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MOLECULAR AND GENOMIC |
1 Department of Physiology and Biophysics
2 Graduate Program in Neuroscience
3 Graduate Program in Physiology, Biophysics and Systems Biology, Weill Medical College of Cornell University, New York, NY 10021, USA
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Abstract |
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(Received 7 March 2006;
accepted after revision 5 May 2006;
first published online 11 May 2006)
Corresponding author L. G. Palmer: Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA. Email: lgpalm{at}med.cornell.edu
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Introduction |
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, ß and 
, each of which contains two membrane-spanning (M1 and M2) regions, and relatively short N- and C-terminal intracellular regions. The channel is characterized biophysically by its slow kinetics of gating, lack of strong voltage dependence and small unitary conductance (Canessa et al. 1994; Garty & Palmer, 1997; Kellenberger & Schild, 2002; Snyder, 2002). Pharmacologically, it is identified by its sensitivity to the potassium-sparing diuretic, amiloride, and its analogues. Channels expressed in Xenopus oocytes, the most common expression system used to study ENaC behaviour, are biophysically and pharmacologically similar to those expressed endogenously in native tissue (Garty & Palmer, 1997; Kellenberger & Schild, 2002).
The epithelial Na+ channel regulates the entry of Na+ ions into cells in which it is expressed. Conversely, entry of Na+ into cells can itself modulate channel activity by a process generically described as feedback inhibition (Garty & Palmer, 1997; Kellenberger & Schild, 2002). The importance of cellular Na+ regulation in fluid homeostasis is emphasized by the findings that deletion or missense mutations of residues after the second transmembrane region of native ß or subunits in the kidney lead to an increase in renal Na+ reabsorption seen in an inheritable form of hypertension called Liddle's syndrome (Shimkets et al. 1994; Hansson et al. 1995; Snyder et al. 1995; Schild et al. 1996; Snyder, 2002) and that this increase may be at least in part attributable to the suppression of feedback inhibition (Kellenberger et al. 1998).
While the general features of ENaC feedback inhibition are well characterized (e.g. reduction of whole-cell Na+ currents when intracellular Na+ levels are high), the mechanisms underlying the inhibition of Na+ entry are still not entirely clear. Some reports have suggested that high intracellular [Na+] ([Na+]i) acts directly on ENaC to curtail Na+ movement into cells (Ishikawa et al. 1998; Awayda, 1999). There is also evidence to suggest that inhibition of ENaC activity by intracellular Na+ is effected via cellular mediators (Frindt et al. 1993; Silver et al. 1993; Komwatana et al. 1996; Abriel & Horisberger, 1999). When intracellular [Na+] is elevated, the rate of endocytic channel retrieval is enhanced (Volk et al. 2004). Channel internalization itself is stimulated by the actions of ubiquitin protein-ligases, such as Nedd4, which are purported to recognize ‘PY’ motifs present in the cytoplasmic, post-M2 region of ENaC subunits and to target them for degradation (Schild et al. 1996; Staub et al. 1997; Dinudom et al. 1998, 2001; Konstas et al. 2002; Fotia et al. 2003). When missense or deletion mutations occur, as in Liddle's syndrome, the interaction between Nedd4 and ENaC is disrupted, leading to increased surface expression of the channel and, finally, to increased absorption of Na+ (Schild et al. 1996; Shimkets et al. 1997; Abriel et al. 1999; Dinudom et al. 2001; Snyder, 2002). In oocytes, the increased activity of Liddle's syndrome channels resulted from relief of inhibition of the channels by high [Na+]i (Kellenberger et al. 1998), again suggesting that intracellular Na+ may activate channel internalization through the Nedd4-dependent mechanism. However, studies comparing the activity of wild-type and Liddle's mutant ENaC in Na+-loaded oocytes showed that increases in amiloride-sensitive macroscopic current in the mutant were not matched by commensurate increases in surface channel expression (Firsov et al. 1996; Kellenberger et al. 1998). This suggests that at least some of the increase in whole-cell current in the mutant is due to an increase in current passed per channel in the membrane. However, direct measures of single-channel kinetics with high or low [Na+]i in oocytes are lacking.
The aim of this study was to describe the effects of high and low intracellular [Na+] on the single-channel properties of wild-type and Liddle's mutant ENaC expressed in oocytes. Our findings support the idea that, in wild-type channels, the process of feedback inhibition relies on reducing both channel open probability and channel number to limit Na+ entry when [Na+]i is high; in Liddle's syndrome ENaC, feedback inhibition is achieved primarily by reducing single-channel open probability.
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Methods |
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The plasmids containing rat ENaC , ß and subunits were linearized with NotI restriction enzyme (New England Biolabs, Ipswish, MA); cRNAs were transcribed with T7 RNA polymerase using the mMESSAGE mMACHINE kit (Ambion, Austin, TX). Pellets of cRNA were dissolved in nuclease-free water and stored at –70°C before use.
Oocytes were harvested from Xenopus laevis. Animals were anaesthetized in water containing tricaine methanesulphonate (1.5 g l–1, adjusted to pH 7.0). Oocytes were removed through a 1 cm incision in the abdomen. After the final collection of oocytes, animals were killed by double pithing while under anaesthesia. All animal procedures were carried out according to the guidelines of and with the approval of the Institutional Animal Use and Care Committee of Weill Medical College of Cornell University. After removal, oocytes were incubated in OR2 solution (in mM: NaCl 82.5, KCl 2.5, CaCl21, MgCl21, Na2HPo41, HEPES 5 pH7.4) with 2 mg ml–1 collagenase type II (Worthington) and 2 mg ml–1 hyaluronidase type II (Sigma-Aldrich) and incubated with gentle shaking for 60 min and then for another 30 min (if necessary) in a fresh enzyme solution at room temperature. Before injection, oocytes were incubated in OR2 solution for 2 h at 19°C. Defolliculated oocytes were selected and injected with 8 ng cRNA of each the , ß and subunits in all experiments. During the expression phase, the oocytes were incubated in either a high- or low-Na+ modified Barth's saline (MBS), pH 7.4. The high-Na+ MBS contained (mM): 85 NaCl, 1 KCl, 0.7 CaCl2l, MgCl2l, Na2 HPo4l, HEPES5 pH1 7.4) 0.8 MgSO4 and 5 Hepes. The low-Na+ MBS contained (mM): 1 NaCl, 40 KCl, 60 NMDG (N-methyl D-glucamine), 0.7 CaCl2, 0.8 MgSO4 and 5 Hepes. All chemicals were from Sigma-Aldrich unless otherwise noted.
Site-directed mutagenesis
Site-directed mutagenesis was performed on rat ß ENaC cDNA using the Pfu enzyme (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Primers were synthesized by Operon Technologies, Inc. (Huntsville, AL) To confirm that the point mutation had been successfully made, sequences of cDNAs were checked by the Cornell University Bio Resource Center (Ithaca, NY, USA).
Electrophysiology
Two-electrode voltage clamp (TEVC).
Oocytes were bathed in either a high-Na+ extracellular recording solution with (mM): 110 NaCl, 2 CaCl2, 1 MgCl2, 2 KCl and 5 Hepes, pH 7.4, or a low-Na+ extracellular recording solution with (mM): 5 NaCl, 105 NMDG, 2 CaCl2, 1 MgCl2, 2 KCl and 5 Hepes, pH 7.4. Whole-cell currents were measured in intact oocytes using a two-electrode voltage clamp (OC-725, Warner Instrument Corp.) with ITC-16 interface (Instrutech) running Pulse software (Heka Elektronik). Pipettes were made from haematocrit capillary tubes (Fisher Scientific) with a three-step vertical pipette puller (Kopf). Pipette resistances were 0.5–1 M
, when filled with 3 M KCl. Steady-state current–voltage curves were generated from a step-voltage protocol consisting of 15 pulses lasting 50 ms from –100 to +40 mV, from a holding potential of 0 mV. Measurements of INa, the amiloride-sensitive Na+ current (difference between Na+ currents obtained in the presence of 10 µM amiloride from those obtained in the absence of amiloride at –100 mV) were made 16–48 h postinjection.
Intracellular [Na+] was calculated using the Nernst equation:
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The effects of the methanethiosulphonate (MTS) reagent were studied by TEVC. [2-(Trimethylammonium)ethyl]methanethiosulphonate bromide (MTSET, Toronto Research Chemicals) was dissolved in the high-Na+ extracellular recording solution to 1 mM and bath applied to the oocyte, as previously described (Snyder et al. 2000). The percentage change in amiloride-sensitive Na+ current was calculated as ((IMTS – Ibasal)/Ibasal) x 100, where IMTS is the amiloride-sensitive current after treatment with MTSET and Ibasal is the amiloride-sensitive current before treatment.
Brefeldin A (Sigma) was added to the MBS incubation solution at a final concentration of 5 µM.
Patch clamp.
Prior to patch clamping the oocyte, the vitelline membrane was mechanically removed in a hypertonic solution containing 200 mM sucrose. The bath solution contained (mM): 110 NaCl, 2 CaCl2, 1 MgCl2, 2 KCl and 5 Hepes, pH 7.4. The pipette solution contained (mM): 110 LiCl, 1 MgCl2 and 5 Hepes, at pH 7.4. Patch-clamp pipettes were prepared from Fisherbrand haematocrit capillary glass (Fisher Scientific) using a vertical puller (Kopf Instruments), coated with Sylgard® (Dow Corning) and fire polished with a microforge to yield resistances of 3–10 M. Currents from patches containing one to eight channels were recorded with an EPC-7 patch-clamp amplifier (Heka Elektronik) for a duration of 1–10 min, and digitized with a Digidata 1332A interface (Axon Instruments). Data were filtered at 1 kHz and analysed with pCLAMP8 software (Axon Instruments).
Data analysis
The probability of a channel being open was calculated from the equation:
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Data are presented as means ± S.E.M. Student's two-tailed t test was used to determine whether differences between groups were significant (P < 0.05) using Microcal Origin 6.0.
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Results |
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ß ENaC mRNA and incubated overnight in a low-Na+ MBS were placed in the high-Na+ recording solution (110 mM; Fig. 1A). Inhibition of ENaC current was monitored by measuring currents every 5 min and correcting for amiloride-insensitive currents measured at the end of the time course. Over 80 min, INa fell to 
40% of its initial value. The reversal potential of the Na+ current was noted in each experiment and used to calculate the [Na+]i using the Nernst equation (Fig. 1B and C). Intracellular Na+ was initially 10 mM, and rose in a sigmoidal manner to approximately 50 mM over the time course of the experiment. The effect of high [Na+] on oocytes expressing a mutant channel with a premature stop codon in the ß-subunit, R564X, was also assessed. Despite the fact that intracellular Na+ levels were roughly the same in mutant and wild-type EnaC-expressing oocytes over the time course of the experiment (Fig. 1C), INa in the mutant stayed the same or even rose slightly (Fig. 1A). Other groups have reported similar differences in the behaviour of Liddle's mutant and wild-type ENaC in response to increasing [Na+]i, presumably reflecting a physiological downregulation of channel activity, which is lacking in the mutant (Kellenberger et al. 1998). However, longer exposure to high extracellular [Na+] did diminish INa even in the Liddle's mutant channels (see below).
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ß ENaC cRNA and incubated overnight in low-Na+ solution. The next day, as shown in Fig. 2A, they were pre-incubated in a high-Na+ solution for approximately 50 min. Over this time, INa declined to approximately 40% of its starting value. The oocytes were subsequently flushed with low-Na+ solution, and changes in INa monitored roughly every 30 min by briefly switching to the high-Na+ recording solution. With this protocol, even after a 60 min low-Na+ perfusion, INa did not recover to its starting value. At best, then, the downregulation of ENaC by high [Na+] is slowly reversible even though the change in [Na+]i induced by the high-Na+ pre-incubation could be entirely reversed within 30 min, achieving levels between 10 and 20 mM over 65 min (Fig. 2B). To examine reversibility over longer time periods, oocytes were first exposed to a high-Na+ solution overnight, and INa measured by TEVC. These oocytes were subsequently switched to a low-Na+ medium, and INa was measured again the following day. Following the low-Na+ incubation, macroscopic current increased but was still lower than in those oocytes that had been incubated continuously for two nights in a low-Na+ solution (Fig. 2C).
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ß ENaC cRNA were co-injected into oocytes, which were then incubated overnight in a low-Na+ MBS. The following day, macroscopic Na+ and potassium currents were measured by TEVC as amiloride-sensitive and Ba2+-sensitive currents, respectively. After a further overnight incubation in high-Na+ solution, whole-cell currents were measured and normalized to the initial current (i.e. current after the first incubation). As Fig. 3 illustrates, high [Na+]i specifically downregulated ENaC current, while the magnitude of ROMK2 current remained unchanged.
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, ß and wild-type ENaC subunits. Injected oocytes were subsequently incubated overnight in an MBS solution containing high [Na+], low [Na+], or high [Na+] with 200 nM ouabain to completely block the endogenous Na+,K+-ATPase (Horisberger & Kharoubi-Hess, 2002). Whole-cell INa was recorded the following day before any attempt was made to patch oocytes. As shown in Fig. 4A, the decrease in ENaC activity after overnight incubation in high [Na+] was similar to that seen after 1 h (Fig. 1A). The INa recorded from oocytes in high [Na+] was –6.9 ± 0.7 µA, while current from those in low [Na+] was –15 ± 2 µA, about a twofold difference. Ouabain application reduced macroscopic currents in oocytes even further to –1.6 ± 0.3 µA. Presumably, these cells suffer a particularly severe rise in [Na+]i as a consequence of inhibiting the Na+,K+ pump which would otherwise work to help extrude intracellular Na+. As a control, ENaC-expressing oocytes were also incubated in a low-Na+ solution containing ouabain. The Na+ current in these cells was not significantly different from those that were incubated in low [Na+] alone (–14 ± 4 µA; n
= 6).
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Single-channel i–V curves were obtained by measuring the amplitude of the current deflections (openings) at various voltages. The single-channel conductance (g) was calculated from the slope of the single-channel i–V curves for each recording. This corresponded to 7.5 ± 0.3 pS for ENaC expressed in oocytes in a high-Na+ medium and 7.4 ± 0.1 pS for those incubated in a low-Na+ medium (Fig. 4C); there was no difference in single-channel conductance between the groups. The conductances were similar to values previously reported for ENaC when Li+ is the charge carrier (Sheng et al. 2000). The shift in i–V relationship is presumably the result of a more positive membrane potential secondary to a larger Na+ gradient in the case of the oocytes which were incubated in low-Na+ medium.
We next calculated N and Po for each of the high-Na+- and low-Na+-treated groups as described in the Methods. N was not significantly different in the two groups (high Na+, 3.8 ± 0.7, n = 13; low Na+, 3.1 ± 0.5, n = 11). This parameter does not necessarily reflect the overall channel density in the membrane, since silent patches or patches with too many channels to resolve were excluded from the analysis. As illustrated in Fig. 4D, there was a significant difference in estimated Po between the two groups; the Po of ENaC expressed in oocytes incubated in high-Na+ solution overnight was 0.30 ± 0.03 (n = 13), while the Po of ENaC in oocytes incubated in low-Na+ solution was 0.57 ± 0.05 (n = 11). In other words, the single-channel open probability appears to be halved as a consequence of Na+ loading.
In our analysis, the biggest assumption was that the number of channels in each patch was known. To check that our estimates for the number of channels in each patch (determined by the number of current levels observed) were reasonable, the aggregate dwell time for each open level was used to compute Pn, or the probability that n channels are open (as described in Methods). These measured, or experimental, Pn values were subsequently compared to Pn values predicted by the binomial distribution. Measured and predicted Pn values for selected traces were directly compared as depicted in Fig. 5A and B, respectively, showing that they are in good agreement. This supports our initial estimates of channel number and, by extension, the estimates of mean single-channel open probability in the high- and low-Na+ groups.
A reduction in Po may be achieved by either decreasing channel open time or increasing channel closed time. A kinetic analysis of opening and closing rate constants was performed as described in the Methods; the results are shown in Table 1. While the values obtained for the rate constant for channel closing were similar in oocytes incubated in either high- or low-Na+ solution, the rate constant for opening was significantly different between the two groups (0.16 ± 0.03 s–1 for high-Na+ solution and 0.81 ± 0.20 s–1 for low-Na+ solution). Thus, it appears that a rise in intracellular [Na+] increases the amount of time ENaC spends in the closed state.
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For these experiments, wild-type (ß) or mutant (ßR564X) ENaC cRNA was injected into oocytes; oocytes were incubated overnight in either a high-Na+ MBS, low-Na+ MBS, or high-Na+ MBS containing ouabain (200 nM). The next day, whole-cell currents were assessed by TEVC; these results are shown in Fig. 6A. The ßR564X channels still responded to changes in intracellular Na+, although the fractional decrease in INa was significantly smaller than that in wild-type channels. Since there was not evidence for a Na+-dependent decline in current after a 1 h challenge (Fig. 1), this decline reflects a slower mechanism of feedback inhibition. We also analysed oocytes expressing ßR564X channels by patch clamp. Figure 6B shows representative 1 min cell-attached patch recordings from oocytes incubated in high- and low-Na+ solution. Single-channel i–V curves were fitted by measuring the amplitude of the current deflections (openings) at various voltages. The single-channel conductance (g) calculated from the slope of the i–V curves was 8.1 ± 0.1 pS for low-Na+ solution and 8.5 ± 0.6 pS for oocytes incubated in high-Na+ solution overnight (Fig. 6C). The difference in single-channel conductance in cells expressing mutant channels was not significant under conditions of high and low [Na+]. However, the conductance of the mutant channels under conditions of low [Na+] was significantly higher that of the wild-type channels (P < 0.05). We next calculated NPo and Po for each of the high-Na+- and low-Na+-treated groups as described in the Methods. Single-channel open probability of ßR564X ENaC expressed in oocytes incubated in low-Na+ solution was 0.81 ± 0.04 (n
= 6), and for channels expressed in oocytes incubated in high-Na+ solution it was 0.52 ± 0.05 (n
= 7; Fig. 6D; P < 0.05). Further analysis of these recordings showed that, while rate constants for channel closing were not significantly different between wild-type and mutant ENaC in high-Na+ solution, rate constants for opening were (0.73 ± 0.19 s–1 for mutant versus 0.16 ± 0.03 s–1 for wild type; Table 1). Moreover, there was also a significant difference in the calculated rate constant for opening for the mutant channels expressed in oocytes incubated in low- compared to high-Na+ solution (2.2 ± 0.5 s–1 for low-Na+ solution versus 0.73 ± 0.19 s–1 for high-Na+ solution). Since the rate constant for opening is inversely proportional to mean channel closed time, it therefore appears that the higher open probability of ßR564X ENaC reflects the shorter amount of time that the channel spends in the closed state.
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and subunits, can, in the presence of the sulfhydryl reagent MTSET, form a channel that is persistently open; that is, when modified with the sulfhydryl reagent, the Po of these channels is almost 1 (Snyder et al. 2000). Oocytes were injected with ßS518C cRNA and incubated in either high- or low-Na+ MBS overnight. Whole-cell INa was measured by TEVC the following day, and was found to be similar to those of wild-type channels subjected to the same incubation conditions (Fig. 7A and B). Subsequent external application of MTSET to the oocyte bath solution upregulated whole-cell currents of oocytes incubated in low-Na+ solution by approximately 210% and in high-Na+ solution by 140%. Despite the increased activity of the single channels, whole-cell currents in oocytes incubated in high-Na+ solution remained substantially lower than those incubated in low-Na+ solution (Fig. 7B). This suggests that a reduced open probability of ENaC accounts for some but not all of the reduced macroscopic current observed in oocytes with high intracellular Na+ compared to low intracellular Na+.
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Discussion |
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As mentioned in the Introduction, ENaC activity is regulated by a negative feedback mechanism that enables the cell to curtail inward Na+ movement as intracellular Na+ rises. At the whole-cell level, feedback inhibition is characterized by a reduction of amiloride-sensitive current that is associated with elevated levels of intracellular Na+. Incubation of oocytes in a high-Na+ medium for 1 h or longer results in macroscopic INa that is two- to threefold lower than that of oocytes incubated in low-Na+ solution. It has been suggested that current ‘run-down’, or the short-term decline (within 1–2 h) of macroscopic current with TEVC, may be related to experimental treatment of the oocytes, such as physical perturbation of the cell membrane by the impaling electrodes (Volk et al. 2004). However, our results suggest that the response is specifically associated with an increase in [Na+]i and may be physiologically relevant.
There are at least three ways in which feedback inhibition might work, none of which is exclusive of the others. First, a cell may reduce the number of active channels at the cell surface. Second, channels which exist in the plasma membrane in an active form can be converted to an inactive state. Third, the percentage of time that an active channel spends in the open state (i.e. the open probability) can be decreased. Unlike the effect of high [Na+] on channel number (N), the effect of high [Na+] on ENaC kinetics or Po has not been studied in oocytes, although qualitatively our results are similar to those reported by Ishikawa and colleagues using excised patches in MDCK cells (Ishikawa et al. 1998). Previous attempts to study ENaC by patch clamp in both native and heterologous systems have been complicated by the fact that from patch to patch, channels display different kinetics and open probabilities (Garty & Palmer, 1997; Kellenberger & Schild, 2002). In our experiments, we decided not to restrict our analysis to patches with a single channel but to estimate the average Po of patches containing one to eight channels. This allowed us to study a larger and perhaps more representative sample of patches. This approach has the disadvantage of uncertainties in the estimates of the number of channels in a patch, which will also affect the estimates of Po. However, we do not think that this potential problem affects the main conclusion that Po is lower in oocytes incubated in high-Na+ solution than those kept in low-Na+ solution. When channels have a Po < 0.5, as in the high-Na+ group, it is more likely that the state in which all the channels are closed is visited than the state in which all channels are open. Thus N will be underestimated while Po would be overestimated. In contrast, when Po > 0.5, as in the low-Na+ group, the opposite occurs, and underestimates of N will lead to underestimates of Po.
The possibility that we had either underestimated or overestimated Po was assessed quantitatively following Marunaka & Eaton (1991). For all the recordings taken from wild-type ENaC-expressing oocytes incubated in low-Na+ solution, the probability that we had missed the level with all channels closed was < 0.05; the probability that we missed the level with all channels open was also < 0.05 for all recordings except one, where it was 0.13. Therefore, it is apparent that N was determined accurately for this group. In oocytes expressing wild-type ENaC incubated in high-Na+ solution, the probability that we missed the level with all channels closed was again very unlikely for all recordings (< 0.05). However, the probability that we missed a level with all the channels open in this group varied from 0.01 to 0.99, with a mean of 0.42. Thus, N is almost certainly underestimated, with the consequence that Po is almost certainly overestimated in this group. If we assume that in every other recording in this batch, one opening was missed, the estimated single-channel Po then falls from 0.30 to 0.26. Consequently, the difference in Po between the high- and low-Na+ groups is likely to be greater, if anything, than what we have estimated.
In oocytes expressing mutant channels, N, and hence Po, was estimated well. In low-Na+ solution, the probability that we missed the level with all channels open or all channels closed was < 0.05 for all recordings. In the high-Na+ group, the probability that we missed a level with all the channels closed was again < 0.05 for all recordings; there was only one instance of a probability greater than 0.05 that we missed the level with all channels open, where it was 0.26.
Further analysis of wild-type Na+ channel rate constants determined that while the rate constants of closing did not vary significantly between high- and low-Na+ incubation groups, rate constants of opening did. Specifically, the rate constant for channel opening of ENaC expressed in oocytes incubated in high-Na+ solution was 0.16 ± 0.03 s–1 compared to 0.81 ± 0.20 s–1 for low-Na+ solution. Thus, it appears that the lower open probability of channels in high-Na+ solution reflects an increased mean closed time for ENaC.
While the decline in INa appears to be triggered by increased Na+ entry, the time course of the decline is not related in a simple way to the intracellular Na+ concentration. This was particularly evident in experiments where attempts were made to reverse inhibition, when intracellular [Na+] fell quickly while current recovered very slowly (Fig. 2). This suggests that the high [Na+]i does not act directly on the channels, which is in agreement with a study by Abriel and Horisberger using the ‘cut-open’ oocyte preparation (Abriel & Horisberger, 1999). Here, the intracellular contents of the oocyte were perfused with solutions containing varying concentrations of Na+, Ca2+ and ATP. In fact, none of these agents was able to induce rundown of macroscopic ENaC current. Thus, rather than Na+ interacting directly with the channel, it seems more likely that modulation of channel activity involves the activity of intracellular factors or proteins which can impinge on channel gating, trafficking, or both (Frindt et al. 1993; Silver et al. 1993; Komwatana et al. 1996).
If a change in ENaC gating could completely account for the difference in macroscopic Na+ current observed when oocytes are incubated in either high- or low-Na+ solution, then activating the channels (i.e. increasing Po) by chemical modifiers might produce greater effects on the channels in high-Na+ solution, diminishing the effects of Na+ loading. To test this hypothesis, an MTS reagent was externally applied to oocytes expressing a mutant channel. MTSET stabilizes the open state of the channel such that the Po approaches one. We found that this manoeuvre reduced but did not abolish the effects of Na+ loading, indicating that changes in Po account for only part of the effects of changing [Na+]i. Indeed, our data support the idea that Na+ entry is also limited by a decrease in channel number at the membrane. In particular, in experiments where BFA was applied externally to oocytes maintained in either high- or low-Na+ solution, it was apparent that although the rate of decay of current in the two conditions was similar, in high-Na+ solution the current decayed to essentially zero, while in low-Na+ solution, even after 10 h, a significant portion of the current remained. This is consistent with a decreased rate of internalization of at least a portion of the channels when intracellular [Na+] remains low.
The time course of decline in channel activity after BFA treatment suggests the presence of two populations of channels, at least with low-Na+ solution. One pool of channels is retrieved or inactivated rapidly, while the other is long lived. This could reflect post-translational processing of the channels, such as phosphorylation of one of the subunits. Previous results showed that phosphorylation of C-terminal residues could alter the affinity of ENaC for ubiquitin protein ligase (Nedd4) binding, which would in turn affect rates of internalization (Shi et al. 2002; Dinudom et al. 2004). It is possible that phosphorylation could also alter Po, providing a link between the two mechanisms of feedback control. Alternatively, the two populations could represent channels with different subunit compositions that could also have different internalization rates. The observations of Weisz and colleagues, who reported differences in lifetimes of different subunits on the surface of A6 cells, are consistent with this possibility (Weisz et al. 2000). If these channels also had different open probabilities, selective depletion of one population from the membrane could give rise to the observed changes in mean Po. Previous results support the possibility that subunit composition can affect single-channel properties (Fyfe & Canessa, 1998).
When compared with wild-type channels in oocytes, Liddle's mutants have a greater macroscopic current amplitude and are less sensitive to downregulation of current in high-Na+ solution. Several studies have shown that the increased ENaC surface expression, presumably caused by impaired internalization of the channel, contributes to increased whole-cell current (Snyder et al. 1995; Firsov et al. 1996; Kellenberger et al. 1998; Volk et al. 2004). Recent data suggest that Liddle's mutations may also increase the fraction of channels at the membrane that are proteolytically cleaved (Knight et al. 2006). Cleavage of ENaC subunits is thought to alter channel gating and increase single-channel Po (Caldwell et al. 2004). This hypothesis is consistent with our experimental data, which show that the increased activity of one Liddle's mutant, ßR564X, results, in part, from a higher single-channel Po. When rate constants for the high- and low-Na+ conditions were analysed, it was apparent that, again, although differences in rates of channel closing were not statistically significant between wild-type and mutant ENaC groups, rates of channel opening were (see Table 1). Taken together, these observations suggest that the increased open probability of channels expressed in cells incubated in low-Na+ solution results from a decrease in the amount of time they spend in the closed state.
Since the Liddle's truncation mutants lack the C-terminal consensus motif for Nedd4-targeted ubiquitination, this suggested to us that the Na+-dependent downregulation of macroscopic current observed in oocytes expressing these channels may result entirely from a reduction in open probability. We thus reasoned that increasing the Po of ßR564X ENaC to 1 might eliminate differences in macroscopic current observed between groups of oocytes incubated in high- and low-Na+ solution. A cysteine at serine 518 was introduced in the ENaC ß-subunit already harbouring the R564X truncation mutation, and MTSET was bath applied to oocytes expressing ßS518C–R564X channels. Indeed, we found this manoeuvre to increase INa such that the difference in whole-cell current between incubation groups was abolished, indicating that a reduction in Po can account for most, if not all, of the downregulation of Na+ current through these mutant channels observed when cells are Na+ loaded.
In summary, in this study we show directly for the first time in intact cells that changes in single-channel kinetics of ENaC can contribute to channel regulation in the case of elevated intracellular [Na+]. Specifically, under these conditions, channels demonstrate a reduced open probability induced by increased channel closed times. Increases in Po can also contribute to channel dysregulation in the case of Liddle's syndrome, although these channels are still susceptible to a Na+-dependent fall in Po. Our results support a physiological scheme where reduced channel Po acts in concert with other processes, such as increased endocytic channel retrieval, to limit Na+ entry when [Na+]i is high.
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References |
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Abriel H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD, Rotin D & Staub O (1999). Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome. J Clin Invest 103, 667–673.[Medline]
Awayda MS (1999). Regulation of the epithelial Na+ channel by intracellular Na+. Am J Physiol Cell Physiol 277, 216–224.
Caldwell RA, Boucher RC & Stutts MJ (2004). Serine protease activation of near-silent epithelial Na+ channels. Am J Physiol Cell Physiol 288, C190–C194.
Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD & Rossier BC (1994). Amiloride-sensitive epithelial Na+ channel is made of 3 homologous subunits. Nature 367, 463–467.[CrossRef][Medline]
Dinudom
A, Fotia
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P < 0.05 compared to high-Na+, then low- Na+ condition.
+
B). In high-Na+ solution, A
= 3.0 µA, 