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J. Biol. Chem., Vol. 279, Issue 11, 10085-10092, March 12, 2004
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From the Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15217
Received for publication, December 11, 2003 , and in revised form, December 29, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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200 nM). In contrast, injection of GST alone or a H3 domain-deleted GST-S1A fusion protein had no effect on INa. In cell-attached patch clamp experiments, GST-H3 acutely decreased ENaC Po by 30%, whereas GST-S1A 
H3 was without effect. Further analysis revealed that ENaC mean closed time was significantly prolonged by S1A. Interestingly, GST-H3 had no effect on channel activity of an ENaC pore mutant that constitutively gates open (Po 
1.0), supporting the idea that S1A alters the closed state of ENaC and indicating that the actions of S1A on ENaC trafficking and gating can be separated experimentally. This study indicates that, in addition to a primary effect on ENaC trafficking, S1A interacts with cell surface ENaC to rapidly decrease channel gating. This rapid effect of S1A may modulate Na+ entry rate during rapid increases in ENaC N. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-, 
-, and 
-ENaC); each consists of two transmembrane domains, short cytoplasmic N and C termini and a large extracellular loop (1). The expression and assembly of all three subunits is required for fully functional ENaC channels, which are characterized by an ionic selectivity of Li+ > Na+ »K+, a low single channel conductance ( 5 picoSiemens), and a high affinity blockade by the diuretic amiloride (2). The channel is thought to associate as a tetramer composed of 2:1;1 (3, 4), although other models have been proposed (5).
The control of whole body Na+ homeostasis is achieved via regulation of the number (N) and open probability (Po) of ENaC channels in the apical membranes of distal nephron principal cells. Recently, many of the important mechanisms that govern ENaC N through endocytic processes have been elucidated (for review, see Ref. 6). However, comparatively little is known regarding the regulation of ENaC insertion into the apical membrane. Previous work suggests SNARE proteins play an important role in the regulated insertion of ENaC (7, 8). The cognate, pairwise interactions of SNARE proteins, are responsible for membrane vesicle docking and fusion in all intracellular trafficking steps (for review, see Ref. 9). Central to the process of vesicle fusion at the nerve terminus are interactions between the coiled-coil domains ("SNARE motifs") of syntaxin, SNAP-25 and VAMP2 (vesicle-associated membrane protein 2), which together form an extremely stable core complex that is thought to promote the membrane fusion event. The SNARE motif of S1A is contained within the third helical (H3) domain, which we have shown to bind the ENaC subunit C termini, resulting in a decrease in cell surface ENaC expression that inhibits Na+ transport (8). S1A has also been shown to interact with other channel proteins to influence their insertion into the plasma membrane, including CFTR (10) and Kv 2.1 (11).
In addition to its effects on ENaC trafficking, the binding of S1A to ion channels in the plasma membrane has been proposed to alter their kinetic properties. Naren et al. (12) demonstrate that S1A acutely decreased whole cell CFTR currents in epithelial cells. The activation rate of Kv 2.1 was significantly prolonged in the presence of S1A (11), and various gating effects have been described for the family of voltage-gated Ca2+ channels (VGCC) (for review, see Ref. 13). The physiological significance of S1A binding on channel gating is unclear, although it has been postulated that, at least in VGCC and Kv 2.1, modification of channel kinetics serves as a further means of controlling the duration and magnitude of vesicular exocytosis (11, 13).
The aim of the present study was to determine whether S1A influences ENaC channel activity. In a previous study injection of a GST-H3 fusion protein into ENaC-expressing oocytes caused a large decrease in current after 1 h that was due to a concomitant decrease in ENaC N (8). In addition to influencing channel insertion, it is also possible that S1A binds to ENaC channels resident in the surface membrane to alter their Po.To investigate this possibility we examined the acute effects of injecting the H3 domain of S1A into ENaC-expressing Xenopus oocytes during whole cell and single channel patch clamp recordings. We demonstrate that S1A causes a rapid, dose-dependent decrease in whole cell current via a decrease in ENaC open probability.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Glutathione S-Transferase-S1A Fusion Proteins—Syntaxin 1A fusion proteins containing GST at the N terminus were generated in BL21-competent cells; conditions for bacterial growth and fusion protein purification were described previously (8). GST-H3 contains the S1A H3 domain (S1A185–265) and GST-H3 (S1A1–189) truncates both the H3 and TM domains from full-length S1A. These fusion proteins were injected into oocytes during electrophysiological recordings using a nanoliter injector (World Precision Instruments, Sarasota, FL).
Whole Cell Electrophysiology—Whole cell Na+ currents were measured by 2-electrode voltage clamp of ENaC expressing oocytes bathed in ND96 solution (96 mM NaCl, 1 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4). In experiments investigating the effect of protein injection, oocytes were clamped at–100 mV until the basal current stabilized. An injection tip was then inserted into the oocyte, and current levels were monitored for another 2 min before injection of 23 nl of a GST fusion protein (see above) or GST alone. Amiloride-sensitive Na+ currents (INa) were recorded as the difference in current before and after the addition of 10 µM amiloride. Amiloride or S1A have no effect on whole cell currents in oocytes not expressing ENaC (8); therefore, the current inhibition produced by S1A is directed at amiloride-sensitive currents arising from ENaC expression.
Single Channel Recording—Before patch clamp recordings, the vitelline membrane of ENaC-expressing oocytes was removed by mechanical dissection after cell shrinkage in a hypertonic solution (200 mM potassium gluconate, 20 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM HEPES; pH was adjusted to 7.4 with KOH). Devitellinized oocytes were immediately transferred to the patch bath (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.8 mM MgSO4, 0.3 mM Ca(NO3), 0.4 mM CaCl2, 10 mM HEPES, pH 7.4) and left to recover a minimum of 10 min before patching. Patch pipettes were pulled from borosilicate capillary glass (G150–3, Warner Instrument Corp., Hamden, CT) using a P-97 micropipette puller (Sutter Instrument Co. Novato, CA). After fire polishing, patch pipettes had resistances between 5 and 10 megaohms when filled with the pipette solution (96 mM NaCl, 10 mM HEPES, 3.4 mM KCl, 0.8 mM MgCl2, 0.8 mM CaCl2; pH adjusted to 7.4 with NaOH).
Single channel currents were recorded in the cell-attached configuration of the patch clamp technique using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and interfaced via a DigiData 1322a acquisition board (Axon Instruments) to a Micron Electronics PC (Nampa, ID). Data were acquired at 1 kHz and stored directly on the hard drive of the PC using pClamp 8.1 (Axon Instruments) software before digitizing at 100 Hz for subsequent analysis and presentation. The product of the number of channels and the channel open probability (NPo) was calculated from amplitude histograms generated using Biopatch software (Version 3.42, Bio-Logic) as the mean total current (I) divided by the single channel current amplitude (i), or NPo = I/i. Open probability (Po) was then calculated by dividing NPo by the total number of channels in the patch (N) estimated from the number of detectable peaks in amplitude histograms generated from records of sufficient duration (typically at least 7 min). This approach provides 95% confidence in determining the correct N value based on methodology developed by Marunaka and Eaton (15). To determine whether experimental modifications alter Po by affecting the channel open or closed state, mean open and closed times of N channels were calculated from
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0.05) was determined using analysis of variance. |
RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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H3. Similar to GST injection, GST-H3 did not significantly affect INa (Fig. 1B), suggesting that the H3 domain of S1A is responsible for this acute inhibitory effect. The rapid inhibition of ENaC by GST-H3 exhibited a dose-dependent relationship with a Ki of 7 ng/µl, corresponding to a half-maximal inhibitory concentration of 200 nM (Fig. 1D). These results suggest that the H3 domain of syntaxin 1A acutely and selectively regulates ENaC channel activity. The rapidity of inhibition (<5 min) is consistent with an alteration in the gating of plasma membrane ENaC channels; however, a larger component of Na+ current remains sensitive to amiloride after this acute effect, suggesting that another action of S1A is responsible for the remainder of the 80% inhibition in Na+ current observed 1 h post-injection. Na+ Current Inhibition Is Composed of Two Components— Results obtained from the short term recordings described above suggest that two processes may be responsible for the overall current inhibition observed in prior studies 1 h post-GST-H3 injection. To test this hypothesis, we measured amiloride-sensitive Na+ currents in oocytes at 10-min intervals for 1 h after injection of GST-H3. Fig. 2 shows that ENaC current inhibition over this period is best fit by a double exponential relation; an initial rapid decrease over the first 10 min accounts for 29 ± 8% inhibition of the initial INa, and a slower, long term decline reduces current to 33 ± 8% of the initial value after 1 h. These results suggest that S1A inhibits ENaC current via two different mechanisms, which are likely composed of an acute decrease in the Po of plasma membrane ENaC followed by a slower reduction in cell surface channel number as was demonstrated previously (8).
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0.5 (2). Channel activity was continuously recorded at the same holding potential after injecting GST-H3. Fig. 3B illustrates a 2-min trace from the same patch recorded 3 min post-GST-H3 injection. Channel activity was significantly reduced compared with the pre-injection period, with a calculated Po of 0.34. Compiling the data from six such recordings yielded a GST-H3-induced decrease in ENaC Po from an average of 0.51 ± 0.02 to 0.36 ± 0.04 (Fig. 3E). This represents an average decrease of 30 ± 7%. This value is within the range observed for the rapid change in whole cell current, demonstrating that the acute reduction in ENaC INa by S1A is due to a decrease in ENaC channel activity.
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H3-GST during cell-attached ENaC recordings. Representative traces of single channel currents are shown before (Fig. 4A) and after (Fig. 4B) injecting H3-GST. Subsequent analysis revealed that Po values were similar in control and treatment conditions (0.48 versus 0.45). The average ENaC Po from several experiments did not change significantly after H3-GST injection (Fig. 4E), suggesting that the H3 domain is responsible for the observed inhibition of channel activity.
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5 channels to determine the basis for S1A modulation of ENaC Po. The mean open time for ENaC recordings made before GST-H3 injection was 6.0 ± 1.0 s. This value was not significantly different from the mean open time calculated from recordings made 5 min after GST-H3 injection (Fig. 5). This indicates that S1A does not reduce ENaC Po by decreasing channel open time. In contrast, the analysis revealed that the mean closed time increased significantly from 5.6 ± 0.6 to 11.1 ± 1.6 s after GST-H3 injection (Fig. 5). Therefore, S1A decreases ENaC Po by significantly prolonging the mean closed time of the channel while having no effect on mean open time.
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subunit. At the so-called DEG position in ENaC-related Caenorhabditis elegans channels, mutating the native alanine to a residue that contains a bulky or charged side chain causes neuronal swelling and degeneration due to enhanced channel activity (17). Snyder et al. (18) have since reported that mutating DEG residues in ENaC channel subunits produces channels with inherent Po fixed near unity. We examined the effect of S1A on the gating properties of mouse ENaC channels expressing a subunit with serine in the DEG position mutated to a lysine (-S518K).
Consistent with data from a similar mutation made in human ENaC (18), oocytes expressing mouse ENaC channels containing -S518K had approximately twice the amiloride-sensitive whole cell current of those expressing wild type ENaC (Fig. 6C). Subsequent patch clamp analysis confirmed that this gain of function was due to an increase in Po from an average of 0.5 in wild type ENaC to 0.97 in -S518K (Fig. 7A). To determine the effect of S1A on this gating mutant we injected GST-H3 into oocytes expressing -S518K ENaC channels. Fig. 6A illustrates a representative trace of whole cell current from this experiment. GST-H3 did not elicit the rapid decrease in INa of -S518K ENaC over the initial 5-min recording period observed in oocytes expressing wt ENaC. In paired wild type controls current decreased by an average of 24 ± 2% in response to H3 domain injection, whereas -S518K ENaC currents exhibited no significant change (Fig. 6B). Interestingly, similar to wild type ENaC, the amiloride-sensitive -S518K ENaC currents measured 1 h after GST-H3 injection were significantly reduced (Fig. 6C). This likely results from the decrease in channel surface expression observed 1 h after GST-H3 injection with wild type ENaC (8), suggesting that S1A still binds to -S518K ENaC to perturb its trafficking but is unable to modify channel activity.
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-S518K ENaC gating, we measured single channel activity in oocytes expressing -S518K ENaC before and after injection of GST-H3. Fig. 7A illustrates that -S518K ENaC channels remain locked open with brief and infrequent closing events after GST-H3 injection. The Po was estimated at 0.97 from the amplitude histogram (Fig. 7C). From the record in Fig. 7B and the corresponding amplitude histogram (Fig. 7D) it is apparent that GST-H3 had no significant effect on -S518K ENaC gating. Channels recorded from the same patch 3 min post-GST-H3 injection remained largely in the open state. These results demonstrate that S1A has no rapid effect on -S518K ENaC currents as it is unable to modify channel kinetics. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-S518K ENaC, which is characterized by constitutively open channel activity. These results suggest that S1A regulates both ENaC trafficking to the plasma membrane and the activity of channels at the cell surface. SNARE proteins play an important role in the trafficking of vesicles to target membranes. Interactions between the SNARE motifs of cognate SNARE proteins on the target (t) and carrier vesicle membranes interact to facilitate vesicle docking, subsequent membrane fusion, and cargo delivery (9). SNARE proteins have been shown to co-ordinate the trafficking of several ion channels and transporters, including aquaporin 2 (19), CFTR (10) ENaC (7, 8), ROMK (renal outer medulla potassium channel) (20), Kv 2.1 (11), glucose transporter 4 (21), N-methyl-D-aspartate receptors (22), and the renal H+-ATPase (23). These studies confirm that regulation of transporter trafficking in a variety of cell types is dependent on the SNARE vesicular transport machinery.
A rapidly growing body of literature indicates that, in addition to their role in trafficking, SNARE proteins functionally interact with many ion channels to control their gating properties (11, 12, 22, 24). The understanding of this process is perhaps most advanced in studies of the VGCC of neuronal tissues. It has been proposed that physical interactions between SNARE components and VGCC serve to regulate presynaptic Ca2+ entry via alterations in gating. This interaction would provide a means to finely regulate vesicle membrane fusion and coordinate this process with Ca2+ entry, which signals secretion (13). This so-called "excitosome" model has been extended to include other voltage-dependent channels of neuronal tissues such as Kv 2.1 (11, 25). In addition, channels from non-excitable cells are regulated via interactions with SNARE proteins. Naren et al. (26) report that the H3 domain of S1A binds to the N terminus of CFTR to inhibit cAMP-stimulated Cl– currents. In addition to their role in trafficking these studies argue for an additional role of SNARE proteins in regulating channel activity.
The results of the present study extend our previous observations, indicating that S1A regulates plasma membrane ENaC density to include an additional effect on channel gating. These actions can be separated temporally into a rapid inhibition of channel activity that occurs within minutes, whereas changes in N take place over a longer time course. The prolonged and progressive rate of decline of current, illustrated by the second exponential component of Fig. 2, is consistent with the expected rate of ENaC endocytosis from the plasma membrane. This time -course agrees with published endocytic rates for ENaC in oocytes treated with brefeldin A to block progression of the channel to the cell surface (27). This finding implies that the S1A-induced decrease in N is due to a block of ENaC channel insertion.
How this trafficking effect of expressed S1A relates to the physiological role of this t-SNARE in apical membrane ENaC insertion and to the physical interaction between ENaC and S1A remains unclear. Overexpression of a syntaxin isoform (or expression of its essential protein interaction motif, the H3 domain) commonly inhibits the vesicle trafficking step in which that isoform serves as the t-SNARE for vesicle fusion (28). This phenomenon is due to selective disruption of the stoichiometric protein interactions required for formation of a specific SNARE fusion complex. Yet, our previous findings argue for an action that is more specific, since elimination of the S1A binding sites on the ENaC subunits abolished the effect of syntaxin on ENaC trafficking (8). This finding suggests that the S1A inhibition is related to the physical presence of ENaC in the trafficking vesicles. Disruption of SNARE protein interactions by overexpressed S1A should lack specificity for vesicle cargo; in this case, ENaC. Rather, the domain-specific interactions of ENaC cytoplasmic tails with S1A may reflect an involvement of the channel in its own trafficking through interactions of ENaC with SNARE proteins. Further understanding of this phenomenon will require definition of whether S1A is the t-SNARE that normally mediates apical ENaC insertion or whether it is a modulator of insertion mediated by another apical t-SNARE. Butterworth et al. (29) find that syntaxins 1A and 3 are expressed at the apical membranes of cortical collecting duct cells, and it will be important to determine whether ENaC insertion shows t-SNARE specificity at this site.
The rapid inhibition of ENaC current (first exponential of Fig. 2) also is likely to be associated with a physical interaction of S1A and the channel subunit C termini. This conclusion is supported by the lack of effect on whole cell ENaC current or single channel activity of a S1A variant in which the H3 domain was deleted. GST-H3 prolonged the mean closed time of the channel, suggesting that the binding of H3 stabilizes the channel closed state, resulting in the observed decrease in Po. The results obtained with the -S518K ENaC mutant support this interpretation rather than an inhibitory effect of S1A on the channel open state. Because -S518K ENaC is essentially locked in the open state, a reduction in open time would have been expected if S1A decreased Po via an interaction with the open conformation. In -S518K ENaC, S1A presumably does not have access to the closed state that is stabilized by its binding, and therefore, it would not be expected to alter ENaC kinetics, as demonstrated.
Nevertheless, -S518K ENaC currents were inhibited 1 h after injection of the S1A H3 domain, indicating that syntaxin has access to its binding sites at the C termini of the mutant channel and that the -S518K mutation does not alter the action of S1A on ENaC trafficking. Thus, the trafficking and gating effects of S1A can be separated. Work by Ganeshan et al. (30) demonstrates that it was possible to identify separate sites on S1A that affected the ability of syntaxin to modulate CFTR activity as opposed to its ability to assemble into SNARE complexes. It will be interesting to determine also whether similar or separate sites on S1A and ENaC mediate the effects of syntaxin on ENaC gating and trafficking. Both effects may require or be influenced by interactions of S1A with other SNARE proteins. For example, Michaelevski et al. (25) report that Kv 2.1 channel activity is differentially regulated depending on whether S1A is expressed alone or in combination with its partner t-SNARE, SNAP-25, and similar data have been obtained for S1A and SNAP-23 interactions with CFTR (31). At present, we do not know whether SNAP-23 modifies the action of S1A on ENaC trafficking or gating.
The action of S1A on ENaC gating may represent a means of controlling overall channel activity during rapid apical insertion of ENaC channels, a process that underlies the regulation of Na+ transport by ENaC agonists (2). A similar concept has been raised in regard to the regulation of neurotransmitter exocytosis at the nerve terminus, where S1A participates in the fusion of vesicles but then may limit further Ca2+ entry via inhibition of VGCC gating (13). The rapid response of ENaC-expressing epithelia to a Na+ transport agonist is often biphasic, a transient increase followed by recovery to a sustained plateau. These kinetics may reflect feedback inhibition of Na+ entry when channel insertion is rapidly stimulated. Increased cellular Na+ concentrations generally elicit a decrease in ENaC-mediated apical membrane conductance in epithelial cells, and this cellular protective mechanism balances apical Na+ entry with basolateral Na+ extrusion (2). The possibility that S1A may be involved in feedback regulation related to ENaC trafficking was suggested by our previous data, which showed that the binding of S1A to the ENaC C-terminal tails was sensitive to elevated Na+ or Ca2+ concentrations in the binding buffer (8).
Our data support the concept that S1A interactions with ENaC serve to regulate both channel activity and channel trafficking. Syntaxin 1A is potentially a component of the apical SNARE machinery that mediates ENaC insertion; alternately, it modulates this process mediated by other SNARE components. Independent of its possible roles in ENaC trafficking, S1A is also a negative regulator of ENaC gating, which may control channel activity in relation to the status of SNARE interactions and the availability of free syntaxin 1A.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S362 BST, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9498; Fax: 412-648-8330; E-mail: frizzell{at}pitt.edu.
1 The abbreviations used are: ENaC, epithelial Na+ channel; S1A, syntaxin 1A; SNAP-25, soluble N-ethylmaleimide factor attachment protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; GST, glutathione S-transferase; INa, amiloride-sensitive Na+ current; VGCC, voltage-gated Ca2+ channel; Kv, voltage-dependent K+ channel; CFTR, cystic fibrosis transmembrane conductance regulator; wt, wild type; t-SNARE, target SNARE.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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