1 Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892; and 2 Department of Oral Physiology, Meikai University, School of Dentistry, Saitama 350-02, Japan
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ABSTRACT |
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Previous studies from our laboratory have shown a close
correlation between increased
Na+-K+-2Cl
cotransporter activity and increased cotransporter phosphorylation after
-adrenergic stimulation of rat parotid acinar cells. We demonstrate here that these effects are paralleled by an increase in
the number of high-affinity binding sites for the cotransporter inhibitor bumetanide in membranes prepared from stimulated acini. We
also show that the sensitivity of cotransporter fluxes to inhibition by
bumetanide is the same in both resting and isoproterenol-stimulated cells, consistent with the hypothesis that
-adrenergic stimulation and the accompanying phosphorylation result in the activation of
previously quiescent transporters rather than in a change in the
properties of already active proteins. In addition, we demonstrate that
the increased phosphorylation on the cotransporter resulting from
-adrenergic stimulation is localized to a 30-kDa phosphopeptide obtained by cyanogen bromide digestion. Immunoprecipitation and Western
blotting experiments demonstrate that this peptide is derived from the
NH2-terminal cytosolic tail of the
cotransporter, which surprisingly does not contain the sole protein
kinase A consensus site on the molecule.
exocrine glands; fluid secretion; stimulus-secretion coupling; cation-chloride cotransporter
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INTRODUCTION |
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THE "SECRETORY" ISOFORM of the
Na+-K+-2Cl
cotransporter (NKCC1 or BSC2) is relatively widely expressed in the
tissues of mammals and other species (2, 24, 29). This transporter is
known to play a central role in the movement of electrolytes and
osmotically obliged water across a number of secretory epithelia (7,
13). It is also thought to be involved in cell volume regulation, the control of intracellular Cl
concentration
([Cl
]i),
and the transport of NH+4 (and thereby
acid/base equivalents) in some cells (4, 7). NKCC1 has now been cloned (8, 13), and sequence analysis demonstrates that it is a member of a
family of cation-Cl
cotransporters, including a "renal" or "absorptive"
Na+-K+-2Cl
cotransporter isoform (NKCC2 or BSC1), a
Na+-Cl
cotransporter (NCC or TSC), and four
K+-Cl
cotransporter isoforms (KCC1, KCC2, KCC3, and KCC4). Hydropathy analyses predict that all of these cation-coupled cotransporters share
a common membrane topology consisting of large hydrophilic NH2- and COOH-termini (15-35
kDa and ~50 kDa, respectively) on either side of a central
hydrophobic transmembrane region (~50 kDa). This transmembrane region
is predicted to consist of 10-12 membrane spanning domains (21)
connected by relatively short intracellular and extracellular loops
(one exception to this is a large extracellular loop between putative
membrane spanning helices 5 and 6 of the KCC's).
In secretory epithelia utilizing NKCC1, salt and water movements
are driven by net transepithelial
Cl transport (7, 13). In
these tissues, the cotransporter is localized to the basolateral
membrane where it concentrates
Cl
in the cytoplasm above
electrochemical equilibrium. Fluid secretory stimuli result in the
opening of an apical Cl
channel that provides the pathway for cellular
Cl
exit and thereby
transepithelial Cl
secretion. There is now good evidence from a number of these tissues
that cotransporter fluxes are also dramatically increased as a part of
the secretory process and that this phenomenon is not simply due to the
changes in electrochemical driving forces for
Na+,
K+, and
Cl
associated with
stimulation (3, 5, 9, 19, 23). Rather, this upregulation appears to
arise from the increased activity of individual cotransporter
molecules, the activation of previously inactive cotransporters, or
possibly both. It has also been shown in several cases that secretory
stimuli can elicit an increase in the number of loop diuretic binding
sites in the cell membrane (11, 15, 19), which is thought to parallel
increased cotransporter activity. Recent studies have provided good
evidence for the involvement of cotransporter phosphorylation in some
of these effects (10, 15, 26, 27). For example, in the shark rectal
gland (15) and the rat parotid gland (23, 26), there are excellent
correlations between the cotransporter phosphorylation state and the
changes in cotransporter activity seen in response to cAMP-generating stimuli.
Recent experiments with shark rectal glands and mammalian airway
epithelia (10, 16) indicate that the cotransporter phosphorylation seen
in response to fluid secretory stimuli in these tissues is, at least in
part, secondary to the decrease in
[Cl]i
resulting from secretory Cl
loss. Thus it has been suggested (16) that
[Cl
]i
itself may act as an intracellular messenger that coordinates Cl
entry and exit during
secretion (e.g., via a
[Cl
]i-dependent
kinase or phosphatase). However, it is highly likely that other
intracellular messenger pathways also play significant roles in the
regulation of
Na+-K+-2Cl
cotransport activity during fluid secretion by these tissues. Indeed,
it has been shown that the cell shrinkage that accompanies secretagogue-induced salt loss in both of these tissues itself increases cotransporter activity and phosphorylation levels (10, 15).
In addition, in airway epithelia, there is good evidence for a direct
stimulation of the cotransporter via a cAMP-dependent cascade (9, 11).
Thus the complexities of the secretory response in these cells
(increased intracellular cAMP, decreased
[Cl
]i,
cell shrinkage) complicate the task of precisely determining the
relative contributions and interactions of these various regulatory events.
In the present paper, we continue our investigation of the upregulation
of NKCC1 in rat parotid acini by -adrenergic stimulation, a
cAMP-generating stimulus in this tissue. As already mentioned, previous
studies from our laboratory have shown a close correlation between
isoproterenol-induced increases in
Na+-K+-2Cl
cotransporter activity and cotransporter phosphorylation in these cells
(23, 26). Specifically, we found that the isoproterenol dose responses
for both of these phenomena were essentially identical (a half-maximal
effect of isoproterenol was seen at ~20 nM isoproterenol in both
cases) and that isoproterenol stimulation was accompanied by the
increased phosphorylation of an ~17-kDa peptide resulting from V8
protease digestion of the cotransporter. In addition, acinar treatment
with permeant cAMP analogs or forskolin also resulted in cotransporter
upregulation and phosphorylation, suggesting the involvement of protein
kinase A in these effects. In contrast to the epithelia discussed
above, however, salivary glands secrete fluid in response to
Ca2+-mobilizing rather than
cAMP-generating agonists (the increased cotransporter activity
resulting from cAMP-generating agonists is thought to account for the
increased fluid secretion observed when
-adrenergic stimulation is
superimposed on muscarinic stimulation in these tissues; see Ref. 23).
Thus
-adrenergic stimulation of salivary acini does not result in
intracellular Cl
loss or
cell shrinkage (18, 23). Because of this and the relatively high level
of NKCC1 activity in the rat parotid gland, this tissue provides an
excellent mammalian system to study the regulation of NKCC1 by
cAMP-generating stimuli in the absence of other confounding effects.
We demonstrate here that the increase in cotransport activity and
phosphorylation resulting from -adrenergic stimulation is
paralleled by an increase in the number of high-affinity bumetanide binding sites in parotid membranes. However, the sensitivity of cotransporter fluxes to inhibition by bumetanide is the same in both
resting and isoproterenol-stimulated cells, suggesting that stimulation
and the accompanying phosphorylation result in the activation of
previously quiescent cotransporters rather than in a change in the
properties of already active proteins. In addition, we show that the
phosphorylation of the cotransporter resulting from
-adrenergic
stimulation is localized to its
NH2-terminal cytosolic tail. This
result was somewhat unexpected, since this region does not
contain the only protein kinase A consensus site on the protein. This
observation may indicate that the signaling cascade after acinar
intracellular cAMP production in these cells is more complex than
originally thought.
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METHODS |
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Materials
Carrier-free 32Pi (10 mCi/ml) and [3H]bumetanide (80.8 Ci/mmol) were obtained from Amersham. Phenylmethylsulfonyl fluoride, L-tosylamido 2-phenylethyl chloromethyl ketone, cyanogen bromide (CnBr), V8 protease, and BSA (no. A6003) were from Sigma. Pepstatin and leupeptin were from Boehringer Mannheim. Calyculin A was from Calbiochem. Multimark and Mark 12 molecular mass standards and prepoured 16% Tricine gels were from Novex. Protein G-Sepharose beads were from Pierce. All other chemicals were from standard commercial sources and were reagent grade or the highest purity available.The composition of the physiological salt solution (PSS) was 135 mM NaCl, 5.8 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 0.73 mM NaH2PO4, 11 mM glucose, 20 mM HEPES (pH 7.4 with NaOH), 2 mM glutamine, and 1% BSA. The stop solution (SS) contained 20 mM HEPES (pH 7.4 with NaOH), 100 mM NaCl, 10 mM Na2ATP, 50 mM NaF, 15 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 0.1 µM calyculin A, 5 mM EDTA, 300 µM phenylmethylsulfonyl fluoride, 100 µM L-tosylamido 2-phenylethyl chloromethyl ketone, 1.5 µM pepstatin, and 1.5 µM leupeptin. Buffer A was 10 mM HEPES buffered with Tris to pH 7.4 plus 100 mM mannitol. Buffer V was buffer A containing 1 mM EDTA and 1 mM sodium orthovanadate.
Preparation of Parotid Acini
Dispersed acini were prepared from the parotid glands of male Wistar rats (Harlan Sprague-Dawley, Indianapolis, IN) by collagenase digestion as previously described (26). After preparation, acini were washed and resuspended in an appropriate solution for subsequent experimental steps (see below). Acini were continuously gassed with 95% O2-5% CO2 or 100% O2, as appropriate.Bumetanide Binding Studies
Membrane preparation. During washing and centrifugation, the volume of the above final acinar pellet was estimated by eye, and acini were suspended in ~10 times this volume of PSS (typically 1.5-2.0 ml/rat). Acini were rested for 20 min at 37°C, and then 200-µl aliquots of the acinar suspension were stimulated with 1 µM isoproterenol for 40 s. Stimulation was terminated by the addition of 500 µl of ice-cold SS containing 1 mM sodium orthovanadate and disruption by sonication as previously described (26). A total of four to five aliquots of sonicated stimulated acini were combined and centrifuged at 1,000 g for 10 min, and the resulting pellet was discarded. The supernatant was centrifuged at 100,000 g for 30 min. This pellet was suspended in ~300 µl of buffer V, centrifuged again at 100,000 g for 30 min, and suspended at a protein concentration of ~2 mg/ml in buffer V (membrane protein was measured using the Bio-Rad Protein Assay Kit using bovine IgG as the standard). A similar number of aliquots of unstimulated acini was treated in the same fashion.[3H]bumetanide binding
assay.
Equilibrium bumetanide binding was measured using a nitrocellulose
filtration assay as described previously (28). Briefly, a 20-µl
aliquot of the membrane preparation described above was combined with
20 µl of incubation medium consisting of buffer V containing 200 mM sodium acetate, 190 mM potassium
acetate, 10 mM KCl, and various concentrations of
[3H]bumetanide (final
concentration range 8-1,000 nM). After a 1-h incubation, the
reaction was terminated by the addition of ice-cold buffer A containing 100 mM sodium
gluconate, 100 mM potassium gluconate, and 1 mM EDTA followed by
Millipore filtration (HAWP 0.45 µm). Other procedures were
essentially as described previously (28).
[3H]bumetanide binding
observed in the absence of K+ and
Cl (replaced by
Na+ and acetate, respectively) was
subtracted from that observed in their presence to yield the
"specific component" of bumetanide binding. In previous studies,
we have demonstrated that both K+
and Cl
are required for the
binding of bumetanide to its inhibitory site on the
Na+-K+-2Cl
cotransporter (28); thus, the binding measured in their absence represents the nonspecific binding and trapping of bumetanide by the
membranes and filters.
Orientation of the Membrane Preparation
The orientation of the membrane preparations from isoproterenol-stimulated and unstimulated acini used for the bumetanide binding experiments described above (cytosolic side out, cytosolic side in, and unvesiculated) was determined by measuring K+-dependent p-nitrophenyl phosphatase (KpNPPase) activity (hydrolysis of p-nitrophenyl phosphate by Na+-K+-ATPase) in the presence and absence of ouabain (2 mM) and digitonin (0.0067%). The rationale for the method is described in the text. Membranes were prepared as described above except that sodium orthovanadate was omitted from buffer V. KpNPPase activity was determined at 25°C in buffer A containing 10 mM KCl, 6 mM MgCl2, 2 mM EGTA, 0.33 mM EDTA, 6 mM p-nitrophenyl phosphate (Sigma 104 phosphatase substrate; Sigma Chemical, St. Louis, MO), and 0.17 mg membrane protein/ml. The reaction volume was 300 µl, and the assay was terminated by the addition of 1.0 ml of 0.2 N NaOH. p-Nitrophenyl concentrations were determined from the absorbance of the sample at 405 nm.Measurement of
Na+-K+-2Cl
Cotransport Activity in Intact Acini
32Pi Labeling of Acini and Preparation of Acinar Membrane Triton Extracts
Acinar 32Pi labeling was essentially carried out as previously described (26). Briefly, after preparation, acini were washed and resuspended in ~10 times their volume of PO4-free PSS (PSS without NaH2PO4), incubated in the presence of 50 µCi/ml 32Pi for 30 min, then washed and resuspended in the same volume of PSS. Aliquots (200 µl) of 32Pi-labeled acini were incubated in the presence or absence of 1 µM isoproterenol for 40 s, 500 µl of ice-cold SS was added, and the cells were disrupted by sonication. Membranes were then prepared in the same manner as described above for bumetanide binding studies. After the 100,000-g spin, the membrane pellet from each 200 µl of acini was resuspended in 700 µl of ice-cold extraction buffer (SS containing 0.3% Triton X-100 and no NaCl), kept on ice for 30 min, then centrifuged at 100,000 g for 30 min. The supernatant from this high-speed spin is referred to as the "Triton extract." In past experiments from our laboratory, we have demonstrated that virtually all of the acinar Na+-K+-2ClAnti-Na+-K+-2Cl
Antibodies
The antibody -wNT(m) was raised in mice against a 6xHis fusion
protein corresponding to amino acids 3-202 of rtNKCC1. This fusion
protein was produced as follows. A Srf
I/Fsp I fragment containing bases
10-604 of the coding region of rtNKCC1 (21) was isolated by
restriction digestion and cloned into the
Sma I site of the
Escherichia coli expression vector
pQE32 (Qiagen, Santa Clarita, CA) "in frame" with a 6xHis
NH2-terminal tag. Subsequent procedures were essentially as recommended by Qiagen for the pQE32 vector. Briefly, TOP10F' cells (Invitrogen, Carlsbad, CA) were transformed with the recombinant plasmid, and ampicillin-resistant colonies were screened for appropriately oriented plasmid inserts by
restriction digestion and for
isopropyl-
-D-thiogalactopyranoside (IPTG)-inducible
6xHis protein production by Western blotting using Penta-His antibody
(Qiagen). A suitable clone was chosen for the production of recombinant
protein, and the identity of the corresponding pQE32 plasmid insert was
confirmed by end-to-end sequencing. These bacteria were grown for 1 h
in the presence of 1 mM IPTG and then were lysed in 8 M urea, 0.1 M
NaH2PO4,
and 0.01 M Tris · HCl, pH 8.0, for 30 min. The lysate
from 200 ml of bacteria was centrifuged at 4,000 g for 10 min, and the supernate was
incubated with 200 µl of Ni-NTA beads (Qiagen) for 60 min with
constant mixing. These beads were then washed two times with the lysis
buffer and two times with the lysis buffer adjusted to pH 6.3. The
6xHis-tagged recombinant protein was then eluted with lysis buffer
adjusted to pH 4.5. The procedure for producing
-wNT(m) in mice was
essentially the same as that described for
-wCT(m) above.
In Western blots of rat parotid plasma membranes (not shown), all of
the above antisera recognized a single ~170-kDa protein, the
molecular mass we have previously established as that of the (fully
glycosylated)
Na+-K+-2Cl
cotransporter in this tissue.
Antibody Conjugation to Protein G Beads
Immunoprecipitation of the
Na+-K+-2Cl
Cotransporter and Phosphopeptide Mapping
CnBr digestion was carried out by adding 200 µl of CnBr (10 mg/ml) in 70% formic acid to the above protein G pellet. After being incubated overnight at room temperature, the sample was dried in a Speed Vac, 200 µl of 70% formic acid were added, and the sample was dried again. This was followed by two additions of 200 µl distilled water with drying after each addition. Finally, the pellet was extracted in sample buffer for Tricine-SDS electrophoresis. In later experiments, we found that an overnight incubation with CnBr was not necessary and that 3 h were sufficient for complete digestion.
Digestion with V8 protease was carried out as previously described (26). Briefly, bands corresponding to rtNKCC1 or one of its CnBr digestion products (see below) were cut from dried Tricine gels, rehydrated in 50 mM NH4HCO3 (pH 8.0) plus 1 mM dithiothreitol, and incubated with V8 protease (20 µg/ml) for 6 h. The digested material recovered from the gel fragments was then dried, taken up in sample buffer, and subjected to Tricine-SDS electrophoresis.
Gel Electrophoresis, Western Blotting, and Autoradiography
Tricine-SDS electrophoresis and autoradiography were carried out as previously described (26). For Western blotting, proteins were transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA), blocking and incubation with primary and secondary antibodies were essentially as previously described (20), and detection was carried out using the enhanced chemiluminescence kit from Amersham. Tricine (16%)-SDS gels were used to obtain all of the results shown. The positions of the Multimark (208, 53, 34, 23, 13, 7, and 4 kDa) or Mark 12 (200, 55, 31, 22, 14, 6, and 3.5 kDa; Fig. 6 only) molecular mass standards are indicated on the left-hand-side of the autoradiographs and Western blots (not all markers are shown because of space limitations).Data Presentation and Analysis
All experiments were repeated three or more times with similar results. Quantitative results are given as means ± SE. Linear and nonlinear least-squares analysis was carried out using the program SigmaPlot for Windows 4.0 (SPSS). ![]() |
RESULTS AND DISCUSSION |
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Effect of Isoproterenol Treatment on Cotransporter-Specific Bumetanide Binding
Figure 1 illustrates the effect of bumetanide concentration on the specific component of bumetanide binding to membranes prepared from isoproterenol-stimulated and unstimulated rat parotid acini; the data are presented as a Scatchard plot. The results obtained with membranes from stimulated acini lie on a good straight line (R2 = 0.95), consistent with the presence of a single high-affinity bumetanide binding site in this preparation. The line drawn through these points in Fig. 1 was obtained by linear regression analysis and yields a dissociation constant (Kd) of 48 ± 5 nM for this site. On the other hand, the results obtained with membranes from resting (unstimulated) acini are clearly curvilinear, indicating the presence of specific bumetanide binding sites with both high and low affinity. The scatter on these data make it difficult to reliably estimate the Kd of the low-affinity site. However, when the low-affinity site is approximated by a nonsaturable component of binding, nonlinear regression analysis (see legend for Fig. 1) yields a Kd of 59 ± 13 nM for the high-affinity site, in good agreement with the Kd observed in membranes from isoproterenol-treated acini. This analysis also indicates that there are 7.8 ± 0.7 times more high-affinity bumetanide binding sites in membranes from stimulated acini than in membranes from unstimulated acini (see legend for Fig. 1).
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We have verified that the orientations of the membrane preparations (relative amounts of cytosolic-side-out, cytosolic-side-in, and unvesiculated membrane fragments) from isoproterenol-stimulated and unstimulated acini used for the experiments shown in Fig. 1 are the same. This was done to confirm that the differences seen in Fig. 1 were due to changes in the properties of the bumetanide binding site itself rather than to a change in its accessibility to [3H]bumetanide in one preparation versus the other (e.g., a higher proportion of cytosolic-side-out vesicles in membranes from unstimulated acini). To do this, the KpNPPase activity of the membrane preparations was measured as described in METHODS: 1) in the presence of digitonin (0.0067%), 2) in the presence of ouabain (2 mM), 3) in the presence of digitonin plus ouabain, and 4) in the absence of both digitonin and ouabain.
The idea here was to take advantage of the facts that
p-nitrophenyl phosphate and ouabain
interact with
Na+-K+-ATPase
at the intracellular and extracellular sides of the membrane, respectively, and that permeabilization by digitonin would allow access
to intravesicular sites. In preliminary experiments (not shown), we
found that KpNPPase activity increased with digitonin concentrations up
to 0.004% and then remained constant over the concentration range
0.004-0.016%. These data indicate that a digitonin concentration
of 0.0067% results in permeabilization of the membranes without
inhibition of KpNPPase activity. Total ouabain-sensitive KpNPPase
activity is then given by the difference between the activities
measured under conditions 1 and
3 above. By similar arguments, the
activity due to unvesiculated membrane fragments is given by
conditions 4-2, that due to
cytosolic-side-out vesicles by conditions 2-3, and that
due to cytosolic-side-in vesicles by conditions
1-4. The averaged results of three experiments of this type are shown in Table 1. These
results indicate that there is no significant difference between the
orientations of the membrane preparations from stimulated and
unstimulated acini.
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Effect of Isoproterenol Treatment on the Bumetanide Sensitivity of
Na+-K+-2Cl
Cotransport
Figure 2 shows the bumetanide sensitivity
of
Na+-K+-2Cl
cotransporter fluxes in isoproterenol-stimulated and unstimulated rat parotid acini. There is no significant difference between the data
points for stimulated and unstimulated cells at any bumetanide concentration, and the results are fit well by an equation that assumes
a single bumetanide binding site with a half-maximal inhibition constant (K0.5)
of 1.2 µM1
(see legend for Fig. 2). This experiment demonstrates clearly that
there is no evidence for a significant component of
Na+-K+-2Cl
cotransporter flux with reduced sensitivity to bumetanide in unstimulated acini.
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Taken together with the past results from our laboratory discussed
above, the experiments shown in Figs. 1 and 2 strongly suggest that the
phosphorylation of the salivary NKCC1 after isoproterenol stimulation
results in the conversion of the unphosphorylated carrier, which has
little or no transport activity (and possibly low-affinity bumetanide
binding), to the phosphorylated carrier, which exhibits significant
Na+-K+-2Cl
cotransport activity and high-affinity bumetanide binding. We should
note, however, that at the present time we cannot exclude the
possibility that phosphorylation of NKCC1 may also play a role in its
membrane trafficking. More specifically, it is possible that, in
addition to any effects on NKCC1 function, phosphorylation could also
result in the translocation of the cotransporter to the cell surface
from an intracellular membrane pool. Additional experiments will be
required to examine this issue.
Evidence that the Major Site of Phosphorylation of rtNKCC1 in
Response to -Adrenergic Stimulation is not the
Protein Kinase A Consensus Site
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To confirm that the 30-kDa phosphopeptide seen in Fig.
3A was truly unrelated to the
predicted CnBr digestion product containing the LL232 epitope (and thus
the protein kinase A consensus site), we carried out the additional
experiments shown in Fig. 4. In these
studies, we immunoprecitated the rtNKCC1 protein from the Triton
extract of
32Pi-labeled
isoproterenol-treated acini and digested with CnBr as usual, then
reprecipitated the CnBr digest with the -wCT(m) antibody. Because this antibody was raised against amino
acids 750-1203 of the rtNKCC1 sequence, it would be expected to
immunoprecipitate the CnBr fragment containing the LL232 epitope (see
above). The results of such an experiment are shown in Fig.
4A. This autoradiogram illustrates
that the 30-kDa phosphopeptide is not, in fact, found in the pellet
from the second immunoprecipitation but remains in the supernatant. In
Fig. 4B, we show that, although the
30-kDa phosphopeptide was not immunoprecipitated by the
-wCT(m)
antibody, the 25-kDa peptide recognized by antibody LL232 is
immunoprecipitated as expected. The results of Figs. 3 and 4 provide
strong evidence that the 30-kDa phosphopeptide is not recognized by
either the LL232 or the
-wCT(m) antibody and thus that it does not
contain the protein kinase A consensus site of rtNKCC1.
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Analysis of the sequence of rtNKCC1 predicts that CnBr digestion will
result in three large peptides with molecular masses of 20.0 kDa
(corresponding to amino acids 2-209), 19.3 kDa (corresponding to
amino acids 894-1063), and 17.0 kDa (corresponding to amino acids
420-578) and a number of smaller peptides with molecular masses
8.7 kDa or less. Because NKCC1 is known to be highly glycosylated (17,
25), we wondered if the 30-kDa phosphopeptide could correspond to a
smaller peptide containing the glycosylated region of rtNKCC1 and if
this could account for its rather high apparent molecular mass.
However, in additional experiments (not shown), we have demonstrated
that deglycosylation of rtNKCC1 does not result in a detectable shift
in the apparent molecular mass of the 30-kDa phosphopeptide.
Accordingly, we next considered the possibility that the 30-kDa
phosphopeptide might correspond to the
NH2-terminal region of rtNKCC1,
specifically the 20.0-kDa fragment predicted from CnBr digestion (amino
acids 2-209). To test this possibility, we raised an antibody,
-wNT(m), against amino acids 3-202 of rtNKCC1 (see
METHODS). In preliminary experiments (not shown), we found
that this antibody recognized a peptide with a relative molecular mass
30 kDa in CnBr-digested rat parotid basolateral membranes. In Fig.
5, we show the results of a double
immunoprecipitation experiment similar to the one shown in Fig.
4B except that in Fig. 5 the second
immunoprecipitation was carried out with
-wNT(m). It is clear from
Fig. 5 that the 30-kDa phosphopeptide is quantitatively immunoprecipitated by
-wNT(m), confirming that it corresponds to an
NH2-terminal CnBr digestion
product of rtNKCC1.
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Several additional observations indicate that the 30-kDa phosphopeptide
is in fact the predicted
NH2-terminal 20-kDa CnBr digestion
product of rtNKCC1 and that the difference between its apparent and
predicted molecular masses is a result of the anomalous mobility of
this peptide in SDS gels. First, it is unlikely that this is a longer
peptide arising from incomplete digestion by CnBr, since we find that
CnBr digestion is complete after 3 h of incubation under our
experimental conditions and digestion was carried out for >12 h for
most of our experiments (see METHODS). Second, the adjacent
predicted CnBr digestion product (amino acids 210-287) is a
8.7-kDa peptide that we have been able to detect in Western blots using
an antipeptide antibody raised against amino acids 238-261 of
rtNKCC1 (21; data not shown); thus, CnBr-mediated cleavage between
Met-209 and Asp-210 has apparently occurred. Last, the recombinant
peptide used to produce the -wNT(m) antibody contains 200 of the 208 amino acids in the 20.0-kDa
NH2-terminal CnBr digestion
product (see METHODS) and has a predicted molecular mass of
22.3 kDa (the additional molecular mass is due to the 6xHis tag and
other vector sequence). However, this protein was also found to migrate
anomalously on SDS gels with an apparent molecular mass
34 kDa (not
shown). In this regard, we would point out that the amino acid
composition of the NH2-terminal
end of rtNKCC1 is rather unusual; of the 208 amino acids contained in the predicted NH2-terminal CnBr
digestion product, 40 are alanines and 36 are glycines. This relatively
large number of small neutral amino acids would have the effect of
making the NH2-terminus very flexible, and this could account for its anomalously high apparent molecular mass on SDS gels.
Finally, it is worth mentioning that, even in highly overexposed
autoradiographs, we have never seen any significant signal at the
25-kDa position, leading us to conclude that the protein kinase A
consensus site on this protein is simply not phosphorylated to any
significant degree under our experimental conditions in the rat
parotid. Interesting, in similar experiments to those presented here
employing mouse submandibular glands, we do, in fact, see some labeling
of an ~25-kDa fragment resulting from CnBr digestion of mouse NKCC1.
However, this labeling is much weaker than that observed for the
corresponding 30-kDa phosphopeptide, and there is no indication that
the phosphorylation of this 25-kDa peptide is affected by isoproterenol
treatment (data not shown). In the mouse, like the rat, it is the
phosphorylation of the 30-kDa CnBr digestion product that is mainly
affected by -adrenergic stimulation (not shown).
Relationship Between the 30-kDa Phosphopeptide and the Previously Identified 17-kDa V8 Protease Digestion Product
As mentioned in the Introduction, previous work from our laboratory identified a 17-kDa peptide resulting from V8 protease digestion of rtNKCC1, the level of phosphorylation of which is also increased after
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Analysis of the results of a series of experiments like those shown in
Figs. 3 and 6 by scanning denistometry indicated that the levels of
phosphorylation of the 30-, 17-, and 14.5-kDa peptides increased 2.72 ± 0.04-, 3.38 ± 0.55-, and 2.89 ± 0.32-fold,
respectively, with isoproterenol treatment (this increase for the
17-kDa peptide is in excellent agreement with our previous
determination of this quantity; see Ref. 26). None of these values is
significantly different from one another; however, all are
approximately one-half of the magnitude of the observed
isoproterenol-induced increases in
Na+-K+-2Cl
cotransporter activity (23) and specific high-affinty bumetanide binding sites (Fig. 1) measured in the rat parotid after the same isoproterenol treatment. This latter observation presumably indicates that all of these peptides contain additional phosphoamino acids whose
level of phosphorylation is not affected by
-adrenergic stimulation.
At the present time, we are unable to purify sufficient quantities of
the rtNKCC1 protein to determine the
NH2- and COOH-terminal amino acids
of the 17- and 14.5-kDa peptides via direct sequencing. However, based
on the apparent molecular masses of the 17- and 14.5-kDa
phosphopeptides and the positions of the possible CnBr and V8 protease
digestion sites (V8 protease is expected to cleave after Glu under the
experimental conditions employed here), we have made a tentative
assignment of their relative locations in the rtNKCC1 sequence in Fig.
7. First, based on the arguments given
above, we have assigned the 30-kDa peptide to the sequence Glu-2 to
Met-209 (as already indicated, Met-209 would actually be converted to a
homoserine by its reaction with CnBr). Next, we note that, even given
the anomalous mobility of peptides derived from the
NH2-terminal region of rtNKCC1, it
is highly unlikely that one could produce a peptide of apparent
molecular mass 14.5 kDa from the 30-kDa phosphopeptide if V8 protease
cleaves at all of its possible cut sites (cf. Fig. 7). Accordingly, the
sequences corresponding to the 17- and 14.5-kDa peptides are expected
to contain some internal Glu residues. On the other hand, the
difference between the 17- and 14.5-kDa peptide must be due to cleavage
at one or more Met residues within the 17-kDa peptide. To accommodate the 17-kDa peptide within the 30-kDa peptide, this clearly must be due
to cleavage at Met-209, resulting in the removal of an ~2.5-kDa
fragment from the COOH-terminal end of the 17-kDa peptide. We have
somewhat arbitrarily assigned Thr-109 to the
NH2-terminal end of the 17- and
14.5-kDa peptides (V8 protease cleavage after Glu-108) since this
results in the 14.5-kDa peptide being approximately one-half the length
of the 30-kDa peptide. We likewise have assigned Glu-227 to the
COOH-terminal end of the 17-kDa peptide. According to this scheme, the
true molecular masses of the 14.5- and 17-kDa peptides are 10.3 and
12.2 kDa, respectively. Although other locations for the
NH2-terminal ends of the 14.5- and
17-kDa peptides and the COOH-terminal end of the 17-kDa peptide are
clearly possible, their positions relative to the 30-kDa peptide seem
clear.
|
The identification of the NH2-terminal phosphorylation sites associated with upregulation of NKCC1 in the rat parotid will require further experimentation. Unfortunately, sequence analysis provides little if any indication of their possible identity. For example, the 30-kDa CnBr digestion product is predicted to contain 21 serines, 13 threonines, and 5 tyrosines; of these, none are protein kinase A or tyrosine kinase consensus sites, and only Ser-37 is a protein kinase C consensus site (21). Interestingly, however, among these possible phosphoacceptors are Thr-208 and Thr-203, two highly conserved residues in all NKCC1's sequenced to date (2, 21, 24, 29, 30). Although neither of these amino acids is found in a recognized protein kinase consensus site, the corresponding two threonines in the shark rectal gland NKCC1 (Thr-189 and Thr-184) have been shown to be phosphorylated in response to a cAMP-generating stimulus (1, 15). However, in a number of additional experiments (not shown), we have been unable to significantly dephosphorylate the 30-kDa phosphopeptide using carboxypeptidase A, a protease that sequentially removes most COOH-terminal amino acids from proteins. Although we cannot exclude the possibility that the 30-kDa phosphopeptide may be resistant to carboxypeptidase A treatment, because of the proximity of Thr-208 and Thr-203 to its COOH-terminal end (Met-209), this result at least suggests that the phosphorylation pattern of NKCC1 resulting from treatment with cAMP-generating stimuli in the rat parotid may not correspond to that found in the shark rectal gland.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Bruce J. Baum for encouragement and many helpful discussions during the course of this work.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1
The difference between the
Kd for
high-affinity bumetanide binding to isolated acinar membranes seen in
Fig. 1 (50 nM) and the
K0.5 for
bumetanide inhibition of cotransporter flux seen in Fig. 2 (
1.2
µM) can be accounted for by the well-documented inhibition of
bumetanide binding by physiological concentrations of
Cl
(e.g., see Ref. 28).
Because of this effect, bumetanide binding experiments are typically
carried out at low Cl
concentration
([Cl
]), as were
the experiments shown in Fig. 1 (5 mM; see METHODS). However, because fluxes via the
Na+-K+-2Cl
cotransporter are markedly reduced at low
[Cl
], these
measurements are typically carried out at physiological [Cl
]. In
previous studies of rabbit salivary acinar membranes, we have shown
that the effect of Cl
is to
decrease the bumetanide binding affinity (28). Inspection of Fig. 5 of
Ref. 28 indicates that a >10-fold decrease in bumetanide binding affinity is expected when
[Cl
] is
increased from 5 mM (as employed in the experiments shown in Fig. 1) to
144 mM (as employed in the experiments shown in Fig. 2). A similar
difference in bumetanide binding
Kd (
100 nM) and bumetanide inhibitory
K0.5 for
cotransporter fluxes (~1 µM) has been documented in experiments
carried out under similar conditions to those presented here with HT-29
cells (6, 14).
Address for reprint requests and other correspondence: R. J. Turner, Bldg. 10, Rm. 1A06, 10 Center Dr. MSC 1190, National Institutes of Health, Bethesda, MD 20892-1190 (E-mail: rjturner{at}nih.gov).
Received 7 June 1999; accepted in final form 16 August 1999.
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