INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Amiloride-sensitive epithelial Na⁺ channel(s) fulfills a variety of physiological roles in different Na⁺-transporting epithelia such as those in the distal nephron, distal colon, lung epithelia, and duct cells of exocrine glands (Garty and Palmer, 1997). Previous electrophysiological studies show that these channels display functional heterogeneity in different tissues in terms of biophysical characteristics, including unitary conductance, kinetics, ion selectivity, and sensitivity to amiloride (Palmer, 1992). It is unclear whether this phenotypic heterogeneity reflects a corresponding genotypic diversity.

The first amiloride-sensitive epithelial Na⁺ channel (ENaC)¹ was cloned from rat (rENaC) colon by functional expression cloning, and shown to be composed of three homologous subunits, , , and  ENaC (Canessa et al., 1993, 1994a; Lingueglia et al., 1993, 1994). ENaC subunits have since been identified in different species, including human (hENaC) (McDonald et al., 1994, 1995; Voilley et al., 1994), Xenopus (xENaC) (Puoti et al., 1995), bovine (bENaC) (Fuller et al., 1995), and chicken (cENaC) (Goldstein et al., 1997). The cloned ENaC subunits show structural and sequence similarities between these species; they all possess two transmembrane domains flanked by a large extracellular loop and two short NH₂ and COOH termini (Canessa et al., 1994b; Snyder et al., 1994; Renard et al., 1994).

ENaC has since attracted a particular attention for the following reasons: (a) this cloned channel expressed in Xenopus oocytes shares several common biophysical properties with those of the highly selective Na⁺ channels that mediate Na⁺ entry into distal segments of the kidney (reviewed in Garty and Palmer, 1997); (b) several heritable mutations in the genes encoding the ENaC subunits cause genetic disorders, including pseudohypoaldosteronism type I and Liddle's syndrome (Shimkets et al., 1994; Chang et al., 1996; Strautnieks et al. 1996); (c) inactivation of ENaC by targeted gene knock out in mice causes an early death due to defective lung fluid clearance at birth, demonstrating that this channel also plays a critical role in lung function (Hummler et al., 1996).

Despite the importance of the cloned ENaCs in epithelial cells, their functional aspects have been studied mainly by heterologous expression in nonepithelial cell systems such as Xenopus oocytes and planar lipid bilayer reconstitution (reviewed in Garty and Palmer, 1997). Single channel conductance of rENaC studied in planar lipid bilayers (13 pS) (Ismailov et al., 1996) is different from that observed in Xenopus oocytes (4-5 pS) (Canessa et al., 1994a), and the existence of a subconductive behavior of rENaC in planar lipid bilayers (Ismailov et al., 1996) has not been observed in patch records of rENaC expressed in Xenopus oocytes (Canessa et al., 1994a). Moreover, cAMP-dependent activation of ENaC was not observed in either Xenopus oocytes or in lipid bilayer systems (Awayda et al., 1996), in contrast to the stimulatory effects of cAMP on most amiloride-sensitive Na⁺ channels in native tight epithelia, including mammalian collecting tubules and amphibian epithelia (reviewed in Garty and Palmer, 1997). The rENaC transfected in NIH-3T3 fibroblasts, however, responded to cAMP (Stutts et al., 1995, 1997). Comparison of ENaC properties obtained in these systems raises the possibility that their biophysical characteristics and regulatory mechanism may depend upon the physical state of the membrane into which they are inserted, and on the cytosolic micro-environment, both heterogenous in different cell types.

Na⁺ channel activity in native tight epithelia is regulated by various hormones such as aldosterone and vasopressin (reviewed in Rossier and Palmer, 1992; Garty and Palmer, 1997). In addition, it has been proposed that inorganic cations such as Na⁺ and Ca²⁺ may also regulate channel activity. Previous studies have demonstrated that increasing luminal Na⁺ concentration resulted in saturation of Na⁺ entry in frog skin (Fuchs et al., 1977), toad urinary bladder (Palmer et al., 1980; Li et al., 1982), and rabbit descending colon (Thompson and Sellin, 1986; Turnheim et al., 1983). This saturation has been explained by the following mechanisms: (a) saturation of the single channel conductance by external Na⁺ (Palmer and Frindt, 1986, 1988); (b) inhibition of Na⁺ channels by extracellular Na⁺ itself ("self-inhibition"); (c) inhibition of Na⁺ channels due to changes in cytosolic composition such as Na⁺ and Ca²⁺ ("feedback inhibition") (Chase, 1984; Garty and Lindemann, 1984; Palmer, 1985; Silver et al., 1993). Since all of the above studies using intact epithelia had several parameters changed simultaneously (e.g., membrane potential, intracellular pH, concentrations of Na⁺ and Ca²⁺), the detailed mechanisms by which intra- or extracellular Na⁺ and/or Ca²⁺ regulate these channels remain unclear. Furthermore, the exact molecular identity of the channels studied in these native epithelia is not known.

Recently, Stutts et al. (1995) have demonstrated, using short circuit current measurements, that stable expression of rENaC in Madin-Darby canine kidney (MDCK) epithelial cells exhibited amiloride-sensitive transepithelial sodium currents, which could be stimulated by cAMP. Such a mammalian epithelial cell line stably expressing rENaC should allow electrophysiological and biochemical characterization of this molecularly known channel. To our knowledge, neither whole-cell nor single-channel currents via rENaC heterologously expressed in any mammalian epithelial cells have been extensively characterized. In the present paper, therefore, we provide a characterization of Na⁺ conductance attributable to rENaC heterologously expressed in MDCK cells using patch-clamp techniques. We first used the whole-cell patch-clamp technique to characterize the biophysical properties of macroscopic currents from populations of rENaC channels in this heterologous expression system. We also performed single channel recording experiments to characterize the properties of the microscopic single channel conductance, which could be responsible for the macroscopic currents. We then studied the effects of Na⁺ and Ca²⁺ on rENaC activity. In this report, we demonstrate that (a) the whole-cell Na⁺ conductance attributable to rENaC activity saturates with increasing extracellular Na⁺ concentration likely due to saturation of the single channel conductance, (b) single channel activity is inhibited by high cytosolic Na⁺ concentrations, and (c) an increase in cytosolic Ca²⁺ has an inhibitory effect on rENaC expressed in MDCK cells.

	METHODS

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rENaC-expressing MDCK Cells

High resistance MDCK cells stably expressing rENaC or rENaC were previously generated (and kindly provided by C. Canessa and B. Rossier, University of Lausanne) by stably transfecting cells with ENaC in pLKneo (Hirt et al., 1992), which contains a dexamethasone-inducible promoter, together with  and ENaC cloned into a cytomegalovirus promoter-based vector and expressed constitutively (Stutts et al., 1995). Expression of the ENaC subunits was confirmed by immunoprecipitation of metabolically labeled channel using antibodies directed against each subunit (Stutts et al., 1995; Staub et al., 1997). MDCK cells stably expressing epitope-tagged rENaC were generated by transfecting FLAG-tagged (DYKDDDDK)rENaC in pCEP4 (Invitrogen Corp., San Diego, CA), followed by triple HA-tagged (YPYDVPDY) rENaC in pLKneo. Both tags were inserted just upstream of the STOP codon. Protein expression was verified by immunoblotting with antibodies to the tags. MDCK cells stably expressing rENaC and rENaC were maintained in DMEM containing fetal bovine serum (FBS, 10%), penicillin (100 U/ml), streptomycin (100 µg/ml), G418 (300 µg/ml), and amiloride (10 µM) at 37°C in 5% CO₂-containing humidified air. Cells expressing rENaC were maintained as above, only with the addition of hygromycin (0.1 mg/ml). Wild-type MDCK cells were maintained in DMEM plus serum and antibiotics (without G418) as above. For patch-clamp experiments, cells were seeded at low density on a cover glass and patched 2-4 d after seeding. Only single cells were used for whole-cell patch-clamp experiments. rENaC-expressing MDCK cells were induced by adding 1 µM dexamethasone and 2 mM butyrate to the culture medium overnight, as described previously (Stutts et al., 1995).

Patch Clamp Analyses

Cells grown on cover glass were rinsed with an appropriate NaCl-rich bath solution (see below), and then transferred to a chamber mounted on a Leitz inverted microscope. Current recordings were made from MDCK cells using the standard whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). The patch-clamp pipettes, which were pulled from glass capillaries (Dagan Corp., Minneapolis, MN) using a horizontal puller (P-97; Sutter Instruments Co., Novato, CA), had resistances of ~2-3 M when filled with a standard Cs glutamate-rich solution.

An Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster City, CA) was used to measure whole-cell and single channel currents. The reference electrode was a Ag/AgCl electrode that was connected to the bath via an agar bridge (10 mg/ml) filled with a NaCl-rich bathing solution. The amplifier was driven by pClamp software to allow the delivery of voltage-step protocols with concomitant digitization of the whole-cell current. The whole-cell currents were filtered through an internal four-pole Bessel filter at 1 kHz and sampled at 2 kHz.

Single-channel activity was recorded in cell-attached, inside-out, and outside-out patch configurations (Hamill et al., 1981) using an Axopatch-1D amplifier. Single-channel currents were filtered at 50-100 HZ with an internal four-pole Bessel filter, sampled at 1-2 kHz and stored directly on the computer's hard disk through the TL-1 DMA interface (Axon Instruments). Subsequent current analysis was performed using programs supplied with Axograph or pClamp software (Axon Instruments).

Current-voltage (I-V) relations were studied using 10-mV pulses, each of 400-ms duration, delivered at voltages ranging between 140 and +90 mV; voltage pulses were separated by 3-10 s, during which the cell potential was held at +40 or 0 mV. Steady state whole-cell currents were measured at 350 ms from the start of each voltage pulse. As an alternative to voltage steps, voltage ramps were applied. Typically, the command voltage was varied from 100 to +80 mV over a duration of 800 ms every 10 or 30 s. Reversal potentials were estimated by fitting linear regression lines at least to the three data points nearest to the zero current potential. Whole-cell conductances were calculated by fitting a linear regression to the data points between 53 and 103 mV. For kinetic analysis of the amiloride-sensitive whole-cell Na⁺ currents, most relaxations were fitted with a single exponential using a least-squares method. To permit capacitive transients to subside, only data collected >10 ms after the potential change were used for the calculations. The amplitude of the relaxation was defined as the ratio of the steady state current (estimated as the asymptote of the exponential) to the initial current immediately after the potential change (estimated by extrapolation of the exponential to the time of the potential change). We have assumed that the relaxation amplitude, as defined here, equals the ratio of the fraction of channels open at the test potential to the fraction of channels open at the holding potential, and hence that it is proportional to the channel open probability.

To analyze titration curves for amiloride inhibition of the macroscopic Na⁺ current (I_Na), the ratio I/I₀ measured in the presence (I) of amiloride to that in its absence (I_o), normalized to the value in the presence of 1 µM amiloride, was described by the following equation:

(1)

where K_i is the inhibitory constant of the blocker, A is the concentration of the blocker, n' is pseudo Hill coefficient, and B is a constant defined as the current remaining in the presence of maximal inhibition by amiloride.

In the case of a voltage-dependent block, K_i(V) is the voltage- dependent inhibitory constant, which has been expressed by Woodhull (1973) as a Boltzmann relationship with respect to the voltage,

(2)

where K_i(o) is the inhibitory constant at 0 mV, z' is a slope parameter, and F, V, R, and T have their conventional meanings. z' is equal to the product of the actual valence of the blocking ion z and the fraction of the membrane potential (or electrical distance)  acting on the ion.

The K_m for external Na⁺ was calculated from the whole-cell Na⁺ current induced by varying the concentration (C) of Na⁺ in the bath solution from 0 to 150 mM. The data were fitted to the Michaelis-Menten equation:

(3)

where I_max is the maximal current and K_m is the half-activation constant using a nonlinear fit program.

The capacitance transient current in most experiments was compensated by using the Axopatch-1D amplifier. The cell capacitance was 33.0 ± 1.3 pF (n = 103). When constructing I-V plots, the steady state whole-cell current was measured at 350 ms after onset of the voltage pulse. The currents were not leak corrected. The series resistance (R_s) in these studies, which was 17.2 ± 0.6 M (n = 103), was not compensated. The pipette potential was corrected for the liquid junction potentials between the pipette solution and the external solution, and between the external solution and the agar bridge, as described by Barry and Lynch (1990).

The compositions of the standard pipette and bath solution were as follows: the pipette solution (pH 7.4) contained (mM): 120 Cs-glutamate, 10 CsCl, 1 MgCl₂, 10 HEPES, and 1 EGTA. When the free Ca²⁺ concentrations of the pipette solution in whole-cell patch-clamp experiments were fixed at 10⁶ M, 10 mM EGTA was used as a Ca²⁺ buffer. The free concentrations of Ca²⁺ were calculated from an equation that takes into account the concentrations of Mg²⁺, Ca²⁺, EGTA (96% purity), and pH (Oiki and Okada, 1987), and the appropriate amount of CaCl₂ was added to the solution. The pH of the solution was adjusted with CsOH. The cells were initially immersed in bath solution (pH 7.4) containing (mM): 140 NaCl, 4.3 KCl, 1 MgCl₂, and 10 HEPES. After the formation of whole-cell configuration, the bath solution was changed to one containing (mM): 145 Na-glutamate, 1 MgCl₂, and 10 HEPES, pH 7.4. Relative permeabilities for various cations were determined using external solutions containing an equimolar amount of the glutamate salts of the test cation. The pH of the bath solutions was adjusted with LiOH, KOH, or NMDG-OH. When the free Ca²⁺ concentrations of the bath solution in inside-out patches were varied between 10⁵ and <10⁹ M, 1 or 5 mM EGTA was used as a Ca²⁺ buffer. In these experiments, the bath solution was K- or NMDG-glutamate rich. The free concentrations of Ca²⁺ were calculated as described above.

All experiments were performed at room temperature (20- 23°C). Bath solution changes were accomplished by gravity feed from reservoirs. The results were reported as means ± SEM of several independent experiments (n), where n refers to the number of cells patched, each from a different dish of cells. Statistical significance was evaluated using the two tailed paired and unpaired Student's t test. A value of P < 0.05 was considered significant.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Comparison of Whole-Cell Na⁺ Conductance in Wild-Type and rENaC-transfected MDCK Cells

Previous studies have shown that MDCK cell membranes contain several types of K⁺ and Cl channels (Lang et al., 1990). Thus, to isolate the Na⁺ currents, the K⁺ and Cl currents were minimized using Cs-glutamate-rich solution in the pipette, and by using glutamate as the major anion in bath solutions containing no K⁺ ions. To compare whole-cell Na⁺ conductance in untransfected vs. rENaC-expressing MDCK cells, we first measured the Na⁺ current by changing the bath solution from a Na-glutamate-rich solution to a solution in which Na⁺ was replaced by equimolar amounts of the impermeant NMDG⁺ cation. Fig. 1 A shows that untransfected MDCK cells exhibited a small Na⁺ conductance, with whole-cell Na⁺ current amplitude of 27.0 ± 14.0 pA (n = 7) at 143 mV. A similar small conductance was also observed in these untransfected cells after overnight treatment with 1 µM dexamethasone and 2 mM butyrate (whole-cell Na⁺ current amplitude at 143 mV was 47.6 ± 10.7 pA, n = 8). In contrast, rENaC-expressing MDCK cells exhibited a much greater Na⁺ conductance, with whole-cell Na⁺ current amplitude of 1,466.2 ± 297.5 pA (n = 14) at 143 mV (Fig. 1 B, left). These results indicate that rENaC-transfected MDCK cells express a functional Na⁺ conductance at the plasma membrane.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   (A) Inward Na+ current amplitude at 143 mV measured in untransfected MDCK cells after overnight treatment with 2 mM butyrate and/or 1 µM dexamethasone. The Na+ current was measured by changing the bath solution from Na-glutamate-rich solution to a solution in which Na+ had been replaced by equimolar amounts of the impermeant NMDG+ cation. Values are mean ± SEM of six to eight experiments. (B, left) Comparison of whole-cell Na+ conductance in untransfected and rENaC-(stably) transfected MDCK cells after overnight treatment with 2 mM butyrate and 1 µM dexamethasone. All the Na+ current in the rENaC-transfected MDCK cells is amiloride sensitive, as shown in Fig. 2. Values are mean ± SEM of 8 and 14 experiments, respectively. (right) Amiloride (10 µM)-sensitive whole-cell currents in - or rENaC stably transfected MDCK cells. Cells were treated overnight with 2 mM butyrate + 1 µM dexamethasone. Inward current amplitude was measured at 143 mV. Values are mean ± SEM of six experiments.

View larger version (58K): [in this window] [in a new window]   Fig. 2.   (A) Representative tracings of whole-cell currents obtained from rENaC-expressing MDCK cells. The cell was held at a potential of +37 mV between test voltage pulses (143 to +87 mV). The pipette was filled with a standard Cs-glutamate-rich solution. Whole-cell I-V relation was measured when the bath contained a Na-glutamate-rich solution (a), and then replacing the bath with a similar solution containing 10 µM amiloride (b), or with an NMDG-glutamate-rich solution (c). (B) Comparison of steady state I-V relation for the amiloride-sensitive whole-cell () and Na+ () currents. (C) Comparison of inward current amplitudes at 143 mV for the amiloride-sensitive Na+ and total Na+ currents.

Amiloride Sensitivity of the Na⁺ Conductance

To determine if the Na⁺ conductance observed in the rENaC-transfected MDCK cells is amiloride sensitive, we examined the effect of amiloride (10 µM) on the whole-cell Na⁺ currents recorded from single cells expressing rENaC (Fig. 2). In the experiment shown in Fig. 2 A, we first measured the whole-cell I-V relation when the bath contained a Na-glutamate-rich solution, and then replaced the bath with a similar solution containing 10 µM amiloride. By subtracting the whole-cell records observed before the addition of the inhibitor from those observed after its addition, we obtained the steady state I-V relation for the component of the whole-cell current attributable to amiloride-sensitive Na⁺ channels. The steady state I-V relation for the amiloride-sensitive whole-cell current was then compared with that for the whole-cell Na⁺ current estimated in the same cell. As seen in Fig. 2, B and C, there was no difference between the amiloride-sensitive currents and the Na⁺ currents at different membrane potentials, indicating that the whole-cell Na⁺ current observed in rENaC-expressing MDCK cells was solely mediated by amiloride-sensitive Na⁺ channels. These results are consistent with a previous report that demonstrated a much greater amiloride-sensitive short-circuit current in rENaC-transfected than in -untransfected MDCK cells (Stutts et al., 1995).

Since it has been demonstrated that partial ENaC subunit combinations ( or ) form amiloride-blockable pores in Xenopus oocytes (Canessa et al., 1994; McDonald et al., 1995; McNicholas and Canessa, 1997), such "partial channels" might significantly contribute to the amiloride-sensitive conductance observed in our rENaC-expressing MDCK cells. To test this possibility, we performed additional whole-cell voltage-clamp experiments using - or rENaC-expressing MDCK cells. Under the present experimental conditions, however, the - and rENaC-expressing cells exhibited a very small amiloride-sensitive conductance, with amiloride-sensitive whole-cell current amplitude of only 115.2 ± 41.4 pA (n = 6) and 52.1 ± 24.5 pA (n = 6) at 143 mV, respectively (Fig. 1 B, right).

We next examined the amiloride sensitivity of the whole-cell Na⁺ currents. Fig. 3 A shows that the inward currents associated with membrane hyperpolarization were inhibited by amiloride in a dose-dependent manner, and that the amiloride-sensitive currents were inwardly rectifying (Fig. 3 B). Fig. 3 C summarizes the effect of amiloride on the whole-cell inward current at 103 mV. The K_i for the amiloride effect at this membrane potential was ~20 nM. Since the amiloride block of epithelial Na⁺ channels is known to be weakly voltage dependent (Palmer, 1985; Warncke and Lindemann, 1985), we further analyzed the voltage dependence of the block. The data with 0.1 µM amiloride were fitted with an equation derived from Eqs. 1 and 2 (Fig. 3 D). According to the Woodhull formalism, this slope parameter equals the valence of the blocking particle times the fraction of membrane potential acting on the particle at its blocking site. A mean slope parameter z' for the voltage dependency of amiloride block was estimated to be 0.20 at 22°C. Therefore, the electrical distance, , was 20% of the transmembrane electric field, suggesting that the amiloride-binding site is located within the outer entrance of the ion conductive pathway. This is consistent with recent site-directed mutagenesis experiments of rENaC, suggesting an involvement of a short segment preceding the second membrane spanning domain, which may be within the transmembrane electric field, in channel block by amiloride (Schild et al., 1997).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Amiloride sensitivity of whole-cell Na+ currents in rENaC-expressing MDCK cells. (A) Representative tracings of the effect of different concentrations of amiloride obtained from a single MDCK cell. The pipette was filled with a standard Cs-glutamate-rich solution, and the bath contained a Na-glutamate-rich solution. The cell was held at a potential of +37 mV between test voltage pulses (143 to +87 mV). (B) The corresponding I-V relations obtained from the same cell shown in A. (C) Dose-inhibition relation for the amiloride effect at 103 mV. Effect of different concentrations of amiloride (I/I0) was normalized to a value in the presence of 1 µM amiloride. Data were fitted with Eq. 1 (see METHODS), where n' = 0.8. Each point represents the mean ± SEM of two to five experiments. (D) Effect of membrane potential on the amiloride inhibition (at 0.1 µM) of the whole-cell current. Data were fitted with an equation derived from Eqs. 1 and 2 (see METHODS). Each point represents the mean of four experiments.

Voltage Dependence of rENaC Expressed in MDCK Cells

rENaC-expressing MDCK cells exhibited slowly activating currents when the membrane was hyperpolarized to potentials more negative than 100 mV. Fig. 4 A shows tracings of the whole-cell currents associated with voltage jumps from a holding potential of +37 mV between 143 and +87 mV before and after addition of amiloride (10 µM) to the bath solution. To characterize the time course of the activation of amiloride-sensitive whole-cell Na⁺ currents in these cells, we digitally subtracted the whole-cell currents recorded in the presence of the inhibitor from those recorded in its absence. A relaxation of the whole-cell current when the membrane was strongly hyperpolarized was truly mediated by amiloride-sensitive conductance (Fig. 4 A, c). To further characterize the relaxation of the whole-cell current, we examined the effect of membrane potential on its relaxation kinetics, which was analyzed as described in METHODS. Fig. 4 B shows a plot of the relaxation time constant as a function of membrane potential. The time constant did not change significantly at voltages between 143 and 33 mV: the values at 143 and 73 mV were 114.4 ± 23.0 ms (n = 7) and 94.9 ± 11.1 ms (n = 6), respectively. The relative amiloride-sensitive current amplitude was shown to be slightly voltage dependent so that the relative value was 1.41 ± 0.08 (n = 7) at 143 mV and 1.16 ± 0.03 (n = 6) at 73 mV (Fig. 4 C).

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Voltage dependence of whole-cell Na+ conductance in rENaC-expressing MDCK cells. (A) An example of tracings of the whole-cell currents (a) before and (b) after the addition of amiloride (10 µM) to the bath solution. (c) Tracings of amiloride-sensitive currents obtained from a and b. Hyperpolarizing and depolarizing pulses 400 ms in duration were applied from a holding potential of +37 mV to potentials between 143 and +87 mV in 20-mV intervals. The pipette was filled with a Cs-glutamate-rich solution. Dependence of the relaxation time constant (B) and amiloride-sensitive current amplitude relative to that at +37 mV (C) on the membrane potential estimated from the whole-cell Na+-current induced by voltage pulses from a resting potential of +37 mV. The relative amiloride-sensitive current amplitude is defined as described in METHODS. Each point represents the mean ± SEM of four to six experiments.

Since amiloride-sensitive outward current showed a time-dependent activation at markedly depolarized intracellular voltages (+80 mV) when rENaC was expressed in Xenopus oocytes (Awayda et al., 1996), we wanted to examine if a similar phenomenon can be observed in rENaC-expressing MDCK cells. To do this, cells were bathed in NMDG-glutamate-rich solution and dialyzed with Na⁺-glutamate-rich pipette solution. Under these conditions, however, we found that amiloride-sensitive outward current exhibited a time-dependent inactivation at markedly depolarized voltages (Fig. 5 A, c). Time constant of the current decay at +68 and +28 mV was 162.8 and 139.4 ms, respectively. The relative amiloride-sensitive current amplitude was 1.25 at +68 mV and 1.13 at +28 mV. These data together support the idea that the channels are activated by membrane hyperpolarization.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   (A) Whole-cell currents recorded from rENaC-expressing MDCK cells using a Na-glutamate-rich pipette solution. Bath solution was NMDG-glutamate-rich in the absence (a) or presence (b) of 10 µM amiloride. (c) Tracings of amiloride-sensitive currents obtained from a and b. Hyperpolarizing and depolarizing pulses 400 ms in duration were applied from a holding potential of 62 mV to potentials between 92 and +68 mV in 40-mV intervals. (B) The corresponding I-V relations of the whole-cell currents obtained from the same cell shown in A. (C) The corresponding I-V relation of amiloride-sensitive Na+ outward currents obtained from the same cell shown in A, c.

Ion Selectivity of the Whole-Cell Na⁺ Conductance

With the standard Cs-glutamate-rich pipette solution and Na-glutamate-rich bath solution, the amiloride (10 µM)-sensitive whole-cell currents had a zero-current potential of +63.2 ± 10.5 mV (n = 16). Assuming that the amiloride-sensitive current was carried by Na⁺ and Cs⁺, the relative permeability of Na⁺ to Cs⁺ (P_Na/P_Cs) was estimated to be 11. To characterize the ion selectivity of the macroscopic currents mediated by rENaC expressed in MDCK cells, we examined the relative permeabilities for various cations of the whole-cell currents. Fig. 6 A shows representative whole-cell current tracings recorded from a single cell. In this experiment, whole-cell currents were recorded in the presence of 150 mM Na⁺, NMDG⁺, K⁺, or Li⁺ as the glutamate salt in the bathing solution. Replacement of Na⁺ with NMDG⁺ or K⁺ failed to support the amiloride-sensitive inward current. When Li⁺ was used as the main charge carrier, however, it produced larger inward currents than Na⁺, which was completely blocked by amiloride (10 µM). Fig. 6 B shows the corresponding I-V relations of the amiloride-sensitive Na⁺ and Li⁺ currents depicted in Fig. 6 A. When the amiloride-sensitive Na⁺ and Li⁺ currents were measured in the same cells, the corresponding current amplitudes at 143 mV were 0.85 ± 0.15 nA (n = 6) and 1.81 ± 0.33 nA (n = 6), respectively. The ratio of the amiloride-sensitive Li⁺ to Na⁺ current at this membrane potential was calculated to be 1.94 ± 0.27 (n = 6) (Fig. 6 C).

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Ion selectivity of whole-cell Na+ conductance in rENaC-expressing MDCK cells. (A) Representative tracings of whole-cell currents recorded from rENaC-expressing MDCK cells in the presence of: (a) Na+, (b) Na+ plus 10 µM amiloride, (c) NMDG+, (d) K+, (e) Li+, or (f) Li+ plus 10 µM amiloride (150 mM of each cation as the glutamate salt in the bathing solution). The pipette solution was filled with a Cs-glutamate-rich solution. Hyperpolarizing and depolarizing pulses 400 ms in duration were applied from a holding potential of +37 mV to potentials between 143 and +87 mV in 10-mV intervals. (B) The corresponding I-V relations of the amiloride-sensitive Na+ and Li+ currents shown in A. (C) Comparison of amiloride (10 µM)- sensitive Na+ and Li+ currents at 143 mV. K+ currents were estimated by subtraction of whole-cell current in the presence of 150 mM K+ from that in the presence of 150 mM NMDG+. The amiloride-sensitive Na+ and Li+ currents were measured in the same cells. Data are the mean ± SEM of six experiments.

We also ensured that NMDG⁺ does not pass through the channel in additional experiments in which the cells were bathed in NMDG-glutamate-rich solution and dialyzed with Na⁺-glutamate-rich pipette solution. Under these conditions, if NMDG⁺ could not permeate the channel, amiloride-sensitive current should be outward at all membrane potentials. A zero-current potential of the amiloride-sensitive current could not be observed even at 100 mV, suggesting an extremely low permeability for NMDG⁺ (Fig. 5 C). Given this extremely low permeability, our results with K⁺ replacement indicate that the permeability to K⁺ is comparable to that of NMDG⁺ (Fig. 6). From these experiments, we estimated the ion selectivity sequence to be Li⁺ > Na⁺ >> K⁺ = NMDG⁺.

Single Channel Characteristics of rENaC Expressed in MDCK Cells

To characterize single channel features of rENaC expressed in MDCK cells, which could be responsible for the whole-cell currents, we performed single-channel current measurement. We first used an outside-out patch configuration to study the single channel conductance attributable to rENaC, because this configuration allows measurement of the single channel current under the same ionic conditions as those performed with whole-cell recordings. The patch pipette was filled with a Cs-glutamate solution and the bath contained Li-glutamate-rich solution as in the whole-cell experiments. Under these conditions, we usually observed multiple openings of Na⁺ channels in patches from the rENaC-expressing cells. Fig. 7 A demonstrates the presence of amiloride-sensitive single channel currents in an outside-out patch. In this patch, single channel current transitions with up to four channel levels could be observed. Application of amiloride (10 µM) abolished channel activity. This effect was reversible upon washout of amiloride. To characterize amiloride-sensitive single channel conductance, we next determined the I-V relation of the channel. Such I-V relation from nine experiments are summarized in Fig. 7 B. Based on these results, the single channel conductance was estimated to be 8.1 ± 0.7 pS (n = 9) for Li⁺ under these experimental conditions. A reversal potential of the current was deviated far from 0 mV, suggesting that Li⁺ permeability of the channel was much greater than Cs⁺, although the relative permeability of Li⁺ to Cs⁺ could not be determined experimentally. We also found that the single-channel conductance was reduced by replacement of Li⁺ with Na⁺ in the bath solution (Fig. 7 C), indicating a greater permeability of Li⁺ over Na⁺. The single channel conductance for Na⁺ was 4.7 ± 0.6 pS (n = 4). We could not detect any current transition when K⁺ was substituted for Li⁺ (data not shown). In cell-attached and inside-out patches, we also found similar single channel currents to those in outside-out patches. In cell-attached patches, single channel conductance of the inward current was 8.6 ± 0.9 pS (n = 3) and 4.7 ± 0.5 pS (n = 7) when Li⁺ and Na⁺ were used as a charge carrier, respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   (A) Amiloride-sensitive single channel recorded in an outside-out patch from rENaC expressed in MDCK cells. The patch pipette was filled with Cs-glutamate solution and the bath contained Li-glutamate-rich solution. Application of amiloride (10 µM) to the bath solution abolished channel activity. Holding potential was 4 mV. (B) Current-voltage relation for the amiloride-sensitive single channel current in outside-out patches. The pipette was filled with a Cs-glutamate-rich solution and the bath contained a Li-glutamate-rich solution. Each point represents the mean ± SEM of nine experiments. (C) Single channel current traces from an outside-out patch in the presence of 150 mM Li+ or Na+ as the glutamate salt in the bathing solution. Holding potential was 4 mV.

As expected from the whole-cell voltage-clamp experiments, we found that channel activity was increased upon membrane hyperpolarization. Fig. 8, A and B, shows representative tracings of a single channel inward current and the corresponding I-V relation in excised inside-out patches. The pipette and bath contained Li-glutamate- and K-glutamate-rich solutions, respectively. In this experiment, the channel activity defined as nP_o at a holding potential of +20, 0, 40, and 80 mV were 0.15, 0.23, 0.40, and 0.78, respectively. Fig. 8 C shows the results of six different experiments where nP_o was determined at various membrane potentials. Although the absolute value of nP_o was variable between each experiment, it was consistently increased upon membrane hyperpolarization. When nP_o value was normalized to a value of 1 at 10 mV, the relative nP_o was 2.02 ± 0.86 (n = 6) at 40 mV (P < 0.034) and 3.19 ± 0.52 (n = 4) at 80 mV (P < 0.025), respectively.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Voltage dependence of single channel activity in inside-out patches. (A) Representative tracings of a single channel inward current at various membrane potentials, and (B) the corresponding I-V relation. (C) Summary of six different experiments where nPo was determined at various membrane potentials. Lines connect data obtained from the same patch. The pipette was filled with a Li-glutamate-rich solution, and the bath contained a K-glutamate-rich solution.

As previously shown in whole-cell patch-clamp experiments (Fig. 4 B), the voltage-dependent relaxation was apparent when the membrane was hyperpolarized. We also confirmed this property using an inside-out patch. With Li-glutamate-rich solution in the pipette and NMDG-glutamate-rich solution in the bath, we performed an ensemble analysis. Membrane potential was held at +24 mV, and then stepped to 136 mV. When this voltage pulse was repeated 18 times, the corresponding averaged current response showed a slowly activating current (Fig. 9), with a time constant estimated to be 102 ms. This relaxation time constant was similar to that observed in the whole-cell experiments (Fig. 4 C). Taken together, these results strongly suggest that this amiloride-sensitive single channel current likely contributes to the amiloride-sensitive whole-cell current observed in the rENaC-expressing MDCK cells.

View larger version (17K): [in this window] [in a new window]   Fig. 9.   An ensemble analysis of single channel current in an excised inside-out patch. The pipette was filled with a Li-glutamate-rich solution and the bath solution was NMDG-glutamate rich. The membrane potential was held at +21 mV and then stepped to 139 mV. Top and middle traces represent the same current response to the voltage-step in different magnification. Lower trace represents the averaged current response when this voltage pulse was repeated 18 times.

Extracellular Na⁺ Dependency of the Whole-cell Na⁺ and Single Channel Conductance

One of the mechanisms proposed for the regulation of luminal Na⁺ entry in native Na⁺ transporting epithelia is a self-inhibition of channels by extracellular Na⁺ (for review see Garty and Palmer, 1997). To test whether extracellular Na⁺ concentrations could regulate Na⁺ channel activity, we first examined the dependence of the whole-cell Na⁺ current on extracellular Na⁺ in our rENaC-expressing cells. Fig. 10 A shows representative tracings of whole-cell currents from a single cell in the presence of increasing concentrations of extracellular Na⁺. The Na⁺ concentration was varied by equimolar replacement with NMDG⁺. In this experiment, whole-cell currents were recorded in the presence of 150 mM Na⁺, and then extracellular Na⁺ concentration was changed to 0, 5, 10, 30, 75, and 100 mM. Our results show that the inward current declined with decreasing extracellular Na⁺ concentration (Fig. 10 A). To obtain I-V relationships for the component of the whole-cell current attributable to the Na⁺ conductance, the steady state I-V relation of the whole-cell currents was measured in the absence of extracellular Na⁺, and then subtracted from those observed in the presence of various concentrations of external Na⁺. Fig. 10 B shows the I-V relations of whole-cell currents in different extracellular Na⁺ concentrations obtained for the same cell shown in Fig. 10 A. When the current amplitude at 53 mV was normalized to the value at 150 mM Na⁺, plotting the normalized inward Na⁺ current amplitude as a function of extracellular Na⁺ concentration revealed a saturating relation (Fig. 10 C). When the data were fitted by the Michaelis-Menten equation, K_m and I_max were estimated to be 30.5 mM and 1.14 pS, respectively. At 103 mV, we also observed a similar saturating relation, which could be described by the Michaelis-Menten equation (K_m and I_max were 23.1 mM and 1.06 pS, respectively). Furthermore, when normalized whole-cell conductances between 53 and 103 mV to the value at 150 mM Na⁺ were plotted as a function of extracellular Na⁺ concentration, a similar relation was also observed (K_m = 14.2 mM and G_max = 0.99) (Fig. 10 D).

View larger version (38K): [in this window] [in a new window]   Fig. 10.   Extracellular Na+ concentration dependency of the whole cell Na+ current. (A) Representative tracings of whole-cell currents from a single cell in the presence of increasing concentrations of extracellular Na+. The concentration was varied by equimolar replacement with NMDG+. (B) The I-V relations of whole-cell currents in different extracellular Na+ concentrations obtained for the same cell shown in A. (C) Plot of the inward Na+ current at 53 mV as a function of extracellular Na+ concentration. The current was normalized to the value at 143 mV. The data were fitted by the Michaelis-Menten equation (solid line). Each point represents the mean ± SEM of three to nine experiments. (D) Plot of the normalized whole-cell Na+ conductance as a function of extracellular Na+ concentration. The data were fitted by the Michaelis-Menten equation (solid line). Each point represents the mean ± SEM of three to nine experiments.

To examine the Na⁺ dependency of the single channel conductance, we performed single-channel current recording using various concentrations of Na⁺ in the pipette solution, and pooled the data from different patches. Since outside-out patches were difficult to maintain for a long enough time to complete the whole protocol, we instead used cell-attached and inside-out patches for this series of experiments. Fig. 11 A shows a representative single channel recording in a cell-attached patch configuration. The pipette solution was filled with a low Na solution (29 mM Na⁺) and the bath solution contained 150 mM K-glutamate. When the single channel conductance of the inward current was plotted against Na⁺ concentrations in the pipette solution, it saturated with increasing Na⁺ concentrations, yielding K_m and I_max values of 24.4 mM and 5.1 pS, respectively (Fig. 11 B). The K_m value observed in these experiments was in agreement with that in the whole-cell experiments above.

View larger version (21K): [in this window] [in a new window]   Fig. 11.   Na+ concentration dependency of the single channel conductance. (A) Extracellular Na+ concentration dependency of the single channel conductance of the inward current. A representative single channel recording in a cell-attached patch. The pipette solution was filled with a low (29 mM) Na+ solution and the bath solution contained 150 mM K-glutamate. Pipette potential was held at +160 mV. (B) Plot of single channel conductance of inward current as a function of Na+ concentrations in the pipette solution. The data were fitted by the Michaelis-Menten equation (solid line). Each point represents the mean ± SEM of three to eight experiments. Intracellular Na+ concentration dependency of the single channel conductance of the outward current: (C) current-voltage relation for single channel outward Na+ current in inside-out patches. The pipette solution was filled with a Cs-glutamate-rich solution and the bathing solution contained 150 mM Na-glutamate. Each point represents the mean ± SEM of nine experiments. (D) Intracellular Na+ concentration dependency of single channel conductance of outward current in excised inside-out patches. Single channel conductance of outward current is plotted as a function of Na+ concentrations in the bathing solution. The pipette solution was filled with a Cs-glutamate-rich solution and the bathing solution contained various concentrations of Na+. The data were fitted by the Michaelis-Menten equation (solid line). Each point represents the mean ± SEM of three to nine experiments, except the data of 50 mM Na+ (n = 1).

Intracellular Na⁺ Dependency of the Single Channel Conductance of the Outward Current

We also characterized cytosolic Na⁺ dependency of the single channel conductance of the outward current using the inside-out patch configuration. In these experiments, the pipette was filled with a Cs-glutamate-rich solution and the bath solution contained increasing concentrations of Na⁺. Single channel conductance of the outward current was 4.2 ± 0.5 pS (n = 8), when Na⁺ was used as a charge carrier (Fig. 11 C). The channel responsible for the outward current was also permeable to Li⁺ (P_Li > P_Na), but not to K⁺ (data not shown). When the single channel conductance of the outward current was plotted against cytosolic Na⁺ and fitted with the Michaelis-Menten equation, the K_m and I_max values were 39.5 mM and 5.1 pS, respectively (Fig. 11 D).

Effect of Cytosolic Na⁺ Concentration on Channel Activity

We next investigated the effects of cytosolic Na⁺ on the gating of single channel currents in excised inside-out patches. The pipette solution was Li-glutamate-rich, and the bath solution contained 0 or 50 mM Na⁺ by substituting with NMDG⁺. 1 mM EGTA was added to the bath solution to ensure low free Ca²⁺ concentrations. Fig. 12, A and B, shows representative tracings for the effect of Na⁺ concentration on channel activity. After excision of the patches, and ensuring that channel activity was stable (see also Fig. 14 A), we systematically changed the cytosolic Na⁺ levels. As shown in Fig. 12 A, increasing cytosolic Na⁺ concentration from 0 to 50 mM decreased channel activity and, conversely, decreasing cytosolic Na⁺ from 50 to 0 mM increased channel activity (Fig. 12 B). Analysis of the current- voltage relationship showed that the single channel conductance of the inward current was not affected by cytosolic Na⁺ concentration (data not shown). Fig. 12 C summarizes these experiments. Despite variability in the absolute nP_o value between experiments, this value was consistently decreased when the cytosolic Na⁺ concentration was increased from 0 to 50 mM. When the nP_o value was normalized to 1 with 0 mM Na⁺, the relative nP_o with 50 mM Na⁺ was 0.49 ± 0.07 (n = 8) (P < 0.0002) (Fig. 12 D). Conversely, the absolute nP_o value was also consistently increased when the cytosolic Na⁺ concentration was decreased from 50 to 0 mM. When the nP_o value was normalized to 1 with 0 mM Na⁺, the relative nP_o with 50 mM Na⁺ was 0.40 ± 0.09 (n = 7, P < 0.0007). With cytosolic 100 mM Na⁺, the relative nP_o was 0.33 ± 0.19 (n = 4, P < 0.038) (Fig. 12 D). Taken together, these results strongly suggest that cytosolic Na⁺ concentrations may regulate the gating properties of rENaC expressed in MDCK cells.

View larger version (35K): [in this window] [in a new window]   Fig. 12.   Effect of cytosolic Na+ concentration on single channel activity. (A and B) Representative tracings in an excised inside-out patch. The pipette was filled with a Li-glutamate-rich solution. (A) The effect of increasing cytosolic Na+ concentration from 0 to 50 mM, and (B) the effect of decreasing cytosolic Na+ concentration from 50 to 0 mM. Holding potentials were 0 and 30 mV, respectively. 1 mM EGTA was added to the bath solution to ensure low Ca2+ concentrations. (C) Summary of the effect of cytosolic Na+ concentration (50 mM) on single channel activity. Data were the mean ± SEM of seven or eight experiments. (D) Effect of increasing cytosolic Na+ concentration from 0 to 50 or 100 mM. Data were the mean ± SEM of four or eight experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 14.   (A) Stability of Na+ channel activity in excised inside-out patches. Channel activity was estimated every 2.5 min for 15 min. Pipette solution was filled with Li-glutamate-rich solution and the bath solution contained NMDG-glutamate-rich solution with 1 mM EGTA. Data represent the mean ± SEM of nine experiments. (B) Representative experiment, where the effect of increasing cytosolic Ca2+ concentration on Na+ channel activity was examined in an excised inside-out patch. The pipette was filled with Li-glutamate-rich solution and the bath solution contained 150 mM K-glutamate. Holding potential was 64 mV. Cytosolic Ca2+ concentration was changed from <1 nM to 1 µM. (B) Representative compressed and expanded scale trace for both conditions. (C) Effect of cytosolic Ca2+ concentrations on channel activity. The nPo value was normalized to 1 with pCa > 9. Data for pCa = 7, 6, or 5 represent the mean ± SEM of four, eight, or eight experiments, respectively.

Effect of Cytosolic Ca²⁺ on the Whole-Cell Na⁺ Conductance and on Single Channel Activity

Extensive work had previously demonstrated the effect of Ca²⁺ on Na⁺ channel activity in native Na⁺-transporting epithelia (reviewed by Chase, 1984). We therefore investigated whether rENaC activity is affected by cytosolic Ca²⁺ levels using whole-cell and single channel current recording techniques. In whole-cell experiments, ramp command voltages were applied from 104 to +76 mV every 30 s. We used this protocol since the reversal potential of the whole-cell current can be estimated, so that any appearance of other conductances, including leak conductance, could be easily identified. First, we performed experiments where ionomycin (1 µM) was used to increase cytosolic Ca²⁺ levels. This concentration of ionomycin has been shown to increase cytosolic Ca²⁺ in MDCK cells under conventional whole-cell patch-clamp configuration (Delles et al., 1995). Fig. 13 A shows the instantaneous current- voltage relation of the whole-cell current obtained from a single MDCK cell expressing rENaC. The reversal potential of the whole-cell current was clearly positive, suggesting that the current was carried by Li⁺. An example of control experiments is shown in Fig. 13 B and summarized in Fig. 13 C. The addition of 1 µM ionomycin and 1 mM Ca²⁺ to the bath solution induced an inhibition of the whole-cell inward Li⁺ current (Fig. 13 D). Time constant of the inhibition was 16.9 ± 1.6 min (n = 3). Since we did not perform simultaneous measurements of inward current and cytosolic Ca²⁺ concentration in the same cells, we could not directly evaluate how much cytosolic Ca²⁺ increase was achieved under the above experimental conditions. We therefore performed additional experiments where free Ca²⁺ concentration in the pipette solution was fixed at 1 µM using 10 mM EGTA to clamp the cytosolic Ca²⁺ concentration. Fig. 13 E shows the representative experiment, where the effect of 1 µM Ca²⁺ (pCa = 6) on the whole-cell current was examined. In this experiment, current recording began 3 min after whole-cell dialysis. Our results show that perfusion of the cytoplasm with a pipette solution containing 1 µM Ca²⁺ induced a biphasic inhibition of the current. When the time course of the inhibition was fitted with a double exponential curve, the mean time constants of the fast and slow components were 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3), respectively.

View larger version (29K): [in this window] [in a new window]   Fig. 13.   Effect of ionomycin on whole-cell current. (A) Instantaneous I-V relation of the whole-cell current obtained from a single MDCK cell expressing rENaC. Ramp command voltages were applied from 104 to +76 mV every 30 s. The pipette was filled with a Cs-glutamate-rich solution with 1 mM EGTA, and the bath solution contained Li-glutamate (nominally Ca2+ free). (B) An example of experiments in unstimulated cells. Ramp command voltages were applied from 104 to +76 mV every 30 s. (C) Summary of control experiments. t = 0 min corresponds to 3.9 ± 0.4 min (n = 9) after whole-cell dialysis. Each point represents the mean ± SEM of three to nine experiments. (D) Effect of the addition of ionomycin (1 µM) and 1 mM Ca2+ to the bath solution (nominally Ca2+ free). Experimental condition was the same as in A. Each point represents the mean ± SEM of three experiments. (E) The effect of cytoplasm perfusion with 1 µM Ca2+. Representative experiment, where the effect of 1 µM Ca2+ in the pipette solution on whole-cell current was examined. In this experiment, the current recording began 3 min after whole-cell dialysis. Ramp command voltages were applied from 104 to +76 mV every 30 s. When time course of the inhibition was fitted with a double exponential, the corresponding time constants of the two components were 1.3 and 64.3 min, respectively. The pipette was filled with a Cs-glutamate-rich solution (pCa = 6), and the bath solution contained Li-glutamate (nominally Ca2+ free).

To examine the effect of cytosolic Ca²⁺ concentrations on the single channel conductance and gating, we performed additional inside-out patch experiments. Although channel activity usually runs down after excision of the patch membrane, it does tend to reach a steady level 10-45 min after excision (Fig. 14 A). Only channels that achieved steady levels were included in the experiment. Fig. 14 B shows that an increase in cytosolic Ca²⁺ concentration led to a decrease of channel activity. In this experiment, nP_o was decreased from 0.5 to 0.15 when cytosolic Ca²⁺ was increased from <1 nM to 1 µM. When the nP_o value was normalized to 1 with pCa > 9, increasing cytosolic Ca²⁺ to 1 or 10 µM led to a significant decrease in nP_o (0.65 ± 0.08, n = 8, P < 0.005), and (0.45 ± 0.13, n = 8, P < 0.005), respectively (Fig. 14 C). However, when cytosolic Ca²⁺ was increased from <1 nM to 0.1 µM, no significant change in nP_o was observed (normalized nP_o value was 1.07 ± 0.11, n = 4, P = 0.58) (Fig. 14 C). Analysis of current- voltage relation showed that increasing cytosolic Ca²⁺ from <1 nM to 1 or 10 µM did not affect the single channel conductance (data not shown). Collectively, these results suggest that cytosolic Ca²⁺ inhibits rENaC activity.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Although a previous study showed that rENaC stably transfected in MDCK cells exhibits a much greater amiloride-sensitive short circuit current than that in untransfected cells (Stutts et al., 1995), detailed electrophysiological properties of rENaC expressed in these cells have not been determined. Using patch-clamp techniques, we have now characterized rENaC heterologously expressed in these cells and demonstrated regulation of rENaC activity by cytosolic Na⁺ and Ca²⁺ concentrations. To our knowledge, this is the first electrophysiological characterization of the cloned ENaC expressed in mammalian epithelial cells.

Comparison of the Biophysical Properties of rENaC Express