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Intrinsic gating mechanisms of epithelial sodium channels

Hong-Long Ji, Catherine M. Fuller, and Dale J. Benos

Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
REFERENCES

The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in alpha -ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal beta - and gamma -ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, alpha -ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC.

Liddle mutant; amiloride; inside-out patch; voltage clamp; post-M2 region


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
REFERENCES

LIDDLE'S SYNDROME is a form of hereditary hypertension produced by mutations within the epithelial sodium channel (ENaC) (1, 17). These mutations result in constitutive channel activation. Both an increase in functional channel number and an increase in single-channel open probability (Po) have been reported (5-9, 14, 15). The initial description of Liddle's syndrome identified truncation mutations in the COOH-terminal polypeptide chain of the beta -ENaC (and subsequently the gamma -ENaC) subunit as being causative for constitutive channel activation (3, 5-9, 14, 15). We proposed the hypothesis that the COOH-terminal chains of beta  and gamma  could act as intrinsic channel blockers by serving as an inactivation moiety. Our evidence, obtained in both bilayers (7, 8) and heterologous expression systems (9), supports this type of mechanism. We tested the hypothesis that the functional gating particle comprised the COOH-terminal tails of both the beta - and gamma -ENaC subunits associated as a two-strand, antiparallel beta -sheet. Support for this idea was threefold: 1) the inhibitory effects of adding COOH-terminal beta - and gamma -ENaC 30-amino acid residue peptides together with ENaC comprising wild-type alpha - and COOH-terminally truncated beta - and gamma -subunits produced a greater than additive inhibition of the channel; 2) circular dichroism studies showed that the 30-mer beta  and gamma  peptides formed a beta -sheet; and 3) when the isoleucines and valines within the 30-mer peptides were replaced by the beta -sheet, breaking amino acids proline or aspartic acid, the resulting peptides were unable to affect basal-activated ENaC (8). The paradigm that we have developed for ENaC gating is as follows. Because alpha -ENaC itself forms a functional sodium channel (4), there must be an intrinsic gating mechanism in alpha -ENaC alone. We hypothesized that calcium is intimately involved in this process and have presented evidence to this effect (2). The overall gating properties of alpha -ENaC vs. alpha beta gamma -ENaC in bilayers do not differ (7). Because the elimination of the cytoplasmic COOH-terminal tails of either or both of the beta - and gamma -subunits substantially increases single-channel Po (5-9), there must be at least two separate gating processes, one inherent to alpha -ENaC alone and one conferred onto the complex by the beta - and gamma -subunits.

To further elucidate the mechanism underlying the COOH-terminal beta - and gamma -ENaC tail block of ENaC, we tested the hypothesis that a highly conserved region following (or at the most distal end of) the second transmembrane domain (M2) in alpha -ENaC may act as a putative receptor region for the negatively charged COOH-terminal beta - and gamma -ENaC tails and, thus, facilitate their interaction with the channel. We have identified a sequence of positively charged amino acids between residues 586 and 591 of human alpha -ENaC (613-624 of the rat alpha -ENaC ortholog) (10) that is identical in all five mammalian alpha -ENaC subunits cloned to date (rat, bovine, human, mouse, and guinea pig) and that is arginine rich (RRFRSRYWSPGR). This region is conserved in delta -ENaC (RRLRRAWFSWPR) (16) but is not present in either the beta - or gamma -ENaC subunit or in the ENaC-related Aplysia sodium channel that is gated by FMRF-amide (phenylalanine-methionine-arginine-phenylalanine) (12). We hypothesized that this concentration of positive charge may form a site for electrostatic interaction of the negatively charged COOH-terminal beta  and gamma  tails and, thus, provide an anchoring point for the peptides somewhere near the internal channel mouth. The peptides may bind at a site away from the actual channel mouth but exert an inhibitory effect via steric hindrance or by inducing a conformational change. In the present experiments, we tested this hypothesis by mutating either two or four of the arginine residues in this sequence to amino acids of opposite charge. Our prediction was that the disruption of the charge distribution in this region would abolish the ability of the COOH-terminal beta - and gamma -ENaC peptides to inhibit the channel.

We expressed the beta -ENaC subunit truncated at amino acid R564 (beta R564X) and the gamma -ENaC truncated at amino acid R574 (gamma R574X) in combination with the wild-type rat alpha -ENaC subunit (i.e., alpha beta R564Xgamma R574X) in Xenopus oocytes. Figure 1 shows representative amiloride-sensitive Na+ current traces obtained in voltage-clamped oocytes before and after 1 mM peptide mixture injection. In this series of experiments, the currents obtained from oocytes injected with wild-type alpha -ENaC and truncated beta - and gamma -ENaC subunits were greater than the corresponding currents recorded from wild-type alpha beta gamma -ENaC injected in oocytes obtained from the same frogs (data not shown). The average inward current levels were approximately twofold increased, as reported earlier (9). Injection of a mixture of beta  plus gamma  COOH-terminal peptides (SP30beta and SP30gamma ; 1:1, 500 µM each) into oocytes expressing the truncated ENaC constructs (i.e., alpha beta R564Xgamma R5764X-ENaC) decreased the current by ~50% at negative potentials. In contrast, the currents in oocytes expressing either of the alpha  double mutations (i.e., alpha R586E,R587E or alpha R589E,R591E) or the quadruple mutation in the alpha -subunit (alpha R586E,R587E,R589E,R591Ebeta R564Xgamma R574X) were not affected by the same concentration of peptide mixture. As controls, the alpha beta R564Xgamma R574X-ENaC associated current was not affected by water injection or by injection of SP30gamma plus a 30-amino acid peptide identical to SP30beta , with substitution of three prolines for one valine and two isoleucines (see Fig. 4). Summary data for the effect of the peptide mixture on wild-type alpha -subunit plus truncated beta - and gamma -subunits, two double alpha  arginine mutations, and the quadruple mutation (all in combination with the truncated beta - and gamma -subunits) are shown in Fig. 2. Of these constructs, only the channel containing wild-type alpha -ENaC was inhibited by 1 mM peptide mixture. In contrast, both double mutants (namely, alpha R586E,R587E and alpha R589E,R591E) and the quadruple mutant, in which the four positively charged arginines (R residues) were replaced with negatively charged Glu (E residues), were unaffected by injection of SP30beta plus SP30gamma (Fig. 2).


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Fig. 1.   Representative current traces showing the effects of a 30-mer peptide mixture injection into Xenopus oocytes on amiloride-sensitive Na+ currents produced by various mutated alpha -ENaC (epithelial sodium channel) constructs (left). Records were made 24 h after cRNA injection for all groups of oocytes. Test voltages were stepped from a holding potential of 0 mV to -100 through + 100 mV in a 10-mV increment, and each step was held for 500 ms. Right: effects of peptide mixture (total concentration 1 mM; 500 µM each of SP30beta and SP30gamma ) on currents. Each experiment was repeated at least 3 times.



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Fig. 2.   Summary of data showing the effect of SP30beta plus SP30gamma (1 mM) injection on macroscopic amiloride-sensitive Na+ conductance in Xenopus oocytes using different ENaC constructs. Data were averaged amiloride-sensitive Na+ conductances; Error bars indicate SE. N is the number of eggs recorded. The conductances in the presence of peptides were normalized to that in the absence of peptides (100%). The percentages of conductance in the presence of peptide mixture (from far left to the far right) are 38.7 ± 8, 48.3 ± 16, 98.8 ± 8, 50.3 ± 19, 103.5 ± 7, 47.2 ± 21, and 96.3 ± 10% of the control before peptide injection. *P < 0.05 compared with the control.

As further controls for these experiments, we made a series of alpha  mutations in the same position as the arginines, except that instead of changing the sign from positive to negative (glutamic acid), we maintained the same charge distribution by substitution with lysine (K). In an additional group of oocytes, 1 mM peptide added to alpha R586K,R587K,R589K,R591Kbeta R564Xgamma R574X, alpha R586K,R587Kbeta R564Xgamma R574X, or alpha R589K,R591Kbeta R564Xgamma R574X inhibited macroscopic currents to the same extent as when added to the wild-type alpha -subunit. These data are supportive of the hypothesis that this arginine-rich region in the most distal portion of the M2 region plays a role in the interaction of the beta -and gamma -subunit tails, causing inhibition of the channel.

We used single-channel analysis to explain more precisely the results that we obtained with these COOH-terminal peptides in macroscopic current measurements in heterologously expressing oocytes. Figure 3 shows representative single-channel current traces for alpha beta R564Xgamma R574X, the two double alpha  arginine mutants, and the quadruple mutant in combination with the truncated beta - and gamma -subunits. The unitary conductances of all of these combinations of mutated and truncated ENaCs averaged 7 pS, demonstrating that there was no effect on the single-channel conductances by introduction of these mutations in the alpha -ENaC subunit. The peptide mixture only inhibited the single-channel activity (NPo) of the wild-type alpha  construct (Fig. 3, horizontal bar above traces). The peptide mixture was without effect in either of the double Glu (Rright-arrowK) alpha  mutants or in the quadruple Glu (Rright-arrowE) alpha  mutant. The value of NPo was decreased to 1.27 ± 0.3 by the peptide mixture from 2.67 ± 0.4 in patches isolated from oocytes expressing alpha beta R564Xgamma R574X-ENaC. In contrast, the value of NPo recorded from excised patches with the quadruple mutant was unchanged following peptide exposure (2.91 ± 0.4 and 2.89 ± 0.3). In contrast, the values of NPo before and after addition of the control peptide mixture were 1.02 ± 0.19 and 1.14 ± 0.22 (n = 3, Fig. 4).


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Fig. 3.   Representative current traces recorded from inside-out patches of oocytes expressing various mutated alpha -ENaC constructs. Horizontal bars above the current traces indicate perfusion of the bath with 1 mM SP30beta  + SP30gamma peptide mixture. The pipette medium consisted of 100 mM lithium chloride, and 100 mM lithium chloride was also included in the bath solution. The pH of the medium was adjusted to 7.4 with HEPES buffer at 23°C. The holding potential was -60 mV for each trace.



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Fig. 4.   Representative current traces recorded from whole cell and single-channel modes with and without the control peptide. Top traces show the whole cell amiloride-sensitive Na+ current before (left) and after (right) the control peptide cytosolic application. Bottom trace was recorded from an inside-out patch. The horizontal bar above the current trace indicates application of 1 mM Pro SP30beta plus SP30gamma peptide mixture.

These direct biophysical measurements of single ENaC demonstrate directly that the COOH-terminal regions of the beta - and gamma -ENaC subunits function as intrinsic gating particles and that the interaction site occurs at an arginine-rich region located at the most distal portion of the M2 segment of the alpha -ENaC subunit. Thus these terminal cytoplasmic domains of beta - and gamma -subunits play an important role in ENaC gating.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
REFERENCES

Construction of alpha -ENaC mutations. Full-length human alpha beta gamma -ENaC cDNA was a gift from Dr. Michael J. Welsh (University of Iowa) (13), and truncated beta - and gamma -ENaC cDNAs were a gift from Dr. Bernard Rossier (Université et Lausanne, Switzerland) (14). Point mutations in the alpha -subunit were constructed by using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA). Each set of primers contained the appropriate base changes required to code for either two or four glutamates instead of the wild-type arginine residues. Plasmid cDNA, PCR, in vitro transcription, and bacterial transformation were all done as described previously (11). cDNA products containing the specific mutations were confirmed by dideoxy sequence analysis as well.

Oocyte preparation and electrophysiological recording. Oocytes were removed from anesthetized adult female Xenopus laevis (Xenopus Express, Beverly Hills, FL) by standard technique (9). Follicle cells were removed in OR-2 calcium-free medium (in mM: 82.5 NaCl, 2.5 KCl, 1.0 MgCl2, 1.0 Na2HPO4, and HEPES 5.0, pH 7.5), with the addition of collagenase. Defolliculated oocytes were washed in both OR-2 (calcium-free) and OR-2 (complete) medium (in mM: 82.5 NaCl, 2.5 KCl, 1.0 MgCl2, 1.0 CaCl2, 1.0 Na2HPO4, and HEPES 5.0, pH 7.4) and allowed to recover overnight in half-strength Liebovitz's medium at 18°C. Stage VI oocytes were injected with 50 nl (8.3 ng of the appropriate alpha -, beta -, and gamma -ENaC cRNA construct; all subunit mixtures were 1:1:1). Two-electrode voltage clamp and/or single-channel measurements were made 24-48 h postinjection as described previously (9). Oocytes were clamped at a holding potential of 0 mV. The current-voltage relationships were acquired by stepping the holding potential in 10-mV increments from -100 to +100 mV. Current-voltage data were recorded after the monitoring currents were stable, before and after the application of 10 µM amiloride to the bath. Data were sampled at a rate of 1 kHz and filtered at 500 kHz. Data analysis was also as described previously (9). Analysis of single-channel data was performed by using FETCHAN and pSTAT programs of pCLAMP version 8.0 software (Axon Instruments, Burlingame, CA) as previously described (9).

Peptide synthesis and purification. Peptides were synthesized by ResGen (Huntsville, AL). After synthesis, the peptides were subjected to reversed-phase, high-performance liquid chromatography to increase their purity to >90%. Only a single peak was observed on the final chromatograph. The peptides were analyzed for amino acid composition by mass spectroscopy. The sequences for the COOH-terminal 30-amino acid-long beta -ENaC (SP30beta ) and gamma -ENaC (SP30gamma ) peptides were as follows: SP30beta , PIPGTPPPNYDSLRLQPLDVIESDSEGDAI; and SP30gamma , PGTPPKYNTLRLERAFSNQLTDTQMLDEL.


    ACKNOWLEDGEMENTS

We thank LaToya Bishop, Susan Copeland, and Hannah Mebane for excellent technical assistance and Isabel Quinones for superb secretarial help. We also thank Dr. J. K. Bubien for helpful comment and for critically reading the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37206.

Address for reprint requests and other correspondence: D. J. Benos, Dept. of Physiology and Biophysics, The Univ. of Alabama at Birmingham, 1918 University Blvd., MCLM 704, Birmingham, AL 35294-0005 (E-mail: benos{at}physiology.uab.edu).

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.

10.1152/ajpcell.00610.2001

Received 26 December 2001; accepted in final form 1 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
REFERENCES

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14.   Schild, L, Canessa CM, Shimkets RA, Gautschi I, Lifton RP, and Rossier BC. A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci USA 92: 5699-5703, 1995[Abstract].

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