EDITORIAL FOCUS
Characterization of a phosphorylation event resulting in upregulation of the salivary Na+-K+-2Clminus cotransporter

Kinji Kurihara1,2, Marilyn L. Moore-Hoon1, Masato Saitoh1, and R. James Turner1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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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 beta -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 beta -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 beta -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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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 beta -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 beta -adrenergic stimulation is superimposed on muscarinic stimulation in these tissues; see Ref. 23). Thus beta -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 beta -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 beta -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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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

Previous work from our laboratory has established that the initial rate of intracellular pH (pHi) recovery of rat parotid acini from an NH4Cl-induced alkaline load provides a measure of Na+-K+-2Cl- cotransporter activity in these cells (22, 23). This is because a significant component of this recovery is due to NH+4 entry into the cytoplasm on the cotransporter as a substitute for K+, resulting in the dissipation of the extracellular to intracellular NH+4 gradient and consequently a recovery of pHi toward resting levels. For these measurements, acini were loaded with the fluorescent pHi indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, washed and resuspended in PSS containing 0.01% BSA, and assayed for pHi using a Spex ARCM-MIC spectrofluorimeter (Spex Industries, Edison, NJ) as previously described (3, 4). The initial rate of pHi recovery from an NH4Cl-induced alkaline load was determined as described (4). The rate of this recovery in the presence of 100 µM bumetanide was taken as a measure of non-Na+-K+-2Cl- cotransporter-related fluxes and was subtracted from all data points.

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+-2Cl- cotransporter is recovered in this Triton extract (26).

Anti-Na+-K+-2Cl- Antibodies

Antibody LL232 (12) was generously provided by Dr. Bruce J. Baum. This antibody was raised in rabbits against a peptide corresponding to amino acids 965-986 of the rat parotid Na+-K+-2Cl- cotransporter (rtNKCC1; see Ref. 21). Antibodies alpha -wCT(r) and alpha -wCT(m) were raised in rabbits and mice, respectively, against a 6xHis fusion protein corresponding to amino acids 750-1203 of rtNKCC1. The production of the fusion protein and alpha -wCT(r) is described (21). To produce alpha -wCT(m), BALB/c mice were immunized intraperitoneally every 3-4 wk with ~30 µg of antigen diluted in PBS and combined with Freund's complete adjuvant for the first injection and Freund's incomplete adjuvant for those thereafter. Serum was collected from immunized mice ~10 days after the third or later injection. alpha -wCT(m) and alpha -wCT(r) were used in the early and later phases of this work, respectively. For the purposes employed here (immunoprecipitation), their properties were indistinguishable.

The antibody alpha -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-beta -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 alpha -wNT(m) in mice was essentially the same as that described for alpha -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

alpha -wCT(m) and alpha -wCT(r) were preconjugated to protein G beads as follows. Protein G beads were first washed three times with 5.5 volumes of PSS per volume of beads (for antibody conjugation experiments 1% ovalbumin replaced 1% BSA in PSS). Next, one volume of serum was combined with three volumes of beads and 17 volumes of PSS and incubated at 4°C for 1 h. The beads were then washed two times in SS containing 1% ovalbumin and 0.3% Triton X-100 and suspended in the same buffer as a slurry of 50% beads and 50% buffer. alpha -wNT(m) was conjugated to protein G beads in the same manner except that two volumes of serum were used with three volumes of beads.

Immunoprecipitation of the Na+-K+-2Cl- Cotransporter and Phosphopeptide Mapping

An aliquot of Triton extract containing 240 µg of protein was combined with 30 µl (slurry) of protein G beads preconjugated with alpha -wCT(m) or alpha -wCT(r) (see above) and incubated for 4 h at 4°C (the protein content of Triton extracts was measured using the Pierce BCA Protein Assay Kit using BSA as the standard). The beads were then collected by centrifugation and washed six times with 500 µl of SS containing 0.3% Triton X-100, changing the tube on the last wash. In control experiments (not shown), we have established that these experimental conditions result in the quantitative immunoprecipitation of rtNKCC1 from the Triton extract. The final pellet of beads was either extracted with sample buffer for Tricine-SDS electrophoresis or subjected to CnBr digestion as described below.

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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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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Fig. 1.   Effect of bumetanide concentration ([bumetanide]) on the specific component of bumetanide binding to membranes prepared from stimulated and unstimulated rat parotid acini. Binding of [3H]bumetanide to membranes prepared from isoproterenol-stimulated (1 µM for 40 s; open circle ) and unstimulated () rat parotid acini was measured as described in METHODS (bumetanide concentration range 0.008-1.0 µM). Nonspecific binding, unrelated to the Na+-K+-2Cl- cotransporter, was subtracted from all data points (see METHODS). Results from 4 independent experiments were averaged to produce the figure. To reduce scatter, the results of each experiment were normalized to the binding observed at 31.25 nM bumetanide in membranes from isoproterenol-treated acini. Data are presented as a Scatchard plot. Line drawn through the open circles was obtained by linear regression analysis and yields Kd = 48 ± 5 nM and x-intercept 2.61 ± 0.13 (R2 = 0.95). Results from membranes from unstimulated acini are fit well (R2 = 0.99) by the sum of a saturable and unsaturable site (line not shown for clarity) that yields Kd = 59 ± 13 nM and x-intercept 0.34 ± 0.03 for the saturable site and 0.00124 ± 0.00002 for the binding coefficient of the unsaturable site. Specific component of bumetanide binding to membranes prepared from isoproterenol-treated acini was 1.5 ± 0.4 pmol/mg protein (n = 4) at 31.25 nM bumetanide. Thus the average number of bumetanide binding sites in membranes from isoproterenol-treated acini is 3.9 ± 1.1 pmol/mg protein.

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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Table 1.   Orientation of membrane preparations from stimulated and unstimulated acini

Effect of Isoproterenol Treatment on the Bumetanide Sensitivity of Na+-K+-2Cl- Cotransport

Previous studies from our laboratory have demonstrated that maximal isoproterenol stimulation results in an approximately sixfold increase in the Na+-K+-2Cl- cotransport activity of rat parotid acini (23). As already stressed, this effect correlates well with cotransporter phosphorylation in these cells (26). The experiments shown in Fig. 1 indicate a similar increase in the number of high-affinity bumetanide binding sites after isoproterenol treatment (within the limits of our experimental uncertainties, these isoproterenol-induced increases in cotransport activity and number of high-affinity bumetanide binding sites are not significantly different). In fact, the results shown in Fig. 1 also suggest that the effect of isoproterenol stimulation is to convert the low-affinity bumetanide binding sites seen in unstimulated cells to high-affinity sites. To explore this effect further, we examined the bumetanide sensitivity of Na+-K+-2Cl- cotransport activity in stimulated and unstimulated acini. Specifically, we wondered whether the low-affinity component of specific bumetanide binding seen in membranes from unstimulated acini (Fig. 1) could be associated with any transport activity. We reasoned that if this were the case we would find a component of cotransporter flux in unstimulated acini that was less sensitive to bumetanide inhibition than fluxes in stimulated acini.

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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Fig. 2.   Bumetanide sensitivity of Na+-K+-2Cl- cotransporter fluxes in isoproterenol-stimulated and unstimulated rat parotid acini. Na+-K+-2Cl- cotransport activity was measured as described in METHODS in isoproterenol-stimulated (1 µM for 40 s; open circle ) and unstimulated () rat parotid acini in the presence of the bumetanide concentrations indicated. To compare the bumetanide dose responses of stimulated and unstimulated acini directly, results from each experimental condition were normalized to fluxes measured in the absence of bumetanide (n >=  3 for all points). Line drawn through the data points is a nonlinear least squares fit to all of the data points to the equation flux = (1 - [bumetanide]/K0.5)-1 for a single bumetanide inhibitory site. This fit yields a half-maximal inhibitory concentration of bumetanide (K0.5) of 1.2 ± 0.2 µM.

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 beta -Adrenergic Stimulation is not the Protein Kinase A Consensus Site

All mammalian NKCC1's sequenced to date contain a single protein kinase A consensus site (LysLysGluSer) located in the highly conserved COOH-terminal region of the molecule (2, 21, 24, 30). In rtNKCC1, this putative phosphoacceptor site is Ser-982. Because of the well-established association of beta -adrenergic stimulation with salivary acinar cAMP production and the resulting activation of protein kinase A, our initial hypothesis was that this protein kinase A consensus site was the phosphorylation site associated with isoproterenol treatment. We also had available an antibody, LL232, raised against a peptide corresponding to amino acids 965-986 of the rat NKCC1 sequence (see METHODS) that includes this protein kinase A consensus site. Analysis of the rtNKCC1 sequence indicated that CnBr digestion of the protein would produce a peptide of molecular mass 19.3 kDa (amino acids 894-1063) that would contain the LL232 epitope (CnBr specifically cleaves Met-X bonds, where X is any amino acid, converting the COOH-terminal methionine to homoserine). Figure 3A shows the phosphorylation pattern of the rtNKCC1 protein immunoprecipitated from unstimulated or isoproterenol-stimulated 32Pi-labeled acini then subjected to CnBr digestion or no CnBr treatment, as indicated. As previously demonstrated in our laboratory (26), isoproterenol stimulation results in an obvious increase in the phosphorylation of the 170-kDa rtNKCC1 protein. As also shown in Fig. 3, the dominant phosphopeptide resulting from CnBr digestion of this protein has an apparent molecular mass approx 30 kDa, and its phosphorylation is likewise increased with isoproterenol treatment. To determine whether this phosphopeptide contained the protein kinase A consensus site, we carried out Western blots on similarly prepared samples using the LL232 antibody. An example of such an experiment is shown in Fig. 3B. Surprisingly, however, inspection of Fig. 3, A and B, reveals that the CnBr digestion product recognized by the LL232 antibody migrates with an apparent molecular mass (approx 25 kDa) that is significantly lower than the phosphopeptide labeled in response to isoproterenol treatment.


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Fig. 3.   Comparison of the apparent molecular masses of the dominant phosphopeptide produced by CnBr digestion of rtNKCC1 and the peptide recognized by the LL232 antibody. Acinar membrane Triton extracts were prepared from resting rat parotid acini (-) or from acini treated with 1 µM isoproterenol (Iso) for 40 s (+). rtNKCC1 protein was then immunoprecipitated from these extracts using the antibody alpha -wCT(m) and either run directly on Tricine-SDS electrophoresis (-) or subjected to CnBr digestion (+) before electrophoresis, as indicated. A: autoradiogram from 32P-labeled acini. Material from 240 µg of Triton extract was used in each lane. Approximate position of the dye front (DF) of the gel is also indicated. B: Western blot from unlabeled acini using LL232 as the primary antibody. Material from 2 µg of Triton extract was used in each lane.

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 alpha -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 alpha -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 alpha -wCT(m) antibody and thus that it does not contain the protein kinase A consensus site of rtNKCC1.


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Fig. 4.   The 30-kDa phosphopeptide is not coimmunoprecipitated with the CnBr digestion product of rtNKCC1 containing the protein kinase A consensus site. Acinar membrane Triton extracts were prepared from acini treated with isoproterenol, and rtNKCC1 was immunoprecipitated and digested with CnBr as described for Fig. 3. A portion of the CnBr digest was solubilized in stop solution containing 0.3% Triton X-100, treated with 1 mM dithiothreitol for 15 min then 2 mM N-ethylmaleimide for 15 min, and then reimmunoprecipitated with alpha -wCT(m) antibody [20 µl of protein G beads preconjugated with alpha -wCT(m) were used to immunoprecipitate the CnBr digest from 180 µg of Triton extract]. A: autoradiogram from 32P-labeled acini. The following samples were run on the gel: immunoprecipitated rtNKCC1 (Co), immunoprecipitated rtNKCC1 digested with CnBr (CB), and the pellet (IP) and supernatant (Sup) from the reimmunoprecipitation of the CnBr digest with alpha -wCT(m). B: Western blot using LL232 as the primary antibody. The pellet (IP) from the reimmunoprecipitation of the CnBr digest and an equivalent background sample obtained by replacing the CnBr digest with buffer in the immunoprecipitation reaction (Bkg) were analyzed.

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, alpha -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 approx 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 alpha -wNT(m). It is clear from Fig. 5 that the 30-kDa phosphopeptide is quantitatively immunoprecipitated by alpha -wNT(m), confirming that it corresponds to an NH2-terminal CnBr digestion product of rtNKCC1.


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Fig. 5.   The 30-kDa phosphopeptide is recognized by an antibody raised against the NH2-terminus of rtNKCC1. Experiment was carried out as described for Fig. 4A except that the first immunoprecipitaion was done with alpha -wCT(r) and the second with alpha -wNT(m). The following samples were run on the gel: immunoprecipitated rtNKCC1 (Co), immunoprecipitated rtNKCC1 digested with CnBr (CB), and the pellet (IP) and supernatant (Sup) from the reimmunoprecipitation of the CnBr digest with alpha -wNT(m).

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 alpha -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 approx 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 beta -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 beta -adrenergic stimulation of the rat parotid. The results of V8 protease digestion of the rtNKCC1 protein immunoprecipitated from isoproterenol-treated and untreated 32P-labeled acini are shown in Fig. 6, lanes on left. The 17-kDa phosphopeptide and its increased phosphorylation in response to isoproterenol treatment are clearly evident. In Fig. 6, lanes on right, we show the results of V8 protease digestion of the 30-kDa phosphopeptides obtained from CnBr digestion of rtNKCC1 from isoproterenol-treated and untreated cells. The result is a 14.5-kDa phosphopeptide whose level of phosphorylation is likewise increased by isoproterenol treatment.


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Fig. 6.   Relationship between 30-kDa phosphopeptide and the previously identified 17-kDa V8 protease digestion product. 32P-labeled rtNKCC1 was immunoprecipitated from resting (-) or isoproterenol-treated (+) rat parotid acini and then either run directly on Tricine-SDS electrophoresis (-) or subjected to CnBr digestion (+) before electrophoresis. Bands corresponding to rtNKCC1 or the 30-kDa phosphopeptide were then cut from the gel, digested with V8 protease (see METHODS), and rerun on a second Tricine gel.

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 beta -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.


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Fig. 7.   Tentative assignment of the location of the 17-kDa and 14.5-kDa peptides in the rtNKCC1 sequence. See text for details of assignments. Locations of the methionines (Met) and glutamic acids (Glu) in the NH2-terminal 320 amino acids of rtNKCC1 are indicated by vertical lines above the bottom horizontal axis.

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.


    ACKNOWLEDGEMENTS

We thank Dr. Bruce J. Baum for encouragement and many helpful discussions during the course of this work.


    FOOTNOTES

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 (approx 50 nM) and the K0.5 for bumetanide inhibition of cotransporter flux seen in Fig. 2 (approx 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 (approx 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 277(6):C1184-C1193