is apparently not
glycosylated(39) . This observation is consistent with the
notion that the transmembrane domain of the chimera is that of NHE3.
Figure 5:
Immunoblotting of the full-length NHE1 and
NHE3 and chimeric NHE1/3 and NHE3/1 exchangers in AP-1 cells. Membranes
(microsomal fraction) isolated from cells stably transfected with the
indicated NHE were subjected to immunoblot analysis and probed with
antibodies raised against NHE1 (NHE1, panel A) or NHE3
(
NHE3, panel B). Specific immunoreactive bands are
indicated by arrowheads. Lighter, nonspecific bands of
75
and 110 kDa were routinely observed in all lanes. Blots are
representative of three independent
experiments.
The immunoreactivity of the NHE3 antibody was weaker and therefore
required longer exposure times. This intensified two nonspecific bands
of 75 and 110 kDa, which were present in all samples but were more
apparent upon extended exposure. Nevertheless, a band of 80 kDa was
also discernible in NHE3 transfectants, as expected. In the NHE1/3
cells, an additional reactive band of 105-110 kDa was seen, but
not in the NHE3/1 transfectant. The higher molecular mass of the latter
band suggests glycosylation of the NHE1 transmembrane domain. These
findings are compatible with the predicted primary structure of the
chimeras.
Additional confirmation of the composition of the chimeric
constructs was obtained using amiloride and a substituted benzoyl
guanidine, compound HOE 694. These inhibitors are thought to act
externally on the N-terminal transmembrane region of the
protein(38) . More importantly, the isoforms are differentially
sensitive to these drugs. As illustrated in Fig. 6, NHE1
activity was almost entirely inhibited by 1 µM HOE 694, in
agreement with the inhibitory constant (K =
0.16 µM) reported in an earlier study(40) .
Similar inhibition was observed when NHE1 transfectants were exposed to
1 mM amiloride (Fig. 6). In contrast, a much higher
concentration of HOE 694 (500 µM) produced only a modest
inhibition of NHE3. The nearly complete inhibition obtained with 1
mM amiloride indicates that the HOE 694-resistant component of
H
transport is mediated by the exchanger (Fig. 6).
Figure 6:
Effects of amiloride and HOE 694 on the
activity of full-length and chimeric exchangers. AP-1 cells transfected
with full-length NHE1 or NHE3 (left column) or with the
chimeric NHE1/3 or NHE3/1 (right column) were loaded with
BCECF in bicarbonate-free medium. The cells were acid-loaded with
ammonium and the pH recovery induced by addition
of Na
was measured fluorimetrically in the presence (open symbols) or absence (solid circles) of
inhibitors. The inhibitors used were amiloride (1 mM throughout; open circles) or HOE 694 (triangles). The concentration of HOE 694 used was 1
µM for full-length NHE1 and the NHE1/3 chimera and 500
µM for NHE3 and NHE3/1. Results are the mean ± S.E.
of at least 20 cells from three separate
experiments.
The inhibitor sensitivity of the NHE1/3 chimera was virtually identical to that of NHE1, implying that the two exchanger constructs share a common transmembrane domain. Conversely, the NHE3/1 chimera was resistant to 1 µM HOE 694, resembling the behavior of NHE3. Together with the immunoblotting data of Fig. 5, these pharmacological findings confirm the composition of the chimeric constructs.
The sensitivity of the chimeras to
forskolin and to ATP depletion was tested next. NHE1/3 transfectants
displayed cooperativity toward H (Hill coefficient
2.3) and, in the range studied, their activity was half-maximal
near pH
6.3. Treatment of this chimera with forskolin
resulted in a moderate inhibition of transport and was particularly
noticeable at more acidic pH
(Fig. 7, A and B). In this figure the data are means ± S.E. of 24/20
(control/treated) individual determinations. The occurrence of a small
yet significant inhibition was confirmed when measuring
Na
uptake (Fig. 9A). In
contrast, the NHE3/1 chimera was not inhibited by cAMP. Only a small,
insignificant stimulation was noted at more alkaline pH
(Fig. 7, C and D). The data are means
± S.E. of 20/24 (control/treated) individual determinations.
Again, determinations of
Na
influx
yielded consistent results (Fig. 9A). These findings
confirm that the transmembrane region of NHE3 is not sufficient to
confer sensitivity to inhibition by cAMP. Moreover, the results of Fig. 7(A and B) imply that while the cytosolic
tail of NHE3 is necessary for the response, the participation of other
regions of the protein is likely required to fully mimic the inhibitory
effect observed in wild type NHE3.
Figure 7:
Effect of forskolin on the activity of
chimeric exchangers: pH determinations. AP-1 cells transfected with
NHE1/3 (A and B) or NHE3/1 (C and D) were loaded with BCECF in bicarbonate-free medium. The
cells were acid-loaded with ammonium and treated with (open
symbols) or without (solid symbols) 10 µM forskolin during the final 10 min of incubation with the dye.
Next, the pH recovery induced by addition of
Na
was measured fluorimetrically. Results in each
panel are the mean ± S.E. of at least 20 cells from three
separate experiments. Other details are as described for Fig. 2.
Figure 9:
Influence of forskolin and ATP-depletion
on the activities of wild type and chimeric
Na/H
exchangers: Na
uptake determinations. AP-1 cell transfectants stably expressing
wild type rat Na
/H
exchanger isoforms
NHE1 and NHE3 and chimeric exchangers NHE1/3 and NHE3/1 were grown to
confluence on 24-well plates. A, the effect of 10 µM forskolin on the transport activities of the exchanger was studied
exactly as described in the legend to Fig. 3. Data are expressed
as the mean percentage of amiloride-inhibitable
Na
influx ± S.E. (n = 16) from four independent experiments. B, the
influence of cellular ATP depletion on the transport activities of the
exchangers was performed as described in the legend to Fig. 3.
Data are expressed as the mean percentage of amiloride-inhibitable
Na
influx ± S.E. (n = 8-12) from 2-3 independent experiments.
Significance of differences between control and experimental
measurements was calculated using two-tailed Student's t test and is indicated by an asterisk (p <
0.001).
The ATP dependence of transport
by the chimeras was also tested. Both the NHE3/1 and the NHE1/3
constructs were severely inhibited when the cells were depleted
metabolically. The data are means ± S.E. of 34/42 and 22/20
(control/treated) individual determinations, respectively. The
inhibition was apparent when both H extrusion (Fig. 8) and Na
influx was measured (Fig. 9B). For both chimeras, inhibition appeared to be
mediated by a leftward displacement of the pH
dependence of
the rate of transport, as found for the full-length parental
exchangers.
Figure 8:
Effect of ATP depletion on the activity of
chimeric exchangers: pH determinations. AP-1 cells transfected with
NHE1/3 (A and B) or NHE3/1 (C and D) were loaded with BCECF in bicarbonate-free medium. The
cells were acid-loaded with ammonium and treated with (open
symbols) or without (solid symbols) a glucose-free medium
containing 5 mM deoxyglucose plus 1 µg/ml antimycin in
order to deplete ATP during the final 10 min of incubation with the
dye. Next, the pH recovery induced by addition of
Na
was measured fluorimetrically. Results in each
panel are the mean ± S.E. of at least 20 cells from three
separate experiments. Other details are as described for Fig. 4.
Sodium and bicarbonate reabsorption in renal and intestinal
epithelial cells is mediated mainly by Na/H
exchange across their apical membranes. The isoform of the
exchanger responsible for this exchange is believed to be NHE3. This
isoform has a higher affinity for Na
and is
substantially less sensitive to amiloride derivatives and HOE 694 than
the housekeeping NHE1(12) . The differential sensitivity to
these inhibitors was confirmed in the heterologous expression model
illustrated in Fig. 6. Moreover, complementary chimeras
constructed with NHE3 and NHE1 indicated that the transmembrane domain
suffices to dictate the degree of sensitivity to these inhibitors. This
conclusion is in good agreement with earlier findings that identified
unique residues within the transmembrane domain as important
determinants of the affinity for amiloride and its
analogs(17, 41) .
Inhibition of exchanger activity persisted in the NHE1/3
chimera, which contains the C-terminal cytoplasmic domain of NHE3, but
was not observed in the reciprocal chimera, NHE3/1. These findings
imply that the cytoplasmic domain of NHE3 is required for cAMP to exert
its inhibitory action. This conclusion was confirmed by the deletion
studies. Truncation of NHE3 at position 579 similarly prevented
modulation by forskolin ( Fig. 2and Fig. 3). However,
only a fraction of the cytoplasmic tail is essential for the cAMP
effect, since the inhibition was intact following truncation of NHE3 at
position 684. Loss of responsiveness occurred in two stages; partial
effects were seen in NHE3638, while complete unresponsiveness was
apparent in NHE3
579. This behavior is suggestive of multiple
target sites for cAMP, resembling the reported behavior of
NHE,
the trout red cell isoform of the exchanger that is also responsive to
cAMP. However, unlike NHE3,
NHE is stimulated by cAMP and
has two distinct sites that are substrates for phosphorylation by
PKA(43) . Elimination of these sites greatly reduces but does
not abolish the stimulatory effect of cAMP(44) . It is
noteworthy that
NHE has highest homology to the mammalian NHE1
isoform which is also stimulated by cAMP in certain cell
types(26) .
NHE3 differs from NHE in that loss of
responsiveness to forskolin occurs upon deletion of residues
579-684, a region that does not contain the optimal consensus
sequence (R-R/K-X-S*/T*) for PKA phosphorylation(45) .
However, phosphorylation of this region by PKA may occur at variant
sites (R-X
-S*/T* or R-X-S*/T*) such as
RLES
or RRRS
IR, which more closely resemble
a classical protein kinase C consensus sequence
((R/K
-X
)-S*/T*-(X
-R/K
) (45) . In this regard, it is noteworthy that Levine et al.(46) recently demonstrated that the same region of NHE3
mediates inhibition of the rabbit homolog by protein kinase C, which
kinetically resembles the effect of PKA. Alternatively, it is
conceivable that the structure or disposition of the 579-684
domain changes upon phosphorylation of potential upstream targets (e.g. RRS
, RRGS
, and
RPS
), thereby contributing to the transduction of the
inhibitory signal. It is also possible that NHE3 may not be directly
phosphorylated by PKA, in which case ancillary cAMP-sensitive
regulatory protein(s) may be the target of the kinase. This notion is
supported by the recent discovery of a soluble cytoplasmic protein
required for inhibition of Na
/H
exchange by cAMP in renal brush border
vesicles(47, 48) . More detailed studies to test these
hypotheses are currently ongoing in order to precisely delineate the
molecular mechanism responsible for cAMP-dependent regulation of NHE3.
Structure-function analysis
of the ATP dependence was also performed by examining progressive
deletions of the C-terminal tail of NHE3. In contrast to the forskolin
response, the inhibitory effect of ATP depletion was preserved even in
the most profound truncation studied, NHE3579. It is concluded
from these findings that the site(s) responsible for the ATP
requirement are situated in the transmembrane domain or in a portion of
the tail near its emergence from the membrane. It is noteworthy that
ATP dependence persisted in NHE3
579 despite deletion of most
consensus phosphorylation sites. For NHE1, it has been suggested that
regulation by ATP is independent of changes in the phosphorylation of
the exchanger itself(32) . Hence, a similar mechanism may
mediate the metabolic dependence of NHE3. No conventional
nucleotide-binding sites are identifiable from the sequence of either
NHE1 or NHE3, suggesting that perhaps associated proteins are required
to confer ATP sensitivity to the exchangers. The cytoskeleton, which is
drastically rearranged upon depletion of ATP, may contribute to this
effect.
In summary, a similar kinetic profile is observed upon
inhibition of NHE3 by cAMP and by depletion of cellular ATP. Both
procedures induce a shift in the pH sensitivity of the
exchanger to more acidic values. Nevertheless, the mutational analysis
reported here implies that the mode of regulation by ATP and cAMP is
unlikely to be identical. The latter required the presence of most of
the cytosolic tail, while the ATP dependence persisted after more
severe deletions. This points to the existence of distinct domains of
NHE3 that are responsible for unique regulatory functions. More precise
definition of the specific inhibitory mechanisms may require detailed
information of the putative accessory proteins and their sites of
interaction with NHE3.