Interaction of Syntaxins with the Amiloride-sensitive Epithelial Sodium Channel*

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na+ entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (α, β, γ subunits) with syntaxin 1A or 3 cRNAs inXenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na ؉ entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (␣, ␤, ␥ subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving proteinprotein interactions.
The epithelial sodium channel (ENaC) 1 provides the ratelimiting step in the absorption of sodium ions in the distal nephron and functions to maintain Na ϩ ion homeostasis (1,2). Such channels have been characterized in amphibian skin and in mammalian tissues, including urinary bladder, renal collecting duct, distal colon, sweat and salivary glands, lung, and taste buds. They mediate the first step of active Na ϩ reabsorption and play a major role in the maintenance of electrolyte and water homeostasis. The ENaC complex is specifically blocked by the diuretic drug amiloride and its analogues (3), is composed of three homologous ␣, ␤, and ␥ subunits (4,5), and is involved in the pathogenesis of human low renin, salt-sensitive hypertension. For example, Liddle syndrome is an autosomal dominant form of human hypertension in which the cytosolic region of either the ␤ or ␥ subunit is truncated or altered by missense mutations in a critical PPPnY domain in their cytosolic carboxyl termini (6,7).
Na ϩ entry through the apical membrane of epithelial cells is tightly regulated by several known mechanisms (8). These include changes in protein expression, the relative distribution of protein between intracellular pools and the apical membrane, and changes in unitary conductance properties of the channel. Mutations associated with Liddle syndrome lead to both an increase in channel density at the cell surface and an increase in open probability (9,10). Channel density at the cell surface could be modulated by retrieval of the channel complex from the plasma membrane and subsequent degradation. The ␣ and ␥ subunits of ENaC are ubiquitinylated and hence targets for degradation through the lysosomal pathway (11). Clathrinmediated endocytosis has also been implicated in the control of ENaC half-life and density of cell surface expression (12).
Another mechanism for ENaC regulation is through proteinprotein interactions, although few data exist to date on this possibility. There is a direct interaction between PPPnY domains in the carboxyl termini of the ENaC subunits and the WW domain of the Nedd-4 protein (13,14). Another family of proteins that could mediate such functional interactions is the syntaxins, plasma membrane localized t-SNARES that are hypothesized to mediate vesicle trafficking (15,16). Several lines of evidence raise the possibility of an interaction between syntaxin and ENaC: (i) each member of the syntaxin family contains several domains predicted to form ␣-helical coiled-coiled structures (16), regions likely to be involved in protein-protein interactions; (ii) several syntaxin isoforms (1A, 3, and 4) have been localized to the apical membrane in cultured epithelial cells (17) and principal cells of the rat cortical collecting tubule (18), and thus could be co-localized with ENaC; and (iii) syntaxin 1A has been shown to interact with and functionally regulate a number of ion channels including Ca 2ϩ channels (19,20), CFTR Cl Ϫ channels (19 -22), and GABA transporters (23). Syntaxins 3 and 4 have a high degree of homology with syntaxin 1A. The role of these syntaxins in plasma membrane fusion reactions and regulation of ion channel activity has not yet been determined.
In the present report, we show that syntaxins 1A and 3 interact with and functionally regulate rat ENaC complexes when they are co-expressed in Xenopus oocytes. We found that these syntaxin isoforms enhance the surface expression of ENaC complexes but had opposite effects on ENaC function. Similar functional results were observed when syntaxin 1A was co-expressed with an ENaC complex containing a COOHterminal truncated ␤ subunit (as occurs in Liddle's syndrome (7)). Co-immunoprecipitation studies demonstrated that syn-taxin 1A or 3 was physically associated with the ENaC complex. These data suggest that ENaC can be regulated functionally by syntaxins, likely through protein-protein interactions, and that the surface expression of the ENaC complex is regulated in part by vesicular trafficking.
K100 Chimera-The COOH terminus truncated chimeric ␤ subunit (rat plus human) was constructed by replacing the COOH-terminal region of the full-length rat ␤ subunit with human COOH-terminal sequences. Human ␤ subunit COOH-terminal sequences were obtained by PCR amplification using cDNAs prepared from peripheral blood lymphocytes from an individual in the original Liddle's kindred (6). PCR primers were designed so that the PCR products contained artificially introduced XhoI or AccI sites at the ends. The forward primer corresponded to nucleotides 1609 -1631 (numbered according to Gen-Bank TM data base, L36592) with one nucleotide substitution (A to C at 1612) to create a XhoI restriction site, and the reverse primer corresponded to nucleotides 1790 -1814 with two nucleotide substitutions (GG to CA at 1807-1808) to create an AccI restriction site. The amplified PCR products were digested with XhoI and AccI and ligated into the full-length rat ␤ subunit cDNA (␤rENaC) in pSPORT. The XhoI site in ␤rENaC was created at the corresponding site of the human ␤ subunit by PCR-directed mutagenesis, and an extra AccI site at nucleotide position 2 (numbered according to GenBank TM , X77932) in brENaC was removed by a restriction with SalI, followed by blunt-end ligation. The sequence of the K100 chimera was confirmed by restriction map analysis as well as DNA sequencing. Nucleotide sequences were determined using an ABI 373 automated DNA sequencer at the University of Alabama Center for AIDS Research Sequencing Core.
RNA Transcription and Oocyte Injection-Vectors containing ENaC and syntaxin inserts were linearized, and complimentary RNAs (cRNA) were synthesized in vitro using the mMessage mMachine™ kit. For most experiments, 5 ng each of the rat ENaC subunit cRNAs (␣, ␤ FLAG , and ␥ FLAG cRNAs) were injected into stage V or VI oocytes. Oocytes were incubated at 18°C for 36 -48 h in ND96 plus horse serum (25) and then assayed.
Electrophysiology-Electrophysiological recordings were made using a standard two-electrode voltage clamp system (Gene Clamp 500, Axon Instruments, Foster City, CA). Oocytes were bathed continuously in ND96. For the current-voltage analysis, oocytes were held at Ϫ40 mV, and the holding potential was stepped for 500 ms, in increments of 20 mV, from Ϫ120 to ϩ20 mV. Experimental controls and data analysis were performed using pClamp software. Amiloride-sensitive currents reported in the text were determined as the difference in currents before and after the addition of 10 M amiloride to oocytes at a holding potential of Ϫ100 mV.
Western Blots-Oocytes were injected with ENaC cRNAs with or without syntaxin 1A or 3. For control assays, some oocytes were injected with wild-type rat ␣, ␤, ␥ cRNAs without inserted FLAG epitopes. Oocytes were homogenized in RIPA buffer with a glass homogenizer on ice. The cell debris was removed by centrifugation at 500 ϫ g for 10 min, and the supernatant fractions were collected. The supernatant fractions were boiled in Laemmli sample buffer (34) and electrophoresed on 10% SDS-polyacrylamide gels. The proteins were transferred to PVDF membrane in Towbin transfer buffer for 2 h. The PVDF membrane was then blocked with 5% non-fat dry milk and probed with M2 monoclonal antibody. The blots were visualized using the enhanced chemiluminescence technique.
Immunoprecipitation-Oocytes were injected with ENaC cRNAs with or without syntaxin 1A or 3. For control assays, some oocytes were injected with wild-type rat ␣, ␤, ␥ cRNAs without inserted FLAG epitopes. Oocytes were homogenized in DIGNAM D buffer with a glass homogenizer on ice. Cell debris was removed by centrifugation, and the supernatant fraction was obtained. The lysates were incubated with anti-syntaxin antibody for 4 -6 h. 50 l of a 50% slurry of protein A-Sepharose beads was added to the sample, and the preparation was gently shaken for 2-4 h. The beads were then washed three times with DIGNAM D. The proteins were then eluted in Laemmli sample buffer and electrophoresed on 10% SDS-polyacrylamide gels and transferred to PVDF membrane in Towbin transfer buffer for 2 h. The PVDF membrane was then blocked with 5% non-fat dry milk and probed with M2 monoclonal antibody directed against the FLAG epitope. The blots were visualized using the enhanced chemiluminescence technique.
Immunofluorescence-Oocytes were fixed in 3% formaldehyde in PBS, pH 7.4, for 30 min. Nonspecific sites were blocked with 1% bovine serum albumin in PBS. The oocytes were then incubated with M2 monoclonal antibody at 37°C for 1 h. After three washes in PBS, the oocytes were incubated with goat anti-mouse IgG-alexa 488 for 1 h at 37°C. The oocytes were washed five times with PBS and examined immediately with fluorescent microscopy.
Digital Confocal Epifluorescence Microscopy-Oocytes were optically sectioned on an Olympus IX70 inverted epifluorescence microscope equipped with step motor filter wheel assembly (Ludl Electronics Products Ltd., Hawthorne, NY) and filter set 83000 (Omega Optical, Brattleboro, VT). Images were captured with a SenSys cooled CCD, high resolution, monochromatic, digital camera (Photometrics, Tucson, AZ). Deconvolution of optical sections and merging of resultant sections were done with a PowerMac 9500/132 computer supplied with IP Lab Spectrum software (Scanalytics, Fairfax, VA) and Power Microtome software (VayTek, Inc., Fairfield, IA).

RESULTS
Effect of Syntaxins 1A and 3 on Amiloride-sensitive Currents-To test the hypothesis that syntaxins functionally regulate ENaC, amiloride-sensitive currents were assayed in Xenopus oocytes expressing ENaC with or without co-expression of syntaxin 1A and 3 ( Fig. 1). At Ϫ100 mV, oocytes expressing ENaC showed basal currents that were inhibited by 10 M amiloride (Ϫ671 Ϯ 121 nA). These amiloride-sensitive currents increased 2-fold (to Ϫ1245 Ϯ 187 nA) in oocytes expressing full-length syntaxin 3, but not with a syntaxin 3 construct (Syn 3⌬C) lacking the 13-amino acid COOH-terminal membraneanchoring domain (Ϫ708 Ϯ 134 nA). The basal currents were inhibited 60% (Ϫ269 Ϯ 72 nA) in oocytes expressing syntaxin 1A. This change was not observed in oocytes expressing ENaC and a syntaxin 1A construct (Syn 1A⌬C) lacking the 13-amino acid membrane-anchoring domain. These data indicate that (i) functional regulation is syntaxin isoform-specific, and (ii) that the membrane anchor of syntaxin is required for it's regulatory effects. Similar functional regulation of CFTR activity, with inhibition by syntaxin 1A and stimulation by syntaxin 3, has been reported previously (21,22).
IV Curve-To understand if syntaxins affect the unitary properties of the amiloride-sensitive sodium channel in oocytes, we monitored the current-voltage relationship in ENaC-expressing oocytes co-injected with or without syntaxin 1A or syntaxin 3. We observed no significant change in either the resting membrane potential or the reversal potential of amiloride-sensitive sodium currents in presence of syntaxin 1A or 3 (Fig. 2). These data indicate that either of syntaxin isoform does not effect these macroscopic channel behaviors.
K100 ϩ Syntaxin-K100 is a carboxyl-terminal deletion mutant of the ␤ subunit of ENaC associated with Liddle's syndrome. This mutation increases amiloride-sensitive currents in Xenopus oocytes (24) (Fig. 3). Consistent with this observation, we found that a mixture of ENaC subunits containing this construct (rat ␣, K100, rat ␥) produced an approximately 2-fold increase in amiloride-sensitive currents (Ϫ912 Ϯ 156 nA) compared with basal ENaC currents recorded with wild-type rat ␣, ␤, and ␥ cRNA (Ϫ443 Ϯ 141 nA). We observed a substantial inhibition of the K100-stimulated currents when syntaxin 1A was co-expressed (Ϫ376 Ϯ 132 nA; Fig. 3, lane 4). In fact, the currents associated with wild-type ENaC were almost the same as the K100 mixture co-injected with syntaxin 1A. After coinjection with syntaxin 3, further increases in the K100-stimulated currents were seen compared with K100 without syntaxin 3 co-expression, but the magnitude of this effect was dependent upon the basal level of ENaC activity.
Dose Dependence on Currents-The regulation of ENaC activity by both syntaxins 3 and 1A was dose-dependent. A total of 5 ng ENaC (1.66 ng each of ␣, ␤, and ␥ ENaC subunit cRNA) was co-injected in the Xenopus oocytes with increasing concentrations of syntaxin cRNA (0, 5, 10, 15, 20, and 25 ng). Fig. 4A shows the dose-dependent activation of ENaC currents with increasing amounts of injected syntaxin 3 cRNA. We observed a saturable increase in amiloride-sensitive currents with increasing syntaxin 3 cRNA. The maximum stimulation of these currents was approximately 2.5-fold. Similar experiments were also conducted with syntaxin 1A. We observed a steady decline in amiloride-sensitive currents with increasing amounts of syntaxin 1A cRNA (Fig. 4B). The maximum reduction of ϳ65% was observed with an ENaC:syntaxin ratio of 1:5. Although the maximum modulatory effects of syntaxin on ENaC activity was observed at the maximum concentration injected, for practical purposes a ratio of 1:3 (ENaC:syntaxin) was found to produce comparable results and hence used in all other reported experiments.
Immunoprecipitation of ENaC with Syntaxins-The functional data suggested an interaction between ENaC and syntaxin proteins. To test this hypothesis, we performed co-immunoprecipitation studies in which we injected Xenopus oocytes with epitope-tagged ENaC and either syntaxin 1A or 3. Oocytes expressing these proteins were immunoprecipitated with antisyntaxin antibody and blotted with M2 monoclonal antibody that recognizes the FLAG epitope (9). ENaC proteins were co-immunoprecipitated with both syntaxins 3 and 1A (Fig. 5A). Similar results were observed when the complex was first precipitated with M2 monoclonal antibody followed by immunodetection with isoform-specific syntaxin antibody (Fig. 5B). Of note, full-length and carboxy-truncated syntaxins co-immunoprecipitated with ENaC.
To examine further this interaction, we co-injected oocytes with increasing quantities of syntaxin cRNA. The homogenates were precipitated with anti-FLAG antibody and detected using isoform-specific anti-syntaxin antibody. With increasing concentrations of syntaxin, we were able to co-immunoprecipitate FIG. 2. Current-voltage relationship of the amiloride-sensitive ENaC currents. Oocytes were injected with ENaC (E), ENaC ϩ syntaxin 3 (Ⅺ), and ENaC ϩ syntaxin 1A (q). The oocytes were held at Ϫ40 mV, and the holding potential was stepped for 500 ms, in increments of 20 mV, from Ϫ120 to ϩ20 mV. The amiloride-sensitive currents were determined as the difference in currents before and after the addition of 10 M amiloride.

FIG. 3. Effect of syntaxins 3 and 1A on K100 construct activity.
Oocytes were injected with wild-type ENaC cRNA (␣, ␤, ␥ subunits) or with the K100 construct (␣, K100, ␥) and with syntaxin cRNAs (Syn3 or Syn1A). The amiloride-sensitive currents were recorded after 24 -48 h post-injection as reported in the text. The data represent a mean of three experiments carried out with three sets of cRNA at different times under comparable conditions. The numbers in parentheses indicate the number of oocytes used. Experimental conditions that resulted in a significant change (p Ͻ 0.05) from the relevant control values are denoted by an asterisk. more bound syntaxin 1A or syntaxin 3 with ENaC (Fig. 6, A  and B). These data suggest that ENaC and syntaxins physically associate in the oocyte expression system.
Immunofluorescence-Since the syntaxin family of membrane proteins has been implicated in membrane trafficking events, we next tested the hypothesis that the increase in amiloride-sensitive ENaC activity in the presence of syntaxin 3, and the decrease in amiloride-sensitive currents in the presence of syntaxin 1A was due to correlated changes in surface ENaC expression. We used FLAG epitope constructs (inserted into the extracellular loop of ␤ and ␥ subunits) of ENaC, which we could label and detect on the oocyte surface by epifluorescence. As expected, we observed an increase in epifluorescence on the oocyte membrane surface of oocytes co-injected with syntaxin 3 (Fig. 7). Surprisingly, syntaxin 1A co-expression also caused an increase in surface epifluorescence, even though the functional activity of ENaC was reduced. The carboxylterminal deletion constructs of syntaxins 1A and 3 did not affect surface labeling of ENaC complexes, confirming the importance of the carboxyl-terminal membrane-spanning region of syntaxins for interactions with membrane transport proteins (21,22). These data demonstrate that both syntaxin isoforms increase the net outward trafficking of ENaC proteins. The fact that syntaxin 1A increased surface ENaC epifluorescence but decreased ENaC currents suggests that factors other than surface expression, per se, can regulate ENaC function.
Total Cell ENaC Determination-Since we observed increased ENaC surface labeling in oocytes co-injected with syntaxins, it was of interest to see if the syntaxins altered the steady-state level of total cell ENaC protein. To answer this question, we extracted total ENaC proteins from oocytes and performed Western blot analysis (Fig. 8). Our data indicate that the protein levels were unchanged in the oocytes preparations expressing ENaC with or without syntaxins 1A or 3. These results suggest that the syntaxins did not affect the ENaC half-life, or synthesis rate, and that the increased surface expression of ENaC observed with co-injection of syntaxins 1A or 3 was due to a redistribution of internal stores of the ENaC complex to the surface membrane. DISCUSSION We used the Xenopus oocyte expression system to explore the role of syntaxins in the expression of amiloride-sensitive ENaC. The principal findings of the studies include: (i) ENaC can be FIG. 4. Dose-dependent modulation of ENaC currents by syntaxins 3 or 1A. The regulation of ENaC activity by both syntaxins 3 and 1A is dose-dependent. 5 ng of ENaC (1.66 ng each of ␣, ␤, and ␥ subunit cRNA) was co-injected in Xenopus oocytes with increasing concentration of syntaxin cRNA (0, 5, 10, 15, 20, and 25 ng). A, the dose-dependent activation of ENaC currents with increasing amounts of injected syntaxin 3 cRNA. B, similar experiments were also conducted with syntaxin 1A. A decline in amiloridesensitive currents was observed with increasing amount of syntaxin 1A fulllength cRNA. The data are from three separate experiments. The numbers in the parentheses indicate the number of oocytes used. Experimental conditions that resulted in a significant change (p Ͻ 0.05) from the relevant control values are denoted by an asterisk.

FIG. 5. Syntaxins co-immunoprecipitate with ENaC.
Oocytes were injected with ENaC (E) cRNAs (rat ␣, ␤ FLAG , ␥ FLAG ), with or without syntaxin cRNA (syntaxin 3, S3; syntaxin 1A, S1A). For control assays, oocytes were injected with rat ␣, ␤, ␥ cRNAs without FLAG constructs. Oocytes were homogenized in DIGNAM D buffer, cell debris was removed, and the supernatant fraction was precipitated with antisyntaxin isoform-specific antibodies. The proteins were eluted in Laemmli sample buffer. A, ␤ FLAG and ␥ FLAG ENaC subunits were visualized by Western blot using M2 monoclonal antibody with enhanced chemiluminescence. B, in another set of experiments, the proteins were first immunoprecipitated with M2 monoclonal antibody, and the blots were developed with syntaxin isoform-specific antibody. functionally modulated by syntaxins 1A and 3, (ii) ENaC proteins physically interact with syntaxins 1A and 3, (iii) syntaxins 1A and 3 both increase the trafficking of ENaC proteins to the plasma membrane, (iv) co-expression with syntaxins 1A or 3 does not alter the steady-state amount of total ENaC protein, (v) enhanced ENaC trafficking may not reflect a parallel increase in ENaC function, and (vi) the ␤ subunit carboxyl terminus does not interact with syntaxin 1A.
Ion channel proteins are targeted to plasma membranes by the translocation of transport vesicles (25). Subsequent vesicular fusion increases channel density at the surface. Important questions remain about the mechanisms by which transport vesicles are targeted to a specific membrane domain, and docking and fusion are regulated. The SNARE hypothesis (26,27), based on the regulated exocytosis of synaptic vesicles, has expanded this concept. Vesicles may be targeted by membraneassociated proteins that behave as vesicle-targeting receptors or SNAREs. These proteins are associated with vesicular membranes (v-SNAREs) or with the target membrane (t-SNAREs). Syntaxins are a group of t-SNAREs, which participate in intracellular trafficking pathways (26,27).
By using the oocyte expression system, we have demonstrated that syntaxins 1A and 3 can modulate amiloride-sensitive currents associated with ENaC. Syntaxin 3 is a positive functional regulator, whereas syntaxin 1A acts as a negative regulator of of ENaC activity. Both syntaxins physically interact with ENaC as demonstrated by the immunoprecipitation experiments. We were able to reciprocally co-immunoprecipitate ENaC proteins with syntaxin 1A and syntaxin 3 and also demonstrate that the functional interaction is critically dependent upon the carboxyl-terminal membrane-spanning regions of the syntaxins. Truncation of these regions abrogated the ability of syntaxins 1A and 3 to affect either the surface expression or the functional activity of the ENaC complex ( Figs. 1 and 7), even though the carboxyl-terminal truncated constructs still co-immunoprecipitated with ENaC (Fig. 5B). This finding is consistent with previous studies, which demonstrated that the carboxyl-terminal membrane-spanning regions of the syntaxins are critical for there localization in the plasma membrane and for their targeting (tSNARE) function (19 -23, 28). In addition, the co-immunoprecipitation with the ⌬C syntaxin constructs demonstrates that the ␣-helical regions interact with the ENaC proteins, rather than the carboxylterminal domains.
The confocal microscopy data (Fig. 7) clearly demonstrate that ENaC density is increased at the oocyte surface when the ENaC subunits are co-injected with syntaxin 1A or syntaxin 3, thereby implying a role for these proteins in ENaC trafficking. The ⌬C syntaxin constructs did not affect the ENaC surface expression. The opposite effects of syntaxins 3 and 1A on ENaC functional activity indicate a complex interaction between the ENaC complex and these syntaxins. We presume that additional components, either of the ENaC complex or other regulator factors, may play a crucial role in regulating ENaC functional activity. It appears likely that the pool of ENaC channels expressed at the membrane surface was labeled in our studies, since our confocal localization used a minor modification of the surface-labeling method described previously (9). Some of the labeled ENaC could be present in subapical vesicles that were unfused with the plasma membrane. Since docking and fusion are distinct functions, the syntaxin-ENaC protein interaction may trigger interactions with other proteins consistent with the large array of proteins associated with syntaxins in SNARE complexes. Interaction between syntaxins and ENaC channel proteins may also result in conformational changes as documented for SNAP-SNARE complex (29) or could affect the stability of interacting components in the ENaC complex once they are assembled and expressed in the surface membrane.
Syntaxin 3 has been cloned and described in various cell systems (16) and enhances CFTR activity when co-injected in the oocyte system (21). Since syntaxin 3 is expressed in principal cells of the collecting tubule (18,30), it could play a role in the trafficking of ENaC and water channels in these cells. A6 cells (an amphibian renal cell line), which functionally express ENaC activity and are used to study its regulation (31), can also be labeled with syntaxin 3 antibody in our laboratory (data not shown). Syntaxin 3 has approximately 65% homology with FIG. 7. Effect of syntaxins on the ENaC expression on oocyte surface. Oocytes were injected with ENaC cRNAs (rat ␣, ␤ FLAG , ␥ FLAG ) with or without syntaxin cRNA. For control assays, some oocytes were injected with wild-type rat ␣, ␤, ␥ cRNAs without FLAG constructs. The oocytes were fixed in formaldehyde and labeled with M2 monoclonal antibody. Fluorescence was detected by incubating the oocytes with goat anti-mouse IgG-alexa 488. The bar represents 0.25 mm.

FIG. 8. Effect of syntaxins on the total ENaC expression in oocytes.
Oocytes were injected with ENaC (E) cRNAs (rat ␣, ␤ FLAG , ␥ FLAG ) with or without syntaxin cRNA. For control assays (C), some oocytes were injected with wild-type rat ␣, ␤, ␥ cRNAs without FLAG constructs. Oocytes were homogenized in RIPA buffer, the cell debris was removed, and the supernatants were collected. The supernatants were boiled in Laemmli sample buffer and electrophoresed on 10% SDS-polyacrylamide gels. The proteins were transferred to PVDF membrane and probed with M2 monoclonal antibody. The blots were visualized using the enhanced chemiluminescence technique. syntaxin 1A and also has ␣-helical domains. Recently, the H3 domain of syntaxin 1A has been shown to interact with CFTR (22). A similar homologous H3 domain is present in syntaxin 3. Our data demonstrate the physical and functional association of syntaxin 3 with ENaC in the oocyte expression system. The presence of this syntaxin isoform in native kidney (16,18) supports the hypothesis that syntaxin 3 could participate in the regulation of ENaC expression. In contrast to cultured epithelial cells where syntaxin 3 is apically expressed (17,32), syntaxin 3 appears to be localized at the basolateral membrane of principal cells (18), while ENaC is primarily expressed in the apical membrane. Of note, syntaxin 4 is clearly expressed at the apical membrane of principal cells of the collecting tubule (33). The potential interactions of syntaxin 4 with ENaC have not yet been defined.
ENaC regulates Na ϩ reabsorption (1) and maintains electrolyte balance. Several mutations in the ENaC subunits cause Liddle's syndrome, with abnormal regulation of blood pressure and electrolyte balance (1, 6, 7). These mutations increase ENaC functional activity and surface expression (9,24). Therefore, identification of the mechanisms involved in the regulation of ENaC activity is critical for our understanding of the pathogenesis of human salt-sensitive hypertension. Truncations of the carboxyl terminus of the ENaC ␤ subunit were the originally described mutations in Liddle's syndrome (6). When such a construct (K100) is co-expressed with wild-type ␣ and ␥ ENaC subunits, and syntaxins 1A, a similar inhibitory effect was observed as with the wild-type ENaC constructs, demonstrating that the functional interaction between syntaxin 1A and ENaC does not involve the carboxyl terminus of the ␤ subunit. Since syntaxin 1A can still down-regulate the enhanced ENaC activity associated with the K100 construct, it is possible that some sort of protein-protein interaction may provide a therapeutic approach to regulating ENaC activity in human hypertension.
In conclusion, ENaC physically and functionally interacts with at least two syntaxins, each of which differentially affect ENaC function. These interactions suggest a role for syntaxins in the trafficking and functional regulation of ENaC complexes and may also directly affect channel function as has been described for N-type calcium channel (20) and CFTR (20 -22). In view of the variety of syntaxin-associated proteins that have been described, it will be important to determine the specific nature of the syntaxin interactions with the ENaC complex.