Munc-18-2 regulates exocytosis of H+-ATPase in rat inner medullary collecting duct cells

Julie A. Nicoletta,1,2 Jonathan J. Ross,1,2 Guangmu Li,1,2 Qingzhang Cheng,1,2 Jonathon Schwartz,1,2 Edward A. Alexander,1,2,3 and John H. Schwartz1,2,4

1Renal Section, Boston University Medical Center, and Departments of 2Medicine, 3Physiology, and 4Pathology, Boston University School of Medicine, Boston, Massachusetts 02118

Submitted 31 December 2003 ; accepted in final form 3 July 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Exocytic insertion of H+-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H+-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H+-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H+-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H+-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H+-ATPase. In a pull-down assay of H+-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H+-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H+-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H+-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H+-ATPase vesicles.

soluble N-ethylmaleimide-sensitive factor attachment protein target receptor; cell pH; acid secretion


THE SOLUBLE n-ETHYLMALEIMIDE-SENSITIVE factor (NSF) attachment protein target receptor (SNARE) hypothesis of vesicle transport postulates that proteins in vesicular and target membranes form a core complex that leads to the docking and fusion of these membranes. Synaptobrevin or vesicle-associated membrane proteins (VAMPs) are primarily located on vesicles, and syntaxins are located on the target membranes. VAMP and syntaxin along with synaptosome-associated protein 23 or 25 (SNAP-23, the isoform expressed in somatic tissue, or SNAP-25, the isoform expressed in neuroexcitable tissue) form a parallel four-helix bundle. VAMP and syntaxin each contribute one helix and SNAP-23 or -25 contributes two helices (16) to form the four-helix fusion complex. The formation of this complex is thought to provide the energy required to overcome the energetic barrier of lipid bilayer fusion (16).

Syntaxin has been found to have three different conformational arrangements: one in isolation, another when bound to Munc, and a third as part of the core SNARE complex (16). It has been proposed that when syntaxin is bound to Munc, it is in a "closed conformation," which does not allow it to bind to either SNAP-23 or VAMP (16). Once Munc and syntaxin dissociate, syntaxin "opens" to a conformation that allows SNAP-23/25 and then VAMP to bind, forming the core complex (16). Munc proteins have been found to bind to either the closed or the open conformation of syntaxin and are thought to function, at times, like a chaperone protein for syntaxin (12).

Munc proteins have been found to regulate vesicle trafficking in other cell systems, presumably by binding or releasing syntaxin to either prevent or favor vesicle fusion. It has been suggested that Munc cycles on and off membranes in a stage-specific manner during vesicle transport (7) and the dissociation of Munc and syntaxin determines the kinetics of postfusion events (9).

The role of SNAREs in targeting vesicles is controversial (17, 19), and it has been suggested that these proteins only serve as the machinery for fusion (5, 8). There is evidence that vesicle (v) and target (t) SNAREs are promiscuous, and in in vitro assays a given v-SNARE can form complexes with many different t-SNAREs (11). In rat inner medullary collecting duct (IMCD) cells, it has been shown that the SNARE complex that forms as a result of acute acidification not only contains v- and t-SNAREs but also contains a subunit of H+-ATPase (4). There is evidence to suggest that the H+-ATPase is specifically required for the targeting and fusion of proton pump-laden vesicles to the apical membrane of rat IMCD cells (7). In these studies, we present evidence that Munc-18-2 plays a regulatory role in the vesicle transport of H+-ATPase to the apical membrane of rat IMCD cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Solutions and reagents. The following solutions were used: NaCl-HEPES buffer (NHB; in mM: 110 NaCl, 50 HEPES acid, 5 KCl, 1 MgCl2, 5 KH2PO4, 1 CaCl2, and 5 glucose, pH titrated to 7.2 with 1 M NaOH); choline chloride-HEPES buffer (CHB; identical to NHB except that 110 mM choline chloride was substituted for NaCl, 10 mM K-acetate was added, and the pH was titrated to 7.2 with 1 M KOH instead of NaOH).

Cell culture. IMCD cells were obtained from rat papillae as described previously (22). Aliquots of the isolations were stored at –70°C and activated as needed. The IMCD cells were transfected with either green fluorescent protein (GFP)-syntaxin 1A as previously described (14) or GFP-Munc-18-2. The transfected cells were grown to confluence on plastic culture dishes in Dulbecco's modified Eagle's medium (DMEM) plus 10% FCS, penicillin, streptomycin, and 800 µg/ml G-418 in an atmosphere of 95% air-5% CO2. Wild-type IMCD cells were grown in a similar medium that did not contain G-418.

Acid loading of cells. IMCD monolayers were subjected to a reduction in cell pH (pHi) by a method previously described (21). This method decreases the pHi of IMCD cells from ~7.2 to ~6.5 because of the reversal of the Na+/H+ exchanger and diffusion of acetic acid into the cells (2, 21, 23). Briefly, DMEM was removed from the confluent IMCD cells, and they were washed twice with PBS to remove the DMEM and washed once with CHB. The monolayers were then incubated in CHB at 37°C for 20 min. As a control, IMCD monolayers were treated similarly but the CHB was replaced by NHB. In a second series of studies cells were acidified by an NH4Cl pulse technique. Cells were initially incubated in NHB and then exposed to NHB containing 10 mM NH4Cl for 10 min, followed by NH4Cl-free NHB (22).

Cell homogenization. Acid-loaded and control IMCD cells were scraped from the plastic dishes with a spatula and a homogenizing buffer. This buffer was made of either CHB or NHB with protease inhibitors [500 µM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) hydrochloride, 150 nM aprotinin, 1 µM E-64 protease inhibitor, 0.5 mM EDTA disodium, and 1 µM leupeptin hemisulfate], 0.5% Triton X-100, and, at times, 1% SDS. The suspended cells were homogenized by ten 1-s strokes in a Teflon-coated Dounce homogenizer. Intact cells and nuclei were removed by centrifuging for 15 min at 4°C and 1,200 rpm. The supernatant was stored at –20°C until being used for protein determination, immunoprecipitation, or immunoblot analysis.

Antibodies. The following antibodies were used in these studies: a mouse monoclonal anti-syntaxin antibody, clone HPC-1 (Sigma-Aldrich); a rabbit anti-Munc-18-2 polyclonal antibody (Calbiochem); a mouse monoclonal anti-Munc-18-2 antibody (BD Transduction Laboratories); a mouse anti-Munc-18-1 antibody (Calbiochem); a goat anti-Munc-18-3 antibody (Santa Cruz Biotechnology); a rabbit polyclonal antibody to GFP (BD Bioscience); a rabbit or mouse anti-GP-135 antibody (Abcham); and a rabbit polyclonal anti-31-kDa subunit of H+-ATPase (a gift from Dr. Dennis Brown, Harvard Medical School, Boston, MA).

RT-PCR and DNA sequencing. RT-PCR was performed to determine whether the major isoforms of Munc-18 are expressed in IMCD cells in culture. IMCD cells were grown to confluence and lysed with a concentrated guanidinium thiocyanate solution. Poly(A) RNA was harvested as per manufacturer's instructions, with an mRNA purification kit (Ambion), and tested for purity. The samples were then stored at –80°C until use. Reverse transcription was performed with avian myeloblastosis virus transcriptase (Roche) at 42°C for 30 min in the presence of 0.2 nM dNTPs, 5 mM DTT, and 5 U RNase inhibitor (Roche). PCR was initiated immediately after reverse transcription, with a mixture of Taq and Tgo DNA polymerases. To ensure amplification of rare mRNAs, 40 PCR cycles were performed, at 94°C for 30 s, 57°C for 30 s, and 68°C for 180 s. Primers utilized were Munc-18-1: 5'-ATG GCC CCC ATT GGC CTC AAG GCG GT-3', 5'-TTA ACT GCT TAT TTC TTC GTC TGT TTT ATT-3'; Munc-18-2: 5'-ATG GCG CCC TTG GGG CTG AAG GCG GTG GTG-3', 5'-CAG GGC AGG GCT ATG TCC TCC AGC TTC TGA-3'; and Munc-18-3: 5'-ATG GCG CCG CCG GTG ACG GAA CGG GG-3', 5'-CTA CTC ATC CTT AAG AAA GGA AAC TTT ATC-3'. Samples were then loaded on a 0.7% agarose Tris, acetic acid, EDTA gel for electrophoretic analysis. Products of appropriate base pair size were cut out of the agarose gel, subjected to glass-milk purification (GeneClean kit), and then subjected to automated sequencing (Center for Advanced Biomolecular Research DNA/Protein Analysis Core, Boston University). The homology between the PCR product and the corresponding sequence of Munc-18-1, -2, and -3 was compared with the National Center for Biotechnology Information (NCBI) BLAST program (National Institutes of Health, Bethesda, MD).

Expression and purification of Munc-18-2 as 6x His tag. Munc-18-2 template cDNA was synthesized with the Titan One Tube RT-PCR kit from mRNA template of IMCD cell origin. The primers used were Munc F (5'-ATG GCG CCC TTG GGG CTG AAG GCG GTG GTG-3') as the forward primer and Munc R (5'-CAG GGC AGG GCT ATG TCC TCC AGC TTC TGA-3') as the reverse primer. mRNA (100-ng template) was added to the reaction mixture, and 40 cycles were run with denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and elongation at 68°C for 3 min. The Munc-18-2 PCR product was ligated with pQE-30UA vector (Qiagen) according to the manufacturer's instructions. The ligated product of pQE-30UA-Munc-18-2 was introduced into Escherichia coli M15 [pREP4] cells and selected on Lennox bacterial (LB)-agar plates containing 25 µg/ml kanamycin and 100 µg/ml ampicillin. Positive clones were analyzed by PCR using pQEF (5'-ATC ACC ATC ACC ATC ACG GAT CCC AC-3') as forward primer and Munc R (5'-CAG GGC AGG GCT ATG TCC TCC AGC TTC TGA-3') as reverse primer. Clones with the positive PCR product were considered to bear the right oriented insert of Munc-18-2 and were chosen for expression of 6x His-Munc-18-2 protein. E. coli transfected with His-Munc-18-2 were grown in LB medium with ampicillin (100 mg/l) until the A600 was 0.6–1.0. Isopropylthiogalactoside (IPTG) was added to a final concentration of 0.1 mM, and the bacteria were incubated for another 3 h in a shaking incubation at 37°C. The bacteria were centrifuged at 4,000 rpm for 8 min. The supernatant was discarded, and the pellet/cells were resuspended in 5 ml/g wet weight of lysis buffer (in mM: 50 NaH2PO4, 300 NaCl, and 10 imidazole, titrated to a pH of 8.0 with NaOH). Lysozyme was added to a concentration of 1 mg/ml, and the mixture was placed on ice for 30 min. The mixture was then centrifuged at 8,000 rpm at 4°C for 25 min. Protease inhibitors were added to the supernatant, and the supernatant was stored at –20°C until the next day. The next day, a Ni-NTA Superflow Column (Qiagen) was rinsed three times with 10 ml of lysis buffer. The lysate was thawed and allowed to filter through the Superflow column at a rate of ~0.5–1 ml/min. The column was washed three times with 10 ml of lysis buffer, followed by three washes with 10 ml of wash buffer (in mM: 50 NaH2PO4, 300 NaCl, and 20 imidazole, titrated to a pH of 8.0 with NaOH). The His-Munc-18-2 was eluted from the beads by washing the Superflow column with an elution buffer (in mM: 50 NaH2PO4, 300 NaCl, and 250 imidazole). The elution was collected and lyophilized. The powder was resuspended in a minimum amount of double-distilled water (ddH2O) and was dialyzed at 4°C against 10 mM Tris titrated to a pH of 7.0 with HCl. The volume of the dialysate used was 300 times that of the elution. After dialysis, the elution was lyophilized a second time. The powder produced was again resuspended in a minimum amount of ddH2O. This was dialyzed a second time to reduce the Tris concentration to 10 mM. Protease inhibitors were added, and the His-Munc-18-2 was stored at 20°C until use.

Expression of Munc-18-2 as a GFP-tagged protein in IMCD cells. Vector:Munc-18-2 template cDNA was synthesized with the Titan One Tube RT-PCR kit from mRNA template of IMCD cell origin. The primers used were Mun FN1 (5'-AGT AGA ATT CCA CCA TGG CGC CCT TGG GGC TGA AGG CGG TG-3') as forward primer and Mun RN1(5'-ACA GGG TAC CAC GGG CAG GGC TAT GTC CTC CAG CTT CTG-3') as reverse primer. One hundred nanograms of template mRNA were added in the reaction, and reverse transcription was performed at 50°C for 30 min; 40 cycles were run with denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and elongation at 68°C for 3 min for PCR.

The PCR product of Munc-18-2 and the vector pECFP-N1 were both cut by EcoRI and KpnI and then ligated with T4 DNA ligase. Ligated product of pECFP N1-munc-18-2 was introduced into E. coli cells and selected on LB-agar plates containing 25 µg/ml kanamycin. Positive clones were analyzed for the correct oriented insert of Munc-18-2 by DNA sequencing and were chosen to transfect the IMCD cells for expression of Munc-18-2-GFP protein.

Expression of Munc-18-2-GFP protein in IMCD cells. IMCD cells were transfected with pECFP N1-munc-18-2 expression plasmid with Lipofectamine. Cells were selected on the DMEM containing 800 µg/ml G418. Clones bearing the antibiotic resistance gene and green fluorescence were chosen and further verified by Western blotting for the expression of Munc-18-2-GFP fusion protein—a band that can be detected by both anti-GFP and anti-Munc-18-2 antibodies with a molecular mass of 87 kDa. The stable cell line with the constant expression of Munc-18-2-GFP fusion protein was used for functional experiments.

Immunoprecipitation. Immunoprecipitation was performed with 800 µg of protein from the syntaxin 1A-transfected IMCD whole cell homogenate, mixed with IP buffer (in mM: 10 Tris·HCl, 2 EDTA, 0.5 DTT, and 0.5 PMSF with 1% SDS, titrated to a pH of 7.5) to a total volume of 1 ml. The homogenate was precleared with 10 µl of nonimmune serum and 20 µl of protein A or L agarose beads. The homogenate, serum, and beads were vortexed and placed on a rocker at 4°C for 30 min. The mixture was centrifuged for 2 min at 4°C and 14,000 rpm. The supernatant was transferred to a clean tube, and 3 µl of primary antibody were added. The mixture was vortexed and placed back on the rocker at 4°C overnight. The next day, 50 µl of either protein A (for rabbit antibodies) or protein L (for mouse antibodies) beads were added. The mixture was vortexed and placed on a rocker at 4°C for 2 h. Afterward, the mixture was centrifuged for 2 min at 14,000 rpm and 4°C. The supernatant was removed, and the beads were washed three times. Between washes, the mixtures were centrifuged for 2 min at 4°C and 14,000 rpm. For the first wash, the beads were resuspended in 750 µl of HS-B (in mM: 20 Tris·HCl, 120 NaCl, 25 KCl, 5 EDTA, 5 EGTA, and 0.1 DTT with 0.1% SDS, 1% deoxycholate, and 0.5% Triton X-100); this was vortexed, and an underlayer of 175 µl of HS-B sucrose (1 M sucrose in HS-B) was placed. The second wash was with 1 ml of a high-salt wash buffer (1 M NaCl in HS-B). The third wash was with 1 ml of a low-salt wash buffer (in mM: 2 EDTA, 0.5 DTT, and 10 Tris·HCl, pH 7.5). Forty microliters of 4x sample buffer were added to the beads after the third wash and then boiled for 5 min, followed by centrifugation for 1 min at 12,000 rpm and room temperature before the supernatant was loaded onto a 10% agarose gel.

Immunoblot. Whole cell homogenate and samples from the three different studies were loaded onto a 10% polyacrylamide-SDS gel and were run under reducing conditions. The protein was electrophoretically transferred to nitrocellulose filters. The filters were blocked with 5% (wt/vol) nonfat powdered milk or bovine serum albumin (BSA) mixed in TBST (50 mM Tris, 141.34 mM NaCl, and 0.2% Tween 20, titrated to a pH of 7.6) for 30–45 min. The filters were incubated with a primary antibody (1:750 for rabbit Munc-18-2, 1:1,000 for mouse Munc-18-2, 1:1,000 for Munc-18-1, 1:1,000 for Munc-18-3, 1:1,000 for syntaxin 1A, 1:1,500 for GP-135, and 1:5,000 for H+-ATPase) in 1% BSA in TBST at 4°C with agitation overnight. The filters were washed three times with TBST and incubated in secondary antibody (horseradish peroxidase antibody, 1:1,000–1:2,000 in TBST with 5% milk or BSA) for 1 h with agitation at room temperature. After three washes with TBST, bound antibody was detected with the enhanced chemiluminescence (ECL) system (Pierce, Rockville, IL).

Apical membrane preparation. IMCD cells transfected with GFP-syntaxin 1A were grown to confluence in 150-mm dishes. The apical membranes were isolated from the IMCD cells by a vesiculation method adapted by our laboratory for polarized epithelial cells (21). The cells were washed once with PBS and twice with either CHB or NHB and were then incubated in 37°C with 5% CO2 for 20 min in CHB or NHB. The CHB or NHB was removed, and 15 ml of vesiculation medium (0.145% paraformaldehyde, 2 mM DTT, and protease inhibitors mixed with either CHB or NHB) were added to each plate. The plates were incubated at 37°C with 5% CO2 for 90 min. The vesiculation medium was removed from the plates, and the plates were washed with either CHB or NHB two or three times. The vesiculation medium and the CHB or NHB washes were centrifuged at 20,000 rpm and 4°C for 90 min. The supernatant was discarded, and the pellet was resuspended in 200 µl of IP buffer per plate of vesicles. A protein assay was performed, and the protein was run on an agarose gel and immunoblotted.

Preparation of rat inner medullary kidney homogenate. The cortices of four rat kidneys were removed, and the medullae were cut into small pieces and rinsed twice with cold PBS. The pieces were placed in a tube with 2.2 ml of homogenate buffer (in mM: 10 HEPES KOH, 1 EGTA, 0.1 EDTA, and 0.3 PMSF with 0.32 M sucrose, titrated to a pH of 7.5), protease inhibitors were added, and the medullae were homogenated manually until dissolved into the buffer. The homogenate was then sonicated on ice with a Bronson sonicator with sixty 1-s pulses. The homogenate was again homogenized manually for another 5 min. The homogenate was centrifuged at 1,200 rpm and 4°C for 10 min to remove the nuclei. The supernatant was saved, Triton X-100 was added to a total concentration of 1%, and DTT was added to a concentration of 1 mM. The mixture was manually homogenated another 50 times and then placed on ice for 60 min. It was centrifuged at 16,000 rpm and 4°C for 30 min. The supernatant was saved and was diluted with homogenate buffer to a protein concentration of 8.65 mg/ml. Protease inhibitors were added, and the inner medullary kidney homogenate was stored at –20°C until use.

Pull-down assay. The pull-down assay was performed with either plain Sepharose beads or Sepharose beads linked to glutathione S-transferase (GST)-syntaxin 1A. The production of GST-syntaxin 1A and its binding to Sepharose beads were described previously (15). The beads were washed three times with cold PBS before use. The beads were incubated with buffer A (in mM: 20 HEPES, 2 CaCl2, 2 MgCl2, and 100 KCl, titrated to a pH of 7.2 with KOH) alone or with varying amounts of His-Munc-18-2 or BSA. Protease inhibitors were added, and the mixture was vortexed and incubated at 4°C on a rocker for 4 h. After this incubation, the beads were washed three times with cold PBS and were mixed with 800 µg of inner medullary kidney homogenate and 10 mM ATP, diluted to 1 ml with buffer A. Protease inhibitors were added. This mixture was incubated at 4°C with agitation overnight. The next morning, the centrifuge tubes were placed on ice and the beads were allowed to fall to the bottom of the tubes. The supernatant was removed, and the beads were washed three times with buffer A, centrifuging at 500 rpm for 2 min between washes. Forty microliters of 4x SDS sample buffer were added to the beads, boiled for 5 min, and centrifuged at 12,000 rpm for 30 s, and the supernatant was loaded onto a 10% agarose gel.

H+-ATPase-mediated proton transport. The rate of Na+-independent pHi recovery (H+-ATPase-mediated H+ transport) was determined in monolayers of wild-type IMCD cells and IMCD cells that were transfected to overexpress Munc-18-2 as previously described by researchers at our laboratory (2, 22). Quiescent IMCD cells grown on glass coverslips were incubated for 1 h at 37°C in NHB containing 10 µM of the acetoxymethyl ester of BCECF (BCECF-AM). The coverslip was then placed in a cuvette containing 1 ml of NHB and secured at a 35° angle to the excitation beam. The monolayer was washed three times with NHB and then suspended in 1 ml of NHB. Fluorescence intensity was measured in a PerkinElmer model LS 650-10 fluorospectrophotometer equipped with a thermostatically controlled (37°C) cuvette holder at excitation wavelengths of 505 and 455 nm with a slit width of 2 nm and an emission wavelength of 560 nm with a slit width of 4 nm. At the end of each experiment, the fluorescence intensity ratio (FIR) was calibrated to pHi with potassium HEPES buffer containing nigericin 10 mg/ml (22). Na+-independent pHi recovery was determined after a 20 mM NH4Cl-induced acid load when incubated in Na+-free CHB.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Identification of isoform of Munc expressed by IMCD cells. To determine which Munc-18 isoform is expressed by wild-type IMCD cells, both RT-PCR (Fig. 1A) and immunoblot analysis (Fig. 1B) were performed. The isoforms of Munc identified in mammalian tissue include Munc-18-1, -2, and -3. RT-PCR identified the expression of Munc-18-2 mRNA by the presence of an appropriate 1,800-bp band (Fig. 1A, lane 3). When this 1,800-bp band was cut out of the gel and analyzed by an automated sequencer, the sequence obtained was 99% identical to that reported for rat Munc-18-2 and <60% identical to either Munc-18-1 or -3. Sequencing errors and/or PCR errors probably account for any differences observed between our product and the published sequence. The presence of neither Munc-18-1 nor Munc-18-3 mRNA could be detected using this method (Fig. 1A, lanes 4 and 5). RT-PCR for actin was also performed to serve as a positive control (Fig. 1A, lane 6).



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Fig. 1. Identification of the isoform of Munc expressed by inner medullary collecting duct (IMCD) cells. To determine which Munc-18 isoform is expressed by IMCD cells, both RT-PCR (A) and immunoblot analysis (B) were performed. The isoforms of Munc identified in mammalian tissue include Munc-18-1, -2, and -3. In A, RT-PCR only identified Munc-18-2 mRNA in IMCD cells by the presence of an appropriate 1,800-bp product (lane 3). Munc-18-3 and Munc-18-1 mRNA could not be detected by this method (lanes 4 and 5). RT-PCR for actin was also performed to serve as a positive control (lane 6). In B, it was also demonstrated by immunoblot analysis of IMCD cell homogenate that only Munc-18-2 can be documented. In lanes 2 and 3 a 67-kDa band was observed when either a primary rabbit (lane 2) or mouse (lane 3) antibody to Munc-18-2 was used. Antibody to Munc-18-1 (lane 1) or Munc-18-3 (lane 4) did not identify protein bands of appropriate molecular mass.

 
In Fig. 1B, it is demonstrated by immunoblot of IMCD whole cell homogenate that only Munc-18-2 can be identified. A 67-kDa band was observed when either a primary rabbit (Fig. 1B, lane 2) or mouse (Fig. 1B, lane 3) antibody to Munc-18-2 was used. Antibody to Munc-18-1 (Fig. 1B, lane 1) or Munc-18-3 (Fig. 1B, lane 4) did not identify protein bands of appropriate molecular mass. Thus IMCD cells primarily express Munc-18-2.

Change in association of Munc-18-2 with syntaxin 1A and syntaxin 1A with H+-ATPase after cell acidification. Acute cell acidification induces regulated exocytic insertion of H+-ATPase-containing vesicles into the apical membrane (20). If the association of syntaxin 1A with Munc-18-2 prevents the targeting-fusion of H+-ATPase-containing vesicles with the apical membrane, cell acidification is likely to reduce the interaction between syntaxin 1A and Munc-18-2. To evaluate this proposal, we examined the coimmunoprecipitation of Munc-18-2 with syntaxin 1A (Fig. 2) and syntaxin 1A with H+-ATPase (Fig. 3). In these studies, IMCD cells transfected with GFP-syntaxin 1A were used. These cells overexpress syntaxin 1A with a GFP tag that enhanced our ability to specifically immunoprecipitate syntaxin. Cell acidification induced by placing cells in a Na+-free acetate-containing buffer reduced the amount of syntaxin 1A coimmunoprecipitated with Munc-18-2 by 51 ± 6% (n = 4; P < 0.05) and the amount of Munc-18-2 coimmunoprecipitated with syntaxin 1A by 59 ± 7% (n = 4; P < 0.05). The amount of Munc or syntaxin 1A directly immunoprecipitated was similar in control and acidified cells (Fig. 2, A and B). In contrast, cell acidification increased the association of syntaxin 1A with H+-ATPase (Fig. 3) by 175 ± 25% (immunoprecipitation of syntaxin 1A) or 164 ± 18% (immunoprecipitation of H+-ATPase) (n = 5; P < 0.05). Qualitatively similar results were obtained when cell acidification was induced by the NH4Cl pulse method or when wild-type cells were used in place of the transfected cell line (data not shown). When normal serum was used instead of specific antibody, we did not detect the presence of either H+-ATPase or syntaxin by immunoblot (Fig. 3, A and B).



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Fig. 2. Reduction in the interaction between Munc-18-2 and syntaxin 1A with cell acidification, a stimulus for H+-ATPase exocytosis. IMCD cells transfected with green fluorescent protein (GFP)-syntaxin 1A were washed with PBS and then exposed to a control solution (NaCl-HEPES buffer, NHB) or to a solution that would acidify the cells (choline chloride-HEPES buffer, CHB). The cells were then harvested, and whole cell homogenate was prepared. Munc-18-2 (A) or syntaxin 1A (B) was immunoprecipitated (IP) from these homogenates, and the immunoprecipitates were subjected to immunoblot (IB) analysis. The immunoprecipitates for Munc-18-2 were blotted for syntaxin 1A and vice versa. The immunoblots were subjected to densitometric analysis; C and D depict the quantitative reduction in the coimmunoprecipitated syntaxin 1A (C) or Munc-18-2 (D) induced by cell acidification. *P < 0.05 (n = 4).

 


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Fig. 3. Enhanced interaction between syntaxin 1A and H+-ATPase with cell acidification. The cells were grown, treated, and harvested as described in Fig. 2. Either syntaxin 1A or the 31-kDa subunit of H+-ATPase was immunoprecipitated or immunoprecipitation was performed with normal rabbit serum and evaluated by immunoblot analysis. The immunoprecipitates for H+-ATPase were blotted for syntaxin 1A (A) and the immunoprecipitates for syntaxin 1A were blotted for H+-ATPase (B). Immunoblot of homogenates immunoprecipitated with normal serum did not identify the 31-kDa H+-ATPase subunit or syntaxin. The immunoblots were subjected to densitometric analysis. C and D depict the quantitative increment in syntaxin 1A coimmunoprecipitated with H+-ATPase and H+-ATPase coimmunoprecipitated with syntaxin 1A. *P < 0.05 (n = 4).

 
Cell acidification decreases amount of Munc-18-2 and increases amount of H+-ATPase in apical membrane of IMCD cells. The dissociation of Munc-18-2 from syntaxin 1A with cell acidification should lead to loss of apical membrane Munc and gain of H+-ATPase. To evaluate this hypothesis we isolated apical membranes from control nontransfected IMCD cells and cells subjected to acute acidification. The apical membranes were isolated from the cells by a method that induces vesiculation and release of the apical membrane from intact cells (1). The isolated apical membrane was evaluated by immunoblot analysis for its content of Munc-18-2 (Fig. 4A), H+-ATPase (Fig. 4B) and GP-135, an apical membrane resident protein (Fig. 4C). With cell acidification, there is a 32 ± 7% (n = 4; P < 0.05) decrease in Munc-18-2, a 246 ± 27% (n = 4; P < 0.05) increase in H+-ATPase, and no significant change in the resident marker GP-135 in the apical membrane. Thus with exocytic insertion of proton pumps into the apical membrane there is a decline in apical membrane Munc-18-2.



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Fig. 4. Cell acidification decreases the amount of Munc-18-2 and increases the amount of H+-ATPase in the apical membrane of IMCD cells. IMCD cells were grown, washed, and treated with NHB (control) or CHB (acid) as described in Fig. 2. The apical membranes were isolated from these cells 20 min after the induction of cell acidification (CHB) by a method that induces release of apical vesicles with exposure of the cells to paraformaldehyde and DTT. The isolated apical membrane was evaluated by immunoblot analysis for its content of Munc-18-2 (A), H+-ATPase (B), and GP-135, an apical membrane resident protein (C). The immunoblots were subjected to densitometric analysis; the quantitative change in apical membrane Munc-18-2 (D), H+-ATPase (E), and GP-135 (F) with cell acidification is displayed. *P < 0.05 (n = 4).

 
Munc-18-2 decreases binding of syntaxin 1A to H+-ATPase. To evaluate the effect of Munc-18-2 on the affinity of syntaxin 1A for H+-ATPase we used an in vitro pull-down assay. In this assay, fusion GST-syntaxin 1A bound to Sepharose beads was used to pull down H+-ATPase from a renal medullary homogenate (Fig. 5). To evaluate the effect of Munc-18-2, the syntaxin 1A-coated beads were preincubated with buffer, 2x or 4x molar excess of His-tag Munc-18-2, or 2–4x albumin (Fig. 5). Compared with control, 2x and 4x Munc-18-2 reduced the amount of H+-ATPase pulled down by syntaxin by 64 ± 9% and 68 ± 11%, respectively (n = 4, P < 0.05; Fig. 5). In contrast, preincubation of the beads with albumin, a protein of molecular mass similar to that of Munc-18-2, had no significant effect on the interaction between syntaxin 1A and H+-ATPase. These results demonstrate that Munc-18-2 interferes with the binding of H+-ATPase by syntaxin 1A.



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Fig. 5. Munc-18-2 decreases the binding of syntaxin 1A to H+-ATPase. To evaluate the effect of Munc-18-2 on the affinity of syntaxin 1A for H+-ATPase we used an in vitro pull-down assay. In this assay, fusion glutathione S-transferase (GST)-syntaxin 1A bound to Sepharose beads was used to pull down H+-ATPase from a renal medulla homogenate. The amount of H+-ATPase pulled down was evaluated by immunoblot analysis (A). To evaluate the effect of Munc-18-2, the syntaxin-1A-coated beads were preincubated with buffer (lane 1), 2x or 4x molar excess of His-tag Munc-18-2 (lanes 2 and 3), or 2x or 4x albumin (lanes 4 and 5). The immunoblots were subjected to densitometric analysis; in B, the quantitative effect of Munc-18-2 on the amount of H+-ATPase pulled down by syntaxin 1A is displayed. *P < 0.05 (n = 4).

 
Effect of PKC inhibitor on Munc-syntaxin dissociation after acute cell acidification. It has been proposed that PKC phosphorylates Munc and that on phosphorylation Munc dissociates from syntaxin (10). This dissociation allows for a change in the tertiary structure of syntaxin such that it can bind other SNAREs. To test the role of PKC in the regulation of Munc-syntaxin complex dissociation and exocytic insertion of H+-ATPase into the apical membrane, we examined the effect of a potent, selective PKC inhibitor (GO-6983; Calbiochem) on the changes induced by cell acidification described above. After acute cellular acidification the decline in the amount of Munc coimmunoprecipitated with syntaxin is blunted by the pretreatment with 20 nM GO-6983 (Fig. 6A). Furthermore, this same inhibitor prevented the reduction in apical membrane Munc and the increment in H+-ATPase observed with cellular acidification (Fig. 6B). To control for other nonspecific effects we also used a second but structurally different PKC inhibitor, 1-(5-iosquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7). H7 at 10 µM produced changes similar to those of GO-6983.



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Fig. 6. Effect of inhibition of PKC on cellular acidification-induced changes in syntaxin-Munc interaction and apical membrane levels of Munc and H+-ATPase. IMCD cells were washed with PBS and then exposed to a control solution, NHB, or to CHB, a solution that acidifies the cells (Acid). One-half of the group of monolayers studied were pretreated 15 min before acidification with a PKC inhibitor (PKCI, GO-6983 or H7; A). After acidification a whole cell homogenate was prepared and syntaxin was immunoprecipitated from an aliquot of this homogenate and immunoblotted for Munc-18-2 (B). Cells were prepared as described in A except that instead of producing a whole cell homogenate, the apical membrane was isolated by the vesiculation method. The isolated apical membrane was immunoblotted for GP-135, Munc-18-2, and the 31-kDa subunit of H+-ATPase. The blots are representative of 3 studies.

 
Effect of overexpression of Munc-18-2 on Na+-independent pHi recovery of IMCD cells after acute acid load. To induce the overexpression of Munc-18-2, cells were transfected with a construct to express a GFP-tagged protein, GFP-Munc-18-2. The transfected cells expressed amounts of wild-type Munc similar to those of nontransfected cells. However, the transfected cells also expressed four times more GFP-Munc than the wild-type protein (Fig. 7A). Although transfected IMCD cells express increased total Munc, the levels of expression of H+-ATPase in wild-type and transfected cells were similar as judged by immunoblot analysis of the 31-kDa subunit in whole cell homogenate. Despite the similar level of expression of proton pump protein, wild-type cells that had lower levels of Munc-18-2 expression had a significantly greater rate of proton pumping after an acute acid load than did cells that overexpressed Munc (Fig. 7B).



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Fig. 7. Effect of overexpression of Munc18-2 on H+-ATPase expression and function. Wild-type IMCD cells and IMCD cells tranfected to express Munc-18-2-GFP were studied in parallel. A: whole cell homogenate immunoblotted with rabbit antibody against Munc-18-2 or against the 31-kDa subunit of the H+-ATPase. B: rate of cytosolic pH recovery in the absence of external Na+ after an acid load induced by a 10 mM NH4Cl pulse.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Acid secretion by the renal collecting duct is mediated by a proton pump (vacuolar H+-ATPase). In {alpha}-intercalated cells and IMCD cells the proton pumps are located in the apical plasma membrane and on subapical vesicle membranes (26). The rate of H+ secretion is regulated primarily by the number of proton pumps inserted into the apical membrane. These pumps are cycled between the apical membrane and a subapical reservoir of vesicles by regulated exo- and endocytosis (6). We have demonstrated that in IMCD cells, regulated exocytosis is activated by a signal cascade that begins with cellular acidification followed by activation of phospholipase C, mediating a rise in inositol trisphosphate and cytosolic calcium (24). The targeting to and fusion of the vesicle with the apical membrane is dependent on intact SNARE proteins (3, 4, 14, 15). With activation of exocytosis a large, clostridial toxin-sensitive complex forms that includes not only the usual proteins found in neurosecretory complexes (syntaxin 1A, SNAP-23, VAMP, NSF, and {alpha}- and {gamma}-SNAP) but also the H+-ATPase itself (4). The H+-ATPase interacts specifically with syntaxin 1A in a VAMP- and SNAP-23-independent manner. This isoform-specific interaction is likely to be an important element in the targeting of the H+-ATPase vesicle to the apical membrane (14).

Munc-18-2 is the Munc isoform expressed in IMCD cells (Fig. 1). Munc has been shown to participate in the regulation of exocytosis in other systems such as Caco-2 cells (18), 3T3-L1 adipocytes (25), adrenal chromaffin cells (9), and skeletal muscle cells (13). When Munc binds to syntaxin, it modifies the conformation of syntaxin to a closed state. In the closed state syntaxin will not bind SNAP-23 or VAMP and thus will not form a fusion complex. On phosphorylation, Munc dissociates from syntaxin and syntaxin can then participate in complex formation (10).

In the present study we have explored the role of Munc-18-2 in regulating the interaction of syntaxin with H+-ATPase. On activation of regulated exocytosis in IMCD cells by cytosolic acidification, the association of Munc-18-2 with syntaxin 1A decreased (Fig. 2) whereas the association between syntaxin 1A and H+-ATPase increased (Fig. 3). These observations are consistent with the proposal that the dissociation of Munc-18-2 from syntaxin increases its affinity for H+-ATPase.

Because syntaxin 1A possesses a membrane-binding domain whereas Munc does not, the dissociation of Munc from syntaxin should result in the dissociation of Munc from the membrane fraction where syntaxin 1A is localized. Indeed, the apical membrane from IMCD cells subjected to cytosolic acidification contained less Munc and increased H+-ATPase but had no measurable change in syntaxin 1A (data not shown) or GP-135 (a resident protein) content (Fig. 4). Thus, in IMCD cells, regulated exocytosis follows a pattern similar to that observed in yeast: on activation of exocytosis, Munc-18 dissociates not only from syntaxin but also from the plasma membrane (7). This dissociation allows syntaxin to bind to the targeting molecule (H+-ATPase) and to form a functional fusion complex.

To directly study the effect of Munc-18-2 on the binding of H+-ATPase to syntaxin 1A we used an in vitro assay. In this in vitro assay, after GST-syntaxin 1A bound to Sepharose beads was exposed to 6x His-Munc-18-2, there was less H+-ATPase pulled down than by syntaxin 1A-bound beads not exposed to fusion Munc (Fig. 5). This provides direct evidence that Munc, in addition to its role in regulating SNARE complex formation (18), also regulates targeting of the vesicles to the apical membrane by altering the affinity of syntaxin 1A for the H+-ATPase. Consistent with this conclusion is our observation that the overexpression of Munc in the intact cells without change in the cellular level of H+-ATPase expression results in a reduction in the rate of proton pumping after an acid load. Thus increased Munc expression in the intact cells appears to reduce functional H+-ATPase in the apical membrane.

Although we have not identified the specific signal that induces the dissociation of Munc from syntaxin in IMCD cells, data from previously published studies provide evidence that phosphorylation is an important regulator of the Munc-syntaxin interaction. It has been documented that when protein phosphatase 1 is inhibited, there is a loss of membrane-associated Munc, which suggests that protein phosphorylation plays a role in the regulation of this cycle (7). The kinase that likely phosphorylates Munc is an isoform of PKC (10). Given that the initiating signal cascade includes activators of PKC (generation of diacylglycerol and a rise in cytosolic calcium) (14), it is reasonable to speculate that phosphorylation of Munc-18-2 by PKC is an important regulator of Munc-18-2 with syntaxin 1A in IMCD cells. Consistent with this proposal is the observation in the current study that two structurally different PKC inhibitors blunt the dissociation of Munc from syntaxin and the amplification of H+-ATPase in the apical membrane after an acute acid load.

Thus we postulate that after acute cell acidification, Munc-18-2 is phosphorylated and, as a consequence, dissociates from syntaxin. The dissociation of Munc-18-2 from syntaxin induces a conformational change in syntaxin that enhances its affinity for H+-ATPase and thereby allows targeting of the H+-ATPase to the apical membrane. Furthermore, this conformational change permits VAMP and SNAP-23 to associate with syntaxin to form the fusion machinery, the SNARE complex.


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 ABSTRACT
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59529.


    ACKNOWLEDGMENTS
 
We appreciate the gift of rabbit antibody to the 31-kDa subunit H+-ATPase from Dr. Dennis Brown.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Schwartz, Evans Biomedical Research Center, 650 Albany St., Boston, MA 02118-2908 (E-mail: jhsch{at}bu.edu)

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. Section 1734 solely to indicate this fact.


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