Antigen retrieval reveals widespread basolateral expression of syntaxin 3 in renal epithelia

Sylvie Breton1, Takeaki Inoue2, Mark A. Knepper2, and Dennis Brown1

1 Program in Membrane Biology and Renal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and 2 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a key role in docking and fusion of intracellular transport vesicles and may regulate apical and basolateral membrane protein delivery in epithelial cells. In a previous study, syntaxin 3 (a target SNARE) protein was detectable in the kidney only in intercalated cells. We now report a more widespread distribution of syntaxin 3 in a variety of renal epithelial cells after antigen retrieval. Sections of rat kidney were treated with SDS and incubated with antisyntaxin 3 antibodies. Strong basolateral membrane staining was seen in descending and ascending thin limbs of Henle, thick ascending limbs of Henle, the macula densa, distal and connecting tubules, and all cells of the collecting duct including A- and B-intercalated cells. The papillary surface epithelium and the transitional epithelium of the ureter were also stained, but proximal tubules were negative. Western blotting revealed a strong signal at 37 kDa in all regions, and the antigen was restricted to membrane fractions. SDS treatment was not necessary to reveal syntaxin 3 in intercalated cells. These data show that syntaxin 3 might be involved in basolateral trafficking pathways in most renal epithelial cell types. The exclusive basolateral location of syntaxin 3 in situ, however, contrasts with the apical location of this SNARE protein in some kidney epithelial cells in culture.

soluble N-ethylmaleimide-sensitive factor attachment protein receptor; urinary tubule; basolateral membrane


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE GENERATION AND MAINTENANCE of distinct membrane domains in all cell types depend on the fidelity of vesicle trafficking and fusion processes. Over the past few years, it has become clear that fusion between donor and target membranes is regulated by a complex machinery that involves the interaction of membrane-associated and soluble proteins. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of transmembrane proteins that are found on target (t-SNAREs) and vesicle membranes, and it is believed that specific interactions between cognate SNAREs represent an important step in vesicle docking and fusion (17, 18). More recent data have resulted in modifications of the original SNARE hypothesis, and the precise way in which SNAREs and other accessory proteins interact before and during vesicle-target membrane fusion is the subject of intense investigation (14, 21, 22). The series of protein-protein interactions envisaged in the SNARE hypothesis provides an elegant way in which the distinct protein and lipid fingerprint of functionally distinct membranes can be tightly controlled and modulated.

The syntaxins represent a major class of t-SNAREs (1, 4). Their intracellular distribution has been examined in different epithelial cells to determine whether they might play some role in generating the polarized epithelial cell phenotype. The function of the urinary tubule, for example, depends on the apical and basolateral expression of a multitude of different transport systems, all of which must be correctly targeted to ensure the vectorial movement of fluid, ions, and other molecules across the lumen-to-blood epithelial interface (8, 9). In Madin-Darby canine kidney (MDCK) cells, derived from dog kidney, transfected syntaxin 3 was restricted to the apical plasma membrane and syntaxin 4 to the basolateral membrane, while syntaxin 2 was present on both membrane domains (11). In contrast, studies on endogenous syntaxins in renal epithelia in situ revealed syntaxin 4 on the apical plasma membrane of principal cells and syntaxin 3 on the basolateral membrane of intercalated cells (13). However, this report also showed that syntaxin 3 mRNA was expressed in a cellular pattern that was more widespread than detectable protein (13). In the present study, therefore, the location of syntaxin 3 protein in the kidney was reexamined using an antigen retrieval technique that has been previously described (7). The results show that, although no apical localization of syntaxin 3 became apparent after antigen retrieval, basolateral syntaxin 3 was detectable in many more segments of the urinary tubule than previously reported. These data indicate that syntaxin 3 may have a more ubiquitous role in basolateral trafficking in the kidney than was originally envisaged.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Polyclonal Antibodies

The production and characterization of polyclonal antibodies against syntaxin 3 have been described previously (13). Briefly, a 23-amino acid peptide corresponding to the NH2 terminus of syntaxin 3 (KDRLEQLKAKQLTQDDDTDEVEC) was coupled to keyhole limpet hemocyanin and used to immunize rabbits by a standard protocol. The antisera from two rabbits were affinity purified with the immunizing peptide using the SulfoLink system (Pierce, Rockford, IL). Preimmune serum was purified on a protein A affinity column (Pierce) and was used for control incubations. Some incubations were also performed using the affinity-purified antibody that had been preabsorbed with the immunizing peptide.

Preparation of Membrane Pellets From Rat Kidneys

Membranes from the cortex, outer medulla, and inner medulla were prepared from rat kidneys, as previously described (13). Adult Sprague-Dawley rats were killed by decapitation, and tissues were homogenized in ice-cold homogenization solution (250 mM sucrose and 10 mM triethanolamine) with protease inhibitors. Plasma membrane-rich pellets were obtained by centrifugation, and the 17,000-g pellet was used for Western blotting. For the inner medulla, the 17,000-g supernatant was further centrifuged at 200,000 g to obtain a pellet of low-density microsomes, as previously described (13).

Electrophoresis and Western Blotting

Membranes were solubilized at 60°C for 15 min in Laemmli buffer, and proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 12% minigels. After transfer of the proteins, nitrocellulose membranes were blocked with blotting buffer (150 mM NaCl, 50 mM NaH2PO4, and 0.05% Tween 20, pH 7.5) that contained 5% nonfat dried milk and were incubated with affinity-purified antisyntaxin 3 antibody (0.5 µg/ml). Donkey anti-rabbit IgG coupled to horseradish peroxidase was then applied at a concentration of 0.16 µg/ml. Staining was revealed by enhanced chemiluminescence (LumiGlo, Kirkegaard and Perry Laboratories, Gaithersburg, MD).

Experimental Animals and Tissue Fixation

Experiments were conducted using mature (300-350 g) male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA). Rats were anesthetized using a pentobarbital sodium (50 mg/ml) injection of 0.15 ml/100 g body wt. The kidneys were fixed via left ventricle cardiac perfusion with 150 ml of Hanks' balanced salt solution followed by paraformaldehyde-lysine-periodate (PLP) fixation. The original PLP recipe (15) was modified to increase the paraformaldehyde concentration from 2% to 4%. The kidneys were removed after 5 min of perfusion in PLP and placed in PLP fixative at room temperature for a further 4-6 h or overnight at 4°C. Tissue was washed three times in PBS and kept at 4°C in PBS containing 0.02% sodium azide before use.

Antigen Retrieval and Immunofluorescence

PLP-fixed tissues were cryoprotected in a solution of 30% sucrose in PBS for >= 2 h at room temperature. They were embedded in OTC Compound 4583 (Tissue-Tek, Miles) and mounted on a cutting block. After freezing in a Reichert Frigocut microtome, the tissue was cut into 3- to 4-µm sections, which were picked up on microscope slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA).

For immunostaining, sections were hydrated for 5 min in PBS and pretreated for 5 min with 1% SDS in PBS, an antigen retrieval technique that we have previously described (7). Slides were washed three times in PBS for 5 min each time and then preincubated in 1% BSA in PBS-0.02% sodium azide for 15 min. Sections were incubated in primary antisyntaxin antiserum (5 µg/ml) for 90 min at room temperature, washed twice for 5 min in high-salt PBS (2.7% NaCl) to reduce nonspecific staining, and washed once in normal PBS. Sections were then incubated for 1 h with secondary antibody, goat anti-rabbit IgG coupled to FITC (Jackson Immunologicals, West Grove, PA), and they were again washed as described above. Double labeling was performed by subsequent incubation of some sections with a chicken anti-H+-ATPase antibody and donkey anti-chicken IgG conjugated to Cy3 (Jackson Immunologicals). The chicken antibody was raised against the COOH terminus of the 31-kDa H+-ATPase subunit (E subunit), and its use has been described previously (3). The slides were mounted in Vectashield (Vector Labs, Burlingame, CA) diluted 1:1 with Tris buffer, pH 8.5, and examined using a Nikon E800 epifluorescence microscope. Syntaxin 3 staining was photographed using TMAX 400 film push-processed to 1600 ASA. Double-stained sections were digitally imaged using a Hamamatsu Orca charge-coupled device camera and IPLab Spectrum software (Scanalytic, Vienna, VA). Final pseudocolored images were imported into and printed from Adobe Photoshop.

Control incubations were performed using preimmune IgG or antibodies that had been preabsorbed with the syntaxin 3 immunizing peptide before the first incubation step. All control incubations were negative, illustrating the specificity of the staining patterns reported here.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Western Blotting of Kidney Membranes

Syntaxin 3 was detected by Western blotting in all kidney regions (Fig. 1A). The recognized band was at 37 kDa. In contrast to previous data (13), an additional weaker 67-kDa band was not detected by the antisyntaxin antibody in the present series of experiments. In whole membranes, the strongest expression was observed in the kidney cortex and outer medulla. A weaker band was present in membranes from the inner medulla. When inner medullary membranes were fractionated further, the 37-kDa band was detectable only in the "plasma membrane-enriched" fraction, and not in the 200,000-g pellet, which is composed of smaller vesicles and light endosomes (Fig. 1B).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 1.   A: immunoblotting of syntaxin 3 in total membrane preparations from different kidney regions. A strong band at 37 kDa is seen in cortical and outer medullary membranes, and a weaker band is seen in membranes from the inner medulla. No staining was seen when preimmune IgG was used. B: enrichment of the 17,000-g membrane pellet in syntaxin compared with the whole homogenate from the inner medulla; the 200,000-g light endosomal fraction contained no detectable syntaxin 3.

Immunofluorescence Staining of Kidney Sections

Cortex. In the kidney cortex, staining was bright in cortical collecting ducts, connecting segments, and cortical thick ascending limbs (Fig. 2). No specific staining was detectable in proximal convoluted tubules or in the glomerulus. Staining in collecting ducts and connecting segments was basolateral, and all cell types were stained. In the cortical thick ascending limb, staining was also basolateral. Double staining with antibodies against H+-ATPase showed that the basolateral plasma membranes of A- and B-intercalated cells were stained for syntaxin 3. Staining was stronger in B-intercalated cells than in cortical A-intercalated cells (Fig. 3), and syntaxin 3 staining of B-cells partially overlapped the basolateral H+-ATPase staining pattern.


View larger version (95K):
[in this window]
[in a new window]
 
Fig. 2.   Immunofluorescence localization of syntaxin 3 in rat kidney cortex after antigen retrieval with SDS. A: basolateral staining in cortical thick ascending limbs (TAL) and much stronger staining in connecting segments (CNT) and cortical collecting ducts (CCD). Proximal tubules (PT) and glomeruli (G) show no specific staining. B: higher magnification of a CCD; all epithelial cells in this segment show basolateral labeling for syntaxin 3. Scale bars: 35 µm (A), 17.5 µm (B).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3.   Double-immunofluorescence staining of rat kidney cortex for syntaxin 3 (A, red) and the 31-kDa subunit of H+-ATPase (B, green). In a cortical collecting duct, many cells, including A-intercalated cells (a, identified by an exclusive apical H+-ATPase staining) and B-intercalated cells (b, identified by basolateral and/or bipolar H+-ATPase staining), show basolateral syntaxin 3 staining. The strongest syntaxin 3 staining was detected in B-intercalated cells. C: merged image in which colocalization of syntaxin 3 and H+-ATPase appears as a yellow basolateral staining. Proximal tubules (PT) show an apical punctate staining for H+-ATPase (B), but syntaxin 3 staining is not detectable (A). Scale bar, 10 µm.

Outer medulla. In the outer medulla, collecting ducts, thick ascending limbs of Henle, and thin limbs of Henle were brightly positive (Figs. 4 and 5A). In all cases, the staining was basolateral. In the case of thin limbs, the resolution was insufficient to determine the polarity of the staining. All collecting duct cells were stained with an approximately equal intensity. Stained thin descending limbs were identified by finding regions of transition between unstained proximal tubule S3 segments (Fig. 4A) and the descending thin limb. Stained thin ascending limbs were identified at regions of transition with the thick ascending limb (Fig. 4B). In addition, the thin ascending limb staining had a punctate appearance because of the highly interdigitated nature of the epithelium in this tubule region.


View larger version (127K):
[in this window]
[in a new window]
 
Fig. 4.   Syntaxin 3 staining at the junction between outer and inner stripes of the outer medulla (A) and the inner stripe and the inner medulla (B). A: transitions (arrows) between negative S3 proximal tubule (PT) segments and positive thin descending limbs of Henle (TDL) and strong basolateral staining of thick ascending limbs of Henle (TAL). B: transitions (arrows) between the ascending thin limbs (ATL) and TAL at the border between the outer medulla (inner stripe) and the inner medulla. ATL have a punctate appearance, reflecting the highly interdigitated nature of this epithelium in cross sections. The basolateral membrane of all cells in collecting ducts (CD) is brightly stained. Scale bar, 10 µm.



View larger version (78K):
[in this window]
[in a new window]
 
Fig. 5.   A: syntaxin 3 staining of the inner stripe of the outer medulla showing basolateral staining of thick ascending limbs of Henle (TAL), staining of thin descending limbs of Henle (TDL), and strong staining of all cells in collecting ducts (CD). B and C: outer third of the inner medulla after SDS pretreatment of tissue sections and without SDS pretreatment, respectively. Without SDS, the strongest staining is seen in CD intercalated cells (C, arrows), as previously described. These cells were identified by double staining sections with anti-H+-ATPase antibodies (not shown). After SDS pretreatment, strong basolateral staining is seen in all CD cells. Thin limbs of Henle (TLH) are also positive in this region. Scale bar, 20 µm.

Inner medulla. In the outer third of the inner medulla, the basolateral membranes of all collecting duct epithelial cells were brightly stained (Fig. 5B). Thin limbs of Henle were weakly positive. For comparative purposes, adjacent sections were stained without SDS pretreatment. As described previously, the only cells showing positive staining under these conditions were intercalated cells (Fig. 5C). However, the intercalated cell staining was somewhat weaker in the absence of SDS. In the middle third and the tip of the papilla, bright basolateral staining of principal cells was observed (Fig. 6A). Principal cells in these regions were weakly stained, even in the absence of SDS pretreatment (Fig. 6B). In addition, papillary surface epithelial cells also showed a marked basolateral staining for syntaxin 3, as did the cells of the transitional epithelium of the ureter (Fig. 6A).


View larger version (108K):
[in this window]
[in a new window]
 
Fig. 6.   A: tip of the papilla (inner medulla) showing strong basolateral staining of collecting duct (CD) principal cells after SDS pretreatment. The basolateral membrane of papillary surface epithelial cells (PE) and the plasma membrane of transitional epithelial cells (TE) of the ureter and pelvic epithelium also show a strong staining for syntaxin 3. B: region similar to A but stained for syntaxin 3 without SDS pretreatment. Staining of principal cells is much weaker under these conditions. Scale bar, 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A previous study reported that the t-SNARE syntaxin 3 was detectable by immunocytochemistry only in A-intercalated cells in the kidney (13). This led to the suggestion that syntaxin 3 was somehow involved in the targeting of basolateral membrane proteins, including the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger AE1, in this specialized acid-secreting cell type. However, the same study also reported that mRNA for syntaxin 3 was much more widely expressed in the kidney when different tubule segments were examined by RT-PCR. This observation prompted us to reexamine renal tissue for the expression of syntaxin 3 protein using an antigen retrieval technique that was developed in our laboratory (7). This method is based on the ability of SDS to denature proteins and expose otherwise cryptic antigenic sites. We have used this approach to detect some other antigens in renal epithelial cells that are undetectable by more conventional immunocytochemical methods (2, 6).

Our present results show that the renal distribution of the syntaxin 3 protein is much more widespread than was originally proposed. Virtually all cells and tubule segments examined showed strong basolateral expression of syntaxin 3. A notable exception was the proximal tubule, which correlates with the previously reported absence of syntaxin 3 message in this tubule segment (13). Thus basolateral syntaxin 3 expression appears to be a common feature of many different epithelial cells in the kidney, and its role in basolateral targeting processes is not restricted to A-intercalated cells. Indeed, A- and B-intercalated cells were basolaterally stained for syntaxin 3, and the brightest staining was in the B-intercalated cells. In their previous study, Mandon et al. (13) did not examine syntaxin 3 in the cortex and, therefore, did not describe B-intercalated cell staining, since these cells are present mainly in cortical collecting ducts. The positive staining after SDS treatment is probably due to the unmasking of cryptic antigenic epitopes that are at least partially unavailable for antibody binding under conventional incubation conditions. SDS could simply result in protein unfolding (i.e., change in the secondary or tertiary structure of a protein), or it could dissociate multiprotein complexes that hinder antibody binding. In cells where the fluorescence was strongest, some staining was detectable in the absence of SDS, e.g., in intercalated cells, as previously described (13). However, even in these cells, the staining intensity was increased after SDS treatment. Although the increased staining of syntaxin 3 in the present study is largely attributed to the use of an antigen retrieval technique, some other methodological differences could also have contributed to the different results. For example, Mandon et al. used paraformaldehyde alone as a fixative, whereas we used PLP in our present study. There may also be differences in sensitivity between peroxidase- and fluorescence-based immunodetection procedures.

Syntaxins belong to a family of proteins that are an integral part of the machinery that controls membrane fusion in all cell types (1, 4). Docking and fusion of transport vesicles with their correct target membrane are strictly regulated by a complex series of protein-protein interactions that together ensure that membrane proteins are delivered to the appropriate membrane domain. So-called SNARE proteins, including the syntaxins, are present on donor and acceptor membranes, and their interaction with other members of the fusion machinery is now beginning to be understood (17, 18, 20, 21). It was originally believed that different SNARE proteins would be expressed only on specific membrane domains and that they would interact with a cognate SNARE protein present on the fusion partner in each case. However, more recent data have indicated that the process is considerably more complex and that the distinction between t-SNAREs, such as syntaxins, and vesicle SNAREs, such as cellubrevin and synaptobrevin/VAMP, is not as clear cut as originally envisaged (19, 23). Both types of SNARE proteins can be located on the same membrane domain (21). Nevertheless, certain SNAREs do have a limited cellular distribution on particular membrane domains (1), which may be important for apical and basolateral polarization in epithelial cells.

In the kidney, we have now found that syntaxin 3 is an exclusively basolateral protein, implying that its role is indeed in regulating processes that occur at the basolateral membrane of these cells. Syntaxin 4, on the other hand, was found on the apical membrane of collecting duct principal cells, where it was proposed to have a role in apical aquaporin 2 (AQP2) targeting in the kidney (12). In contrast, evidence from MDCK cells clearly locates syntaxin 3 on the apical plasma membrane and syntaxin 4 on the basolateral membrane (11), precisely the opposite distribution to that found in the kidney in situ. The data in MDCK cells were obtained from transfected cells overexpressing the various syntaxin isoforms, and this must be borne in mind when these results are interpreted. However, the authors discuss this issue extensively in their report and concluded that the observed polarity of syntaxin expression probably does not result from an overexpression artifact.

With regard to the function of these syntaxin isoforms, it has been proposed that syntaxin 4 is involved in apical AQP2 targeting in the kidney (12). However, MDCK cells transfected with AQP2 also show apical AQP2 expression (10), despite the fact that the apical SNARE is syntaxin 3 in these cells (11). In addition, recent data from our laboratory confirm previous reports that AQP2 can be expressed basolaterally in inner medullary principal cells in the kidney in situ (5, 16), where syntaxin 3 is expressed. Thus assigning specific sorting properties to the syntaxin family of SNARE proteins is not a simple task and is likely to be dependent on the cell type under examination. From the present and previous data, it must be concluded that syntaxin 3 and syntaxin 4 are localized such that either might contribute to the polarized membrane insertion of AQP2. Functional studies will ultimately be required to dissect the complex series of events that lead to polarized insertion of membrane proteins in renal epithelial cells.


    ACKNOWLEDGEMENTS

We thank Valerie Beaulieu for technical help.


    FOOTNOTES

These studies were supported by National Institutes of Health grant DK-42956 to D. Brown and S. Breton. Part of the funding was from the Intramural Budget of the National Heart, Lung, and Blood Institute (project no. Z01-HL-01282-KE to M. A. Knepper).

Address for reprint requests and other correspondence: D. Brown, Program in Membrane Biology/Renal Unit, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129 (E-mail: brown{at}receptor.mgh.harvard.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.

10.1152/ajprenal.00128.2001

Received 19 April 2001; accepted in final form 3 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Advani, RJ, Bae HR, Bock JB, Chao DS, Doung YC, Prekeris R, Yoo JS, and Scheller RH. Seven novel mammalian SNARE proteins localize to distinct membrane compartments. J Biol Chem 273: 10317-10324, 1998[Abstract/Free Full Text].

2.   Alper, SL, Stuart-Tilley AK, Biemesderfer D, Shmukler BE, and Brown D. Immunolocalization of AE2 anion exchanger in rat kidney. Am J Physiol Renal Physiol 273: F601-F614, 1997[Abstract/Free Full Text].

3.   Bagnis, C, Marshansky V, Breton S, and Brown D. Remodeling the cellular profile of collecting ducts by chronic carbonic anhydrase inhibition. Am J Physiol Renal Physiol 280: F437-F448, 2001[Abstract/Free Full Text].

4.   Bennett, MK, Garcia-Arrarras JE, Elferink LA, Peterson K, Fleming AM, Hazuka CD, and Scheller RH. The syntaxin family of vesicular transport receptors. Cell 74: 863-873, 1993[ISI][Medline].

5.   Bouley, R, Breton S, McLaughlin M, and Brown D. Vasopressin (VP) stimulates apical and basolateral membrane insertion and clathrin coated pit localization of AQP2 in collecting duct principal cells (Abstract). J Am Soc Nephrol 11: 14A, 2000.

6.   Breton, S, Lisanti MP, Tyszkowski R, McLaughlin M, and Brown D. Basolateral distribution of caveolin-1 in the kidney. Absence from H+-ATPase-coated endocytic vesicles in intercalated cells. J Histochem Cytochem 46: 205-214, 1998[Abstract/Free Full Text].

7.   Brown, D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, and Alper S. Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol 105: 261-267, 1996[ISI][Medline].

8.   Brown, D, and Stow JL. Protein trafficking and polarity in kidney epithelium: from cell biology to physiology. Physiol Rev 76: 245-297, 1996[Abstract/Free Full Text].

9.   Caplan, MJ. Membrane polarity in epithelial cells: protein sorting and establishment of polarized domains. Am J Physiol Renal Physiol 272: F425-F429, 1997[Abstract/Free Full Text].

10.   Deen, PM, Rijss JP, Mulders SM, Errington RJ, van Baal J, and van Os CH. Aquaporin-2 transfection of Madin-Darby canine kidney cells reconstitutes vasopressin-regulated transcellular osmotic water transport. J Am Soc Nephrol 8: 1493-1501, 1997[Abstract].

11.   Low, SH, Chapin SJ, Weimbs T, Komuves LG, Bennett MK, and Mostov KE. Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells. Mol Biol Cell 7: 2007-2018, 1996[Abstract].

12.   Mandon, B, Chou CL, Nielsen S, and Knepper MA. Syntaxin-4 is localized to the apical plasma membrane of rat renal collecting duct cells: possible role in aquaporin-2 trafficking. J Clin Invest 98: 906-913, 1996[Abstract/Free Full Text].

13.   Mandon, B, Nielsen S, Kishore BK, and Knepper MA. Expression of syntaxins in rat kidney. Am J Physiol Renal Physiol 273: F718-F730, 1997[ISI][Medline].

14.   Mayer, A. Intracellular membrane fusion: SNAREs only? Curr Opin Cell Biol 11: 447-452, 1999[ISI][Medline].

15.   McLean, IW, and Nakane PK. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem 22: 1077-1083, 1974[ISI][Medline].

16.   Nielsen, S, DiGiovanni SR, Christensen EI, Knepper MA, and Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90: 11663-11667, 1993[Abstract].

17.   Pfeffer, SR. Transport vesicle docking: SNAREs and associates. Annu Rev Cell Dev Biol 12: 441-461, 1996[ISI][Medline].

18.   Rothman, JE, and Wieland FT. Protein sorting by transport vesicles. Science 272: 227-234, 1996[Abstract].

19.   Scales, SJ, Chen YA, Yoo BY, Patel SM, Doung YC, and Scheller RH. SNAREs contribute to the specificity of membrane fusion. Neuron 26: 457-464, 2000[ISI][Medline].

20.   Schiavo, G, Gmachl MJ, Stenbeck G, Sollner TH, and Rothman JE. A possible docking and fusion particle for synaptic transmission. Nature 378: 733-736, 1995[ISI][Medline].

21.   Waters, MG, and Pfeffer SR. Membrane tethering in intracellular transport. Curr Opin Cell Biol 11: 453-459, 1999[ISI][Medline].

22.   Weber, T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Sollner TH, and Rothman JE. SNAREpins: minimal machinery for membrane fusion. Cell 92: 759-772, 1998[ISI][Medline].

23.   Yang, B, Gonzalez L, Jr, Prekeris R, Steegmaier M, Advani RJ, and Scheller RH. SNARE interactions are not selective. Implications for membrane fusion specificity. J Biol Chem 274: 5649-5653, 1999[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 282(3):F523-F529