From the
NHE3, a cloned intestinal and renal brush border
Na
A family of Na
The isoforms NHE1, 2,
and 3 have 40-60% overall amino acid identity, with the membrane
spanning domains, which transport Na
It seemed likely that
NHE3 might be regulated in a manner different from NHE1 as the
kinetics, second messenger regulation, and presumptive physiological
function of NHE3 are different from those of NHE1. NHE3 is activated by
serum and FGF via an increase in V
We previously cloned the NHE3
cDNA into the NheI and SaII sites of pMAMneo to
create the NHE3/pMAMneo construct, which was stably transfected into
PS120 cells
(1) . To create NHE3 C terminus truncation mutants,
the NHE3/pMAMneo construct was linearized with SalI, followed
by a ``fill-in'' reaction with
The
rate of Na
The computer program used to analyze the data
establishes a line which best fits the data based on the kinetic model.
Estimates for K` and V
A series of PS120/NHE3 truncation mutants with portions of
the C-terminal cytoplasmic domain removed was generated; these mutants
and the number of putative consensus sites for protein kinase C in each
truncation are shown diagrammatically in Fig. 1.
To determine whether
the effects of W13 and KN-62 were additive, the effects of a maximum
concentration of W13 (45 µM), were determined in cells
pretreated for 10 min with a concentration of KN-62 which maximally
stimulated NHE3 (50 µM). In these studies, cells exposed
to KN-62 for 10 min had initial Na
Truncation mutant E3/756 was
studied to determine if amino acids 757-832 were required for the
W13 and KN-62 stimulation of NHE3. Pretreatment with W13 (50
µM, 10 min) (Fig. 2B) and KN-62 (50
µM, 10 min) (Fig. 2D) failed to cause
significant stimulation of E3/756. The presence of an inhibitory domain
between amino acids 756 and the C terminus is indicated by the much
faster Na
All the truncated NHE3 isoforms studied were
stimulated by 10% FBS, with an increase in
Na
In this study, we show that the C-terminal domain of the
cloned brush border Na
Phosphorylation of the exchanger or of intermediate
factors appears to play a role in establishing basal
Na
CaM inhibits NHE3 under basal conditions, 1) acting by both CaM
kinase-dependent and -independent mechanisms and 2) with both effects
requiring amino acids 757-832 in the C terminus of NHE3. Both W13
and KN-62 caused concentration-dependent stimulation of NHE3, while
W12, the hydrophobic control for W13 did not affect
Na
Calmodulin
acting by kinase-independent mechanisms in regulation of transport
proteins is well characterized, including regulation of
Ca-ATPase
(25, 26) and the recent demonstration of
Ca
Not known is how CaM affects NHE3. Amino acids
757-832 (the very C terminus of NHE3) of NHE3 are required for
both the CaM kinase-dependent and -independent effects, but this area
of NHE3 does not contain a CaM kinase consensus sequence. This suggests
either involvement of at least one accessory protein or that CaM binds
to this area of NHE3 and activates a CaM-dependent kinase which then
phosphorylates a CaM kinase phosphorylation consensus sequence either
in another part of the C terminus of NHE3 or in a regulatory protein.
Of note, while NHE3 is a phosphoprotein under basal
conditions,
Direct
binding of CaM to NHE3, especially between amino acids 757-832
has not been demonstrated. However, by visual inspection, there is a
region between amino acid 777 and 791 of NHE3 which has some
characteristics in common with several documented calmodulin-binding
sites in several other CaM-binding proteins
(30, 31) .
This region of NHE3 contains hydrophobic residues and also several Arg
(charged) residues but is atypical in that it also contains a Glu and
several Pro residues
(2) . Demonstration of direct CaM binding to
NHE3 and determination of CaM effects on NHE3 phosphorylation are
required to further understand the mechanism of CaM regulation of NHE3.
These effects of CaM demonstrated here with NHE3 in PS120
fibroblasts may explain the previously reported finding that under
basal conditions CaM inhibits ileal NaCl absorption
(32) and
that in brush border membrane vesicles made from rabbit ileum,
W13
(19) , and a specific Ca
Phosphorylation also appears involved in growth factor/kinase
regulation of NHE3 since NHE3 is stimulated by FBS and FGF and
inhibited by phorbol esters. It is likely that the kinase consensus
sites identified in the different regions of the cytoplasmic tail are
important for regulation of the exchanger; however, it is unclear which
are involved in mediating the effects of the agonists. The effect of
PKC is mediated by a region in which there are five putative protein
kinase C consensus sequences, and future studies will be necessary to
determine if one or more of these sites are phosphorylated with PMA
addition.
Within the stimulatory domain of NHE3, there are several
distinct regions which activate the exchanger with an increase in
V
Both NHE1 and NHE3 are activated by FGF, OA, and
FBS
(4) ; however, we reported previously that there are
differences in the mechanism of activation of the two isoforms as
reflected by changes in different kinetic parameters. NHE1 is activated
via an increase in affinity for H
This model of a
single regulatory region for NHE1 has not been confirmed in all
reports. A study by Winkel et al.(33) using
microinjection of an antibody directed at the 658-815 region of
NHE1 found that this antibody altered regulation by endothelin-1 and
In
addition to insights concerning NHE3 regulation, these truncation
studies also give insight concerning the location of the allosteric
site involved in Na
We propose a structure-function model for NHE3 which is shown in
Fig. 7
. In this model there are distinct stimulatory and
inhibitory regions within the cytoplasmic tail of the exchanger, and
there are separate sites for regulation by the different agonists and
inhibitors studied here. The nature of the interaction of the
cytoplasmic tail of NHE3 with the membrane spanning region is unknown.
However, since K`(H
n, represents number of separate coverslips studied for
control and inhibitor.
/H
exchanger, has previously been
shown to be both stimulated and inhibited by different protein
kinases/growth factors. For instance, NHE3 is stimulated by serum and
fibroblast growth factor (FGF) and inhibited by protein kinase C. In
the present study, we used a series of NHE3 C terminus truncation
mutants to identify separate regions of the C-terminal cytoplasmic tail
responsible for stimulation and inhibition by protein kinases/growth
factors. Five NHE3 C terminus truncation mutant stable cell lines were
generated by stably transfecting NHE3 deletion cDNAs into PS120
fibroblasts, which lack any endogenous Na
/H
exchanger. Using fluorometric techniques, the effects of the
calcium/calmodulin (CaM) inhibitor W13, calcium/CaM kinase inhibitor
KN-62, phorbol myristate acetate, okadaic acid, FGF, and fetal bovine
serum on Na
/H
exchange were studied
in these transfected cells. Inhibition of basal activity of full-length
NHE3 is mediated by CaM at a site C-terminal to amino acid 756; this
CaM effect occurs through both kinase dependent and independent
mechanisms. There is another independent inhibitory domain for protein
kinase C between amino acids 585 and 689. In addition, there are at
least three stimulatory regions in the C-terminal domain of NHE3,
corresponding to amino acids 509-543 for okadaic acid,
475-509 for FGF, and a region N-terminal to amino acid 475 for
fetal bovine serum. We conclude that separate regions of the C terminus
of NHE3 are involved with stimulation or inhibition of
Na
/H
exchange activity, with both
stimulatory and inhibitory domains having several discrete subdomains.
A conservative model to explain the way these multiple domains in the C
terminus of NHE3 regulate Na
/H
exchange is via an effect on associated regulatory proteins.
/H
exchangers
(NHE)
(
)
has been cloned from multiple mammalian
species, and the structural and functional properties of three isoforms
(NHE1, NHE2, and NHE3) have been characterized in detail (1-7).
The Na
/H
exchangers cloned to date
have certain common structural features, including an N-terminal
transmembrane domain with a hydrophobicity profile which predicts
10-12 transmembrane segments, and a long C-terminal cytoplasmic
domain, that contains multiple potential consensus sites for protein
phosphorylation by a variety of protein kinases. Although the NHE
isoforms have similarities in amino acid sequence and in structure as
deduced from hydropathy plots, as well as similarities in some
transport properties such as sodium kinetics and the allosteric nature
of proton transport, they differ in second messenger regulation,
amiloride sensitivity, tissue distribution, and presumably in
physiological function. The first isoform cloned, designated NHE1, is
activated by growth factors, phorbol esters, and cell shrinkage and is
highly sensitive to inhibition by amiloride
(8, 9) . NHE1
is present in almost all cell types, is located on the basolateral
membrane in polarized cells, and is thought to function in maintenance
of cell pH and cell volume, and perhaps in cell division. NHE2 is also
activated by growth factors and phorbol esters, although the kinetics
of this activation are different than for NHE1
(3, 6) .
NHE2 message is found in rabbit kidney, intestine, and adrenal gland,
and this isoform is less sensitive to inhibition by 5-amino substituted
forms of amiloride compared with NHE1
(3) . The NHE3 isoform is
activated by growth factors similarly to NHE2; however, it is inhibited
by phorbol esters
(2, 4, 5) . In addition, this
amiloride-resistant exchanger has been found primarily on the apical
membrane of intestinal and kidney proximal tubule epithelial cells and
is thought to carry out transcellular sodium
absorption
(2, 5, 10) .
and
H
, being most highly conserved and the C-terminal
cytoplasmic domains, which regulate rate of transport, having very
little homology among the exchanger isoforms, especially concerning the
many putative protein kinase consensus sequences found there.
Understanding of how the C-terminal cytoplasmic domain regulates the
rate of Na
/H
exchange is best
understood for NHE1, in which all regulation is by an increase in
affinity for intracellular [H
]. It has been
proposed that there are phosphorylation sites within the most
C-terminal domain of NHE1 (amino acids 635-815), which are
necessary for full activation by a variety of agonists, and a single
region within the cytoplasmic tail (amino acids 567-635) which is
required for the effect of all agonists, including those acting via
different signal transduction pathways, activation via cell shrinkage,
and growth factor regulation in the absence of changes in
phosphorylation of NHE1
(9, 11) . The model described for
NHE1 suggests that effects on Na
and H
transport are the consequences of this single regulatory region
within the cytoplasmic tail causing an increase in proton affinity of
the allosteric site in the N terminus, with a resultant decrease in
K
(H
) and activation of
transport
(11, 12, 13) .
, with no
change in intracellular proton affinity, while phorbol esters cause a
marked inhibition of exchanger activity, also with no change in proton
affinity
(4) . We hypothesized that it was likely that for NHE3
there was more than one region of the C-terminal cytoplasmic domain
involved in the activation and inhibition by second messengers. To
explore the relationship between the structure (amino acid sequence)
and the regulation of the NHE3 isoform, we studied the second messenger
regulation of mutant forms of the NHE3 protein which were truncated at
various sites along the cytoplasmic tail.
Cell Culture
Transfected PS120 fibroblasts were
grown in Dulbecco's modified Eagle's medium supplemented
with 25 mM NaHCO, 10 mM HEPES, 50 IU/ml
penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum in a
5% CO
, 95% air incubator at 37 °C. Geneticin (400
µg/ml) was used to maintain selection pressure and was added
immediately after each subculturing. In addition, cells were exposed to
an acid load consisting of 50 mM NH
Cl/saline
solution for 1 h, followed by 1 h in an isotonic 3 mM
Na
solution. Surviving cells were then placed in
normal culture medium and allowed to reach 30-50% confluence. The
acidification process was initially repeated every 2-3 days until
>50% of cells survived and was then repeated every other week to
maintain high Na
/H
exchange activity.
Construction and Expression of NHE3 Truncation
Mutants
The amino acid sequence of the C-terminal cytoplasmic
tail of NHE3 was mapped for putative protein kinase consensus sites
using a computer program, PC/GENE (PROSITE)
(14) . Identified
putative protein kinase consensus sequences included: 10 protein kinase
C, four calmodulin kinase II, four cAMP-dependent protein kinase, and
two tyrosine kinase sites. This formed the strategy for studying which
region(s) of the C terminus of the exchanger was involved in regulation
by protein kinases and growth factors.
-phosphorothioate
nucleotides and Klenow, and then digested with EcoRV. This
EcoRV restriction site was introduced between the 3`-end of
the NHE3 cDNA and the SalI restriction site during the
construction of the NHE3/pMAMneo construct. The NHE3 cDNA was then
digested from its 3`-end by exonuclease III/mung bean nuclease,
followed by self-ligation and transformation into Escherichia coli to generate NHE3 cDNA deletion clones. These clones were then
sequenced by the dideoxy method of Sanger to determine the extent of
deletion from the 3`-end and to be sure that they were in frame with
the TGA sequence (stop codon) 15 base pairs downstream of the
SalI site. Four constructs of NHE3/pMAMneo were selected (the
notation used was E3/X/pMAMneo, where X is the amino
acid number at the truncation site): E3/509/pMAMneo, E3/585/pMAMneo,
E3/686/pMAMneo, and E/756/pMAMneo. The construct E3/475/pMAMneo was
obtained by cloning into the NheI and XhoI sites of
pMAMneo the NHE3 cDNA fragment (nucleotides 24-1422) which was
obtained by polymerase chain reaction using NHE3/pBluescript as the
template and the primers (5`-primer: GCCGCTCTAGAACTAGTGG (flanking
Bluescript); 3`-primer: GCAGCTTCTCGAGCAGCTTGGGCTCCC (flanking NHE3)).
These five NHE3 deletion constructs were then stably transfected into
PS120 fibroblasts to generate corresponding NHE3 truncation mutants.
Measurement of Na
For fluorometry experiments, cells were
seeded on glass coverslips, grown overnight in serum-free medium, and
studied when they reached 50-70% confluence. The cells were
loaded with the acetoxymethyl ester of
2`,7`-bis(carboxymethyl)5-6-carboxyl-fluorescein (BCECF-AM, 5
µM) in ``Na/H
Exchange Activity
medium''
(containing 130 mM NaCl, 5 mM KCl, 2 mM
CaCl
, 1 mM MgSO
, 1 mM
NaH
PO
, 25 mM glucose, 20 mM
HEPES, pH 7.4) for 60-90 min at 23 °C, then washed with
``TMA
medium'' (containing 130 mM
tetramethylammonium-Cl, 5 mM KCl, 2 mM
CaCl
, 1 mM MgSO
,
NaH
PO
, 25 mM glucose, 20 mM
HEPES, pH 7.4) to remove the extracellular dye, and the coverslip was
mounted at an angle of 60° in a 100-µl fluorometer cuvette
designed for perfusion as described
(15) and thermostatted at 37
°C. The cells were pulsed with 40 mM NH
Cl in
TMA-Cl for 15-20 min; removal of NH
Cl and perfusion
with TMA
medium resulted in acidification of the
cells. In the okadaic acid experiments, cells were incubated with
okadaic acid (OA) (1 µM) in TMA
medium
for 15 min at 37 °C before addition of Na
medium
containing OA, and measurement of pH
recovery. In
other separate experiments,
N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13),
N-(4-aminobutyl)-naphthalene-sulfonamide (W12),
1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine
(KN-62), and phorbol myristate acetate (PMA, 1 µM) were
added 10, 10, 10, and 5 min, respectively, before Na
was introduced, and fibroblast growth factor (FGF 10 ng/ml) and
dialyzed fetal bovine serum (FBS, 10%) were added simultaneously with
the Na
medium. Concentrations of agonists were chosen
as those which had been shown in earlier studies to elicit maximal or
near maximal responses. Amiloride-sensitive
Na
-dependent pH recovery was measured in the presence
and absence of the various agonists. Fluorescence measurements,
determination of intracellular buffering capacity, and calibration of
experiments were performed as previously reported
(16) .
-dependent alkalinization was obtained by
calculating the first order derivative of the
Na
-dependent pH recovery curve. Data points were
recorded from every 3- to 12-s interval during the rapid phase of pH
recovery, with longer (30 s) intervals between data points as the rate
of alkalinization slowed. To minimize variability, a similar number of
data points were collected in control and test cells in each
experiment. Hydrogen ion efflux rates (µM
H
/s), equivalent to rate of
Na
/H
exchange, were then determined
by multiplying the rate of change in intracellular pH by the cellular
buffering capacity at the corresponding pH values. Scatter plots of
H
efflux rate versus intracellular
[H
] were then constructed. Control cells (no
agonist) were studied at the same time in parallel with treated cells
to control for variability in basal exchange rate among cells from
different cell passages and acid selection. All quantitative
comparisons presented are based on analysis of equal number of
control/test cells studied at the same time.
Data Analysis
Kinetic analyses did not include
data from cells with intracellular pH greater than 7.1, as there is an
endogenous acidification process in PS120 cells only when the
intracellular pH is over 7.1, a phenomenon previously described by Tse
et al. (5). At pH values <7.1, the pH
recovery was entirely Na
dependent and
amiloride-sensitive. Na
/H
exchange
rate data were analyzed using a nonlinear regression data analysis
program (ENZFITTER, Biosoft Corp.) (17) which allowed fitting of data
to a general allosteric model described by the Hill equation (v = V
*
[S]
/K`+[S]
)
with estimates for V
and K`, as well as
fitting to a hyperbolic curve, such as expected with Michaelis-Menten
kinetics. Standard errors generated were used as an indication of the
accuracy of the parameter estimates, as suggested in the program
manual. In this analysis, K` estimates were used to compare
the relative affinities of the exchangers for intracellular
[H
]. Apparent Hill coefficients
(n
) were calculated using data from the velocity
range around 1/2 V
, as described by
Segel
(18) .
with standard
errors represent the 95% confidence interval for the fitted line. Lack
of overlap of the 95% confidence intervals of the lines indicates
significant differences exist between groups being compared (treatment
and control). This can be assessed visually from the figures where
there is minimal or no overlap of data points at V
and from the kinetic parameter estimates (± S.E.)
reported.
Materials
PS120 fibroblasts used for transfection
were originally provided by J. Pouyssegur. Fetal bovine serum was
obtained from Hyclone Corp (Logan UT) and was dialyzed to remove
constitutents M < 60,000; Dulbecco's
modified Eagle's medium was from Life Technologies, Inc., and TMA
was from Fluka Chemical Corp (Ronkonkoma, NY). BCECF-AM and nigericin
were from Molecular Probes (Eugene, OR), okadaic acid (sodium salt) was
from LC Laboratories (Woburn, MA), FGF (basic) was from Boehringer
Mannheim, W13, W12, and KN-04 from Seikagaku America, Inc. (Rockville,
MD), and KN-62 was from Calbiochem (La Jolla, CA), while other reagents
were purchased from Sigma.
Figure 1:
Wild type and truncation mutant forms
of NHE3. The membrane spanning portion of the NHE3 protein is shown as
a hatched bar, while the portion of the C-terminal cytoplasmic
portion remaining is shown as an open bar, with the amino acid
position at the truncation site indicated for each mutant. The number
of putative protein kinase C sites predicted to be present in the
cytoplasmic tail of each truncation mutant is
shown.
Second Messenger Regulation of Truncation Mutants: Inhibitory
Domain
Second messenger regulation of the truncation mutants
stably expressed in PS120 cells was investigated, and from these
studies functionally distinct inhibitory and stimulatory regions of the
C-terminal cytoplasmic domain of NHE3 were identified.
Effect of W13, W12, and KN-62
Previous studies
have demonstrated regulation of ileal brush border
Na/H
exchange under basal conditions
by calmodulin (CaM)
(19, 20) . Furthermore, a putative
CaM-binding site was identified by protein sequence analysis at the
distal C-terminal end of NHE3 (see ``Discussion''). Thus the
effects of inhibitors of CaM were studied on NHE3. Pretreatment of
PS120/NHE3 cells with the CaM inhibitor W13 (50 µM, 10
min) caused a 71% increase in Na
/H
exchange activity compared with untreated controls
(V
918 ± 81 µM/s versus 537 ± 55 µM/s for W13-treated and control
cells, respectively), with no change in K` (0.14 ± 0.06
µMversus 0.15 ± 0.07 µM for
W13 and control cells, respectively) or n
(2.0
and 1.8 for W13 and control cells, respectively)
(Fig. 2A). In a separate series of experiments
(), the W13 effect was shown to be concentration dependent:
10 µM W13 did not affect NHE3 (V
1048 ± 69 µM/s versus 1023 ±
46 µM/s) for W13-treated and control cells, respectively;
25 µM W13 increased the V
by 76%
(2156 ± 164 µM/s) compared to untreated controls
(1225 ± 51 µM/s). The magnitude of the stimulation
of V
was similar when 45 µM W13 was
studied (75% increase) (2395 ± 531 µM/s versus 1369 ± 143 µM/s for W13-treated and control
cells, respectively). Preincubation of the cells for 10 min with the
W13 hydrophobic control, W12 (45 µM), did not affect NHE3
activity (1771 ± 229 µM/s versus 1704
± 151 µM/s for W12-treated and control cells,
respectively) ().
Figure 2:
Effects of calmodulin inhibitors W13 and
KN-62 on NHE3. A, the calmodulin antagonist W13 stimulates
Na/H
exchange in PS120/NHE3 cells.
Cells were acidified by incubation with NH
Cl, followed by
perfusion with either TMA
solution for control cells
(
), or with TMA
solution containing 50
µM W13 (
) for 10 min. Cells were then allowed to
recover to steady state pH in Na
medium. In this
figure, H
efflux rates, equivalent to
Na
/H
exchange, are plotted against
intracellular [H
].
Na
/H
efflux rates were calculated at
various pH, and lines were fit to the data using an allosteric model,
and kinetic parameters (V
,
K`(H
), and n
) were
estimated. W13-treated cells had a 71% higher V
(918 ± 81 µM/s) compared with untreated
control cells (V
537 ± 55
µM/s), with no change in K` or
n
. These data were obtained from 15 similar
experiments. B, effects of W13 (50 µM) were
determined in E3/756 cells studied under the same conditions described
in Fig. 2A. W13 had a minimal effect on E3/756 compared with
its effect on wild type NHE3. Results are from six similar experiments.
C, effects of KN-62 (50 µM) under same conditions
used to study W13 in Fig. 2A on PS120/NHE3. KN-62-treated
cells had a 51% higher V
(2135 ± 138
µM/s versus 1418 ± 114 µM/s
in KN-62-treated and control, respectively) with no change in
K` or n
. These data were obtained from
four similar experiments. D, effects of KN-62 (50
µM) under same conditions used to study W13 in Fig. 2A on E3/756. KN-62 did not affect E3/756. Results are from four
similar experiments.
In order to investigate whether the
Ca/CaM-induced inhibition of NHE3 was mediated
through Ca
-CaM-dependent protein kinase II, we
studied the effect of the Ca
-CaM-dependent protein
kinase II inhibitor KN-62 (). Pretreatment of the cells
with KN-62 (5 µM, 10 min) stimulated the NHE3
V
by 36% (1771 ± 230 µM/s)
versus control (1301 ± 104 µM/s). Similar
preincubation with 25 µM KN-62 stimulated
V
by 51% (2135 ± 138 µM/s)
compared to controls (1418 ± 114 µM/s) (no change
occurred in K` or n
K`: 0.10
± 0.03 µMversus 0.16 ± 0.05
µM in KN-62-treated and control cells, respectively;
n
: 2.1 ± 0.2 versus 2.1 ±
0.2 in KN-62-treated and control cells, respectively)
(Fig. 2C), and the magnitude of stimulation of
V
was not further affected by increasing the
concentration of KN-62 to 50 µM (2013 ± 42
µM/s versus 1369 ± 143 µM/s
for KN-62 and untreated control, respectively; 47% increase).
Pretreatment of the PS120/NHE3 cells with KN-04 (50 µM, 10
min), the negative control substance for KN-62 did not affect the
kinetic parameters of NHE3 (data not shown).
/H
exchange rates determined with or without W13 (45
µM). W13 caused an increase in
Na
/H
exchange rate of 26% in KN-62
exposed cells (2521 ± 316 µM/s versus 2006
± 380 µM/s in W13 plus KN-62 treated cells
versus KN-62 treated alone; studied at the same time,
Na
/H
exchange rate in W13-treated and
control cells was 2553 ± 127 and 1403 ± 30
µM/s, respectively).
/H
exchange rate for E3/756,
although the amount of NHE3 protein has not been considered.
Effect of PMA
A second inhibitory site within the
C-terminal cytoplasmic domain of NHE3 was identified in experiments
involving pretreatment of the cells with phorbol ester to activate
protein kinase C. As we have previously reported
(4) ,
full-length NHE3 was inhibited by 5 min of preincubation with PMA (1
µM) with a 64% decrease in V (890
± 81 µM/s for control and 318 ± 42
µM/s for PMA-treated cells) but no change in K`
(0.19 ± 0.07 µM for control and 0.16 ± 0.12
µM for PMA-treated cells) or n
(2.0
for control and 1.9 for PMA) (Fig. 3A). E3/756, with two
putative protein kinase C sites removed and eight remaining, was
similarly inhibited by PMA (data not shown), as were cells transfected
with truncation 689, with four protein kinase C sites removed (six
remaining) (Fig. 3B). The inhibition of E3/689 by PMA
was reflected by a 39% decrease in V
from 1525
± 96 µM/s for untreated to 924 ± 56
µM/s for PMA-treated cells, with no change in K`
(0.09 ± 0.04 µM for untreated and 0.07 ±
0.03 µM for PMA treated cells) or in the apparent Hill
coefficient (1.8 untreated, 1.9 PMA treated). In contrast, E3/585 cells
showed no inhibition (or stimulation) of
Na
/H
exchange by PMA, with
V
estimates of 1233 ± 94
µM/s for control and 1293 ± 73 µM/s
for PMA-treated cells (Fig. 3C) and no change in
K` (0.14 ± 0.06 and 0.18 ± 0.06 µM
for control and PMA-treated cells, respectively) or in
n
(1.7 for both groups). These results indicate
that the regulatory site(s) for PMA were located between amino acid 585
and amino acid 689. There are five putative PKC consensus sites within
this region.
Figure 3:
Inhibition of NHE3 by phorbol ester occurs
at sites between amino acids 585 and 689. A, phorbol ester
inhibits Na/H
exchange in PS120/E3
cells. Cells were acidified with NH
Cl followed by
TMA
medium, then perfused with either Na
medium (
), or with Na
medium containing
phorbol myristate acetate (
-PMA, 1 µM, added 5 min
before perfusion with sodium medium) and their
Na
-dependent pH recovery in HCO
-free
medium measured. PMA-treated cells had a 64% lower V
(318 ± 42 µM/s) compared with untreated
control cells (V
890 ± 81
µM/s) with similar K`(H
) and
Hill coefficients. B, phorbol ester also inhibits
Na
/H
exchange in E3/689 cells. In a
similar experiment as described in A above, acidified cells
pretreated with PMA (PMA (
), 1 µM) showed a slower
rate of pH recovery than did untreated control cells (
).
PMA-treated cells had a 39% lower V
(924
± 56 µM/s) than did the controls (1525 ± 96
µM/s), while K`(H
) and
n
estimates were not different. C,
phorbol ester does not alter Na
/H
exchange in PS120 E3/585 cells. Control (
) and PMA-treated
cells (
) were acidified and their Na
-dependent
pH recovery measured. There was no difference in
Na
/H
exchange rates for untreated or
treated cells, with similar V
,
K`(H
), and n
estimates
for control and PMA-treated cells. These data are from at least 12
experiments with each cell line. D, the inhibitory effect of
PMA on PS120/NHE3 cells was independent of the effect of W13. Cells
were incubated with W13 (50 µM) for 10 min (
) as
described in Fig. 2, or with W13 for 10 min and PMA (1 µM)
for the last 5 min (
), then allowed to recover to steady state
pH in Na
medium. PMA following incubation with W13
caused a reduction in V
by 47% (from 1110
± 37 to 585 ± 48 µM/s) compared with cells
incubated with W13 alone, with no effect on K` or
n
. These data represent six separate experiments
with W13- and W13/PMA-treated cells.
When PS120/NHE3 cells were pretreated with 50
µM W13, the degree of inhibition by PMA was similar to
that seen in cells not pretreated with W13 (Fig. 3D),
with a decrease in V from 1110 ± 37 to
585 ± 48 µM/s in cells only treated with W13
compared to cells treated with W13 followed by 1 µM PMA,
respectively. This result suggests that regulation by PMA is
independent of the CaM effect.
Second Messenger Regulation of Truncation Mutants:
Stimulatory Domain
In addition to the inhibitory regions,
several distinct but clustered stimulatory regions of the C-terminal
cytoplasmic tail of NHE3 were identified through experiments using OA,
FGF, and FBS. Okadaic acid is a serine/threonine phosphatase 1 and 2a
inhibitor that was used to examine the role of basal phosphorylation in
the activity of the exchanger. Preincubation with OA (1
µM) for 15 min, stimulated
Na/H
exchanger activity in PS120/NHE3
cells, with a 26% increase in V
from 1550
± 172 µM/s for control to 1948 ± 62
µM/s for OA-treated cells (Fig. 4A), with
no change in K` (0.19 ± 0.08 and 0.16 ± 0.06
µM for control and OA-treated cells, respectively). In
E3/585 cells, in which the two identified inhibitory domains of NHE3
were removed, a much greater percent stimulation by OA was seen. There
was a 68% increase in V
from 557 ± 69
µM/s in untreated controls to 936 ± 104
µM/s in OA-treated cells (Fig. 4B), and no
change in K` (0.15 ± 0.06 and 0.21 ± 0.08
µM for control and OA-treated cells, respectively). In
contrast, there was minimal, if any, stimulation by OA in E3/509 cells
(Fig. 4C), with little difference in V
or K` estimates for control (V
87
± 13 µM/s, K` 0.24 ± 0.10
µM) and for OA-treated cells (V
98
± 16 µM/s, K` 0.18 ± 0.10
µM). This suggested that a response element(s) for okadaic
acid was located between amino acids 509 and 585.
Figure 4:
Stimulation of NHE3 by okadaic acid occurs
between amino acids 509 and 585. A, OA stimulates
Na/H
exchange in PS120/NHE3 cells.
Experiments were performed as described above, with cells treated with
OA (
, 15 min preincubation, 1 µM) compared to
untreated cells (
). OA stimulated the V
by
26% (1948 ± 62 versus 1550 ± 172
µM/s) without affecting the
K`(H
) or n
. At 3
µM, OA caused similar stimulation of PS120/NHE3 and also
did not affect the K(H
) or n
of
the exchanger (data not shown). B, OA stimulates
Na
/H
exchange in PS120 E3/585 cells.
Experiments were performed as described above, with cells treated with
OA ((
), 15 min preincubation, 1 µM) compared with
untreated cells (
). OA-treated cells had a 68% higher
Na
/H
exchange rate that did controls,
with V
936 ± 104 and 557 ± 69
µM/s, respectively, with no change in
K`(H
). C, OA does not significantly
stimulate Na
/H
exchange in PS120
E3/509 cells. In similar experiments as with E3/585 cells, E3/509 cells
showed no difference in Na
/H
exchange
or in V
estimates compared with control cells
(98 ± 16 and 87 ± 13 µM/s, respectively).
These results represent data from six separate experiments for each
cell line.
Fibroblast growth
factor caused a 51% stimulation of exchanger activity in PS120/NHE3
cells, with a V 404 ± 36
µM/s for FGF- (10 ng/ml) treated cells compared with
untreated control cells (V
267 ± 28
µM/s) with no change in K` (0.10 ± 0.07
and 0.09 ± 0.06 µM for FGF-treated and control
cells, respectively) or n
(FGF-treated 1.9
versus 2.0 for control) (Fig. 5A). E3/509 cells
(Fig. 5B) also responded to FGF with a 76% increase in
V
(153 ± 9 µM/s versus 87 ± 13 µM/s FGF-treated and control cells,
respectively), also without a change in K` (0.16 ± 0.03
µM for FGF-treated and 0.24 ± 0.10 µM
for control). In contrast, there was no longer a response to FGF in
E3/475 cells as shown in the representative fluorometer trace in
Fig. 5C. The basal rate of E3/475 was so low that it was
not possible to perform quantitative analysis of the initial rate, so
FGF was added during pH recovery for qualitative assessment of the
effect. There was no change in the rate of pH recovery after FGF was
added to E3/475 cells. This indicates that the regulatory site for FGF
is in the region between amino acids 475 and 509.
Figure 5:
Stimulation of NHE3 by fibroblast growth
factor occurs between amino acids 475 and 509. A. FGF
stimulates Na/H
exchange in PS120/E3
cells. Cells were acidified with NH
Cl followed by
TMA
solution and their rate of pH recovery measured in
the presence (
) or absence (
) of FGF (10 ng/ml).
FGF-treated cells showed a 51% increase in V
compared with control (404 ± 36 versus 267
± 28 µM/s for FGF-treated and control cells,
respectively) and no change in K`(H
) or
n
. B, FGF also stimulates E3/509 cells.
Cells treated with FGF (
) showed a 76% increase in
Na
/H
exchange rate compared with
untreated control cells (
) (153 ± 9 versus 87
± 13 µM/s for FGF and control cells, respectively),
also with no significant change in K`(H
).
C, FGF has no effect on E3/475 cells. This figure shows a
representative fluorometer trace with FGF (10 ng/ml) added to E3/475
cells during pH recovery following acidification, as the exchange rate
was too slow to allow kinetic analysis. There was no change in the rate
of pH recovery after addition of FGF. The kinetic plots for PS120/NHE3
and E3/509 represent at least six separate experiments, while the
E3/475 experiment was repeated five times.
In separate
studies, the interaction of two stimulatory domains was studied. In
PS120/NHE3 cells which were exposed to OA (1 µM) for 15
min, subsequent addition of FGF at the steady state caused a further
increase in exchanger activity as indicated by further alkalinization
(data not shown).
/H
exchange rate. E3/475 cells,
with only 19 amino acids remaining of the putative cytoplasmic tail,
showed an increase in pH recovery when 10% dialyzed FBS was added
during pH recovery. The stimulation by FBS was inhibited by 3
mM amiloride, indicating that the response was indeed due to
an increase in Na
/H
exchanger
activity (Fig. 6). This suggests that the regulatory site for FBS
may be located in the putative membrane spanning portion of the protein
or in the small portion of cytoplasmic tail remaining in E3/475.
Figure 6:
Fetal bovine serum stimulates the NHE3
exchanger truncated at amino acid 475. This representative fluorometer
trace shows the response of acidified E3/475 cells to FBS (10%,
dialyzed) added during perfusion with Na medium.
Amiloride (3 mM) was then added at the time indicated to
confirm that the response was amiloride sensitive
Na
-dependent alkalinization. These tracings are
representative of five similar experiments.
/H
exchanger
isoform NHE3 is involved in its regulation by protein kinases and
growth factors. This is similar to what has been found for the
housekeeping isoform NHE1. In contrast to NHE1, in which all regulation
is stimulatory, we have identified distinct stimulatory and inhibitory
regions in the C terminus of NHE3. Moreover, both these domains contain
multiple subdomains with separate sites within the stimulatory region
for the effects of OA, FGF, and FBS and within the inhibitory region
for the effects of CaM and PMA (Fig. 7).
Figure 7:
The stimulatory and inhibitory regions of
the NHE3 cytoplasmic tail are indicated in this model of the NHE3
Na/H
exchanger. The cytoplasmic tail
contains separate sites for stimulation by FGF and OA and for
inhibition by PMA and calmodulin, while the effect of FBS may be
mediated either directly through the most proximal part of the
cytoplasmic tail or the membrane spanning portion of the exchanger or
possibly through an intermediate or accessory factor which then
interacts with the exchanger. The mechanism by which the cytoplasmic
tail interacts with the membrane spanning portion to cause the changes
in Na
/H
exchange rate
(V
) is not known. This interaction may be due to
conformational changes within the NHE3 molecule, perhaps in response to
phosphorylation of amino acid residues in the cytoplasmic tail, or may
involve intermediates, shown here as postulated accessory proteins
R
and R
, which might mediate the stimulatory
and inhibitory effects of the second
messengers.
The stimulatory and
inhibitory regions regulate NHE activity separately, and the exchanger
can be both up- and down-regulated simultaneously. This is best shown
by the response of NHE3 to FBS which contains both growth factors to
stimulate the exchanger and protein kinase C activators to inhibit the
exchanger. We previously reported PS120/NHE3 cells pretreated with the
protein kinase C inhibitor H7 showed a greater stimulation by FBS
compared with cells treated only with FBS
(4) . This indicates
that while the overall effect of FBS on NHE3 is stimulatory, this
effect is modulated by a concurrent inhibition mediated by protein
kinase C.
/H
exchanger activity. Evidence for
this includes the effect of OA and KN-62 to increase exchanger
activity. Okadaic acid, a phosphatase 1 and 2A inhibitor would only be
expected to increase phosphorylation and
Na
/H
if there were basal kinase
activity. KN-62 is a CaM kinase inhibitor, originally described as
specifically inhibiting CaM kinase II, and subsequently as also
inhibiting CaM kinase III and V
(21, 22, 23) .
KN-62 would only be expected to alter basal
Na
/H
exchange if CaM kinase were
inhibiting basal Na
/H
exchange.
/H
exchange. The W13
concentration-dependent stimulation was in the concentration range
demonstrated for other CaM-dependent processes
(24) . That W13
stimulated NHE3 above that stimulated by a maximum concentration of
KN-62 demonstrates that W13 is causing an effect in addition to that of
KN-62; and thus we suggest that CaM acts by both CaM kinase-dependent
and -independent mechanisms. Both forms of CaM regulation occur at
basal [Ca
]
. This is
not surprising for the kinase-independent regulation, since elevating
Ca
acts by increasing the affinity for CaM, and CaM
effects at basal [Ca
]
are known to occur. However, CaM kinase II is normally activated
by elevated Ca
, and further studies are required to
understand whether and how CaM kinase(s) regulates NHE3.
/CaM inhibition of the olfactory
cyclic-nucleotide-gated cation channel, in which
Ca
/CaM inhibits cyclic-nucleotide activation by an
ATP-independent mechanism
(27, 28) . Of note, this
Ca
/CaM inhibition involved a decrease in response to
cAMP and not an effect on basal activity
(27) . In a recent
report, calmodulin and/or CaM kinase II was shown to inhibit
Na
/H
exchange in LLC-PK1 cells, a
piglet renal epithelial cell line, although in this study the
inhibition followed stimulation by calcitonin, and there was no effect
on basal activity
(29) . The inhibition of
Na
/H
exchange activity by calmodulin
reported here differs from the effect of calmodulin on the plasma
membrane Ca
-ATPase where calmodulin is thought to
interact with the CaM-binding domain to free the active site of the
pump and allow access to the substrates, thereby activating
transport
(26) . Thus our study demonstrates a newly recognized
function for CaM, that of causing basal inhibition of a transport
protein.
(
)
it is not known whether there are
changes in its phosphorylation as part of CaM regulation.
/CaM kinase II
inhibitory peptide
(20) both caused an increase in basal
Na
/H
exchange activity. In those
studies, CaM kinase II activity was implicated in the inhibition of
basal exchanger activity because the peptide inhibitor was designed to
inhibit kinase activity by binding to the CaM kinase II autoinhibitory
domain
(20) . An additional, direct role of calmodulin was not
tested. The fact that at least partially similar regulation of NHE3 by
CaM occurs in a fibroblast and in the highly specialized brush border
domain of an intestinal epithelial cell suggests that this aspect of
regulation is specific to the Na
/H
exchanger isoform rather than to the cell in which the
Na
/H
exchange occurs.
, although it is not known if these sites are
phosphorylated. The region between amino acids 509-585 appears to
be activated under basal conditions as seen from the effect of OA, and
the site between amino acids 475 and 509 appears to mediate the effect
of FGF. Although the latter region does not contain a
``classical'' tyrosine kinase consensus sequence, there is a
tyrosine as well as 2 serines present in the region. It is less clear
which region of the protein N-terminal to amino acid 475 mediates the
response to FBS, as there is only a single serine remaining within the
cytoplasmic tail, and no sites which fit a computer algorithm (PCGENE)
for a kinase consensus site motif. Serum may regulate NHE3 through an
intermediate regulatory protein, but it is also possible that the
N-terminal domain alone mediates activation by FBS. It is interesting
that the exchanger is activated by FBS via a different site than that
for FGF and suggests that either the different growth factors have
separate binding sites, or that there is another factor in serum which
is responsible for the activation. Further evidence that prediction of
the effect of various second messengers on NHE activity based entirely
on the putative phosphorylation sites is not possible comes from the
observation that although there are four cAMP-dependent kinase
consensus sequences within the cytoplasmic domain of NHE3, cAMP has no
effect on the activity of the exchanger when transfected into either
PS120 fibroblasts or Caco-2 intestinal epithelial cells (4 and data not
shown).
as a consequence of
treatment with the agonists, whereas NHE3 responds with an increase in
V
with no change in H
affinity.
Both NHE1 and NHE3 require ATP for regulation, which is consistent with
the exchangers or an intermediate being phosphorylated during
regulation, although ATP-dependent non-phosphorylation-dependent
control of transport processes are well recognized. Pouyssegur et
al.(11) have studied the regulation of NHE1 in detail
using a similar strategy to that presented here with deletion of
various portions of the C-terminal cytoplasmic domain. NHE1 is
stimulated by growth factors, thrombin, and phorbol esters. Although
these agonists act via different second messenger pathways (receptor
tyrosine kinase for EGF and a PKC-dependent pathway for thrombin and
phorbol esters), they all stimulate the same increases in
phosphorylation of NHE1 based on phosphopeptide maps. This implies that
the second messenger pathways converge on a common activating kinase,
which then phosphorylates the exchanger; this phosphorylation has been
shown to be only on the portion of NHE1 C-terminal to amino acid 635.
In addition, Wakabayashi et al.(11) identified a
critical region in NHE1 between amino acids 567-635, without
which no regulation of NHE1 occurs. In the absence of the portion of
NHE1 which is phosphorylated, this region is sufficient to mediate part
of the growth factor response and to preserve high pH
sensitivity, leading to speculation that there must be additional
regulatory factors which activate NHE1 through interaction with the
cytoplasmic domain N-terminal to amino acid 635.
-thrombin, but that PKC and osmotic induced activation, as well as
pH sensing, were mediated via a separate, more proximal region of the
cytoplasmic tail. In addition we found the NHE1/519, a truncated form
of NHE1 having only 19 amino acids at the end of the N-terminal portion
could be stimulated by serum.
(
)
These results
suggest that sites in addition to the crucial regulatory region
(567-635) are involved in hormonal regulation of NHE1.
/H
exchange.
Removal of all but
19 amino acids in the cytoplasmic tail
(truncation at amino acid 475) resulted in greatly reduced
Na
/H
exchange activity, as seen by
the extremely slow rate of recovery shown by E3/475 cells in
Fig. 5C. It is not known if this reflects less plasma
membrane location of the exchanger or less function of a normal amount
of E3/475 in the plasma membrane. Although the basal rate of this
truncation mutant was too slow for kinetic analysis, when the E3/475
was activated by FBS, the kinetic plot of
Na
/H
exchange had a sigmoidal shape
(data not shown) similar to that of the full-length NHE3
(Fig. 2A, 3A, and 4A). This suggested
that the allosteric site(s) for H
binding was
preserved, and therefore most likely resides within the membrane
spanning region. This is further supported by the finding that the
other truncation mutants showed the same degree of allosteric
interaction, with a Hill coefficient, n
2
for all the truncated exchangers, as well as for the full-length NHE3.
) is not altered during
regulation, the tail probably does not affect affinity of the proton
binding site(s). We postulate that the regulatory regions of the
cytoplasmic tail of NHE3 interact either directly or indirectly with
effector sites on the membrane-spanning domain to increase or decrease
activity by changing exchanger activity or turnover number. As shown in
Fig. 7
there may be intermediate ``regulators'' which
interact with the regulatory areas in the C terminus identified in this
study and/or with the effector site(s) on the N-terminal part of NHE3.
We suggest the involvement of a stimulatory and an inhibitory protein
or a single protein with stimulatory and inhibitory domains. The nature
of these regulatory proteins is unknown, but analogy to the
subunits of the class of heterotrimeric guanine nucleotide-binding
proteins, which have isoforms which both stimulate and inhibit
adenylate cyclase, is one possible model. We favor the existence and
involvement of the intermediate protein(s) in regulation of NHE3 since
we think it would be difficult for at least five separate regulatory
parts of the C terminus of NHE3 to interact directly with the transport
domain of NHE3.
Table:
Effect of W13, W12, and KN-62 on PS120/NHE3
/H
exchanger; FGF, fibroblast growth factor; FBS, fetal bovine
serum; BCECF-AM, 2`7`-bis(carboxyethyl)-5(6)-carboxyfluoroscein
(acetoxymethyl ester); PMA, phorbol 12`-myristate 13-acetate; OA,
okadaic acid; CaM, calmodulin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.