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

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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 SP_30</