HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 11, 9743-9749, March 12, 2004

This Article Abstract Full Text (PDF) All Versions of this Article: 279/11/9743    most recent M311952200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sheng, S. Articles by Kleyman, T. R. Search for Related Content PubMed PubMed Citation Articles by Sheng, S. Articles by Kleyman, T. R. Social Bookmarking                      What's this?

Extracellular Histidine Residues Crucial for Na⁺ Self-inhibition of Epithelial Na⁺ Channels^*

Shaohu Sheng, James B. Bruns, and Thomas R. Kleyman

From the Renal-Electrolyte Division, Department of Medicine and the Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, October 31, 2003 , and in revised form, December 15, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Epithelial Na⁺ channels (ENaC) participate in the regulation of extracellular fluid volume homeostasis and blood pressure. Channel activity is regulated by both extracellular and intracellular Na⁺. The down-regulation of ENaC activity by external Na⁺ is referred to as Na⁺ self-inhibition. We investigated the structural determinants of Na⁺ self-inhibition by expressing wild-type or mutant ENaCs in Xenopus oocytes and analyzing changes in whole-cell Na⁺ currents following a rapid increase of bath Na⁺ concentration. Our results indicated that wild-type mouse ENaC has intrinsic Na⁺ self-inhibition similar to that reported for human, rat, and Xenopus ENaCs. Mutations at His²³⁹ (H239R, H239D, and H239C) in the extracellular loop of the ENaC subunit prevented Na⁺ self-inhibition whereas mutations of the corresponding His²⁸² in ENaC (H282D, H282R, H282W, and H282C) significantly enhanced Na⁺ self-inhibition. These results suggest that these two histidine residues within the extracellular loops are crucial structural determinants for Na⁺ self-inhibition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Epithelial Na⁺ channels (ENaC)¹ mediate Na⁺ transport across apical membranes of high resistance epithelial cells in the kidney, colon, and lung. The regulation of Na⁺ transport in collecting ducts has an important role in extracellular fluid volume homeostasis and in the control of blood pressure (1). Alterations in the functional expression of this channel have been observed in association with several disorders, including inherited forms of hypertension (Liddle syndrome), volume depletion associated with hyperkalemia (pseudohypoaldosteronism type 1), and cystic fibrosis (2, 3). Three subunits (, , and ENaC) have been cloned from different species and are members of the ENaC/degenerin gene superfamily (4). All three subunits participate in formation of the channel pore and share a similar membrane topology with two transmembrane domains (M1 and M2) that are connected by a large extracellular loop (ECL), and cytoplasmic N and C termini (5).

ENaC activity is regulated by a variety of extracellular and intracellular factors including hormones, proteases, other proteins, as well as monovalent and divalent cations (6, 7). Na⁺ exerts two distinct types of inhibitory effects on ENaC activity: self-inhibition and feedback inhibition. The inhibitory effects of Na⁺ are distinguished by their sites of action, modes of action, and time course. Na⁺ self-inhibition was originally observed in studies of native epithelial tissues in the setting of a rapid increase in extracellular Na⁺ concentration (6, 8–10). It is not dependent on Na⁺ influx, nor on intracellular Na⁺ concentration. On the other hand, increases in intracellular Na⁺ concentration result in feedback inhibition of ENaC (11–14). The latter is likely due to a reduction of channel density at the plasma membrane (2). The inhibition of ENaC by extracellular or intracellular Na⁺ occurs on different time scales, and may have an important role in limiting increases in intracellular Na⁺ concentration and cell volume when cells are exposed to physiological conditions that would otherwise dramatically increase rates of cellular Na⁺ influx via ENaC (10). Both Na⁺ self-inhibition and feedback inhibition contribute to the complex regulatory network that tightly controls ENaC activity.

ENaCs cloned from human, rat, or Xenopus and expressed in Xenopus oocytes exhibit Na⁺ self-inhibition (15). We previously reported that mouse ENaC (mENaC) is blocked by external Ni²⁺, and identified key extracellular His residues (His²⁸² and His²³⁹) within conserved regions of the - and -subunits that were required for channel block by Ni²⁺ (16). As both Ni²⁺ and Na⁺ are extracellular inhibitors of ENaC, we speculated that extracellular regions that participate in Ni²⁺ block may also have a role in Na⁺ self-inhibition.

We have examined whether specific residues within ENaC ECLs have a role in Na⁺ self-inhibition by comparing the inhibitory responses to external Na⁺ of wild-type and mutant mouse ENaCs in the oocyte expression system. Wild-type mENaC displayed typical Na⁺ self-inhibition. In contrast, we observed that Na⁺ self-inhibition was dramatically altered in channels containing mutations at either of two His residues within the extracellular loops of and ENaC that dramatically decreased Ni²⁺ block. Channels with mutations of His²³⁹ showed no Na⁺ self-inhibition, whereas channels with a mutation of the corresponding His residue in the -subunit (His²⁸²) showed enhanced Na⁺ self-inhibition.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Site-directed Mutagenesis—All ENaC clones used in this study were mouse ENaC subunits whose cDNAs were inserted into pBluescript SK-vector (Stratagene, La Jolla, CA) (17). Point mutations were generated previously by a PCR-based method (16).

ENaC Expression and Two Electrode Voltage Clamp—ENaC expression in Xenopus oocytes and two electrode voltage clamp were performed as previously reported (16). Defoliculated oocytes were injected with 1–4 ng of cRNA for each mENaC subunit (per oocyte) and incubated at 18 °C in modified Barth's saline (MBS, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 15mM HEPES, 0.3 mM Ca(NO₃)₂, 0.41 mM CaCl₂, 0.82 mM MgSO₄, 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, 100 µg/ml gentamycin sulfate, pH 7.4). All experiments were performed 20–50 h following cRNA injections at room temperature (20–24 °C). Oocytes were placed in an oocyte recording chamber from Warner Instruments (Hamden, CT) and perfused with constant flow rate of 12–14 ml/min.

Procedures for Observing Na⁺ Self-inhibition—To examine Na⁺ self-inhibition, a low Na⁺ bath solution (NaCl-1; containing 1 mM NaCl, 109 mM NMDG, 2 mM KCl, 2 mM CaCl₂, 10 mM HEPES, pH 7.4) was replaced rapidly by a high Na⁺ bath solution (NaCl-110; containing 110 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 10 mM HEPES, pH 7.4) while the oocytes were continuously clamped to –60 mV (or –100 mV in some experiments). Bath solution exchange was performed with a 6-channel teflon valve perfusion system from Warner Instruments. At the end of the experiment, 10 µM amiloride was added to the bath to determine the amiloride-insensitive component of the whole cell current. Currents remaining in the presence of 10 µM amiloride were generally less than 200 nA. Results from oocytes that showed unusually large amiloride-insensitive currents (>5% of total currents) were discarded to minimize current contamination from endogenous channels and membrane leak. To ensure consistency, each batch of oocytes injected with wild-type or mutant ENaC cRNAs were examined in an alternating manner.

The first 40 s of current decay was fitted with an exponential equation by Clampfit 9.0 (Axon Instruments Inc.). The peak current (I_peak) was the measured maximal inward current immediately after bath solution exchange from low Na⁺ to high Na⁺ concentration. The steady state current (I_ss) represented the measured current at 40-s post-I_peak. The current ratio of I_ss/I_peak was calculated from amiloride-sensitive I_ss and I_peak that were obtained by subtracting amiloride-insensitive currents from I_ss and I_peak.

To estimate the Michaelis constants (K_m) for Na⁺ concentration-current relationship, both I_peak and I_ss were measured in the same cell after the bath Na⁺ concentration was raised from 1 to 3, 10, 30, 60, and 110 mM and were normalized to their maximal values. The relative I_peak and I_ss were plotted against the Na⁺ concentrations. K_m and V_max (maximal relative current) were obtained by best fitting of the current-concentration data according to Equation 1 with least square non-linear curve fitting using Origin Pro 7.0 (OriginLab Corporation, Northampton, MA).

(Eq. 1)

In the above equation, I is the relative I_peak or I_ss and the C refers to the Na⁺ concentration used to initiate self-inhibition.

We estimated the apparent inhibitory constant (K_i) of Na⁺ self-inhibition from the results of the above experiments. The ratios of I_ss and I_peak representing the amplitudes of self-inhibition at the different Na⁺ concentrations were plotted against the external Na⁺ concentrations. A K_i value was estimated from a best fitting of the data with Hill (Equation 2).

(Eq. 2)

In Equation 2, C is the Na⁺ concentration and the n is the Hill coefficient.

Statistical Analysis—Data are presented as mean ± S.E. Significance comparisons between groups were performed with unpaired Student's t tests. A p value of less than 0.05 was considered statistically different.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Na⁺ Self-inhibition of mENaC—We examined Na⁺ self-inhibition of mENaC expressed in Xenopus oocytes by tracking the current decay following a rapid increase in the extracellular Na⁺ concentration from 1 to 110 mM. The inward current measured at –60 mV quickly reached its maximal level (I_peak) and then relaxed exponentially to a relatively steady level (I_ss) (Fig. 1). Two parameters were used to describe the speed and amplitude of Na⁺ self-inhibition, the time constant () for the current decline and the ratio of the steady state (I_ss) to peak (I_peak) whole cell amiloride-sensitive Na⁺ current. Current decay was observed with a time constant of 8.14 ± 0.38 s (n = 20) (Fig. 1). The ratio of steady state current to peak current was 0.66 ± 0.02 (n = 20). The time course and amplitude of the self-inhibition we observed in mENaC are comparable, though not identical, to those reported for human, rat and Xenopus ENaCs (15, 18). Na⁺ self-inhibition did not appear to exhibit voltage-dependence, as we observed similar responses at clamping voltages of –40, –60, –80, and –100 mV.² A similar self-inhibition response was seen in oocytes expressing different levels of currents, varying from –1 to –20 µA at a holding potential of –60 mV. The intracellular Na⁺ concentration did not significantly affect the speed or amplitude of Na⁺ self-inhibition, as the responses were similar in Na⁺-loaded oocytes that were incubated in 88 mM Na⁺ MBS and in oocytes that were kept in 10 mM Na⁺ MBS following cRNA injection. It has been reported that Na⁺ self-inhibition is not dependent on the current level, nor on intracellular Na⁺ concentration (15).

View larger version (20K): [in this window] [in a new window]   FIG. 1. Na+ self-inhibition of mENaC expressed in oocytes. A, typical recording of Na+ self-inhibition. The oocyte was injected with wild-type mENaC cRNAs and clamped continuously at –60 mV throughout the experiment. A 1-min recording was made in 110 mM Na+ bath solution before testing self-inhibition to examine the stability of the whole cell current. Gray bars and open bar indicate the high [Na+] (110 mM) and low [Na+] (1 mM) bath solutions, respectively. The current decay following switching from 1 mM Na+ to 110 mM Na+ solution represents Na+ self-inhibition. The maximum observed inward current was referred to as peak current (Ipeak), whereas the current measured at 40 s was used as a measure of the steady state current (Iss). The light gray dotted line was obtained from exponential fitting of the current decay, and the time constant from the fitting is presented as in seconds (see "Experimental Procedures"). B, sequence alignments of the segments containing the His282 and His239 (boxed) within the ECL of mENaC subunits. The first residue number in each sequence is indicated in parentheses. M1 and M2 indicate the first and second transmembrane domains, and P denotes the putative P region of ENaC. C, representative recordings of the Na+ self-inhibition responses of , H282R, and H239R mENaCs. Relative currents were obtained by normalizing the currents respectively to the Ipeak in each trace. Open and gray bars have same meaning as in A.

Substitutions of His²³⁹ within the Extracellular Loop Eliminated Na⁺ Self-inhibition—One of the peculiar features of ENaC architecture is that each subunit possesses a very large extracellular loop composed of about 450 residues. While its role in the regulation of channel activity is still unclear, recent studies suggest that the ECL may be involved in inhibitor binding (amiloride and Ni²⁺), channel gating, and subunit assembly and processing (19–23). A six residue tract (WYRFHY) within the ECL of the -subunit was identified as a putative amiloride binding site based on its homology with the antigen binding domain of an anti-amiloride antibody (19, 24). Specific mutations within this tract altered the sensitivity of -subunit channels to amiloride, and altered gating characteristics of both channels as well as channels (16, 19, 20). We and other investigators (16, 25) recently reported that Ni²⁺ is an external inhibitor of ENaC expressed in Xenopus oocytes. The His²⁸² within this tract and the corresponding residue in the -subunit (His²³⁹) were identified as putative Ni²⁺ binding sites (16). As both external Ni²⁺ and high concentrations of extracellular Na⁺ inhibit ENaC, we hypothesized that they might share a similar mechanism for their inhibitory effects on ENaC. This prompted us to examine the possibility that sites responsible for Ni²⁺ block may participate in Na⁺ self-inhibition. We examined Na⁺ self-inhibition of mutant mENaCs containing point mutations at either H282 or H239. To our surprise, Na⁺ self-inhibition was not observed in oocytes expressing H239R, H239D, or H239C mENaC. As shown in Figs. 1C and 2, the currents reached their maximal level and essentially stayed at the same level, with minimal loss of current over a 1-min period.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Mutations at His239 eliminated Na+ self-inhibition. Representative recordings from oocytes expressing H239R (A), H239D (B), or H239C (C) mENaCs are shown. Identical responses of the whole cell currents to rapid increases in the external Na+ concentration were observed in at least 10 oocytes for each mutant. Open and gray bars indicate the time when the cells were bathed in 1 and 110 mM Na+ solutions, respectively.

View larger version (40K): [in this window] [in a new window]   FIG. 3. Mutations at His282 increased Na+ affinity for self-inhibition. Na+ self-inhibition was examined in individual oocytes expressing wild-type or mutant channels with extracellular Na+ concentrations of 110, 60, 30, 10, or 3 mM. A, recordings of current changes in response to the alterations of external Na+ concentration in oocytes expressing , H282D, H282R, H282W, or H282C, were representative of at least four experiments for each channel type. The short black lines above each trace indicate the periods of time when the cell was bathed in a 1 mM NaCl solution. The numbers below each current decay are the Na+ concentrations (in mM) used for inducing self-inhibition. B, relationship of the relative Ipeak and external Na+ concentrations. Relative Ipeak represents the individual peak current at a specific Na+ concentration that was normalized to the maximal Ipeak observed in the same cell. Lines are from the best fitting with Equation 1. C, relationship of the relative Iss (normalized to maximal Iss measured in the same cell) and external Na+ concentration. Solid lines are from best fitting with Equation 1 and dashed lines are from the best fitting with Equation 3. D, the relationship of the ratio of Iss and Ipeak and Na+ concentrations. Lines are from the best fitting of the data with the Hill equation. E, the relationship of the time constants of Na+ self-inhibition and Na+ concentrations (30, 60, and 110 mM).

(Eq. 3)

where K_i is an inhibitory constant for Na⁺ that reflects Na⁺ self-inhibition. Curve fitting of I_ss versus Na⁺ concentration with Equation 3 showed average correlation coefficients of greater than 0.97 for both wild-type and mutant channels. The estimated K_i values for wild-type and mutant channels (H282D, H282R, and H282C) with Equation 3 were comparable to those obtained by fitting the I_ss/I_peak data with Hill equation (see Equation 2 and see Table II). The results suggest that substitution of His²⁸² with Asp, Arg, or Cys increased apparent affinity of Na⁺ for self-inhibition.

The estimated K_m values for channels containing mutations at His²³⁹ were in the range of 72 to 100 mM, much higher than that of wild-type ENaC. This observation suggested that current saturation of wild-type ENaC (particularly for I_ss) at low Na⁺ concentrations was the result of Na⁺ self-inhibition.

As mutations of His²⁸² and His²³⁹ resulted in opposite effects on Na⁺ self-inhibition, we examined the self-inhibition response of a channel with mutations at both sites (H282R--H239R mENaC). Oocytes expressing these channels responded to an increase in the bath Na⁺ concentration from 1 to 110 mM with an extremely slow ( = 16.87 s) and small decline in whole cell Na⁺ current (I_ss/I_peak = 0.9, Fig. 4 and Table I). These results indicate that the H239R mutation has a dominant effect on Na⁺ self-inhibition, supporting a key role of His²³⁹ in channel response to increases in the concentration of extracellular Na⁺.

View larger version (18K): [in this window] [in a new window]   FIG. 4. The H239R mutation has a dominant effect on Na+ self-inhibition of H282RH239R mENaC. The Na+ self-inhibition response was examined in oocytes expressing H282RH239R channels and clamped at –60 mV by rapidly changing bath solutions from 1 mM Na+ (open bar) to 110 mM Na+ (gray bar). Control currents were recorded for 1 min prior to testing for Na+ self-inhibition to demonstrate that basal currents were stable in the 110 mM Na+ bath. The maximal current immediately after switching to the high Na+ concentration bath was slightly greater than the basal current and was followed by a modest decline in the whole cell current. This recording is representative of six experiments.

Na⁺ Self-inhibition Is Absent When ENaC Is Gated Open—It is likely that self-inhibition reflects a reduction of ENaC open probability (Po) initiated by an increase in the concentration of extracellular Na⁺ (10, 15, 27), although direct evidence at the single channel level is still lacking. We examined whether locking ENaC in the fully open state would affect Na⁺ self-inhibition. We previously reported that covalent modification of S580C mENaC with the sulfhydryl reagent MTSET converts channels to a nearly constant open state (28). MTSET had no effect on Na⁺ self-inhibition of wild-type channels.² The Na⁺ self-inhibition response of S580C prior to MTSET was moderately faster and deeper than that of WT mENaCs (Fig. 5A and Table I). However, following MTSET treatment Na⁺ self-inhibition was absent, suggesting that locking the channel in an open state prevents ENaC inhibition by external Na⁺ (Fig. 5). We also examined whether MTSET treatment led to a loss of Na⁺ self-inhibition in a channel containing S580C and H282R, a mutation that enhanced self-inhibition. As shown in Fig. 5B, Na⁺ self-inhibition was absent following MTSET treatment of H282R-S580C channels. Interestingly, prior to MT-SET treatment these two mutations (i.e. H282R and S580C) appeared to have an additive effect on Na⁺ self-inhibition, as double mutant channels (H282R/S580C) showed a response to external Na⁺ that was significantly faster ( = 2.61 ± 0.10 s) and greater in magnitude (I_ss/I_peak = 0.32 ± 0.01) than that of , S580C, and H282R mENaCs (Fig. 5B and Table I). These data suggest that a previously characterized gating domain preceding the second transmembrane domain, including Ser⁵⁸⁰, may be associated with the process of Na⁺ self-inhibition. They also raise a question of whether the two domains within the ECL and the pore region of ENaC subunits that affect Na⁺ self-inhibition are located in proximity to each other. Future studies may test this possibility.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Na+ self-inhibition of ENaC was eliminated following MTSET modification of S580C (A) and H282R-S580C (B). These recordings were representative of six experiments with each mutant. Open and gray bars indicate the 1 and 110 mM Na+ bath solutions, respectively. Bath perfusion with 1 mM MTSET is indicated by the black bar. The stimulated current was relatively constant following washout of MTSET.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The rate and amplitude of Na⁺ self-inhibition we observed in oocytes expressing wild-type mENaCs were similar to that reported by Chraibi and Horisberger (15) using oocytes expressing human and rat ENaCs. All cloned ENaCs examined to date display inhibition in response to sudden increases in extracellular Na⁺ concentration, confirming the notion that self-inhibition is an intrinsic property of the channel rather than mediated by other factors (10). However, the speed and amplitude of Na⁺ self-inhibition responses differ among species (see "Results" and Refs. 15 and 18).

It has been speculated that there exist a regulatory site or "allosteric receptor" for Na⁺ on the extracellular side of the ENaC complex that is responsible for Na⁺ self-inhibition (6, 29). Our data support this hypothesis, as we observed that mutations of His²³⁹ eliminated Na⁺ self-inhibition and mutations of the corresponding His²⁸² enhanced Na⁺ self-inhibition. To our best knowledge, this is the first study to demonstrate altered Na⁺ self-inhibition by point mutations within ENaC subunits. A novel -subunit was recently cloned from Xenopus that is most closely related to the -subunit, and forms functional channels when co-expressed with -and -subunits. The Na⁺ self-inhibition response of channels was significantly more robust than channels, and analyses of / chimeras suggested that a region in the proximal portion of the ECL of the and ENaC subunits that includes the His residue corresponding to mouse His²⁸², was primarily responsible for the difference in Na⁺ self-inhibition of and channels (18). Our results are compatible with the view that the proximal ECL has an important role in Na⁺ self-inhibition.

The changes in Na⁺ self-inhibition that we observed with His²⁸² or His²³⁹ mutants suggest that both His residues are involved in Na⁺ self-inhibition, although the exact roles of these His residues in this process remain to be determined. The complete loss of Na⁺ self-inhibition in channels with mutations of His²³⁹ strongly suggests that His²³⁹ is essential for Na⁺ self-inhibition. We speculate that this residue may be located within a region of the channel that plays a key role in conferring the Na⁺ self-inhibition response. Our previous sequence alignments among ENaC subunits revealed several conserved residues near His²⁸² and His²³⁹ (16), and we have observed that mutation of one of these conserved residues (S227C) greatly slowed Na⁺ self-inhibition.² It is conceivable that this His residue (His²³⁹) may directly participate in Na⁺ binding to a putative receptor site on the channel, participate in conformational changes induced by Na⁺ binding to its receptor that result in a change in channel gating, or directly participate in ENaC gating. Our observation that the self-inhibition response is lost following locking S580C mENaC in open state by covalent MTSET modification indicates that Na⁺ self-inhibition reflects changes in channel gating.

The role of His²⁸² in Na⁺ self-inhibition must be different from that of His²³⁹, as substitutions of the residue with Arg, Asp, Cys, and Trp enhanced Na⁺ self-inhibition. All of these mutations at His²⁸² also reduced or eliminated Ni²⁺ inhibition of ENaC activity (16). These data suggest that Na⁺ self-inhibition and Ni²⁺ inhibition of ENaC may be related in some manner. Indeed, we found that pretreatment of oocytes expressing wild-type mENaCs with 1 mM Ni²⁺ in the low Na⁺ bath solution essentially prevented the current decline in response to an increase in bath Na⁺ concentration.² A recent study concluded that external Ni²⁺ stimulated short circuit current in A6 cells expressing endogenous ENaCs by relieving channels from Na⁺ self-inhibition through competition with extracellular Na⁺ (32). The mechanism by which H282 mutants enhance Na⁺ self-inhibition is unknown. The process of Na⁺ self-inhibition may include a minimum of three distinct events: (1) Na⁺ binding to a "receptor," (2) a conformational change induced by Na⁺ binding and (3) a reduction of channel open probability. Theoretically, augmented self-inhibition may occur as a result of changes in one or more of these events. As Na⁺ self-inhibition was observed with each of the His²⁸² mutants, we propose that His²⁸² is not required for Na⁺ self-inhibition, but functions as an important modulator of the inhibitory response to acute increases in extracellular Na⁺. For example, His²⁸² may be involved in maintaining Na⁺ self-inhibition as a low affinity process, such that ENaC activity is not inhibited by low (i.e. less than 30 mM) concentrations of extracellular Na⁺. This proposed role of His²⁸² is supported by our observation that the His²⁸² mutants we analyzed, except for H282W, decreased the apparent K_i for Na⁺.

Our data suggest that His²⁸² and His²³⁹ have different roles in conferring Na⁺ self-inhibition. Oocytes expressing both H282R and H239R showed a very slow and small current decline (I_ss/I_peak = 0.9) in response to a rapid increase in bath Na⁺ concentration, indicating a dominant role of the His²³⁹ mutation in the double mutant channels. The I_ss/I_peak of 0.9 observed with H282RH239R was significantly lower than that of H239R (0.99, Table I, p < 0.001). The residual inhibition by external Na⁺ suggests that the H282R mutation was still capable of "enhancing" the Na⁺ inhibition response of channels with the H239R mutation. These results support the idea that His²³⁹ has an essential role, and His²⁸² has a regulatory role in Na⁺ self-inhibition. It is not surprising that His residues may be involved in Na⁺ self-inhibition, given their ability to interact with other amino acid residues via aromatic interactions with His, Tyr, Phe, and Trp, and via cation-pi interactions with the side chains of Arg and Lys. His residues also interact with metal ions (33–36). His residues have been found to have key functional roles in enzymes, receptors and ion channels (37–40). The changes in Na⁺ self-inhibition due to mutations at both His sites raise a question whether they may interact with each other at an - subunit interface and whether this proposed interaction is important for ENaC activity and channel regulation by extracellular factors including metals, amiloride, pH, temperature, and fluid flow (16, 19, 20, 25, 41–43). In the context of previous studies and our current results, we suggest that a site in the proximity of these His residues has a role in modulating ENaC function. We propose that this region functions as an extracellular allosteric regulatory site (EARS). The moderate augmentation of self-inhibition by S580C and the loss of self-inhibition of S580C and H282R-S580C following MTSET treatment suggest that regions within ENaC subunits, in addition to the "extracellular allosteric regulatory site," may contribute to the complex process of Na⁺ self-inhibition.

In summary, mutations of specific His residues within the extracellular loops of and ENaC subunits dramatically affect the time constant and degree of Na⁺ self-inhibition. The results suggest that His²³⁹ is critical for Na⁺ self-inhibition and His²⁸² may be a negative regulator of Na⁺ self-inhibition.

	FOOTNOTES

 
^* This work was supported by grants from the National Institutes of Health (DK54354) and the Cystic Fibrosis Foundation (Kleyma03PO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Renal-Electrolyte Division, University of Pittsburgh, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9295; Fax: 412-648-9166; E-mail: shaohu{at}pitt.edu.

¹ The abbreviations used are: ENaC, epithelial Na⁺ channel; ECL, extracellular loop; Po, open probability; K_i, inhibitory constant; WT, wild type.

² S. Sheng, J. B. Bruns, and T. R. Kleyman, unpublished data.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Rossier, B. C., Pradervand, S., Schild, L., and Hummler, E. (2002) Annu. Rev. Physiol. 64, 877–897[CrossRef][Medline] [Order article via Infotrieve]
2. Snyder, P. M. (2002) Endocr. Rev. 23, 258–275[Abstract/Free Full Text]
3. Hummler, E. (2003) Curr. Hypertens. Rep. 5, 11–18[Medline] [Order article via Infotrieve]
4. Benos, D. J., and Stanton, B. A. (1999) J. Physiol. 520 Pt 3, 631–644[Abstract/Free Full Text]
5. Kellenberger, S., and Schild, L. (2002) Physiol. Rev. 82, 735–767[Abstract/Free Full Text]
6. Garty, H., and Palmer, L. G. (1997) Physiol. Rev. 77, 359–396[Abstract/Free Full Text]
7. Schafer, J. A. (2002) Am. J. Physiol. Renal Physiol 283, F221–235[Abstract/Free Full Text]
8. Fuchs, W., Larsen, E. H., and Lindemann, B. (1977) J. Physiol. 267, 137–166[Abstract/Free Full Text]
9. Li, J. H., and Lindemann, B. (1983) J. Membr. Biol. 75, 179–192[CrossRef][Medline] [Order article via Infotrieve]
10. Turnheim, K. (1991) Physiol. Rev. 71, 429–445[Abstract/Free Full Text]
11. Komwatana, P., Dinudom, A., Young, J. A., and Cook, D. I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8107–8111[Abstract/Free Full Text]
12. Komwatana, P., Dinudom, A., Young, J. A., and Cook, D. I. (1998) J. Membr. Biol. 162, 225–232[CrossRef][Medline] [Order article via Infotrieve]
13. Kellenberger, S., Gautschi, I., Rossier, B. C., and Schild, L. (1998) J. Clin. Investig. 101, 2741–2750[Medline] [Order article via Infotrieve]
14. Abriel, H., and Horisberger, J. D. (1999) J. Physiol. 516, 31–43[Abstract/Free Full Text]
15. Chraibi, A., and Horisberger, J. D. (2002) J. Gen. Physiol. 120, 133–145[Abstract/Free Full Text]
16. Sheng, S., Perry, C. J., and Kleyman, T. R. (2002) J. Biol. Chem. 277, 50098–50111[Abstract/Free Full Text]
17. Ahn, Y. J., Brooker, D. R., Kosari, F., Harte, B. J., Li, J., Mackler, S. A., and Kleyman, T. R. (1999) Am. J. Physiol. 277, F121–129
18. Babini, E., Geisler, H. S., Siba, M., and Grunder, S. (2003) J. Biol. Chem. 278, 28418–28426[Abstract/Free Full Text]
19. Ismailov, II, Kieber-Emmons, T., Lin, C., Berdiev, B. K., Shlyonsky, V. G., Patton, H. K., Fuller, C. M., Worrell, R., Zuckerman, J. B., Sun, W., Eaton, D. C., Benos, D. J., and Kleyman, T. R. (1997) J. Biol. Chem. 272, 21075–21083[Abstract/Free Full Text]
20. Kelly, O., Lin, C., Ramkumar, M., Saxena, N. C., Kleyman, T. R., and Eaton, D. C. (2003) Am. J. Physiol. Renal. Physiol. 285, F1279–F1290[Abstract/Free Full Text]
21. Firsov, D., Robert-Nicoud, M., Gruender, S., Schild, L., and Rossier, B. C. (1999) J. Biol. Chem. 274, 2743–2749[Abstract/Free Full Text]
22. Hughey, R. P., Mueller, G. M., Bruns, J. B., Kinlough, C. L., Poland, P. A., Harkleroad, K. L., Carattino, M. D., and Kleyman, T. R. (2003) J. Biol. Chem. 278, 37073–37082[Abstract/Free Full Text]
23. Bruns, J. B., Hu, B., Ahn, Y. J., Sheng, S., Hughey, R. P., and Kleyman, T. R. (2003) Am. J. Physiol. Renal. Physiol. 285, F600–F609[Abstract/Free Full Text]
24. Kieber-Emmons, T., Lin, C., Foster, M. H., and Kleyman, T. R. (1999) J. Biol. Chem. 274, 9648–9655[Abstract/Free Full Text]
25. Segal, A., Cucu, D., Van Driessche, W., and Weber, W. M. (2002) FEBS Lett. 515, 177–183[CrossRef][Medline] [Order article via Infotrieve]
26. Schulz, A. R. (1994) Enzyme Kinetics: From Disease to Multienzyme Systems, pp. 38–41, Cambridge University Press, Cambridge, UK
27. Van Driessche, W., and Lindemann, B. (1979) Nature 282, 519–520[CrossRef][Medline] [Order article via Infotrieve]
28. Sheng, S., Li, J., McNulty, K. A., Kieber-Emmons, T., and Kleyman, T. R. (2001) J. Biol. Chem. 276, 1326–1334[Abstract/Free Full Text]
29. Van Driessche, W., and Zeiske, W. (1985) Physiol. Rev. 65, 833–903[Abstract/Free Full Text]
30. Palmer, L. G., Sackin, H., and Frindt, G. (1998) J. Physiol. 509, 151–162[Abstract/Free Full Text]
31. Gilbertson, T. A., and Zhang, H. (1998) J. Gen. Physiol. 111, 667–677[Abstract/Free Full Text]
32. Cucu, D., Simaels, J., Van Driessche, W., and Zeiske, W. (2003) J. Membr. Biol. 194, 33–45[CrossRef][Medline] [Order article via Infotrieve]
33. Creighton, T. E. (1983) Proteins: Structures and Molecular Principles, pp. 14–16, W. H. Freeman, New York, NY
34. Meurisse, R., Brasseur, R., and Thomas, A. (2003) Biochim. Biophys. Acta 1649, 85–96[Medline] [Order article via Infotrieve]
35. Thomas, A., Meurisse, R., Charloteaux, B., and Brasseur, R. (2002) Proteins 48, 628–634[CrossRef][Medline] [Order article via Infotrieve]
36. Bhattacharyya, R., Saha, R. P., Samanta, U., and Chakrabarti, P. (2003) J. Proteom. Res. 2, 255–263
37. Kossiakoff, A. A., and Spencer, S. A. (1980) Nature 288, 414–416[CrossRef][Medline] [Order article via Infotrieve]
38. Sherwood, A. L., Upchurch, D. A., Stroud, M. R., Davis, W. C., and Holmes, E. H. (2002) Glycobiology 12, 599–606[Abstract/Free Full Text]
39. Okada, A., Miura, T., and Takeuchi, H. (2001) Biochemistry 40, 6053–6060[CrossRef][Medline] [Order article via Infotrieve]
40. Gordon, S. E., and Zagotta, W. N. (1995) Neuron 14, 177–183[CrossRef][Medline] [Order article via Infotrieve]
41. Awayda, M. S., Boudreaux, M. J., Reger, R. L., and Hamm, L. L. (2000) Am. J. Physiol. Cell Physiol. 279, C1896–C1905[Abstract/Free Full Text]
42. Askwith, C. C., Benson, C. J., Welsh, M. J., and Snyder, P. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6459–6463[Abstract/Free Full Text]
43. Satlin, L. M., Sheng, S., Woda, C. B., and Kleyman, T. R. (2001) Am. J. Physiol. Renal Physiol. 280, F1010–1018[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. B. Maarouf, N. Sheng, J. Chen, K. L. Winarski, S. Okumura, M. D. Carattino, C. R. Boyd, T. R. Kleyman, and S. Sheng Novel Determinants of Epithelial Sodium Channel Gating within Extracellular Thumb Domains J. Biol. Chem., March 20, 2009; 284(12): 7756 - 7765. [Abstract] [Full Text] [PDF]

				D. M. Collier and P. M. Snyder Extracellular Protons Regulate Human ENaC by Modulating Na+ Self-inhibition J. Biol. Chem., January 9, 2009; 284(2): 792 - 798. [Abstract] [Full Text] [PDF]

				K. K. Knight, D. M. Wentzlaff, and P. M. Snyder Intracellular Sodium Regulates Proteolytic Activation of the Epithelial Sodium Channel J. Biol. Chem., October 10, 2008; 283(41): 27477 - 27482. [Abstract] [Full Text] [PDF]

				V. Bhalla and K. R. Hallows Mechanisms of ENaC Regulation and Clinical Implications J. Am. Soc. Nephrol., October 1, 2008; 19(10): 1845 - 1854. [Abstract] [Full Text] [PDF]

				V. Bize and J.-D. Horisberger Sodium self-inhibition of human epithelial sodium channel: selectivity and affinity of the extracellular sodium sensing site Am J Physiol Renal Physiol, October 1, 2007; 293(4): F1137 - F1146. [Abstract] [Full Text] [PDF]

				S. Sheng, A. B. Maarouf, J. B. Bruns, R. P. Hughey, and T. R. Kleyman Functional Role of Extracellular Loop Cysteine Residues of the Epithelial Na+ Channel in Na+ Self-inhibition J. Biol. Chem., July 13, 2007; 282(28): 20180 - 20190. [Abstract] [Full Text] [PDF]

				L. Yu, D. C. Eaton, and M. N. Helms Effect of divalent heavy metals on epithelial Na+ channels in A6 cells Am J Physiol Renal Physiol, July 1, 2007; 293(1): F236 - F244. [Abstract] [Full Text] [PDF]

				M. D. Carattino, W. Liu, W. G. Hill, L. M. Satlin, and T. R. Kleyman Lack of a role of membrane-protein interactions in flow-dependent activation of ENaC Am J Physiol Renal Physiol, July 1, 2007; 293(1): F316 - F324. [Abstract] [Full Text] [PDF]

				S. Sheng, M. D. Carattino, J. B. Bruns, R. P. Hughey, and T. R. Kleyman Furin cleavage activates the epithelial Na+ channel by relieving Na+ self-inhibition Am J Physiol Renal Physiol, June 1, 2006; 290(6): F1488 - F1496. [Abstract] [Full Text] [PDF]

				H.-L. Ji, X.-F. Su, S. Kedar, J. Li, P. Barbry, P. R. Smith, S. Matalon, and D. J. Benos {delta}-Subunit Confers Novel Biophysical Features to {alpha}beta{gamma}-Human Epithelial Sodium Channel (ENaC) via a Physical Interaction J. Biol. Chem., March 24, 2006; 281(12): 8233 - 8241. [Abstract] [Full Text] [PDF]

				J. Loffing and L. Schild Functional Domains of the Epithelial Sodium Channel J. Am. Soc. Nephrol., November 1, 2005; 16(11): 3175 - 3181. [Full Text] [PDF]

				M. D. Carattino, S. Sheng, and T. R. Kleyman Mutations in the Pore Region Modify Epithelial Sodium Channel Gating by Shear Stress J. Biol. Chem., February 11, 2005; 280(6): 4393 - 4401. [Abstract] [Full Text] [PDF]

				S. Sheng, C. J. Perry, and T. R. Kleyman Extracellular Zn2+ Activates Epithelial Na+ Channels by Eliminating Na+ Self-inhibition J. Biol. Chem., July 23, 2004; 279(30): 31687 - 31696. [Abstract] [Full Text] [PDF]

				H.-L. Ji and D. J. Benos Degenerin Sites Mediate Proton Activation of {delta}{beta}{gamma}-Epithelial Sodium Channel J. Biol. Chem., June 25, 2004; 279(26): 26939 - 26947. [Abstract] [Full Text] [PDF]

				L. Chen, C. M. Fuller, T. R. Kleyman, and S. Matalon Mutations in the extracellular loop of {alpha}-rENaC alter sensitivity to amiloride and reactive species Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1202 - F1208. [Abstract] [Full Text] [PDF]

				R. P. Hughey, J. B. Bruns, C. L. Kinlough, K. L. Harkleroad, Q. Tong, M. D. Carattino, J. P. Johnson, J. D. Stockand, and T. R. Kleyman Epithelial Sodium Channels Are Activated by Furin-dependent Proteolysis J. Biol. Chem., April 30, 2004; 279(18): 18111 - 18114. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 279/11/9743    most recent M311952200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sheng, S. Articles by Kleyman, T. R. Search for Related Content PubMed PubMed Citation Articles by Sheng, S. Articles by Kleyman, T. R. Social Bookmarking                      What's this?