Serine protease activation of near-silent epithelial Na+ channels

Ray A. Caldwell, Richard C. Boucher, and M. Jackson Stutts

The Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599-7248

Submitted 6 August 2003 ; accepted in final form 7 September 2003

ABSTRACT

The regulation of epithelial Na+ channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/retrieval of channels, by changes in channel open probability (Po), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na+ transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outside-out patches from fibroblasts (NIH/3T3) stably expressing rat {alpha}-, {beta}-, and {gamma}-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity (NPo) < 0.03, where N is the number of channels. Within 1–5 min of 3 µg/ml bath trypsin superfusion, NPo increased ~66-fold (n = 7). In patches observed to contain a single functional channel, trypsin increased Po from 0.02 ± 0.01 to 0.57 ± 0.03 (n = 3, mean ± SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na+/Li+ current ratio with trypsin were similar to control values. Modulation of ENaC Po by endogenous epithelial serine proteases is a potentially important regulator of epithelial Na+ transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels.

silent channels; protease; epithelial Na+ transport; cystic fibrosis; hypertension


THE REGULATION of epithelial Na+ channel (ENaC) function is key to understanding the physiology of epithelial Na+ transport. The epithelium-derived serine proteases channel activating protease (CAP) and prostasin are expressed in tissues containing ENaC and augment amiloride-sensitive currents when coexpressed with ENaC in Xenopus oocytes (1, 5, 19, 20). Moreover, aprotinin, a serine protease inhibitor, decreases the amiloride-sensitive short-circuit current in mouse cortical collecting duct and human bronchial epithelia (2, 20). These results suggest that proteases may regulate epithelial Na+ transport in vivo, but the precise functional role of endogenous protease activation of ENaC has remained elusive (16). Cell surface labeling of ENaC combined with amiloride-sensitive current measurements in oocytes provided evidence for an abundance of ENaC having very low channel activity (6). These "silent" channels were estimated to have a whole cell Po of ~0.02, much lower than the average Po reported with direct patch-clamp measurements (14, 15). Thus activation of silent channels by endogenous epithelium-derived serine proteases is a potentially important physiological regulatory mechanism of epithelial Na+ transport.

Here, we functionally identify a near-silent ENaC population in the plasma membrane of 3T3 cells by their activation with brief exposure to extracellular trypsin. Figure 1 shows a representative outside-out patch recording obtained from a fibroblast stably expressing rat {alpha}-, {beta}-, and {gamma}-subunits (rENaC). Only brief and infrequent single transitions were observed during baseline measurement (Fig. 1A, sweep 6). Within 1.5 min of 3 µg/ml bath trypsin exposure, up to four channels were open simultaneously (Fig. 1A, sweep 11). As in this instance, when more than one channel was detected, channel activity was defined as the product of NPo, where N is the number of channels and Po is the open channel probability. Near-silent channels, defined as having NPo < 0.03, were observed in 11 of 23 recordings (or ~48%). The robust increase of the trypsin-induced NPo was specific for the 7.2 pS ENaC (also see Fig. 1A, amiloride), because trypsin activation of endogenous channels was not observed. The open channel diary of the recordings shown in Fig. 1A is illustrated in Fig. 1B. NPo increased 102-fold during the 3-min trypsin superfusion, relative to baseline. Figure 1C shows the summary data from seven experiments in which channel activity was recorded during 1- to 5-min trypsin exposure and compared with baseline activity from the same patch. Trypsin induced an ~66-fold increase of NPo. The trypsin-induced increase of NPo was not reversed despite extensive washout of the enzyme from the bath for up to 20 min.



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Fig. 1. Extracellular trypsin reveals otherwise "near-silent" channels in outside-out patches. A: rat epithelial Na+ channel (ENaC) recordings at –60 mV during superfusion with the standard Li+ bath solution, with and without trypsin (3 µg/ml). The sweep number indicates trace sequence. A single channel with brief and infrequent openings was observed before enzyme exposure (sweep 6). Trypsin (applied at arrow, sweep 8) induced a large increase in average channel activity (NPo) during the 3-min exposure (sweeps 9–13). ENaC identity and patch configuration were confirmed with reversible channel inhibition by using bath amiloride (10 µM; applied at + arrow and removed at – arrow, sweep 14). Dashed lines represent fully closed channel (or amiloride-inhibited current level). B: open channel diary (note log ordinate) of recording in A. NPo increased from 0.02 ± 0.01 to 2.04 ± 0.57 (mean ± SD) during the 3-min trypsin superfusion. C: summary of effects of 3 µg/ml trypsin on NPo. Solid lines connect NPo data obtained from the same patch. Control NPo was 0.02 ± 0.01 and increased ~66-fold (to 1.31 ± 0.36, n = 7) during trypsin superfusion. All experiments were performed at ~23°C.

 

To test whether trypsin catalytic activity was required for activating ENaC through a direct or second messenger-mediated mechanism, patches were exposed to trypsin in the presence of soybean trypsin inhibitor (SBTI). As shown in Fig. 2, an outside-out patch was observed to contain a single functional channel having brief and infrequent openings during the control period and also during a subsequent 5-min trypsin superfusion with SBTI. However, within 1 min of removal of SBTI from the bath, in the continued presence of trypsin, Po increased from 0.003 to 0.621. NPo measured in trypsin with SBTI was indistinguishable from the control NPo (Fig. 2). These experiments required an additional 5-min recording time before superfusion of trypsin alone. In no instance did we observe a time-dependent increase of NPo in these and other extended control recordings (>5 min), which could explain our observations with trypsin.



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Fig. 2. Trypsin-induced channel activity requires catalytic activity. Outside-out patch recording before (control, sweep 3) and after 5-min combined trypsin and 19-fold excess soybean trypsin inhibitor (SBTI) exposure (sweep 18) (see EXPERIMENTAL METHODS). Trypsin increased Po from 0.003 to 0.621 after SBTI removal (–SBTI, arrow at sweep 19). NPo during bath superfusion of trypsin with SBTI was not different from control (control NPo = 0.66 ± 0.27; trypsin + SBTI NPo = 0.67 ± 0.33; P = 0.9, n = 4). The unitary current amplitude (control i = 0.47 ± 0.01 pA; trypsin + SBTI i = 0.50 ± 0.04 pA; –60 mV; P = 0.4, n = 4) was also not affected by enzyme superfusion with SBTI. Channel activity was reversibly inhibited with amiloride (10 µM). Dashed lines indicate fully closed state.

 

Because 3T3 cells contain trypsin-sensitive G protein-coupled proteinase-activated (PA) receptors (12), we evaluated whether PA receptors were responsible for the trypsin-induced increase of ENaC NPo. G protein activation was inhibited by complete replacement of GTP in the pipette solution with equimolar GDP{beta}S. Patches were allowed to dialyze at least 5 min before recordings were initiated. Despite GTP replacement with GDP{beta}S, trypsin increased NPo, as shown in Fig. 3. Similarly, trypsin activation of amiloride-sensitive currents in oocytes also did not involve G protein signaling (3). The inability to wash out the trypsin effect on ENaC activity (for >20 min) is also consistent with a non-receptor-mediated action of the enzyme on NPo. These findings suggest that trypsin catalytic activity likely involves ENaC itself or a closely associated ENaC-regulatory protein present in 3T3 cells (see below).



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Fig. 3. G protein coupled proteinase-activated (PA) receptors are not involved in the trypsin-induced activation of ENaC. A: representative recording showing control (sweep 7) and 3-min trypsin exposure (sweep 16) illustrating the enzyme-induced increase of NPo despite complete replacement of intracellular GTP with GDP{beta}S. Reversible amiloride inhibition of channel activity also is shown (sweeps 17 and 18). B: summary data from 4 experiments in which trypsin increased NPo with GDP{beta}S in the pipette [GDP{beta}S log(NPo) = –1.12 ± 0.42 vs. trypsin + GDP{beta}S log(NPo) = 0.085 ± 0.326; P = 0.035]. Solid lines connect data obtained from the same patch. Channel activity was recorded in 150 mM bath Li+ at –60 mV.

 

The trypsin-induced increase of ENaC NPo could result from a change in N, Po, or a combination of these parameters. In records where only a single channel was observed (i.e., Fig. 2), trypsin induced a 32.8-fold increase in Po (from 0.02 ± 0.01 to 0.57 ± 0.02, P = 0.002, n = 3). The increased Po resulted from an increased open time and a decreased closed time for the channel. The infrequent channel transitions in control, and the long openings (i.e., >20 s) during trypsin exposure, precluded a quantitative analysis and comparison of channel dwell times, however. We found no evidence to support a trypsin-induced fusion of ENaC-containing vesicles with the outside-out patch of membrane (i.e., increased N). The patch-clamp technique is a highly sensitive detector of vesicular fusion with the ability to detect individual vesicle fusion events (10, 11). The large-conductance fusion pore (i.e., 70–250 pS) created during the redistribution of membrane charge that occurs with addition of patch-surface membrane (8) could be readily detected with our recording conditions if it had occurred, but it was never observed (ENaC conductance is 0.03–0.1 of the fusion pore conductance; see below). Moreover, for exocytosis to occur, vesicles along with other proteins necessary for fusion (i.e., synaptobrevin) (8) must remain intact with the excised remnant of plasma membrane. Also, patch excision with the high-resistance pipettes (i.e., 14 M{Omega}) substantially limits the membrane surface area (estimated ≤1 µm2) (17) available for vesicle fusion. Finally, in view of the outside-out configuration with the large Ca2+-buffering capacity of the pipette solution, Ca2+-dependent exocytosis via Ca2+ release from intact stores (i.e., endoplasmic reticulum) is unlikely. Although we cannot exclude the possibility of localized Ca2+ entry via Ca2+ conductance in the plasma membrane, the trypsin-induced increase of amiloride-sensitive currents in oocytes was also not Ca2+ dependent (3). Taken together, these findings indicate that a mechanism whereby trypsin increases NPo through surface membrane fusion of ENaC-containing vesicles (i.e., increased N) is highly unlikely with our recording conditions.

The single-channel conductance, obtained from the least-squares fit of the average unitary current measurements (voltage range: –80 to –40 mV; 5–27 recordings at each voltage) with Li+ as the charge carrier, was 7.2 pS, and during trypsin exposure it was 7.4 pS. The Na+/Li+ current ratio (control = 0.48 vs. trypsin = 0.50; –60 mV) was also similar. Collectively, these results strongly suggest that serine proteases increase ENaC-mediated currents by increasing the Po of near-silent channels, not by increasing the number of channels or single-channel conductance.

Interestingly, despite the robust protease-induced increase of macroscopic amiloride-sensitive currents reported in oocytes, enzyme effects on rENaC NPo were inconclusive (1, 3). This lack of effect was ascribed to the characteristic highly variable ENaC Po and the selection of patches containing channels with moderate control channel activity (i.e., Po ~0.5) (15). Indeed, we also observed wide variability of control NPo from sweep to sweep in the same patch (Fig. 1C, sweeps 1–7). However, trypsin consistently increased NPo. We believe our ability to observe the enzyme-induced increase of channel activity stemmed from measurements of NPo made before and during trypsin exposure from the same patch, coupled with amiloride superfusion to unambiguously identify the fully closed/blocked channel current level. In fact, in some experiments, we observed large baseline channel activity without ever observing the fully closed channel current level in the absence of amiloride. Confirmation of the fully closed/blocked current level is essential for accurate analysis of NPo, which is not always straightforward with the cell-attached patches used in previous studies that could underestimate serine protease-induced effects on NPo. Alternatively, the protease effect on single active channels (i.e., Po ~0.5) is expected to be minimal, if any.

Is proteolytic cleavage of ENaC required for expression of active channels? Recently, Hughey et al. (9) showed that during channel maturation, the extracellular domains in some of mouse {alpha}- and {gamma}-subunits were cleaved by an endogenous, aprotinin-insensitive protease(s). Moreover, in A6 epithelia (18) and human bronchial epithelia (2), a sizable amiloride-sensitive current persists after a lengthy preincubation (>1 h to overnight) with aprotinin or the recombinant Kunitz-type serine protease inhibitor BAY 39-9437. Frindt et al. (7) also reported cleavage of the {gamma}-subunit in rat cortical collecting tubule (CCT) after animals were subjected to ≥15 h of Na+ restriction (13). Interestingly, {gamma}-subunit cleavage correlated with expression of amiloride-sensitive whole cell currents in CCT (7), but the effect of aprotinin on whole cell currents or on the {gamma}-subunit band pattern was not reported. In view of these findings and the results described here, it is tempting to speculate that during maturation, some fraction of {alpha}- and/or {gamma}-subunits are cleaved by aprotinin-insensitive protease(s) and, along with the {beta}-subunit, emerge onto the cell surface membrane as active channels, whereas subunits not cleaved during maturation make up the near-silent pool of channels that are activated by aprotinin-sensitive extracellular proteases (i.e., CAP-1, CAP-2, trypsin). Future studies are needed to identify protease-sensitive residues in the extracellular domains of ENaC and determine how mutating these sites affects channel function. Results from such studies should be informative for understanding the physiological regulation of epithelial Na+ transport.

EXPERIMENTAL METHODS

Cell culture. NIH/3T3 cells were infected with retrovirus encoding cDNAs for rENaC {alpha}-, {beta}-, and {gamma}-subunits (18). Clones stably expressing ENaC subunits were grown at 37°C, under an atmosphere of humidified 5% CO2, in Dulbecco's modified Eagle's medium with 10% bovine calf serum, 10 µM amiloride, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 300 µg/ml G418, and 1 µg/ml puromycin. Cells for patch-clamp experiments were subcultured on 35-mm culture dishes and visually selected using a Nikon inverted microscope equipped with Hoffman modulation contrast optics.

Electrophysiology. Outside-out patches were studied. Patchclamp signals (EPC-7; List, Darmstadt, Germany) were filtered at 0.1 kHz (–3 dB, Bessel), digitized at 1 kHz (16-bit, ITC; Instrutech, Long Island, NY), and acquired with a Pentium computer running HEKA-PULSE acquisition software (Bruxton, Seattle, WA). Patch pipettes (borosilicate; Warner Instruments, Hamden, CT) were fabricated from thin-walled glass using a two-stage pull routine (Sutter Instruments, Novato, CA). Pipette resistance was 13.8 ± 0.3 M{Omega} (mean ± SE, n = 51). A Ag-AgCl electrode connected to the bath via a 3% agar bridge containing 1 M KCl served as the ground electrode. The diffusion potential, measured between pipette and bath solution, was 2.0 ± 0.1 mV (n = 4), and all voltages have been corrected by this amount.

Single-channel activity was recorded at membrane potentials from –100 to 0 mV with flowing bath conditions. Exchange of bath solution occurred in <100 ms with the use of a Fast-step solution exchanger (Warner Instruments). Solution flow rate was 0.2 ml/min. Bath amiloride inhibition of single channels was used to confirm ENaC identity and patch configuration. Channels observed as amiloride insensitive were not included for analysis.

Bath and pipette solutions. The standard bath solution contained (in mM) 150 Li(or Na)-aspartate, 2 MgCl2, 1 CaCl2, and 5 HEPES, titrated to pH 7.30 with LiOH (or NaOH). The standard pipette solution contained (in mM) 120 Tris-aspartate, 20 NaCl, 3 MgATP, 0.2 Na2GTP, 0.1 CaCl2, 1 EGTA, and 5 HEPES, titrated to pH 7.10 with NaOH. In selected experiments, GTP was replaced with equimolar GDP{beta}S. Amiloride (10 µM), trypsin (type I, 10,800 U/mg, <4 U/mg chymotrypsin; Sigma), and SBTI (type I-S; Sigma) were dissolved in the bath solution. The amount of SBTI was 19-fold larger than the amount necessary to inhibit 756 units of trypsin catalytic activity (N{alpha}-benzoyl-L-arginine ethyl ester, substrate).

Data analysis. For records identified as containing a single channel, Po was measured as the total open time of the channel, normalized to the total time of the recording at a particular test voltage. Channel transitions and open times were measured from idealized records based on 50% threshold criterion. For patches containing multiple channels, evident as two or more simultaneously opened transitions, average channel activity (NPo) was analyzed and obtained by integrating the area under the Gaussian curves fitted to the all-points current-amplitude histogram and normalized to the peak area of the baseline current level (i.e., closed channel current level) (4). Data analysis was performed with single-channel analysis software (TAC and TACFit; Bruxton).

Statistics. All results are reported as means ± SE, with n = no. of patches, unless otherwise stated. Because baseline ENaC activity is highly variable (15) from patch to patch, each patch served as its own control. Comparisons of NPo were performed using a paired t-test. The effect of trypsin on patches containing GDP{beta}S in the pipette solution (Fig. 3) was evaluated on log-transformed NPo data. A P value < 0.05 was considered statistically significant.

ACKNOWLEDGMENTS

We thank Dr. Robert Rosenberg (UNC-CH Dept. of Pharmacology) for use of the pipette puller. Also, we thank Daniel Gillie and Lisa Lowenstein for excellent technical assistance.


Address for reprint requests and other correspondence: R. A. Caldwell, Cystic Fibrosis/Pulmonary Research and Treatment Center, CB#7248, Univ. of North Carolina, Chapel Hill, NC 27599-7248 (E-mail: ray_caldwell{at}med.unc.edu).

FOOTNOTES

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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