1Renal Section, Boston University Medical Center, and Departments of 2Medicine, 3Physiology, and 4Pathology, Boston University School of Medicine, Boston, Massachusetts; and 5Department of Nephrology, First Affiliated Hospital, Zhongshan University, Guangzhou, China
Submitted 2 February 2005 ; accepted in final form 27 April 2005
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ABSTRACT |
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soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins; exocytosis; H++ transport
In the kidney, inner medullary collecting duct (IMCD) cells are in part responsible for acid-base homeostasis. H+ secretion by these cells is primarily mediated by an apical membrane H+-ATPase (9). These multisubunit enzymes reside in high density on vesicles that undergo regulated exocytotic insertion into the apical membrane when the cells are acidified and endocytotic retrieval to vesicles when cell pH recovers (1). Previous work from our laboratory (4) showed that SNAREs are involved in the regulation of exocytotic insertion of H+-ATPase-laden vesicles into the apical membrane, affecting the rate of H+ transport by these cells. H+-ATPase forms complexes with SNARE proteins (syntaxin 1A, SNAP23, and VAMP2) both in vitro and in vivo. In addition, the rate of H+ secretion is inhibited either by a cytosolic form of syntaxin 1A or by clostridial toxins that specifically cleave the SNARE proteins syntaxin 1A and SNAP23 (4). These observations confirm that syntaxin 1A and SNAP23 are the t-SNAREs that regulate exocytotic insertion of H+-ATPase containing vesicles. In an in vitro binding assay, solid-phase glutathione S-transferase (GST)-syntaxin fusion proteins were used to pull down syntaxin binding candidates from the kidney homogenate. Several syntaxin isoforms (syntaxin 1A, 1B, 2, 4, 5) were able to form complexes with other SNARE proteins (SNAP23 and VAMP 2) through a conserved H3 domain. However, only syntaxin 1A, also mediated by the H3 domain, bound H+-ATPase (10). On the basis of this latter observation we hypothesize that a specific H+-ATPase binding site is present in the H3 domain of the syntaxin 1A molecule but not in the H3 domain of other syntaxin isoforms.
Syntaxin 1A, like other syntaxin isoforms, contains three domains: an amino-terminal SNARE N, which is believed to have inhibitory regulatory function in SNARE-mediated fusion (17), an H3 domain, which provides one -helix to form the synaptic fusion core complex with the cytoplasmic domain of synaptobrevin II (the second helix of the core) and the amino- and carboxy-terminal domains of SNAP25 or SNAP23 (the third and fourth helices) (6, 18, 23), and a carboxy-terminal transmembrane domain (TMD), which enables syntaxin to anchor to the plasma membrane. The H3 domain is the domain responsible for both SNARE and H+-ATPase binding and consists of amino acid residues 161264 (10).
The purpose of the current study was to identify the regions of the H3 domain of syntaxin 1A responsible for H+-ATPase and SNARE (SNAP23 and VAMP) binding. To this end, a series of pull-down assays were performed with truncated constructs of this H3 domain. In addition, we constructed chimeric forms of syntaxin 1A in which the SNARE N was ligated to fragments of the H3 domain. Our results indicate that the amino-terminal half of the H3 domain of syntaxin 1A has elements that bind SNAP23 and VAMP, whereas the carboxy-terminal half has elements that bind H+-ATPase.
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MATERIALS AND METHODS |
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Kidney Inner Medullary Homogenate
Rat kidney inner medulla was homogenized with a Teflon-coated Dounce homogenizer in HEPES buffer (HB) consisting of (in mM) 20 HEPES, pH 7.5, 100 KCl, 2 MgCl, and 320 sucrose. Just before use, 1 mM DTT, 2 µg/ml pepstatin, 10 µg/l leupeptin, and 0.3 mM phenylmethylsulfonyl fluoride were added. The homogenate was centrifuged at 1,000 g and 4°C for 20 min to obtain a postnuclear supernatant. Triton X-100 was added to the supernatant to a final concentration of 1% and homogenized a second time with a Dounce homogenizer. This 1% Triton X-100-treated homogenate was centrifuged at 13,000 g for 45 min at 4°C. The final supernatant was collected and used for binding assays.
Sample Preparation of Postnuclear, Cytoplasm, and Membrane Fractions
Cultured IMCD cells were homogenized in buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) with a Teflon-coated homogenizer, and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4oC to remove whole cells, nuclei, and mitochondria. This postnuclear supernatant was centrifuged at 200,000 g for 1 h to produce a pellet (membrane fractions enriched for both plasma membranes and intracellular membrane) and a supernatant (cytoplasm fraction).
RNA Purification
Messenger RNA was purified from IMCD cells and rat renal medulla with a commercial poly(A) pure kit (Ambion), and this RNA was analyzed by RT-PCR to identify mRNA expression of syntaxins in the IMCD cells.
RT-PCR
Reverse transcription was accomplished by using 0.1 µg of mRNA as template and oligo(dT)16 as reverse transcriptase primer in a total volume of 20 µl. After incubation at room temperature for 10 min, the reaction temperature was raised to 42°C for 15 min, denatured at 99°C for 5 min, and then cooled to 5°C for 5 min. Upstream and downstream PCR primer (0.1 nmol each) and other components for PCR were then added to a total volume of 100 µl (GeneAmp RNA PCR kit, PerkinElmer Life Sciences). PCR was performed on a MiniCycler with the time and temperature as follows: melting point 95°C, 60 s; annealing 60°C, 30 s; extending 72°C, 120 s for 30 cycles followed by a 420-s extension at 72°C. PCR products were electrophoresed on 1% agarose gels and stained with ethidium bromide.
Expression and Purification of GST-Syntaxin Fusion Protein
GST-syntaxin fusion proteins were expressed in Escherichia coli BL 21 strains with the recombinant pGEX 4T2 and purified as reported previously (10).
Preparation for Syntaxin 1A Truncated and Chimeric Proteins
For truncated syntaxin 1A and syntaxin 1A fragments, rat syntaxin 1A cDNA [Gene Bank Identifier (GI):6665796] fragments were cloned into the plasmid pGEX-5x-1 EcoRI and XhoI cloning site. The primer pairs used in PCR amplification of the cDNA fragments were Syn F1 combined with Syn1264R (1), Syn 1250R (
2), Syn 1235R (
3), Syn 1220R (
4), Syn 1205R (
5), Syn 1190R (
6), and Syn 1161R (
7), Syn F190/F230 combined with Syn1264R (F8/F9), or Syn F190 with Syn 1220R (F10). In making the chimeric molecule, XhoI-cut PCR products of primer pairs Syn F1/Syn1161R, F190N/R264N (C190), or F220N/R264N (C220) were ligated by T4 DNA ligase. Ligated DNA fragments were subjected to EcoRI/NotI digestion and cloned into the corresponding sites of plasmid pGEX-5X-1. Recombinant vectors were transformed into E. coli BL 21 for expression and purification of GST-syntaxin 1A fragment fusion proteins as shown above.
The primer sequences were as follows: Syn F1: cac gaa ttc atg aag gac cga acc cag gag ctc; Syn F190: cac gaa ttc gcc ctc agt gag atc gag acc agg c; Syn F230: cac gaa ttc gac agg atc gag tac aat gtg gaa c; Syn1264R: taa ctc gag ctt cct gcg tgc ctt gct ctg gta c; Syn1250R: taa ctc gag gtc aga cac ggc cct ctc cac gta; Syn 1235R: taa ctc gag gta ctc gat cct gtc aat cat ctc; Syn 1220R: taa ctc gag ggc cat gtc cat gaa cat atc gtg t; Syn 1205R: taa ctc gag caa ctt gat gat ctc act gtg cct; Syn1190R: taa ctc gag ctg ctt cga gat gct gga gtc cat; Syn1161R: taa ctc gag ggt cgt ggt ccg gcc agt gat ctc; F190N: aga ctc gag gcc ctc agt gag atc gag acc ag; F220N: aga ctc gag atg ctg gtg gag agc cag ggg ga; R264N: taa gcg gcc gcc ttc ctg cgt gcc ttg ctc tg.
All the primer DNA-oligo samples were synthesized by Integrated DNA Technologies. Constructs were sequenced to ensure that each construct was correct.
Cloning of Syntaxin 1A Chimeric Construct C220 into Pc DNA3.1/NT-GFP-TOPO for Expression of Green Fluorescent Protein-Syntaxin 1A Chimeric Protein
The primer pair 5'-atg aag gac cga acc cag gag-3' (upstream) and 5'-cta tgc ctt gct ctg gta ctt g-3' (downstream) was used in the PCR reaction to amplify the template of the previous recombinant construct C220. DNA fragments from PCR coding the chimerical SNARE N (aa 1161) and residues 220264 of syntaxin 1A were ligated into the pc DNA3.1/NT-GFP-TOPO vector (Invitrogen). The final clone of the recombinant chimeric syntaxin 1A vector was sequenced to ensure that the construct was correct.
Transfection and Selection of Stable Transfected Cell Lines
Purified recombinant plasmid pc DNA3.1/NT-GFP-syntaxin 1A C220 was used to transfect IMCD cells by calcium phosphate precipitation. Transfected cells were selected in medium containing 800 µg/ml G418. Surviving cells were further selected to yield monoclonal cell lines, using the method of limited dilution. Stable cell lines that expressed GFP-syntaxin 1A C220 protein were confirmed by directly viewing the green fluorescent protein (GFP) fluorescence, immunostaining, and immunoblotting with GFP antibody. Positive clones were used for immunoprecipitation experiments and functional assay.
In Vitro Pull-Down Binding Assay
GST-syntaxin isoform fusion proteins (25 µg) fixed on beads and 800 µg of kidney homogenate protein were incubated at 4°C overnight in HB buffer (20 mM HEPES, pH 7.2, 100 mM KCl, and 2 mM MgCl2, containing 1 mM DTT, 2 µg/ml pepstatin, 10 µg/ml leupeptin, and 0.3 mM phenylmethylsulfonyl fluoride added just before use) with gentle agitation. Proteins bound to beads were recovered by centrifuging at 500 g for 5 min and washed three times with buffer (HB containing 0.5% Triton X-100). The protein attached to the washed beads was solubilized in an equal volume of 2x SDS sampler buffer (final concentration: 50 mM Tris, pH 6.8, 100 mM DTT, 10% glycerin, 2% SDS, and 0.1 bromphenol blue), heated for 5 min at 95°C, and then subjected to SDS-PAGE and immunoblot analysis.
Immunoprecipitation
Cell homogenates [800 µg in 1 ml immunoprecipitation (IP) buffer] were precleared with 30 µl of suspended protein A beads plus 5 µl of normal rabbit serum for 1 h at 4°C. Precleared supernatants were incubated with primary rabbit antibody (1:200) for 2 h at 4°C, followed by addition of 30 µl of protein A beads, and incubated overnight at 4°C with gentle agitation. Bound proteins were washed three times with IP buffer, dissolved in 30 µl of 2x SDS sample buffer, and subjected to immunoblot analysis.
Immunoblots
After SDS-PAGE, the proteins were transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline-Tween 20 and incubated overnight at 4°C with primary antibody. After incubation with appropriate secondary antibodies conjugated to horseradish peroxidase, the blots were developed by the enhanced chemiluminescence method. The film was scanned and then subjected to band densitometry and quantification (NIH Image).
Antibodies used in this study are as follows: rabbit antibody to the 31-kDa subunit of the H+-ATPase (gift of Dr. Dennis Brown, Harvard University) and rabbit polyclonal antibody to the 116-kDa subunit (Synaptic Systems), rabbit antibody to SNAP23 (Synaptic Systems), mouse monoclonal antibody to synaptobrevin/VAMP (Synaptic Systems), and mouse monoclonal antibody to syntaxin-1A, clone HPC-1 (Sigma). The secondary antibodies for the immunoblot were peroxidase-conjugated goat anti-rabbit and anti-mouse IgG and were purchased from Jackson ImmunoResearch.
H+-ATPase-Mediated Proton Transport
The rate of Na+-independent intracellular pH (pHi) recovery (H+-ATPase-mediated H+ transport) was determined in monolayers of wild-type IMCD cells and transfected IMCD cells that express GFP-Syntaxin 1A C220 chimeric protein. Quiescent IMCD cells grown on glass coverslips were incubated for 1 h at 37°C in Na+-HEPES buffer (NHB) [in mM: 110 NaCl, 50 HEPES acid, 5 KCl, 1 MgCl2, 5 KH2PO4, 1 CaCl2, and 5 glucose (pH 7.2)] containing 10 µM BCECF-AM. 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 5 nm and an emission wavelength of 560 nm with a slit width of 10 nm. At the end of each experiment, the fluorescence intensity ratio was calibrated to pHi with potassium HEPES buffer containing 10 mg/ml nigericin. Na+-independent pHi recovery after a 20 mM NH4Cl-induced acid load when incubated in choline-HEPES buffer [in mM: 110 choline chloride, 50 HEPES acid, 5 KCl, 1 MgCl2, 5 KH2PO4, 1 CaCl2, 5 glucose, and 10 potassium acetate (pH 7.2)] was determined as previously described (2, 21).
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RESULTS |
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To demonstrate that the proton-secreting IMCD cells express the t-SNARE syntaxin 1, RT-PCR and Western blot analysis were performed. Positive PCR products with the expected size for syntaxin 1A (882 bp) and syntaxin 1B(984 bp) were obtained by using the IMCD cell mRNA as a template for RT and the appropriate primers for PCR amplification (Fig. 1). These data demonstrate that syntaxin 1 (1A and 1B) is expressed in the IMCD cell at the mRNA level and confirm our earlier observation that syntaxin 1A is expressed by this cell line (11).
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Syntaxin 1A is the main t-SNARE on the presynaptic membrane of neurons. It mediates the targeting and fusion of synaptic vesicles and therefore transmitter release. The plasma membrane localization of syntaxin 1A in the IMCD cell suggests a role for this t-SNARE in mediating the fusion of the cellular vesicles such as H+-ATPase-coated vesicles (10). We showed previously that protein-protein interactions between syntaxin 1A and H+-ATPase occur in both in vitro and in vivo systems (10). It also appears that the H3 domain in syntaxin 1A is critical to this interaction (10).
The SNARE theory for synaptic fusion during exocytosis is based mainly on the fact that the SNARE core complex formation occurs when a t-SNARE pairs with a v-SNARE. Our previous in vitro studies (10) showed that although GST-syntaxin isoforms 1A, 1B, 2, 4, and 5 all form SNARE complexes with SNAP23 and VAMP, only syntaxin 1A, through its H3 domain, not only binds to SNAP23 and VAMP but also binds to H+-ATPase. From this observation we infer that syntaxin 1A encodes a specific binding site in its H3 domain for H+-ATPase. To explore this possibility, a series of truncation and chimeric experiments were performed.
Effect of truncation of H3 domain on binding.
Syntaxins consist of three domains: SNARE N, SNARE binding (H3), and carboxy-terminal TMD. The TMD does not contribute to the binding of syntaxin 1A to either H+-ATPase or SNARE proteins (SNAP23 and VAMP) (Figs. 3 and 4). GST-syntaxin 1A 1264 (1, TMD-deleted construct) bound H+-ATPase and SNARE proteins to the same extent as GST-full-length syntaxin 1A (1A). Deleting the first 15 amino acids from the carboxy-terminal end of the H3 domain (construct
2: syntaxin 1A 1250) did not impair the ability of syntaxin 1A to bind either H+-ATPase or SNARE proteins, but when residues 220250, especially 235250, were deleted (
3,
4), syntaxin 1A lost its ability to complex with H+-ATPase while it retained its ability to complex with SNAP23 and VAMP. Therefore, it is likely that residues 220250 are the subdomain for H+-ATPase binding. Further deletion of the H3 domain (
5,
6, and
7) severely impaired the remaining ability to bind SNAP23 and VAMP, suggesting that residues 161235, especially residues 190220, are the segment responsible for SNAP23 and VAMP binding.
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To document in vivo that the syntaxin 1A H3 subdomain binds the H+-ATPase we evaluated our ability to coimmunoprecipitate the H+-ATPase along with the H3 chimera from homogenate derived from cells that were transfected to express this fusion protein. With GFP antibody to immunoprecipitate the GFP-syntaxin 1A C220 chimera, the 31-kDa subunit of H+-ATPase was detected by immunoblot in the immunoprecipitate. This observation indicates that protein-protein interaction takes place between GFP-syntaxin 1A C220 and H+-ATPase in cells (Fig. 8A).
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DISCUSSION |
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Our previous observation that many syntaxin isoforms can form a SNARE core complex but only syntaxin 1 interacts with H+-ATPase suggests that the SNARE binding domain is conserved throughout the syntaxin family whereas the H+-ATPase binding domain is specific for a single isoform, syntaxin 1. This assumption was examined by performing a BLAST sequence analysis of the NCBI protein data base with syntaxin 1A residues 190220, the SNARE binding domain, and with residues 235250, the H+-ATPase binding domain. The peptide sequence of the SNARE binding domain of syntaxin 1A had homology with H3 domain sequences in syntaxin 1B (96%), 2 (93%), 3 (93%), 4 (80%), and 11 (86%). On the other hand, the peptide sequence of the H+-ATPase binding domain of syntaxin 1A had homology with only syntaxin 1B. Thus the H+-ATPase binding domain is a unique sequence in syntaxin 1.
It was reported that the amino-terminal domain of syntaxin (SNARE N) has a negative regulatory function in the fusion of the phospholipid vesicles (17). Removal of the SNARE N domain of syntaxin accelerated the rate of vesicle fusion but did not affect the rate of v-/t-SNARE assembly (17). In our study, the fragment of syntaxin H3 SNARE binding domain (F8) bound SNAP23 and VAMP to an equivalent degree whether or not it was fused with the SNARE N domain (C190). This demonstrates that SNARE N has no effect on the rate of SNARE core complex formation. However, the H3 fragment encoding the H+-ATPase binding domain (F8 and F9) formed a complex with its partner (H+-ATPase) only when this fragment was ligated to the SNARE N (C220 or C190). These results suggest that SNARE N might play a critical role in facilitating the syntaxin 1A-H+-ATPase interaction. The exact mechanism requires further study.
SNARE proteins may confer the specificity of vesicle targeting, docking, and tethering as well as fusion. However, more recent functional data favor a role only in fusion (7). The interaction among SNARE proteins to form a SNARE complex appears to be critical for synaptic fusion. However, a novel binding partner of the syntaxin family, taxilin, has been recently implicated in intracellular vesicle traffic. Although its exact binding property for the syntaxin family is uncertain, it has been shown that this protein interacts with syntaxin family members without forming the SNARE complex (16). This binding pattern may provide a new way for syntaxin to play a role in the specificity of vesicle targeting and docking. The fact that syntaxin 1A, through a domain not implicated in SNARE binding, interacts with H+-ATPase may be one of the examples in which interactions between cargo protein of the vesicle (H+-ATPase) and the target SNARE protein provide the information for specificity of targeting and/or docking. We propose that the binding of syntaxin 1A to H+-ATPase pilots the H+-ATPase-laden vesicles to the apical membrane in IMCD cells. After targeting of the vesicles, subsequent cascades may take place to form a SNARE complex, leading to the fusion between the apical membrane and the vesicle membrane resulting in the insertion of H+-ATPase into the apical membrane.
Syntaxin 1A was initially believed to be involved only in neurotransmitter release at synaptic clefts in the central nervous system (5). In recent years, evidence has accumulated documenting that syntaxin 1A also plays various roles in a variety of cells in other organs. It interacts with the cystic fibrosis transmembrane conductance regulator Cl channel, inhibiting its activity by means of hydrophilic interactions that are distinct from the hydrophobic interactions that mediate SNARE complex formation (8, 1214). Syntaxin 1A was also reported to increase the net externalization of the epithelial Na+ channel (ENaC) complex through the SNARE machinery (19, 20) and to interact with the LD subtype of voltage-gated Ca2+ channels in pancreatic -cells (25). These data, along with our previous results that indicate that the exocytic insertion of H+-ATPase into the plasma membrane involves SNARE fusion machinery (3, 4, 10, 15) and the finding in this study that syntaxin 1A interacts with H+-ATPase in an isoform-specific manner, indicate that syntaxin 1A has wide diversity in both tissue distribution and function.
In IMCD cells, the interaction between syntaxin 1A and H+-ATPase involves both non-SNARE and SNARE proteins to mediate and regulate the insertion of the proton pump into the apical plasma membrane of these cells. The initial coupling between syntaxin 1A and H+-ATPase through the specific H+-ATPase binding domain takes place by a non-SNARE mechanism, whereas subsequent events (fusion) may involve the formation of SNARE core complex between t-SNARE, syntaxin 1A, and SNAP23 in the plasma membrane and v-SNARE and VAMP from the H+-ATPase-containing vesicles. Thus syntaxin 1A subserves a dual role in the process of H+-ATPase apical membrane amplification: targeting of the H+-ATPase to the apical membrane via a specific cassette in the H3 domain and fusion via a second H3 cassette that mediates SNARE interaction.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Alexander EA, Shih T, and Schwartz JH. H+ secretion is inhibited by clostridial toxins in an inner medullary collecting duct cell line. Am J Physiol Renal Physiol 273: F1054F1057, 1997.
3. Banerjee A, Li G, Alexander EA, and Schwartz JH. Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells. Am J Physiol Cell Physiol 280: C775C781, 2001.
4. Banerjee A, Shih T, Alexander EA, and Schwartz JH. SNARE proteins regulate H+-ATPase redistribution to the apical membrane in rat renal inner medullary collecting duct cells. J Biol Chem 274: 2651826522, 1999.
5. Bennett MK. SNAREs and the specificity of transport vesicle targeting. Curr Opin Cell Biol 7: 581586, 1995.[CrossRef][ISI][Medline]
6. Burkhard P, Stetefeld J, and Strelkov SV. Coiled coils: a highly versatile protein folding motif. Trends Cell Biol 11: 8288, 2001.[CrossRef][ISI][Medline]
7. Chen YA and Scheller RH. SNARE-mediated membrane fusion. Nat Rev Mol Cell Biol 2: 98106, 2001.[CrossRef][ISI][Medline]
8. Ganeshan R, Di A, Nelson DJ, Quick MW, and Kirk KL. The interaction between syntaxin 1A and cystic fibrosis transmembrane conductance regulator Cl channels is mechanistically distinct from syntaxin 1A-SNARE interactions. J Biol Chem 278: 28762885, 2003.
9. Gluck SL, Lee BS, Wang SP, Underhill D, Nemoto J, and Holliday LS. Plasma membrane V-ATPases in proton-transporting cells of the mammalian kidney and osteoclast. Acta Physiol Scand Suppl 643: 203212, 1998.[Medline]
10. Li G, Alexander EA, and Schwartz JH. Syntaxin isoform specificity in the regulation of renal H+-ATPase exocytosis. J Biol Chem 278: 1979119797, 2003.
11. Li X, Low SH, Miura M, and Weimbs T. SNARE expression and localization in renal epithelial cells suggest mechanism for variability of trafficking phenotypes. Am J Physiol Renal Physiol 283: F1111F1122, 2002.
12. Naren AP, Di A, Cormet-Boyaka E, Boyaka PN, McGhee JR, Zhou W, Akagawa K, Fujiwara T, Thome U, Engelhardt JF, Nelson DJ, and Kirk KL. Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl currents. J Clin Invest 105: 377386, 2000.
13. Naren AP, Nelson DJ, Xie W, Jovov B, Pevsner J, Bennett MK, Benos DJ, Quick MW, and Kirk KL. Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms. Nature 390: 302305, 1997.[CrossRef][ISI][Medline]
14. Naren AP, Quick MW, Collawn JF, Nelson DJ, and Kirk KL. Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein-protein interactions. Proc Natl Acad Sci USA 95: 1097210977, 1998.
15. Nicoletta JA, Ross JJ, Li G, Cheng Q, Schwartz J, Alexander EA, and Schwartz JH. Munc-182 regulates exocytosis of H+-ATPase in rat inner medullary collecting duct cells. Am J Physiol Cell Physiol 287: C1366C1374, 2004.
16. Nogami S, Satoh S, Nakano M, Shimizu H, Fukushima H, Maruyama A, Terano A, and Shirataki H. Taxilin; a novel syntaxin-binding protein that is involved in Ca2+-dependent exocytosis in neuroendocrine cells. Genes Cells 8: 1728, 2003.
17. Parlati F, Weber T, McNew JA, Westermann B, Sollner TH, and Rothman JE. Rapid and efficient fusion of phospholipid vesicles by the alpha-helical core of a SNARE complex in the absence of an N-terminal regulatory domain. Proc Natl Acad Sci USA 96: 1256512570, 1999.
18. Poirier MA, Xiao W, Macosko JC, Chan C, Shin YK, and Bennett MK. The synaptic SNARE complex is a parallel four-stranded helical bundle. Nat Struct Biol 5: 765769, 1998.[CrossRef][ISI][Medline]
19. Qi J, Peters KW, Liu C, Wang JM, Edinger RS, Johnson JP, Watkins SC, and Frizzell RA. Regulation of the amiloride-sensitive epithelial sodium channel by syntaxin 1A. J Biol Chem 274: 3034530348, 1999.
20. Saxena S, Quick MW, Tousson A, Oh Y, and Warnock DG. Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel. J Biol Chem 274: 2081220817, 1999.
21. Selvaggio AM, Schwartz JH, Bengele HH, Gordon FD, and Alexander EA. Mechanisms of H+ secretion by inner medullary collecting duct cells. Am J Physiol Renal Fluid Electrolyte Physiol 254: F391F400, 1988.
22. Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, and Rothman JE. SNAP receptors implicated in vesicle targeting and fusion. Nature 362: 318324, 1993.[CrossRef][ISI][Medline]
23. Sutton RB, Ernst JA, and Brunger AT. Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca+2-independent snare complex interaction. J Cell Biol 147: 589598, 1999.
24. Ungar D and Hughson FM. SNARE protein structure and function. Annu Rev Cell Dev Biol 19: 493517, 2003.[CrossRef][ISI][Medline]
25. Yang R, Puranam RS, Butler LS, Qian WH, He XP, Moyer MB, Blackburn K, Andrews PI, and McNamara JO. Autoimmunity to munc-18 in Rasmussens encephalitis. Neuron 28: 375383, 2000.[CrossRef][ISI][Medline]
26. Zhong P, Chen YA, Tam D, Chung D, Scheller RH, and Miljanich GP. An alpha-helical minimal binding domain within the H3 domain of syntaxin is required for SNAP-25 binding. Biochemistry 36: 43174326, 1997.[CrossRef][ISI][Medline]