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J Biol Chem, Vol. 274, Issue 53, 37693-37704, December 31, 1999

Peptide Inhibition of Constitutively Activated Epithelial Na⁺ Channels Expressed in Xenopus Oocytes^*

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

From the Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The hypothesis that 30-amino acid peptides corresponding to the C-terminal portion of the - and/or -rat epithelial sodium channel (rENaC) subunits block constitutively activated ENaC was tested by examining the effects of these peptides on wild-type (wt) rENaC (-rENaC), truncated Liddle's mutants (_T-, _T-, and _TT-rENaC), and point mutants (_Y-, _Y-rENaC) expressed in Xenopus oocytes. The chord conductances of _T-, _T-, and _TT-rENaC were 2- or 3-fold greater than for wt -rENaC. Introduction of peptides into oocytes expressing _T-, _T-, and _TT-rENaC produced a concentration-dependent inhibition of the amiloride-sensitive Na⁺ conductances, with apparent dissociation constants (K_d) ranging from 1700 to 160 µM, depending upon whether individual peptides or their combination was used. Injection of peptides alone or in combination into oocytes expressing wt -rENaC or single-point mutants did not affect the amiloride-sensitive whole-cell currents. The single channel conductances of all the mutant ENaCs were the same as that of wild type (-). The single channel activities (N·P_o) of the mutants were ~2.2-2.6-fold greater than wt -rENaC (1.08 ± 0.24, n = 7) and were reduced to 1.09 ± 0.17 by 100 µM peptide mixture (n = 9). The peptides were without effect on the single channel properties of either wt or single-point mutants of rENaC. Our data demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (_TT) expressed in Xenopus oocytes but not the single-point mutants (_Y or _Y). Moreover, the blocking effect of both peptides in combination on _TT-rENaC was synergistic.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hypertension is a common multifactorial disease imparting an increased risk of myocardial infarction, stroke, and end-stage renal disease. Epidemiological studies suggest that up to 30% of human hypertension may have a genetic basis (1). Epithelial sodium channels (ENaC)¹ play a key role in regulating salt and water homeostasis by controlling sodium reabsorption in the distal nephron. The cDNAs encoding ENaC have been identified, and a heteromultimeric structure of the channel, comprised of three homologous , , and  subunits, has been proposed (2-4). Each subunit contains a large extracellular loop, located between two transmembrane domains, and two short intracellular N- and C-terminal domains. ENaC activities are controlled by insulin, corticosteroids, aldosterone (Ref. 5; for reviews see Refs. 6 and 7), protein kinases (8), proteases (9), cations (10-12), the cytoskeleton (13), and osmotic pressure (14). Shimkets et al. (8) confirmed that protein kinase A, kinase C, and insulin could phosphorylate the C-terminal regions of both - and -ENaC subunits. It is likely that the cytoplasmic N- and C-terminal segments of each subunit may contain the regulatory sites of many of the other aforementioned ENaC modulators.

The role of ENaC in the pathogenesis of genetic hypertension, namely Liddle's syndrome (pseudo-hyperaldosteronism), has been demonstrated by linkage analyses of the genes encoding the ENaC subunits in families with a history of salt-sensitive, low-renin, volume-expanded hypertension. These studies suggested that Liddle's syndrome resulted from truncating or missense mutations deleting a PPPXY motif in the cytoplasmic C terminus of either the - (15-20) or -ENaC subunits (21). Systemic mutagenesis of the rat ENaC homolog, rENaC, suggested that normal ENaC activity could be modified by altering the consensus PPPXY sequence in an individual - or -ENaC subunit (22). Each of these mutations resulted in increased channel activity when co-expressed with the other wt rENaC subunits in Xenopus oocytes, consistent with increased sodium reabsorption in the distal nephron (22, 23). Similarly, stimulated amiloride-sensitive Na⁺ currents in oocytes expressing the Liddle's mutants of human ENaC were also observed (20, 24).

At present, two non-mutually exclusive mechanisms have been posited to account for the enhanced macroscopic Na⁺ channel activity resulting from Liddle's mutations, namely an increased surface density of channel protein and/or increased channel open probability (P_o) (24-26). While an increased residence time of ENaC at the cell surface has been attributed to defective internalization of the channel protein for ENaC constructs containing Liddle's mutations, the mechanisms by which these mutations in the - or -ENaC subunits lead to an increase in single channel P_o are not well understood. Interestingly, the currents associated with Liddle's mutants expressed in oocytes were not down-regulated by elevated intracellular Na⁺ concentration in contrast to wt ENaC currents (27). Furthermore, Dinudom et al. (28) suggested that elevated intracellular Na⁺ promotes the interaction of Nedd4 with the PY motif of -rENaC. Hence, feedback inhibition of ENaC by intracellular Na⁺ would be lost with Liddle's mutations. The differences between the regulation of wt ENaC and Liddle's mutants have not been explored in detail. However, like other cation channels (29-31), the activity of truncated Liddle's -ENaC mutants immunopurified from human lymphocytes were also inhibited by synthesized peptides when reconstituted into planar lipid bilayers (32).

We had previously demonstrated in planar lipid bilayer studies that truncation of the C termini of - or -rENaC led to increased channel P_o (32, 33). We wished to test further the hypothesis that the C-terminal peptides of these subunits could function as inhibitory peptides of rENaC expressed in Xenopus oocytes. In the present study, we investigated the effect of the 30-amino acid C-terminal peptides of - or -rENaC subunits on the currents associated with the truncation and point mutants found in Liddle's syndrome using whole-cell and single channel recording techniques. The results showed that both the individual peptides SP₃₀ or SP₃₀ and a mixture of both peptides decreased the macroscopic currents of truncated Liddle's mutants expressed in oocytes but had no effect on the currents associated with expression of the Liddle's point mutants. The single channel activity (N·P_o) of the truncated Liddle's mutant was down-regulated by the peptides due to decreases in P_o and the number of channels per patch. The conclusions of the present study were that the increased channel P_o of the truncated Liddle's mutants expressed in Xenopus oocytes could be reversed by the synthetic peptides, thus the C termini cytoplasmic tails of the  and  subunits act in concert to form part of the normal gating mechanism of a functional ENaC.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Peptide Synthesis and Purification-- rENaC constructs, including wt -rENaC and  and  subunits of Liddle's mutations _R564 (_T), _R574 (_T), _Y618A (_Y), and _Y628A (_Y) were kind gifts of Drs. Cecilia Canessa (Yale University) and Bernard Rossier (Universität de Lausanne). In vitro translation of each rENaC subunit and their mutations were performed as described previously (34).

Peptides SP₃₀, SP₃₀, and Pro-SP₃₀ were synthesized by Research Genetics (Huntsville, AL). Following synthesis, the peptides were purified (to >90% purity) by reversed-phase high performance liquid chromatography. The amino acid sequence of each peptide was verified by mass spectroscopy. The primary amino acid sequences of the synthetic peptides were as follows: SP₃₀, ⁶¹¹PIPGTPPPNYDSLRLQPLDVIESDSEGDAI⁶⁴⁰; SP₃₀, ⁶²⁰PGTPPPKYNTLRLERAFSNQLTDTQMLDEL⁶⁴⁹; Pro-SP₃₀, ⁶¹¹PPPGTPPPNYDSLRLQPLDPPESDSEGDAD⁶⁴⁰. Peptide concentration was calculated based upon the exact molecular mass of each peptide. The 1:1 peptide mixtures were equimolar, i.e. a 276 µM mixture was a 1:1 mix of two solutions of 276 µM, yielding a final concentration of each peptide of 138 µM.

Oocyte Expression of ENaC-- The methods for isolation of oocytes and expression of rENaC cRNA were as described previously (34). Briefly, ovarian tissue was removed from frogs (Xenopus Express, Burley Hills, FL) under anesthesia through a small incision in the lower abdomen. Oocytes were digested with 2-4 mg/ml collagenase in Ca²⁺-free OR-2 solution for 2 h on a shaker. Following several washes in Ca²⁺-free or regular OR-2 media, oocytes were manually defolliculated. Healthy oocytes at maturation stage V and VI were identified and maintained in L-15 medium at 18 °C. Oocytes were incubated overnight before cRNA injection. A Nanoject microinjector (Drummond Scientific Co., Broomall, PA) was used for injection of 50 nl of rENaC cRNA per oocyte in the ration of 1:1:1 (::) or the same volume of RNase free water (control). The total cRNA of all 3 subunits injected into each oocyte varied from 5 to 25 ng. Injected oocytes were transferred to MBS medium or L-15 medium and incubated at 18 °C for 48 h before recording whole-cell and single channel data.

Solutions and Chemicals-- Ca²⁺-free OR-2 medium was used to wash oocytes before and after digestion with collagenase. This medium contained (in mM) 85.2 NaCl, 2.5 KCl, 10 NaHEPES, 1.0 Na₂HPO₄, 0.5% streptomycin, pH 7.5. Calcium chloride (1.8 mM) was added to Ca²⁺-free OR-2 medium to make regular OR-2 medium. The incubation medium was modified Barth's solution (MBS) with the following composition (mM): 85 NaCl, 1 KCl, 2.4 NaHCO₃, 0.4 CaCl₂, 0.3 Ca(NO₃)₂, 0.8 MgSO₄, 10 HEPES, 1% antibiotic/antimycotic, pH 7.4. An alternative medium for incubation was L-15 medium containing 1/2 strength of Leibovitz-15 powder, 15 mM NaHEPES, 5% heat-inactivated horse serum, 1% antibiotic/antimycotic, pH 7.5. L-15 powder, antibiotic/antimycotic, and heat-inactivated horse serum were from Life Technologies, Inc. Collagenase was purchased from Roche Molecular Biochemicals, and a fresh solution was prepared for each experiment. Amiloride was obtained from Research Biochemicals Int. (Natick, MA), and 5 mM stock (1:1 Me₂SO:H₂O, v/v) was stored at 4 °C in a container wrapped with aluminum foil. The perfusate for two-electrode voltage clamp experiments consisted of (mM) 116 NaCl, 2.0 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 5.0 NaHEPES, pH 7.4.

For patch clamp experiments, we used a hypertonic solution for stripping the vitelline membrane from the oocytes composed of (in mM), 200 potassium aspartate, 20 KCl, 1 MgCl₂, 10 EGTA, 10 NaHEPES, pH 7.4. The basal extracellular medium contained (in mM) 100 LiCl, 10 HEPES, pH 7.4. To obtain outside-out patches, 1.8 mM CaCl₂ was included in the extracellular medium. In order to measure the permeability ratio of Li⁺:K⁺, equimolar potassium was used to replace lithium. Peptides were dissolved in water to 100 mM and stored at 4 °C until used. All other reagents were obtained from Sigma.

Two-electrode Voltage Clamp-- Whole-cell currents were measured in oocytes held at room temperature (22-25 °C) 24 h after cRNA injection. The currents were analyzed using pCLAMP software (Axon Instruments, Foster City, CA). Voltage clamp potentials were evoked using a TEA-200 voltage clamp (Dagan Corp., Minneapolis, MN) controlled by a personal computer connected via a TL-1 interface (Axon Instruments). The injected oocyte was placed in a small holding chamber (<50 µl) and was initially superfused with the desired solution at a flow rate of 1.0 ml/min for at least 5 min before recording. Microelectrodes were filled with 3 M KCl and had a tip resistance of 0.5-2.0 M. The bath electrodes consisted of two chloride-coated silver wires that made electrical contact with the bath through 3% agar bridges immersed in 3 M KCl. For peptide injection, a third glass electrode (Drummond Scientific Co.) filled with a stock solution of peptide (usually 100 mM) was inserted into an oocyte subsequent to impalement with the two recording electrodes. The desired concentration of peptides within an oocyte was defined by injecting a known volume of the peptide stock using an adjustable nanoliter injector (WPI, Sarasota, FL) and calculating the oocyte volume by assuming the oocyte was a prolate spheroid. The oocytes were clamped at a holding potential of 100 mV. Data were filtered at 0.5-1 KHz, digitized, and stored on hardware for off-line analysis. For ramp I-V recordings, test voltages were elicited from a holding potential of 100 to 140 mV through +60 mV at a rate of 10 mV/25 ms for 500 ms. Currents at 100 mV were sampled at 20-s intervals. Capacitance neutralization was done for each recording using the circuit built into the TEA-200 voltage-clamp amplifier. In addition, the small volume of the holding chamber (<50 µl) also helped minimize the capacitance at the input of the head stage. The resting membrane potentials were read directly from the monitor window of the voltage clamp before and after application of amiloride.

Single Channel Recordings-- The oocytes used for patch clamp experiments were first placed in a hypertonic medium at room temperature for 5-20 min. The vitelline membrane was then manually removed from the shrunken oocytes using fine forceps (35). The devitellinized oocyte was immediately transferred to a recording chamber (Warner Instruments, Hamden, CT) and perfused with isoosmotic extracellular solution before patch clamping the cell.

All three configurations (cell-attached, excised inside-out, and outside-out) of patch clamp recordings were used (36). The reference electrode was an Ag/AgCl pellet bathed in the same solution as the bath. For constructing current-voltage (I-V) curves and calculating P_o, data obtained from cell-attached, inside-out, and outside-out patches were included. For plotting concentration dependence curves of amiloride inhibition, the outside-out patch configuration was employed, and a series (from 0 to 10 µM) of concentrations of amiloride was applied. Patch pipettes were made of borosilicate glass, pulled in two steps with a PP83 vertical puller (Narishige, Japan). The tip resistance of the electrodes was 1-10 M. Single channel currents were recorded with an Axopatch 1-B current-voltage clamp amplifier (Axon Instruments, Inc., Burlingame, CA). The current traces were displayed both by CLAMPEX version 7.0 associated Axoscope software on the monitor and on an oscilloscope and were stored on a hard drive connected with the working computer through the internet. By convention, for the outside-out configuration, the intracellular potential corresponds to the pipette potential (V_p), and negative (downward) single currents correspond to cation influx from the extracellular to the intracellular side of the membrane. For the cell-attached configuration, the membrane potential should be close to the actual potential across the membrane patch because the oocyte membrane potential is close to 0 mV measured by two-electrode voltage clamp under our experimental conditions.

Current signals were filtered at 1 kHz with an 8-pole Bessel filter (Frequency Devices Inc., Haverhill, MA) and digitized at 2 kHz using a Digidata 1200 interface and CLAMPEX software (Axon Instruments, Inc., Foster City, CA). For display, currents were low-pass filtered at 100-200 Hz.

Data Analysis-- Amiloride-sensitive macroscopic whole-cell currents were calculated by subtracting the currents in the presence of 10 µM amiloride from the currents in the absence of amiloride. Student's t test was used to analyze the differences of amiloride-sensitive currents among each group. The results were presented as mean ± S.E., and the degree of significance was assessed using the Student's t test. Data from rENaC expressing oocytes for quantifying the inhibitory effect of peptide were normalized to the maximum current in the absence of peptide. The apparent inhibition dissociation constants K_d were computed according to Equation 1,

(Eq. 1)

where K_d is the apparent dissociation constant and n is the Hill coefficient. I and I₀ represent the amiloride-sensitive Na⁺ currents in the presence or absence of the C-terminal peptides, respectively. [Pep] indicates the concentration of individual peptide or the peptide mixture injected into oocytes. Thus, a plot of log(I_Pep/I  I_Pep) versus log[Pep] should yield a straight line with slope n (see Ref. 37 for discussion).

Analysis of single channel data was performed using FETCHAN and pSTAT program of software CLAMPEX version 7.0 (Axon Instruments). The holding potentials (or the polarity of the single channel currents) were corrected accordingly. Assuming the lowest level during more than 3 and up to 30 min of continuous recordings was the closed level, the single channel open probability (P_o) was calculated from using Equation 2,

(Eq. 2)

where N is total number of channels, is the mean current over the period of observations, and i is the main state unitary current determined from all points current amplitude histograms produced by FETCHAN. The mean current over the period of observation was calculated using the events list files generated by CLAMPEX version 7.0 software and Equation 3,

(Eq. 3)

where i_m is an event current (all level including the 0 level); t_m is an event dwell time, and M is the total number of events. The parameters calculated according to the above method may be underestimated due to the long open and closed transitions and multichannel nature of the patches of ENaC expressed in oocytes. Data are expressed as mean value ± S.E. for n patches. Because the pSTAT program could not used to analyze the results of multichannel patches with more than five conductance levels, the program GAUSS (supplied to us by Dr. James Kenyon of the Department of Physiology, University of Nevada) was used to analyze channel data consisting of six or more discrete current levels (38).

The permeability ratios of wt -rENaC and Liddle's mutants expressed in oocytes were computed from the measured reversal potentials using the Goldman-Hodgkin-Katz (GHK) equation.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Macroscopic Currents of Truncation Mutants Are Inhibited by Peptide Injection-- In order to test the hypothesis that peptides corresponding to the C termini of the - and/or -rENaC subunits (SP₃₀ and SP₃₀) inhibit Liddle's mutations inserted into rENaC, the -rENaC subunit truncated at amino acid position Arg-564 (_T) and the -rENaC subunit truncated at amino acid position Arg-574 (_T) were expressed in oocytes in combination with the wt -rENaC subunit (_TT). Fig. 1 shows representative current traces in oocytes evoked by a voltage ramp from 140 mV to 60 mV. The chord conductance of _TT-rENaC-associated currents at hyperpolarizing potentials was ~3-fold greater than that of wt -rENaC. Injection of a mixture of  +  C-terminal peptides (1:1, 138 µM each) into oocytes expressing the truncated ENaC constructs (_TT-rENaC) decreased the current by approximately 63% at hyperpolarizing potentials. In contrast, the current in oocytes expressing wt -rENaC was not affected by the same concentration of peptide mixture. The _TT-rENaC-associated current was not decreased by water injection or by injection of either of two control peptides, one comprised of the last 13 amino acids of the C terminus of CFTR (KEETEEEVQDTRL, final concentration = 1.7 mM; n = 4) or a 30-mer peptide identical in all respects to SP₃₀ except for substitution of 3 prolines for the single valine and two isoleucines (Pro-SP₃₀). Fig. 2 shows the time course of peptide inhibition of inward Na⁺ current in _TT-rENaC-expressing oocytes (Fig. 2A) and the lack of effect of the control Pro-SP₃₀ peptide (Fig. 2B). Although Pro-SP₃₀ and the CFTR peptides carried the same or a greater total number of negative charges as the C-terminal peptides SP₃₀ and SP₃₀, neither the CFTR nor Pro-SP₃₀ peptides had any effect on the whole-cell Na⁺ currents induced by _TT-rENaC expression. Similarly, in parallel patch clamp experiments the addition of the C-terminal CFTR peptide to the bath (n = 3, 500 µM) or Pro-SP₃₀ (n = 3, 1.7 mM) failed to alter the gating behavior of _TT-rENaC in inside-out patches (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Representative recordings showing effect of 30-mer peptide mixture injection into Xenopus oocytes on amiloride-sensitive Na+ currents induced by expression of - and TT-rENaC. Recordings were made 24 h after cRNA injection for both - and TT-rENaC. Currents were elicited with a ramp voltage pulse from 140 to +60 mV starting at a holding potential of 100 mV. Amiloride-sensitive Na+ currents shown in this figure were calculated by subtracting the currents recorded in the presence of amiloride from those in the absence of amiloride (10 µM). The left panels show amiloride-sensitive currents of wt - and TT-rENaC, and the right panels show effect of peptide mixture (276 µM; 1:1 of SP30 + SP30) or water as a control on the corresponding currents in the same oocyte. Injection of the peptide mixture does not affect the current of -rENaC (B) but significantly inhibits TT-rENaC-associated Na+ current by approximately 63% (F). The amplitude of TT-rENaC-associated Na+ current (C and E) at 100 mV is approximately 3-fold greater than that of -rENaC (A). Injection of same volume of nuclease-free water (13.8 nl) into an oocyte expressing TT-rENaC (D) does not show an inhibitory effect on the currents of TT-rENaC. These experiments were each repeated four times.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of 30-mer peptide mixture on macroscopic currents in TT-rENaC expressing oocytes. A, continuous recording of inward current at 100 mV from an TT-rENaC-expressing oocyte, showing the effects of both amiloride (10 µM) and 100 µM SP30 + 100 µM SP30. B, continuous current recording from an TT-rENaC-expressing oocyte showing the lack of inhibitory effect of a control peptide Pro-SP30 at 1.7 mM. C, typical ramp current-voltage curves for TT-rENaC-expressing oocytes without injection of Pro-SP30. D, typical ramp current-voltage relation for TT-rENaC-expressing oocytes previously injected with 1.7 mM Pro-SP30.

Summarized data showing inhibition of wt and _TT-rENaC-induced macroscopic Na⁺ currents both for the individual peptides and a 1:1 mixture of SP₃₀ and SP₃₀ peptides (138 µM each) are plotted in Fig. 3. The mean chord conductance of _TT-rENaC-expressing oocytes was 123.7 ± 12.6 µS (n = 11). This value was more than twice that of wt -rENaC-expressing oocytes (51.9 ± 50 µS, n = 5; p < 0.001). Injection into oocytes of the mixture of SP₃₀ plus SP₃₀ peptides decreased the _TT-rENaC-associated Na⁺ conductance to 37.6 ± 10.7 µS (n = 11), a value statistically indistinguishable from wild-type (p > 0.4). The individual peptides, at the same final concentration as the mixture, i.e. 276 µM, also inhibited the macroscopic inward Na⁺ currents but were not nearly as effective as the peptide mixture. In contrast, neither the individual peptides nor the peptide mixture inhibited wt -rENaC-associated Na⁺ conductance (Fig. 3).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Summarized data showing the effect of the individual peptides or the peptide mixture injection on wt - and TT-rENaC conductance in oocytes. The data are averaged amiloride-sensitive Na+ chord conductances determined as the slope of the current-voltage relation between 140 and 0 mV. Each oocyte was injected with a total of 276 µM peptide (individual or in combination) or an equivalent volume of water. n stands for the number of eggs recorded, and the standard errors (S.E.) are shown. Without injection of the C-terminal peptides, the amiloride-sensitive Na+ conductances for - and TT-rENaC were 52.0 ± 5.0 µS (n = 6) and 123.7 ± 12.6 µS (n = 11), respectively. Injection of the individual peptides SP30, SP30, or the peptide mixture decreased the conductances of TT-rENaC by 103.4 ± 3.7 µS (n = 5), 79.8 ± 5.7 µS (n = 4), and 37.6 ± 10.7 µS (n = 11), respectively. In contrast, the amiloride-sensitive Na+ conductance of -rENaC did not decline after injection of either the peptide mixture (53.0 ± 4.3 µS, n = 6) or injection of the individual peptides SP30 (51.8 ± 5.4 µS, n = 6) and SP30 (48.1 ± 6.4 µS, n = 5).

To study the inhibitory kinetics of the individual peptides and their mixture, increasing concentrations of the SP₃₀ and/or SP₃₀, varying from 92 to 1732 µM, were injected into the cytoplasm of oocytes expressing _T-, _T-, and _TT-rENaC. The concentration-response curves of peptide(s) on amiloride-sensitive Na⁺ currents in oocytes injected with cRNA of rENaC constructs associated with Liddle's syndrome (Fig. 4A) were well described by the Hill equation (Fig. 4B).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of the C-terminal peptides inhibition on macroscopic Na+ currents of T, T, or TT-rENaC expressed in oocytes. A, the normalized amiloride-sensitive Na+ currents of TT-rENaC are plotted as a function of peptide concentrations injected into oocytes (from 0 to 1732 µM). The individual concentration of peptide was varied from 0 to 1732 µM by repeated injections into the same oocyte. The dose-response curves were fit with the Hill equation: I/I0 = 1/{1+([Pep]/Kd)n}. The two far right dashed lines are the double truncation mutant (TT-rENaC) injected with SP30 (closed squares) and SP30 (closed circles). The remaining lines (open symbols) represent single truncation mutant T-rENaC injected with SP30 peptide (open squares) and T injected with peptide SP30 (open circles), respectively. The open triangles represent data obtained from TT-rENaC-expressing oocytes injected with a 1:1 combination of SP30 and SP30 peptides. The apparent dissociation constants (Kd) and the corresponding Hill coefficient constants determined from the associated Hill plots (B) are listed in Table I.

The K_d values and the corresponding Hill coefficient are summarized in Table I. The Hill coefficients for the individual peptides and the peptide mixture range from 0.7 to 1. These results suggested that the individual C-terminal peptides and their mixture interact with one rENaC subunit or with a single binding site.

View this table: [in this window] [in a new window]   Table I Estimated parameters of peptide inhibition on rENaC constructs by fitting with Hill equation The numbers in the parentheses in the 1st column indicated the number of oocytes used. Kd stands for the apparent equilibrium dissociation constant in µM. The individual peptides (SP30 or SP30) or the peptide mixtures (SP30 ( + )) were injected into oocytes expressing single (T or T) or double (TT) mutation rENaC constructs. Means ± S.E. are presented.

Macroscopic Currents of Point Mutants Are Not Inhibited by Peptides-- Among the Liddle's ENaCs, both  and  C-terminal truncated (nonsense) mutants and the single amino acid point mutants resulted in greater amiloride-sensitive Na⁺ currents compared with the wild-type construct when expressed in Xenopus oocytes (22). Because the C-terminal peptides inhibited the currents associated with _T-, _T-, and _TT-rENaC expressed in oocytes, the question arose as to whether this specific effect was based on the ability of the peptides to restore the wild-type nature of these truncation mutants through the specific interaction with the truncated channel or by involvement of intracellular secondary components. To test this hypothesis, subunits containing point mutations associated with Liddle's syndrome, _Y618A (_Y) or _Y628A (_Y), combined with the two complementary subunits of wt rENaC, were expressed in oocytes, and the effect of the peptides was examined.

In oocytes injected with wt -rENaC and point mutant constructs, _Y and _Y, three parallel groups of oocytes from the same frog were injected on the same day. The amiloride-sensitive conductance in oocytes injected with _Y-rENaC and _Y-rENaC were 45.77 ± 3.8 µS (n = 6) and 43.5 ± 3.9 µS (n = 7), respectively. In comparison, the chord conductance of wt -rENaC was 15.7 ± 4.0 µS (n = 6, p < 0.001). The reversal potentials among each group did not show a statistically significant difference, indicating that the cationic selectivity was not modified by point mutagenesis of the tyrosines in the - (in position 618) or - (in position 628) rENaC subunit. The reason why the absolute value of Na⁺ conductance in this batch of oocytes was lower than that measured in the experiments presented in Fig. 3 was because less ENaC cRNA was injected (7.5 versus 25 ng/oocyte).

As shown in Fig. 5, following an injection of either 276 µM SP₃₀ or SP₃₀ into oocytes expressing _Y-rENaC, the current amplitudes were slightly increased to 108.91 ± 2.5 and 114.07 ± 1.83%, respectively. For oocytes expressing _Y-rENaC, the Na⁺ currents were decreased slightly to 85.31 ± 2.4 and 91.53 ± 2.4%, respectively, following - or -peptide injection. No significant inhibitory effects of the individual peptides were found (variation less than 15%) on _Y- and _Y-rENaC-associated amiloride-sensitive Na⁺ currents in oocytes (Fig. 5). Similarly, injection of the peptide mixture into oocytes expressing either _Y- or _Y-rENaC did not significantly affect the current amplitudes (Fig. 5). The above experiments were repeated using the peptides at a concentration of 1732 µM; comparable results, i.e. no significant current inhibition (within 15% of the preinjection control), were observed (n = 3 each; data not shown).

View larger version (61K): [in this window] [in a new window]   Fig. 5.   Inhibition of macroscopic Na+ currents of wt and  and  point mutations of rENaC by individual peptides and the peptide mixture. The protocol for peptide injection into wt and point mutations (Y-, Y-rENaC) is described under "Materials and Methods." The amiloride-sensitive Na+ currents in oocytes without peptide injection were normalized to 100%. Injection of SP30, SP30, or the peptide mixture (276 µM) showed no significant inhibitory effect on wt - (top), Y- (middle), and Y-rENaC (bottom) expressed in oocytes. Variation of amiloride-sensitive currents in the presence of peptides was less than 15% of the control post-peptide injection for each group.

Amiloride Blockade of rENaCs Expressed in Oocytes-- One of the defining features of ENaC is its sensitivity to the potassium sparing diuretic amiloride, which reversibly blocks the channel at nanomolar to micromolar concentrations (for review see Ref. 7). To identify that the currents were carried by exogenous rENaC expression, inhibition of current by amiloride was studied in rENaC-injected oocytes and in H₂O-injected oocytes as a control. Under voltage clamp conditions, amiloride-blockable Na⁺ currents could not be detected in H₂O-injected oocytes (I₁₄₀ = 100 nA). In comparison to the small currents observed in uninjected or water-injected oocytes, large amiloride-sensitive inward Na⁺ currents of 5-10 µA in oocytes injected with cRNA of wt rENaC were seen, indicative of the fact that the currents recorded were indeed produced by exogenous rENaC expression (data not shown).

In order to verify that the currents we observed were produced by exogenous cRNAs of rENaC rather than by activation of an endogenous cation channel, amiloride was applied to outside-out patches. Outside-out patches from oocytes expressing rENaC (wt or mutant varieties) invariably contained multiple channels. Similar continuous recordings were made on oocytes expressing all of the other Liddle's truncation constructs used in these experiments (Fig. 6). When perfused with 100 nM amiloride, the multichannel currents of rENaC and the Liddle's mutants became flickery. After washing out amiloride, the original multichannel traces were recovered, indicating that the inhibitory effect of amiloride was reversible. In contrast, no amiloride-sensitive single channel currents could be seen in 10 outside-out patches of H₂O-injected oocytes.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Amiloride blockade of single channel currents of wt -rENaC and the Liddle's mutations. A, outside-out patches were isolated from oocytes expressing - (top left), TT- (bottom left), Y- (bottom left), and Y-rENaC (bottom right). In the absence of amiloride, each patch contained around five channels for the Liddle's mutations but less than three channels for wt -rENaC. Superfusion of amiloride from 0 to 10 µM to the extracellular side of rENaC channels caused the current traces to flicker due to decrease in open time constant, especially when the concentrations of amiloride are above 100 nM. The blocking of amiloride on rENaCs was reversible and concentration-dependent. For illustration purposes, the effects of only a single concentration of amiloride are shown, and only partial records are presented for simplicity. B, log dose-response curves of amiloride inhibition of wt and Liddle's mutations. The data are expressed as percent inhibition of NPo determined from channel activity in outside patches of oocyte membrane. The data points and the error bars represent the mean and the S.E. for at least 3 separate experiments.

In order to determine whether the efficacy of amiloride was changed by any of these truncation mutations, we constructed dose-response curves for amiloride inhibition of channel activity (N·P_o) in these outside-out patches (Fig. 6B). K_d for wt -, _TT-, _Y-, _Y-rENaC at a holding potential of 20 mV were 55.4, 71.8, 76.4, and 96.7 nM, respectively (p > 0.1, analysis of variance). These results indicate that there was no difference in amiloride sensitivity between wt -rENaC and the Liddle's mutants. Furthermore, amiloride inhibited single channel activity (N·P_o) by acting as an open channel blocker (39).

Effect of a 1:1 Peptide Mixture on Single Channel Currents of Truncated Mutants-- We also used single channel analysis to assay the inhibitory effect of the C-terminal peptides on wt -rENaC and the Liddle's constructs when expressed in oocytes. Fig. 7 shows the single channel current traces for (top) and _TT-rENaC (middle) and their respective I-V curves (bottom) in inside-out patches of oocytes membrane under bi-ionic conditions. The unitary conductances of both wt (n = 9) and _TT-rENaC (n = 6) averaged around 7 pS. The reversal potentials under bi-ionic conditions averaged 97 mV, indicating a P_Li/P_K of 42. 

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Current-voltage relationship curves of wt - and TT-rENaC expressed in oocytes. A and B show the original current traces recorded from inside-out patches displaying single channel activities of wt - and TT-rENaC. The pipette medium consisted of 100 mM LiCl, and 100 mM KCl was included in the bath solution. The pH of the media was adjusted to 7.4 with HEPES buffer at 25 °C. C, the current data for wt - (open circles) and TT-rENaC (closed squares) are the averaged values came from 6 separate experiments, and the error bars indicate the S.E. The data were fit by the Goldman-Hodgkin-Katz current equation for the bi-ionic situation to estimate the unitary conductance, ionic permeability, and the reversal potential. No significant changes were detected in either the conductance 7 pS (n = 6) or the reversal potential (97.4 ± 3.1 mV, n = 6) for TT-rENaC expressed in oocytes, as compared with wt rENaC.

The effects of 1000 µM peptide mixture on the kinetics of the double-truncated Liddle's mutant (_TT) are shown in Fig. 8. It can be seen that the peptide mixture decreased NP_o, but not the conductance of _TT-rENaC, and was completely reversible (Fig. 8C). The inhibition produced by the peptides on _TT-rENaC was very different than that seen for amiloride (cf. Fig. 6A), i.e. the peptides did not appear to act as open channel blockers.

View larger version (51K): [in this window] [in a new window]   Fig. 8.   Single channel inhibition of TT-rENaC by the peptide mixture. Continuous single channel records (inside-out patches) of wt -rENaC (A) or TT-rENaC (B and C) expressed in oocytes are shown. Excised inside-out patches were prepared in order to apply the C-terminal peptides to the cytosolic side of the rENaC channels. The desired concentration of peptide was added directly to a small (0.3 ml) chamber. The effect of SP30 + SP30 on the wt (A) and TT channel (C) and the lack of effect of 200 µM Pro-SP30 peptide on TT-rENaC (B) are depicted. The holding potential was 40 mV. Each condition was replicated six times.

If the C-terminal regions of the - and -ENaC subunits act as intrinsic gating particles, exogenously added  and  C-terminal peptides should not block wt -rENaC because of the presence of intact - and -ENaC C termini. The effect of the peptide mixture on wt -rENaC was thus explored following application of peptides to inside-out patches (Fig. 8A). In contrast to _TT-rENaC, the C-terminal peptides had no effect on the gating of wt -rENaC currents nor did 1000 µM Pro-SP₃₀ peptide produce any inhibition of _TT-rENaC (Fig. 8B), consistent with the macroscopic current recordings (Fig. 1). The single channel activity (N·P_o) and P_o of each group are summarized in Fig. 9. N·P_o of _TT was approximately 3-fold greater than wt -rENaC at a holding potential of 40 mV, consistent with whole-cell current measurements. The corresponding P_o of _TT-rENaC increased by 41.5% compared with wt -rENaC.

View larger version (36K): [in this window] [in a new window]   Fig. 9.   Inhibition of the peptide mixture on the number of channel per patch, single channel activity, and open probability of TT-rENaC. The summarized data on single channel activity (normalized N·Po of wt -rENaC as 1.0, middle), the number of channel per patch (N, bottom), and open probability (Po, top) for wt - and TT-rENaC are averaged from the results of five experiments. The peptide mixture of SP30 and SP30 (1:1, 100 µM) was applied to the isolated inside-out patches from oocytes expressing wt - and TT-rENaC. All three investigated parameters for TT-rENaC exceeded those for wt -rENaC by the order of 2- or 3-fold, in accordance with the macroscopic currents depicted in Figs. 1 and 2 before the addition of the peptide mixture.

Effect of SP₃₀:SP₃₀ Peptides on Single Channel Current of Point Mutants-- Fig. 10 presents representative single channel current traces for wt -, _Y-, and _Y-rENaC in cell-attached patches of Xenopus oocyte membranes. The Liddle's point mutants expressed in oocytes had similar biophysical characteristics to those of wt -rENaC. The channel number (N), the single channel activity (N·P_o), and mean P_o are summarized in Table II. N·P_o of _Y and _Y were 2.65- and 2.18-fold over that of wt -rENaC, respectively, at a holding potential of 40 mV. The corresponding P_o of _Y and _Y increased only slightly, namely by 6.8 and 4.5%, respectively (Table II), values not significantly different than that for the wild-type channel. The number of channels per patch and the single channel activity of _Y- and _Y-rENaC on the other hand were significantly increased compared with wt rENaC, p < 0.01. 

View larger version (34K): [in this window] [in a new window]   Fig. 10.   The unitary current-voltage curve for wt -, Y-, and Y-rENaC expressed in oocytes. The top panels show the single channel recordings elicited from cell-attached patches from oocytes expressing wt -, Y-, and Y-rENaC. The traces were recorded at the sampling rate of 2 kHz and filtered at 200 Hz. The data illustrated at the bottom of the figure are the averaged unitary currents for wt - (closed circles), Y- (closed squares), and Y-rENaC (open squares). The unitary conductances for Y- and Y-rENaC are 6.61 ± 0.006 pS (n = 5) and 6.68 ± 0.002 pS (n = 10), respectively.

View this table: [in this window] [in a new window]   Table II Effect of the peptide mixture on the single channel features of Liddle's mutations in excised inside-out patches The channel number per patch (n), the open probability (Po), and the single channel activity (N · Po) in the absence (control) or presence (+ peptide) of the peptide mixture (100 µM SP30 + 100 µM SP30) are included. The numbers in parentheses in the 1st column are the number of patches. Data are presented as mean ± S.E. of 4-15 experiments and were calculated at a holding potential of 40 mV.

The unitary conductances of _Y and _Y-rENaC were 6.61 ± 0.01 (n = 5), and 6.68 ± 0.02 (n = 10), respectively (Fig. 10), indicating that these single-point mutations in the C terminus did not influence single channel conductance. The cation permeability ratios (P_Li/P_K) for _Y- and _Y-rENaC were similar to that of wt -rENaC when expressed in Xenopus oocytes, i.e. around 40 (Fig. 10, bottom).

The effect of peptides on _Y- and _Y-rENaC was also assessed using the inside-out patch configuration. As shown in Fig. 11, no change in channel activity was recorded for both _Y- and _Y-rENaC. Unlike the truncated Liddle's mutant, the number of channels per patch, the single channel activity, and the P_o were not affected by application of the peptide mixture to the cytosolic side of the patch from the oocytes expressing _Y- or _Y-rENaC (Fig. 12 and Table II). Taken together, these data indicated that the C-terminal peptides inhibited the amiloride-sensitive macroscopic Na⁺ currents and the single channel activity of _TT-rENaC but not those of _Y- and _Y-rENaC, consistent with a role for the C terminus acting as an intrinsic inhibitory particle of ENaC.

View larger version (45K): [in this window] [in a new window]   Fig. 11.   No response to the peptide mixture treatment of single channel currents in oocytes expressing Liddle's missense mutations. Single channel traces of Y- (top) and Y-rENaC (bottom) are shown obtained from the excised inside-out patches. The right half of each trace shows the effect of the peptide mixture (SP30 + SP30 in a ratio of 1:1, 100 µM) application. The single channel behavior of both Y- and Y-rENaC is not sensitive to superfusion of the peptide mixture.

View larger version (47K): [in this window] [in a new window]   Fig. 12.   Summarized data of the C-terminal peptides on the single channel activity, the number of channels per patch, and open probability of Liddle's point mutations. The protocols for recording and analysis of Liddle's point mutations are identical to those described in Fig. 10. The data are expressed as mean ± S.E. The averaged results of N·Po, Po, and N in the absence of the peptide mixture (control) for Y-rENaC (left panels) are 2.87 ± 0.37, 0.44 ± 0.03, and 6.53 ± 0.36, respectively (n = 5). Similar results are observed for Y-rENaC (right panels). The cytoplasmic membrane in inside-out patches perfusion with the peptide mixture (100 µM) seems not to influence the preceding parameters of Y- (left) and Y-rENaC (right).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The hypothesis that the cytosolic tails of - or -ENaC subunits may function as inherent inhibitory particles involved in the normal gating behavior of epithelial Na⁺ channels arose from our previous observations made in planar lipid bilayers that synthetic peptides made up of the last 10 or the last 30 amino acids of either - or -ENaC could inhibit Na⁺ channels whose P_o was increased because they possessed Liddle's truncation mutations (40, 41). In the present study, we extended these initial observations to the oocyte expression system, using a combination of two-electrode voltage clamping and excised patch clamping, to confirm the inhibitory actions of these peptides on ENaCs containing truncations in their  and/or  subunits when expressed in a more native environment.

Expression of Liddle's Mutants-- Two non-mutually exclusive mechanisms have been proposed to account for the basal-activated, macroscopic amiloride-sensitive Na⁺ currents induced by expression of the Liddle's mutants: the retrieval theory (i.e. decreased channel internalization) and/or enhanced single channel activity (42). Identification of specific Nedd4 and spectrin binding domains in the cytosolic region of the ENaC subunits (43, 44) and the interactions of ENaC with actin and other cytoskeletal elements (45) supported the idea that the fundamental problem in Liddle's disease could be inefficient internalization of the channel from the cell surface. Furthermore, observations by Snyder et al. (24) that suggested that >90% of the basal-activated Na⁺ currents are correlated to an increase in ENaC expression at the cell surface. This finding was verified in oocytes expressing a rENaC construct containing a truncated version of the  subunit (R564X). Cell surface expression of _R564X-rENaC was increased over wt rENaC expression, corresponding to a 5.6-fold increase in ENaC currents (25). However, we previously reported that a 35% increase in the fluorescence intensity in oocytes injected with _R564X-rENaC was accompanied by a 4.4-fold increase in amiloride-sensitive Na⁺ current (26). Thus, a smaller increase in cell surface channel density was associated with a large increase in current, suggesting that the retrieval theory alone did not adequately explain the large macroscopic Na⁺ current seen in Liddle's mutants (46). It is possible that the differences between these studies could be due to different responses produced by rENaC and hENaC (24).

The idea that ~90% of the ENaC channels at the cell surface are inactive and only 10% are active (27) underscores the methodological limitations in quantitatively determining not only active ENaC channel number at the cell surface but also single channel P_o. Electrophysiological recordings, including two-electrode voltage and patch clamping, report only electrically active ENaC channels at the cell surface and cannot reveal the existence of quiescent channels within the plasma membrane. The assessment of single channel activity, specifically open probability, can only be done if the total number of channels (active plus inactive) per patch is known. The relationship between macroscopic currents and single channel activity is given by  = i·N·P_o where is the mean current amplitude; i indicates the amplitude of the unitary current; N represents the number of active channels at the cell surface, and P_o is the open probability of the channel. Therefore, in a case where the majority of channels are quiescent, the true P_o will be very low. Thus, a significant component of the basal-activated ENaC currents that is not due to increased ENaC channel expression at the cell surface can result from altered single channel behavior. Such alterations may include an increase in P_o or an increase in single channel unitary conductance.

The findings of the present study showed that the unitary conductances for both the truncation mutants (_TT-rENaC) and the missense Liddle's mutants (_T- and _T-rENaC) did not differ from that of wt -rENaC, in accordance with the observations of others (23, 24). However, the electrically active ENaC number at the cell surface of Liddle's mutants described as the number per patch were 1.8-2.5-fold over that of wt -rENaC. Significantly, in our experiments, P_o for _TT-rENaC was 40% higher as compared with wt -rENaC (Table II). The single channel activity (N·P_o) for all the mutant channels showed a ~3-fold increase, paralleling the macroscopic ENaC currents in oocytes expressing ENaC-containing Liddle's mutations. Moreover, the P_Li/P_K permeability ratios for all of the mutant channels were comparable to those of wt -rENaC expressed in oocytes or of the native ENaC in epithelia (47-49), as was the amiloride sensitivity. Thus, like Firsov et al. (25) and Ismailov et al. (32), we propose the open probability of Liddle's mutations producing truncations of the  or  subunit carboxyl tails is also an important parameter contributing to enhanced macroscopic ENaC currents seen in this disease.

It is important to reconcile our findings with those of other laboratories claiming that single channel P_o does not change when a  or  subunit truncating Liddle's mutation is present in ENaC (24, 50). As indicated above, analyses of P_o from single channel data recorded by patch clamp are difficult because the true number of channels in the patch must be known. Moreover, appropriate observation times must also be attained. Fyfe and Canessa (50) concluded that subunit composition of ENaC primarily determines channel kinetics, yet only two, not three, subunit channels were used in their studies. As they and others (50-53) indicate, the P_o of ENaC is quite variable, ranging from 0.01 to 0.9. This variability in basal P_o reflects uncertainties in channel number within any given patch. If the channel number is underestimated, for example, due to the presence of quiescent channels, then higher values of P_o will be calculated. More reliable estimates of P_o for wild-type ENaC would be obtained because there would be on average fewer quiescent channels present. In our experiments, wide variability in P_o was not observed from patch-to-patch. Although our experiments also suffer from lack of certainty of the absolute number of channels per patch, we only performed our analyses on multichannel patches (2-7 channels), not "single" channel patches, so that a more representative (and potentially more reliable) determination of channel number could be made.

Peptide Inhibition of Liddle's Mutants-- By using the whole-cell configuration of the patch clamp technique, the ability of a 10-amino acid polypeptide (200 µM) identical to either the cytosolic tail of -rENaC or -hENaC to inhibit amiloride-sensitive inward Na⁺ currents in lymphocytes of Liddle's disease was found (41). This finding was confirmed in planar lipid bilayers reconstituted with the Na⁺ channel complex immunopurified from lymphocytes derived from patients affected with Liddle's disease (32). Unresolved questions concern the molecular mechanism underlying peptide inhibition of ENaC and whether peptides comprising the missing segments of the intracellular C-terminal tails of the  or  subunit can inhibit basal-activated Na⁺ currents caused by both single and double truncation mutations (_T-rENaC, _T-rENaC, and _TT-rENaC) and missense mutations (_Y- and _Y-rENaC) identified in Liddle's syndrome cases. In the present study, a combination of the two peptides markedly inhibited _TT-rENaC-associated amiloride-sensitive Na⁺ currents in a dose-dependent fashion. Whereas the individual peptides were also inhibitory, their efficacy was much reduced (Fig. 4). Expression of only one truncation mutant of either the  subunit (_T) or the  subunit (_T) combined with complementary wt ENaC subunits also induced a 3-fold increase in macroscopic amiloride-sensitive Na⁺ currents (Refs. 23, 24, and 27 and present study). The significantly lower inhibitory effect of the individual peptides compared with the peptide mixture on _TT-rENaC suggests that both the  and  subunit C termini are required for efficient inhibition of an activated ENaC. This idea is supported by our data showing that the efficacy of either SP₃₀ or SP₃₀ used individually was enhanced in the single subunit truncation mutant (_T and _T constructs as compared with the double truncation constructs (_TT)). Furthermore, the fact that these peptides did not block the point mutant channels (_Y or _Y) and the fact that these channels did not have an increased P_o is consistent with the argument that the cytoplasmic C-terminal tails of - and -ENaC are involved in the normal gating of ENaC. We hypothesize that there is -sheet formation between the C-terminal tails of  and  that, in essence, forces the tails to act as a single binding peptide. Evidence supporting such an interaction comes from intrinsic fluorescence and circular dichroism studies of these synthetic peptides in solution (33). Measured Hill coefficients of 1 for the peptide mixture are also consistent with this hypothesis.

The specificity of the peptide inhibition on Liddle's ENaC (truncation mutants) was verified in experiments using non-ENaC peptides (e.g., a 14-amino acid-mer of CFTR or Pro-SP₃₀). Cytoplasmic injection or perfusion of either of these peptides at the same concentrations used for the peptide mixture did not cause any decrease of _TT-rENaC-associated amiloride-sensitive Na⁺ currents. Because rENaC activity is down-regulated by hypo-osmotic stress (14) and peptide injection may have increased the oocyte volume (the cell volume increased ~13% assuming the mean liquid volume of oocyte is 500 nl), it is also possible that the decreased channel activity could be due to oocyte swelling. However, injection of the same volume of distilled water had no effect on the current amplitude (Fig. 1). The observations of Kellenberger and colleagues (27) excluded the possibility that the inhibitory effects of the C-terminal peptides of rENaC on the basal-activated currents of Liddle's mutations result from rundown. Their examination of the rundown phenomenon suggested that the wt rENaC-associated, rather than the Liddle's mutant-associated, Na⁺ current shows a time-dependent decrease (by 27% in 25 min). In the present study, the C-terminal peptides did not exert any effect on wt rENaC or the point mutant constructs (_Y, _Y-rENaC), indicating that they did not have untoward nonspecific effects in this expression system. The amiloride-sensitive Na⁺ currents generally achieved stabilization after ~10 min of perfusion under our experimental conditions, and the rundown of wt rENaC currents was correlated to the accumulation of intracellular Na⁺ (27). Comparable experiments were performed on excised inside-out patches of oocyte membranes containing active ENaC.

Our data show that the _TT-rENaC number per patch, P_o, and thus overall channel activity (N·P_o) were inhibited to wild-type levels by the peptide mixture, similar to that seen in the macroscopic experiments. The inhibition of the channel by peptide occurs only from the cytoplasmic side and is dissimilar to that produced by amiloride, in that a "flickering"-type block was not evident. These results are essentially identical to those seen in the bilayer, in that increasing concentrations of the peptides increase a second slow component of channel closed time distribution, leaving the open time distribution unaffected (33), again consistent with the reconstituted planar lipid bilayer system (33).

Deletion of the C termini of the - and -ENaC subunits leads to loss of the PPPXY conserved sequence. The PPPXY motifs located within the C termini have been found to play an important physiological role in controlling Nedd4-induced Ca²⁺-dependent retrieval and clathrin-mediated endocytosis of ENaC (44, 53). Thus, the possibility that the applied C-terminal peptides can function as a sensor for ENaC internalization signaling pathways and thereby result in a decrease in the number of channels per unit at the cell surface cannot be excluded. More likely, however, is that the decrease in the active channel number per patch seen subsequent to peptide addition is due to a decrease in the fraction of the electrically detected rENaC per unit of cell surface, simply by a reduction in P_o. Because the C-terminal domains of the - and -ENaC subunits contain phosphorylation sites for protein kinase A and protein kinase C (8), in vivo the C-terminal regions may interact with some target for protein kinase A- or protein kinase C-mediated phosphorylation on rENaC (subunits or associated cytoskeletal elements) and therefore regulate the activity of rENaC by altering its phosphorylation state (32, 46). The enhanced sensitivity to the activating effects (via protein kinase A) of normal endogenous cAMP levels in affected lymphocytes of Liddle's disease (41) is consistent with this possibility. Also, based on the finding that the channel activity of truncated Liddle's mutants is no longer down-regulated in a feedback manner by the increasing intracellular Na⁺ concentration and that <10% of ENaC population located at the cell surface is in the active channel pool, it has been proposed that a greater fraction of "silent" rENaCs has escaped from the inactive pool due to the rise in cytosolic Na⁺ concentration and thus has contributed to the increased electrically detected number of channels per patch (27).

In summary, our results demonstrate that both a greater ENaC number of channels at the cell surface and an increased P_o contribute to the increased basal ENaC currents associated with expression of ENaC truncation mutants identified in Liddle's disease. In addition, C-terminal peptides structurally identical to the cytoplasmic tails of individual ENaC subunits afforded us an opportunity to examine the regulatory function imposed by the C-terminal tails of rENaC and thus further elucidate the pathogenesis of Liddle's disease. The results in the present study confirmed that the constitutively activated Na⁺ absorption through truncation mutants of the ENaC channel located on the apical membrane of kidney collecting duct correlates to the loss of the inhibitory tails of the  and/or  subunits. Physical addition of the missing regions for both subunits of the truncation Liddle's mutations could achieve successful correction of currents in vitro, both at the macroscopic and single channel levels. Combined with similar observations obtained from patch clamping on human B lymphocytes (41), dual-electrode voltage clamping on oocytes expressing ENaC (26), and planar lipid bilayer incorporation experiments (32, 33), our results provide an experimental basis for understanding inherent ENaC function. Our results also support the idea that different Liddle's mutations, e.g. truncation versus point mutations, produce enhanced macroscopic Na⁺ currents through different mechanisms.

	ACKNOWLEDGEMENT

We thank Eddie J. Walthall (Department of Physiology and Biophysics, University of Alabama, Birmingham) for the superb technical assistance in the construction of the patch clamp setup. We thank Drs. C. Canessa (Department of Physiology, Yale University) and L. Schild (Institute of Pharmacology and Toxicology, Lausanne, Switzerland) for their kind gifts of ENaC, and Drs. S. Kellenberger (Lausanne, Switzerland) and Dr. Y. L. Zhu (Physiology, State University of New York, Buffalo) for their helpful discussion. We thank C. Guy for excellent secretarial service.

	FOOTNOTES

^* This work was supported by NIDDKD Grant DK37206 from the National Institutes of Health.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.

To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Alabama at Birmingham, MCLM 704, Birmingham, AL 35294-0005. Tel.: 205-934-6200; Fax: 205-934-1445; E-mail: Benos@phybio.bhs.uab.edu.

	ABBREVIATIONS

The abbreviations used are: ENaC, epithelial sodium channels; rENaC, rat epithelial sodium channels; hENaC, human epithelial sodium channels; wt, wild type.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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