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A Segment of ENaC Mediates Elastase Activation of Na⁺ Transport

¹ Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261
² Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064
³ School of Pharmacy, Texas Tech. University Health Sciences Center, Amarillo, TX 79106
⁴ Novartis Horsham Research Centre, Horsham, West Sussex RH12 5AB, UK

Correspondence to Robert J. Bridges: bob.bridges{at}rosalindfranklin.edu

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The epithelial Na⁺ channel (ENaC) that mediates regulated Na⁺ reabsorption by epithelial cells in the kidney and lungs can be activated by endogenous proteases such as channel activating protease 1 and exogenous proteases such as trypsin and neutrophil elastase (NE). The mechanism by which exogenous proteases activate the channel is unknown. To test the hypothesis that residues on ENaC mediate protease-dependent channel activation wild-type and mutant ENaC were stably expressed in the FRT epithelial cell line using a tripromoter human ENaC construct, and protease-induced short-circuit current activation was measured in aprotinin-treated cells. The amiloride-sensitive short circuit current (I_Na) was stimulated by aldosterone (1.5-fold) and dexamethasone (8-fold). Dexamethasone-treated cells were used for all subsequent studies. The serum protease inhibitor aprotinin decreased baseline I_Na by approximately 50% and I_Na could be restored to baseline control values by the exogenous addition of trypsin, NE, and porcine pancreatic elastase (PE) but not by thrombin. All protease experiments were thus performed after exposure to aprotinin. Because NE recognition of substrates occurs with a preference for binding valines at the active site, several valines in the extracellular loops of and ENaC were sequentially substituted with glycines. This scan yielded two valine residues in ENaC at positions 182 and 193 that resulted in inhibited responses to NE when simultaneously changed to other amino acids. The mutations resulted in decreased rates of activation and decreased activated steady-state current levels. There was an 20-fold difference in activation efficiency of NE against wild-type ENaC compared to a mutant with glycine substitutions at positions 182 and 193. However, the mutants remain susceptible to activation by trypsin and the related elastase, PE. Alanine is the preferred P₁ position residue for PE and substitution of alanine 190 in the subunit eliminated I_Na activation by PE. Further, substitution with a novel thrombin consensus sequence (LVPRG) beginning at residue 186 in the subunit (_Th) allowed for I_Na activation by thrombin, whereas wild-type ENaC was unresponsive. MALDI-TOF mass spectrometric evaluation of proteolytic digests of a 23-mer peptide encompassing the identified residues (T¹⁷⁶-S¹⁹⁸) showed that hydrolysis occurred between residues V193 and M194 for NE and between A190 and S191 for PE. In vitro translation studies demonstrated thrombin cleaved the _Th but not the wild-type subunit. These results demonstrate that subunit valines 182 and 193 are critical for channel activation by NE, alanine 190 is critical for channel activation by PE, and that channel activation can be achieved by inserting a novel thrombin consensus sequence. These results support the conclusion that protease binding and perhaps cleavage of the subunit results in ENaC activation.

Abbreviations used in this paper: CAP, channel activating protease; ENaC, epithelial sodium channel; FRT, fisher rat thyroid; NE, human neutrophil elastase; PE, porcine pancreatic elastase; TFA, trifluoroacetic acid; TH, human alpha thrombin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The regulated transepithelial transport of Na⁺, critical for maintaining body fluid homeostasis in terrestrial organisms, is attributable to the expression of the heteromeric epithelial Na⁺ channel (ENaC) (Garty and Palmer, 1997). ENaC consists of , ß, and subunits (Canessa et al., 1994; McDonald et al., 1994). It was previously observed that exogenous aprotinin, a serine protease inhibitor, inhibited Na⁺ transport in cortical collecting duct cells from the kidney (Margolius and Chao, 1980; Orce et al., 1980; Vallet et al., 1997; Nakhoul et al., 1998; Vuagniaux et al., 2002) and human bronchial epithelial cells from the lung (Bridges et al., 2001). Epithelial-derived serine proteases such as the channel activating proteases (CAPs, e.g., prostasin) activated ENaC-mediated Na⁺ transport when coexpressed in Xenopus oocytes (Vallet et al., 1997; Donaldson et al., 2002; Vuagniaux et al., 2002). The ability of some of these proteases, CAP1 and prostasin, to increase Na⁺ transport in the coexpression studies was inhibited by aprotinin. In addition to the CAPs, which represent endogenous proteases, ENaC-mediated Na⁺ transport can also be activated by exogenous trypsin, chymotrypsin, neutrophil elastase, and cathepsin G (Chraibi et al., 1998; Caldwell et al., 2005; Harris et al., 2007).

The notion that protease activation of Na⁺ transport results from cleavage of ENaC arises from the correlation between the appearance of cleaved forms of the and subunits and the measured ENaC activity. First, salt restriction or aldosterone infusion (stimuli for increasing Na⁺ reabsorption in the kidney) were associated with the appearance of a lower molecular weight subunit in the rat cortical collecting duct while control rats had only the heavier subunit (Masilamani et al., 1999; Ergonul et al., 2006). Two molecular weight species of the and subunit were found in whole rat kidneys where up-regulation of Na⁺ transport in collecting duct cells correlated with an increase of a lower molecular weight fragment of the subunit and decrease of the higher molecular weight species suggesting a conversion (Ergonul et al., 2006). Aldosterone up-regulation of prostasin in the cortical collecting duct cells (Narikiyo et al., 2002) may generate the subunit cleavage. Further, preventing internalization of ENaC in epithelial cells heterologously expressing ENaC increased the lower molecular weight isoforms of the and subunits at the apical membrane and these changes correlated with decreased sensitivity to exogenous trypsin activation (Knight et al., 2006). Second, mutation of potential furin cleavage consensus sequences found in the and subunits prevented endogenous cleavage of these subunits and reduced Na⁺ transport rates (Hughey et al., 2004a; Bruns et al., 2007).

The single channel studies by Caldwell et al. (2004, 2005) provided evidence that exogenously added serine proteases could activate the channel by direct proteolysis. These results suggest that proteases can directly bind to the channel and induce activation by cleavage. Based on the observations that trypsin and human neutrophil elastase could maximally activate silent channels, we tested the hypothesis that proteases directly interact with ENaC, causing activation of Na⁺ transport in a model epithelial cell line. Site-directed mutagenesis and analysis of the kinetics of current activation by the related proteases neutrophil elastase and porcine pancreatic elastases as well as thrombin were used to show that specific protease interactions with ENaC occur in a segment of the subunit.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
DNA Constructs
Construction of Human , ß, ENaC Tripromoter Expression Vector.
Standard molecular biology methods were used to construct the ENaC tripromoter expression vector. The human , ß, and ENaC subunit cDNAs were isolated from human lung epithelial cells. All three cDNA sequences were identical to the sequence reported by McDonald et al. (1994) (GenBank/EMBL/DDBJaccession nos. L29007, L36592, and L36593). The cDNA was subcloned into pECFPN1 and a stop codon introduced at the 3' end of the subunit to prevent the expression of the CFP. The ß and subunits were subcloned into the pEGFPN1 vector (CLONTECH Laboratories, Inc.) to obtain pEGFPN1/ß and pEGFPN1/. Appropriate start and stop codons were inserted to allow for the expression of the ß and subunits without the C-terminal expression of EGFP. The DNA fragments from pEGFPN1/ß and pEGFP/ vectors comprising the CMV promoters and the subunit cDNA without the GFP sequences were isolated and then subcloned into pECFPN1/ to generate a 13.4-kb tripromoter ß hENaC vector (hENaC). The important features of the hENaC vector are that all three ENaC subunits are arranged in tandem and expression of each subunit is under an independent CMV promoter allowing for the separate expression of each subunit without the expression of ECFP or EGFP. Site-directed mutagenesis was performed using Quick Change II XL (Stratagene) for single and double amino acid substitutions. Overlap PCR was performed using Phusion high-fidelity DNA polymerase (Finnzymes) to introduce a human thrombin consensus sequence (LVPRG) by multiple contiguous substitutions in the _186-190 sequence of hENaC. Overlapping fragments were synthesized by PCR with forward and reverse primers 5'-CTCGTGCCAAGAGGCTCAATGTCATGCACATCGAGTCC-3' and 5'-GCTCCTCAGCAGAATAGCTCATGTTGATTTTCTTCTCC-3'. A second set of forward and reverse primers, 5'-CTGCAGGCCACCAACATCTTTGCACAGGTGCCACAGC-3' and 5'-GCCTCTTGGCACCAACATCTTTGCACAGGTGCCACAGC-3', containing Sbf I and BbvC I restriction sites were used in a second round of PCR to yield the blunt end product that was subcloned into the pCR 4Blunt-TOPO vector (Invitrogen), digested with Sbf I/BbvC I, and ligated to the appropriate Sbf I/BbvC I digest fragment of hENaC. V5 C terminal epitope–tagged ENaC was generated in pc DNA 3.1 hygromycin (Invitrogen) using a standard two-step overlapping PCR method. The sequences of all mutations were verified by DNA sequencing.

RNA Isolation and RT-PCR Experiments
Total RNA was prepared from parental or hENaC FRT cells, using the ThermoScript RT-PCR System (Invitrogen) according to manufacturer's instructions. cDNA was then subjected to PCR amplification using Finnzymes' Phusion High-Fidelity DNA Polymerase (New England Biolabs, Inc.). Primers were designed for identical sequences in human and rat ENaC. Primer sequences were as follows: forward 5'-GGA GAG AAG ATC AAA GCC AAA ATC-3', reverse 5'-GCA TCT CAA TAC TGT TGG CTG GGC-3'. PCR products were separated on a 0.9% agarose gel and visualized by ethidium bromide staining. Amplified products were confirmed by DNA sequencing.

Cell Culture and Transfection
Fisher rat thyroid (FRT) cells were grown in sodium bicarbonate–buffered Hams's F-12 media (Sigma-Aldrich) modified with 10% FCS (Hyclone), 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C and 5% CO₂ as previously described (Sheppard et al., 1994). The cells were expanded in plastic tissue culture flasks. Confluent monolayers were stably transfected with hENaC or mutant plasmids using Lipofectamine 2000 (Invitrogen) and Opti-MEM media (Invitrogen) as described by the manufacturer's instructions and selected with G418 (500 µg/ml; Invitrogen). The selected cells were fed with media containing 50 µM amiloride. FRT cells were seeded onto permeable tissue culture inserts (Transwell Clear, 0.4 µM pore size, 6 mm diameter; Corning) and fed three times a week with or without supplementation with dexamethasone (30 nM) or aldosterone (30 nM). The cells matured for at least 10 d and amiloride was removed from the feeding media two feeding cycles before short-circuit current measurements.

Short-Circuit Current (I_SC) Measurements
Current measurements were performed 10–14 d after seeding. The epithelial cell monolayers were studied by placing the Transwell tissue culture inserts into Costar Ussing chambers with apical and basolateral bathing solution containing (in mM) 120 NaCl, 25 NaHCO₃, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 1.2 CaCl₂, and 10 glucose as previously described (Bridges et al., 2001). The apical and basolateral solutions were continuously circulated with a gas lift. The pH of the solution was 7.4 at 37°C when gassed with a mixture of 95% O₂:5% CO₂. The monolayers were continuously short circuited with a voltage clamp (University of Iowa, Iowa City, IA), and transepithelial resistance was monitored by periodically applying a 4-mV bipolar pulse and calculating resistance from the current change. The amiloride-sensitive I_SC (I_Na) obtained from the difference in current with and without amiloride (50 µM) in the apical bath was taken as a measure of ENaC-mediated electrogenic Na⁺ transport. The amiloride-insensitive current was minimal (<5% of the baseline I_SC). Untransfected FRT cells did not show an I_Na. The proteases trypsin (Sigma-Aldrich), human neutrophil elastase (NE; EPC), porcine pancreatic elastase (PE; EPC), and human thrombin (TH; Sigma-Aldrich) were added to the apical bath and mixed by rapid pipetting with a transfer pipette.

Protocol for I_Na Activation by Proteases
To observe significant and reproducible protease activation it was necessary to pretreat the cells with the serine protease inhibitor aprotinin. Several epithelia appear to have constitutive activation of Na⁺ transport that can be inhibited by aprotinin in a time-dependent manner and the transport rate rapidly reverts to control levels upon addition of exogenous trypsin (Vallet et al., 1997; Bridges et al., 2001; Adebamiro et al., 2005; Myerburg et al., 2006). The slow time dependence of aprotinin inhibition and rapid reversal by excess protease suggests that in the presence of aprotinin, active channels are continually retrieved or degraded while new inactive channels are inserted. This pool of newly inserted channels can then be activated by exogenous proteases. The time course of aprotinin inhibition of current in mammalian airway cells has a half life of 45 min (Bridges et al., 2001). To ensure maximal inhibition for all constructs examined in experiments involving pre-protease inhibition, the protease inhibitor, aprotinin (10 µM; Sigma-Aldrich) or a vehicle control (PBS) was added to the apical surface of the monolayers overnight as previously reported (Vallet et al., 1997) in the modified Ham's F-12 media 1 and remained in the apical bath solution during I_SC measurements. Overnight treatment reduced the variability in the inhibition of I_Na by aprotinin and improved the quantitative analysis of the results.

Data Evaluation and Analysis
The effects of the proteases were assessed in two ways. First, steady-state I_Na was assessed before protease addition (baseline I_Na), then after the I_Na reached a steady-state 20–30 min after addition of NE, PE, or TH (I_PR). Trypsin was then added in excess over the aprotinin concentration to obtain I_Tryp. To compare the effects of NE, PE, and TH to that of trypsin we used the ratio I_PR/I_Tryp and the normalized value I_PR^Norm where I_PR^Norm = (I_PR – baseline I_Na)/(I_Tryp – baseline I_Na). I_PR was measured 20–30 min after the addition of protease (NE, PE, or TH) and I_Tryp was measured 5 min after the addition of trypsin. Second, following addition of NE, PE, or TH the time course of the I_PR^Norm in each experiment was analyzed according to two reactions. The first reaction is shown in Eq. 1.

(1)

The quantity C is the inactive channel at the membrane, E is the added enzyme concentration, and A is the channel activated by proteases. The rate coefficients k_on and k_off have their usual meanings. The differential equation from Eq. 1 is

(2)

Under the assumption that the enzyme concentration is constant due to being in excess and there is no autolysis, the differential equation is solved to yield exponential functions in time that can be fitted to the whole time course of current activation (see online supplemental material, available at http://www.jgp.org/cgi/content/full/jgp.200709781/DC1). The time courses for individual experiments were fitted using Matlab (The Mathworks) to Eq. 3.

(3)

A linear term was added to Eq. 3 to account for a slow decreasing trend in the current records. The reciprocal of the time constant was taken as a measure of the apparent relaxation rate constant k_obs. It is shown in the supplemental material that the k_obs is given by

(4)

The second reaction is of the form shown in Eq. 5.

(5)

The differential equations from Eq. 5 are

(6)

(7)

The k_off and k_on have their usual meanings and k_cat is simply used to express a rate-limiting enzyme concentration-independent step. It is shown in the online supplemental material that the time course of activation can also be approximated by Eq. 3 but here the k_obs is given by Eq. 8:

(8)

where

Linear and nonlinear regression analysis of the k_obs versus enzyme concentration was performed in OriginPro (OriginLab).

Matrix-assisted Laser Desorption Ionization-Time of Flight Analysis (MALDI-TOF)
The sites of cleavage by NE, PE, and TH were searched by MALDI-TOF mass spectrometry on a 23-mer peptide designated peptide T¹⁷⁶-S¹⁹⁸ (Ac-TGRKRKVGGSIHKASNVMHIES-NH₂) and corresponding to the amino acid sequence T176 to S198 of the subunit of ENaC. Additional peptides with glycine substitutions at V182, V193, and A190 as well as a peptide substituted with a TH consensus sequence (LVPRG) beginning at residue 186 were evaluated. Enzymatic digestions were performed at 37°C with NE, PE, and TH at final enzyme concentrations of 200 nM and peptide T¹⁷⁶-S¹⁹⁸ at 200 µM in 200 mM Tris acetate, pH 7.4. Following the incubation period, 100 µl of the reaction was quenched by lowering the pH to 3 with addition of 1.0% trifluoroacetic acid (TFA). The reaction without and with the enzymes were subjected to MALDI-TOF measurements. A ZipTip C₁₈ solid-phase extraction sorbant (Millipore) was washed with 10 µl of acetonitrile/water (1:1, vol/vol) and then equilibrated with aqueous 0.1% TFA. Peptide samples acidified with 5% aqueous TFA to enhance peptide retention on the stationary phase of the C₁₈ sorbant were loaded onto the tip and eluted with 0.1% TFA in acetonitrile/water (1:1, vol/vol). The matrix solution was prepared by dissolving 10 mg of cyano-4-hydroxycinnamic acid in 1 ml of acetonitrile/water (1:1, vol/vol) containing 0.1% TFA. 0.5 µl of the matrix solution was mixed with 0.5 µl of peptide solution. About 0.5 µl of the mixture was deposited onto the MALDI stage and air dried. Positive ion MALDI-TOF Mass spectra were acquired on an Applied Biosystems Voayger DE-Star mass spectrometer operated in reflector mode. Following time-delayed extraction, the ions were accelerated to 20 kV for TOF analysis. A total of 100 laser shots were acquired and signal averaged per spectrum.

In Vitro Translation and Western Blots
cDNAs were transcribed and translated in vitro in the presence of canine pancreatic microsomal membranes using the TnT-coupled reticulocyte lysate systems (Promega). Total DNA templates (0.5 µg) containing 0.25µg ß and 0.25µg -V5 or 0.25 µg of _Th-V5 ENaC subunits were added to an aliquot of the TnT T7 Quick Master mix containing 2 µl microsomal membranes and incubated at 30°C for 60 min. The translation products were collected by centrifuging for 15 min at 10,000 g. and subjected to lysis in 1% Triton X-100 buffer (1% Triton X-100, 500 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.5) at 4°C . The lysates were incubated for 5 min at 37°C in the presence or absence of 200 nM human thrombin (Sigma-Aldrich). All samples in 1x SDS-PAGE sample buffer containing 100 mM DTT were heated for 10 min at 70°C before loading. Proteins were separated on NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and were transferred to Immobilon-P membrane (Millipore). Proteins were detected by Western blot with mouse monoclonal anti-V5 antibody (Invitrogen) targeting a V5 epitope on the C terminus of the ENaC subunit. The signal was developed with the Supersignal west femto maximum sensitivity substrate (Pierce Chemical Co.) and detected with X-Omat Blue XB-1 imaging film (Kodak).

Statistics
Effects of the proteases on steady-state I_Na for the wild-type hENaC were determined using paired t tests of (1) I_Na at baseline versus I_PR or I_Tryp and (2) I_PR versus I_Tryp. Comparison of the ratio of I_PR^Norm and across multiple mutants was performed with one-way ANOVA followed by individual comparisons versus wild-type ENaC using the Bonferroni corrected t test in Origin . P < 0.05 was considered significant.

Online Supplemental Material
The online supplemental material (available at http://www.jgp.org/cgi/content/full/jgp.200709781/DC1) provides the derivations of Eqs. 3 and 8 where the solutions to differential Eqs. 2, 6, and 7 are given.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression of ENaC-mediated Na⁺ Transport in FRT Cells and Regulation by Corticosteroids and Protease
FRT epithelial cells stably transfected with the , ß, and human ENaC subunits in a single tripromoter vector (hENaC) had an I_SC that was blocked by addition of amiloride (50 µM) to the apical side (Fig. 1, A and B). The magnitude of the amiloride-sensitive I_SC (I_Na) averaged 3.4 ± 0.12 µA/cm² (n = 11) and is similar to what has been typically observed by transiently transfecting FRT cells simultaneously with the three ENaC subunits in separate plasmids (Snyder, 2000). Feeding the cells in media supplemented with either aldosterone (30 nM) or dexamethasone (30 nM) for 72 h or more before short circuiting significantly increased I_Na with no increase in the amiloride-insensitive I_SC. Aldosterone induced a 1.5-fold (n = 7) increase in I_Na while dexamethasone induced an eightfold (n = 11) increase in I_Na (Fig. 1 B). The half maximal inhibitory concentration (K_i) for amiloride was 570 ± 86 nM with a maximal inhibition of 96 ± 1.2%. To determine whether proteases regulate Na⁺ transport in FRT cells stably expressing hENaC, the effect of exogenously added trypsin and protease inhibitor were examined. The cells were first preincubated either with the protease inhibitor aprotinin (10 µM) or a vehicle control (PBS) on the apical side for 16–24 h. The cells that were preincubated with aprotinin had decreased I_Na compared with cells preincubated with PBS. During short circuiting, addition of trypsin (15 µM) to the apical bath, in a 5 µM excess over the aprotinin concentration, caused no statistically significant increase in the I_Na of PBS-preincubated cells (3.9 ± 0.87 to 4.5 ± 1.03 µA/cm²; n = 11) but significantly increased I_Na in aprotinin-pretreated cells. The baseline I_Na in the aprotinin-pretreated cells was 3.1 ± 0.68 and the maximal I_Na after addition of trypsin (I_Tryp) was 5.9 ± 0.68 µA/cm² (n = 12). No differences were found in the trypsin-activated current between PBS- and aprotinin-treated cells. Consequently, aprotinin unmasks a portion of I_Na that can be regulated by extracellular protease activity similar to the previously reported effects of aprotinin and trypsin. The absolute values of the aprotinin and trypsin effects on I_Na were greatly enhanced in dexamethasone-treated cells but displayed the same qualitative pattern (Fig. 2, A and B). As already noted, dexamethasone treatment greatly increased the I_Na (37.0 ± 1.19 µA/cm², n = 18). Trypsin addition had no effect on I_Na (37.1 ± 1.14 µA/cm², n = 18) in PBS-pretreated dexamethasone-stimulated cells. Pretreatment with aprotinin caused a 50% decrease in the baseline I_Na (21.3 ± 0.85 µA/cm², n = 18) and in aprotinin-inhibited cells trypsin doubled the I_Na (40.8 ± 2.09 µA/cm², n = 18) to the value observed in PBS-pretreated cells (Fig. 2, A and B). An excess of trypsin above the aprotinin concentration was required to stimulate I_Na. In addition, the stimulatory effects of trypsin on I_Na in aprotinin-treated cells was blocked by soy bean trypsin inhibitor (I_Na with SBTI 0.7 ± 1.22 µA/cm², n = 6). Because the dexamethasone-stimulated hENaC-expressing FRT cells gave a more robust and reproducible I_Na and the protease regulatory pattern persists in dexamethasone-treated cells, dexamethasone-treated cells were used in the remainder of the study. Dexamethasone treatment of untransfected FRT cells did not produce amiloride- or trypsin- sensitive I_SC. In untransfected parental cells treated with PBS, the I_SC before trypsin, after trypsin, and after amiloride addition were 0.1 ± 0.06, 0.2 ± 0.06, and 0.2 ± 0.07 µA/cm² (n = 12), respectively. For cells treated with aprotinin, the I_SC were 0.7 ± 0.3, 0.5 ± 0.16, and 0.5 ± 0.16 µA/cm² (n = 12) before trypsin, after trypsin, and after amiloride, respectively. Furthermore, in cells treated with 30 nM dexamethasone that were stably transfected with the and ß hENaC subunits but without the hENaC subunit there was no I_Na before trypsin (0.2 ± 0.12) µA/cm², after trypsin (0.3 ± 0.08 µA/cm²), and amiloride (0.4 ± 0.09 µA/cm², n = 6). RT-PCR analysis of RNA derived from parental FRT cells without or with dexamethasone treatment did not detect any PCR product for rat ENaC but the expected hENaC product was detected with RNA derived from the hENaC-transfected FRT cells (Fig. 3). Thus dexamethasone-stimulated expression of endogenous rat ENaC and complementation with and ß hENaC subunits cannot explain the I_Na observed in our studies. The importance of this assertion will become apparent in subsequent results.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Expression of ENaC and the corticosteroid effect on Na+ transport in FRT epithelial cells. FRT cells stably transfected with the tripromoter vector containing the , ß, and human ENaC subunits were short-circuited in Ussing chambers with symmetrical NaCl, NaHCO3 buffered ringers. (A) Representative current traces of unstimulated (Ctrl), aldosterone (30 nM), and dexamethasone (30 nM) stimulated cells. The vertical deflections are current responses to 4-mV bipolar pulses for monitoring transepithelial resistance. Amiloride (50 µM), was added to the apical side for the indicated period (bars) to determine INa, the amiloride-sensitive component of the ISC. (B) Summary of the amiloride-sensitive INa (mean ± SEM; n = 7–11).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Na+ transport regulation by aprotinin and trypsin in FRT epithelial cells. (A) Representative ISC traces in FRT cells expressing ENaC, preincubated with and kept in vehicle (PBS) control or aprotinin (APR; 10 µM) during short-circuiting as indicated by the bars. Also shown are traces from cells prestimulated with dexamethasone (30 nM). Trypsin (15 µM) and amiloride (50 µM) were added to the apical side as indicated (horizontal bars). (B) INa (mean ± SEM, n = 11–18) before (open bars) and after (solid bars) addition of trypsin in PBS-preincubated cells and aprotinin-preincubated cells without or with dexamethasone stimulation as indicated. *, P < 0.05 by paired Student's t test comparison of INa at baseline and after addition of trypsin (ITryp).

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 3. RT-PCR analysis of ENaC expression in parental and hENaC-FRT cells. RNA was isolated from parental or hENaC-FRT cells. PCR products were obtained with specific primers spanning identical nucleotide sequences in rat and human ENaC. PCR products were separated on a 0.9% agarose gel and visualized by ethidium bromide staining. Lane 1, standard DNA marker with 1-kb Plus DNA ladder; lanes 2 and 3, RT-PCR of RNA derived from parental cells; lanes 4 and 5, RT-PCR of RNA derived from hENaC FRT cells. Cells for lanes 3 and 5 were treated with 30 nM dexamethasone. The expected PCR product of 1569 bp was detected only in hENaC-FRT cells.

Submicromolar Neutrophil and Porcine Elastase Activate ENaC in FRT Cells
It was previously shown that, like trypsin, NE can activate ENaC-mediated Na⁺ current (Caldwell et al., 2005; Harris et al., 2007). We examined the effect of NE and PE, a similar elastase (Baugh and Travis, 1976; Travis et al., 1979), on the I_SC in FRT cells expressing hENaC. Elastase is not inhibited by aprotinin (Baugh and Travis, 1976; Ohlsson and Olsson, 1976), therefore an excess over the aprotinin concentration was not required to stimulate I_Na. Addition of NE (300 nM) or PE (300 nM) to the apical side of FRT cells expressing hENaC did not alter the I_Na in cells pretreated with PBS (Fig. 4 and Table I). Subsequent addition of trypsin did not further increase the I_Na in PBS pretreated cells. Similar to the effects of trypsin NE and PE clearly increased the I_Na in cells that were pretreated with 10 µM aprotinin as shown in Fig. 4 (B and D). The rise in the I_Na following elastase addition occurred over a 5-min period and increased to a plateau value equal to the baseline I_Na of uninhibited cells that were preincubated with PBS. Addition of trypsin subsequent to NE or PE produced small further increases in the I_Na in aprotinin-treated cells (P < 0.01). The irreversible NE inhibitor MeOSuc-Ala-Ala-Pro-Ala-CMK at 4 µM nearly completely prevented the stimulation of I_Na by NE (300 nM) (I_Na without inhibitor 24.1 ± 4.21 µA/cm², n = 6; I_Na with inhibitor 2.7 ± 0.60 µA/cm², n = 6) and confirm the results of Harris et al. (2007) using a novel NE inhibitor in Xenopus oocyte studies. Thus, NE or PE increased I_Na in aprotinin-inhibited cells to the values observed in control PBS-treated cells and only a small additional further stimulation was seen with trypsin addition in the case of NE-stimulated cells (Fig. 4, E and F, and Table I).

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Elastase-mediated activation of ENaC in FRT cells. (A–D) Representative ISC in FRT cells expressing ENaC that were either pretreated with PBS (A and C) or with 10 µM aprotinin (B and D). (A and B) NE (300 nM) or (C and D) PE (300 nM) was added to the apical bath of short-circuited cells. Trypsin (15 µM) and amiloride (50 µM) were subsequently added to the apical baths in all experiments as indicated (horizontal bars). (E) INa (mean ± SEM, n = 16–24) before (open bars), after 300 nM of NE or (F) PE (stripped bars), and after 15 µM trypsin (shaded bars). *, P < 0.05 in paired Student's t tests of baseline INa and after NE or PE addition. , P < 0.05 in paired student's t tests of INa after NE/PE and after trypsin.

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Two point mutations in ENaC inhibit NE activation of INa in FRT cells. (A) Representative ISC of cells expressing V182G, (C) V193G, and (E) V182G;193G that were pretreated with 10 µM aprotinin before short circuiting. NE (300 nM) was added to the apical bath as indicated (horizontal bars). Subsequently, trypsin (15 µM) and amiloride (50 µM) were added. (B, D, and F) Mean (±SEM, n = 13–39) of baseline INa (open bars), IPR (stripped bars), and ITryp (shaded bars) in cells pretreated with PBS or aprotinin that were expressing ßV182G (B), ßV193G (D), and ßV182G;193G (F) mutants. *, P < 0.05 in Student's t test comparison of amiloride-sensitive ISC before and after NE. , P < 0.05; , P < 0.01 in Student's t test comparison of amiloride sensitive ISC after NE and after trypsin.

View larger version (10K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Effect of mutating residues 182 and 193 in the extracellular domain of ENaC on NE activation of INa. NE (300 nM) was added to short-circuited FRT cells expressing the indicated ENaC mutants that were pretreated with 10 µM aprotinin. 30 min after addition of NE, trypsin (15 µM) was added in excess over aprotinin to achieve a reference point for maximal protease stimulation. (A) Representative traces of IPRNorm for NE activation of wild type (solid squares), ßV182G (open squares), ßV193G (solid circles), and ßV182G; 193G (open circles) ENaCs. The solid lines are the exponential fits of the data. (B) Steady-state values of IPRNorm for the amino acid substitution at 182 and 193 in ENaC (mean ± SEM, n = 6–36). *, P < 0.05, one way ANOVA and t test comparison with wild type. (C) Summary of the from experiments described above (mean ± SEM, n = 4–10) for several amino acid substitutions at residues 182 and 193 in ENaC. *, P < 0.01, one way ANOVA.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Effect of mutations at 182, 190, and 193 in the extracellular domain of ENaC on PE activation of INa.. PE (300 nM) was added to short-circuited FRT cells expressing the indicated ENaC mutants that were pretreated with 10 µM aprotinin. 20 min after addition of PE, trypsin (15 µM) was added in excess over aprotinin to achieve a reference point for maximal protease stimulation. (A and B) Representative ISC trace of FRT cells expressing wild type or ßV182G;V193G mutant. (C) Representative IPRNorm response to PE for wild type (solid squares) and ßV182G;V193G (open squares) mutant with exponential fits (solid lines). (D) Summary of the (±SEM, n = 4–11) from experiments described above for double substitutions at residues 182 and 193 in ENaC. (E) The steady-state IPRNorm (±SEM, n = 8–23) ratios for the double substitutions at 182 and 193 in ENaC. (F and G) Representative ISC trace for FRT cells expressing wild type, ßV182G;V193G, and ßA190G ENaC, respectively. 1,000 nM PE was used. (H and I) (±SEM, n = 5–8) and steady-state IPRNorm (±SEM, n = 5–8) for responses to 1,000 nM PE in cells expressing wild type (open bars), ßV182G;193G (light gray bars), and ßA190G (dark gray bars). *, P < 0.05, one way ANOVA and t test comparison with wild type.

Concentration Dependence of Protease Activation
Because the previous results suggest direct protease–ENaC interaction, a concentration dependence of activation was used to delineate two mechanistic possibilities of current activation: (1) that direct binding was sufficient for channel activation or (2) that binding plus a secondary step is necessary. As can be seen from the progress curves of I_PR^Norm (Fig. 8 A) the rates of current activation as well as the amplitudes of the increase were concentration dependent. Increasing NE concentration from 30 to 1,000 nM reduced for cells expressing wild type from 11.3 ± 1.5 min (n = 8) to 1.6 ± 0.04 min (n = 8). The I_PR^Norm increased with increasing concentration of NE from 0.22 ± 0.02 at 30 nM to 0.75 ± 0.05 at 1,000 nM (Fig. 8 D). The and I_PR^Norm for cells expressing the ß_{V182G; V193G} mutant were also concentration dependent (Fig. 8, B and D). Time constants decreased from 13.8 ± 3.2 min (n = 8) at 30 nM NE to 5.9 ± 0.3 min (n = 8) at 1,000 nM NE, and I_PR^Norm increased from –0.02 ± 0.04 to 0.52 ± 0.02. The concentration dependence of current activation suggests a first approximation of the mechanism of activation of current by NE and PE. Because only one exponential component was present in the fitting of the time courses, the reciprocal of the derived time constants were taken as a pseudo-first-order rate coefficient, k_obs. For wild-type ENaC, the pseudo-first-order rate constant (k_obs) showed saturation behavior with respect to the added enzyme concentration (Fig. 8 C). From the saturation at relatively slow rates, assuming that the enzyme concentration in the chamber was not altered by concentration-dependent autolysis and that the enzyme concentration is in a large excess over its target ENaC, apparent kinetic parameters were derived based on Eq. 8 by nonlinear regression of k_obs against the added enzyme concentration.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 8. NE concentration dependence of the activation of INa in FRT cells expressing wild type and ßV182G;V193G mutant ENaC. (A) Representative IPRNorm responses in FRT cells expressing wild-type ENaC to 30 nM NE (solid squares), 100 nM NE (open squares), 300 nM NE (solid circles), 600 nM NE (open circles), and 1,000 nM NE (solid triangles). (B) Representative IPRNorm responses in FRT cells expressing ßV182G;V193G to 30 nM NE (solid squares), 100 nM NE (open squares), 300 nM NE (solid circles), 600 nM NE (open circles), and 1,000 nM NE (solid triangles). (C) Mean (±SEM, n = 7–8) of kobs (=1/) with respect to the NE concentration for FRT cells expressing wild type (solid squares) and ßV182G;V193G (open squares) ENaC. The solid line through the solid squares is the predicted values from a fit of the wild-type dataset to a kinetic model (see Results) were kcat = 14.2 10–3 s–1 and KM = 570 nM. The solid line through the open squares is a linear regression of the ßV182G;V193G dataset with slope 1,300 M–1s–1 (P < 0.05). (D) Steady-state IPRNorm (±SEM, n = 7–8) ratios for wild type (solid squares) and ßV182G;193G mutant (open squares). Solid lines are fits of the datasets to saturation kinetics. K1/2 was 89 and 470 nM, respectively.

where (I_PR^Norm)^max is the maximum increase and K_1/2 is the enzyme concentration at half maximal stimulation. For NE on wild-type ENaC the K_1/2 was 100 ± 18 nM, and (I_PR/I_Tryp)^Max was 0.84 ± 0.04. For NE on the ß_V182G;V193G mutant, K_1/2 was 510 ± 162 nM and (I_PR^Norm)^Max was 0.94 ± 0.14. Consequently, the apparent affinity for NE activation of I_Na is 5-fold weaker for the ß_V182G;193G mutant compared with wild-type ENaC.

A concentration dependence of activation was also observed for PE activation of the I_Na. The same analysis as described above was performed for the PE datasets. For wild-type ENaC, the saturation behavior of the activation rate (Fig. 9 A) had a K_M of 204± 140 nM and k_cat was 11.4 ± 1.34 x 10^–3 s^–1. For the mutant ß_V182G;V193G, the dependence of k_obs on PE concentration was well defined, in contrast to what was observed for NE. Unlike NE's effect on this mutant, there was saturation with PE demonstrating a K_M of 290 ± 51 nM and a k_cat of 8.7 ± 0.39 x 10^–3 s^–1. Therefore, unlike for NE activation of I_Na these mutations had a minimal effect on PE activation of I_Na. The parameters for the I_PR^Norm versus concentration were K_1/2 of 77 ± 30 nM and 35 ± 9.2 nM for both wild type and ß_V182G;V19G, respectively, and (I_PR^Norm)^Max were 0.7 ± 0.2 and 1 ± 0.05, respectively. Clearly for both wild type and ß_V182G;V193G, activation saturation occurred at low enzyme concentrations (Fig. 8 B) and no difference could be discerned between the two constructs. In contrast to these minor effects, the concentration dependence of PE activation of I_Na in the ß_A190G mutant was significantly impaired. The k_obs increased slowly with enzyme concentration, showing no apparent saturation (Fig. 9 A). Linear regression gave k_cat/K_M of 550 M^–1s^–1. Thus PE activation of wild-type ENaC was 100-fold more efficient than PE activation of ß_A190G. This loss in efficiency is accompanied by a 15-fold increase of the K_1/2 for I_PR^Norm to 1,140 nM (Fig. 9 B).

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 9. PE concentration dependence of the activation of INa in FRT cells expressing wild type, ßV182G;V193G mutant, or ßA190G ENaC. (A) Mean (±SEM, n = 8) of kobs with respect to the PE concentration for FRT cells expressing wild type (solid squares) and ßV182G;V193G ENaC (open squares), and ßA190G ENaC (solid circles). The solid lines are the kinetic fits of the data as described in Materials and methods; fitted parameters kcat and KM were 11.4 x 10–3 s–1 and 204 nM for wild-type ENaC; 8.7 x 10–3 s–1 and 294 nM for ßV182G;V193G. Linear regression of the kobs versus PE concentration for ßA190G gave a slope of 550 ± 89 M–1s–1 (P < 0.05). (D) IPRNorm (mean ± SEM, n = 8) ratios for wild type (solid squares) and ßV182G;V193G (open squares) mutant. Solid lines are fits to saturation kinetics. K1/2 was 77 ± 35, 35 ± 9.2, and 1140 ± 221 nM, respectively.

NE and PE Cleavage of the ENaC Segment

View this table: [in this window] [in a new window]   TABLE III Peptide Products Derived from a Segment of Human ENaC after Protease Digestion and Analysis by MALDI-TOF Mass Spectrometry