Mineralocorticoid regulation of epithelial Na+ channels is maintained in a mouse model of Liddle's syndrome

Anke Dahlmann,1 Sylvain Pradervand,2 Edith Hummler,2 Bernard C. Rossier,2 Gustavo Frindt,1 and Lawrence G. Palmer1

1Department Physiology of Biophysics, Weill Medical College of Cornell University, New York, New York 10021; and and 2Institut de Pharmacologie et de Toxicologie, Université de Lausanne, 1005 Lausanne, Switzerland

Submitted 13 January 2003 ; accepted in final form 23 March 2003


    ABSTRACT
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 ABSTRACT
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Currents through epithelial Na channels (ENaCs) were measured in the cortical collecting tubule (CCT) of mice expressing truncated {beta}-subunits of ENaC, reproducing one of the mutations found in human patients with Liddle's syndrome. Tubules were isolated from mice homozygous for the Liddle mutation (L/L) and from wild-type (WT) littermates. Amiloride-sensitive currents (INa) from single cells were recorded under whole cell clamp conditions. CCTs from mice kept under control conditions and fed a diet with normal levels of Na had very small INas (WT: 18 ± 13 pA; L/L: 22 ± 8 pA at Vm = –100 mV) that were not different in WT and L/L animals. However, the L/L mice had much larger currents when the animals were fed a low-Na diet (WT: 256 ± 127 pA; L/L: 1,820 ± 330 pA) or infused with aldosterone (WT: 285 ± 63 pA; L/L: 1,600 ± 280 pA). Currents from L/L mice were also larger when animals were pretreated with a high-K diet but not when the CCTs were stimulated in vitro with 8-CTP-cAMP. Noise analysis of amiloride-induced fluctuations in INa showed that single-channel currents at Vm = 0 mV were slightly smaller in L/L mice (WT: 0.33 pA; L/L: 0.24 pA). This difference could be attributed to a decrease in driving force since current-voltage analysis indicated that intracellular Na was increased in the L/L animals. Analysis of spontaneous channel noise indicated that the open probability was similar in the two genotypes(WT: 0.77; L/L: 0.80). Thus the increase in whole cell current is attributed to a difference in the density of conducting channels.

kidney; sodium transport; aldosterone; hypertension; amiloride


EPITHELIAL NA CHANNELS (ENaCs) are important in the regulation of renal salt excretion and hence to the regulation of blood pressure (24). Liddle's syndrome, a Mendelian form of hypertension, involves truncations or mutations of the COOH termini of {beta}- or {gamma}-subunits of ENaC (13, 32). These alterations increase the current through ENaCs expressed in Xenopus laevis oo-cytes, a result due at least in part to an increase in surface expression of the subunits (6). Because the mutations usually affect a COOH-terminal PPPxY motif thought to act as an internalization signal (13), it is possible that the increased expression is the result of a decreased rate of removal of channels from the plasma membrane. Recently, a mouse model of Liddle's syndrome was developed (24). When fed a high-Na diet, these animals have a phenotype that reproduces most of the features of the human syndrome.

In the cortical collecting tubule (CCT), Na channels are tightly regulated by aldosterone. Tubules isolated from normal, salt-replete rats have almost no channel activity as measured by transepithelial Na fluxes (25, 31), single-channel (20), or whole cell currents (7, 9). Channel activity is strongly elevated by salt depletion or treatment with aldosterone. Thus low levels of mineralocorticoids can completely suppress the channels, at least in rodents. However, in humans with Liddle's syndrome, aldosterone levels in plasma are below normal. This suggests that in this disease mineralocorticoid control of the channels is lost and channel activity is at least partly aldosterone independent. The aim of the present study was to examine whether the channels are as strongly controlled in the mouse CCT and whether control was abolished or reduced in the animals with the Liddle's syndrome mutation.


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Animals. Initial experiments were carried out using a commercial C57BL/6 mouse strain (Charles River Laboratories, Kingston, NY). Mice with the {beta}-ENaC subunit truncated at position R564 were generated as described previously (24). For the present set of experiments, these animals were backcrossed onto the C57BL/6J mouse background (N10). Mice homozygous mutant for the Liddle mutation (L/L) and their littermate controls were obtained by crossbreeding heterozygous mutant (+/L) mice. Genotyping was carried out by PCR as described (24).

Animals were fed either a standard lab chow (Na content 2.8 g/kg; K content 11 g/kg; Purina), a low-Na diet (Na content 3.8 mg/kg; K content 8.6 g/kg; ICN, Cleveland, OH), or a high-K diet (Na content 5 g/kg; KCl content 100 g/kg; Harlan Teklad, Madison, WI). The low-Na diet was given for either 2 days or for 6–8 days. The high-K diet was given for 6–8 days. Some animals were infused with aldosterone via subcutaneous osmotic minipumps (Alzet model 1002, Alza, Palo Alto, CA) for 6–7 days. Aldosterone was dissolved in polyethylene glycol-300 at a concentration of 2 mg/ml. The infusion rate was 10 µg/day.

Tissue preparation. After the animals were killed, the kidneys were removed, and CCTs were dissected free and opened manually to expose the luminal surface. Under these conditions, the tissues retain their epithelial structure and the cells are presumed to remain polarized. The split tubules were attached to a small plastic rectangle coated with Cell-Tak (Collaborative Research, Bedford, MA) and placed in a perfusion chamber mounted on an inverted microscope. The chamber was continuously perfused with solution pre-warmed to 37°C consisting of (in mM) 135 Na methane sulfonate, 5 KCl, 2 CaCl2, 1 MgCl2, 2 glucose, 5 BaCl2, and 10 HEPES adjusted to pH 7.4 with NaOH.

Electrophysiology. The patch-clamp pipettes were filled with solutions containing (in mM) 7 KCl, 123 aspartic acid, 20 CsOH, 20 TEAOH, 5 EGTA, 10 HEPES, 3 MgATP, and 0.3 NaGDP{beta}S with the pH adjusted to 7.4 with KOH. Basic protocols for measuring whole cell amiloride-sensitive current were previously described (8, 9).

For amiloride-induced noise analysis, the cell membrane potential was clamped to 0 mV and currents were recorded at a gain of 10–100 pA/mV onto videotape using a digital data recorder (VR-10, Instrutech). The cells were superfused with bath media containing no amiloride, one or more submaximal concentrations of amiloride (0.25–1.5 µM), and 10 µM amiloride, a dose considered to be maximum. Data were then filtered at 500 Hz, digitized at 1 kHz, and intervals of 10–30 s were transformed using the fast Fourier transform application within the PClamp 8 software package (Axon Instruments). The resulting spectra were fit with Lorentzians over a frequency range of 0.5 to 200 Hz

The single-channel current (i) was then computed from the fit parameters according to the relationship (15)

(1)
where So is the plateau of the Lorentzians, {tau} = 2{pi}fc where fc is the corner frequency, INa is the macroscopic current through the channels, and PA is the probability that the channel is blocked by amiloride and is given by

where INa (A) and INa(0) are the leak-corrected currents in the presence and absence of the submaximal dose of amiloride.

To estimate the open probability (Po) of the channels, the digitized records in the absence of amiloride and in the presence of a maximal dose were converted to all-points histograms and fitted with Gaussian functions, also using PClamp 8 software. The variance of the currents through the channels ({sigma}Na) was calculated from

where {sigma}T2 and {sigma}A2 are the variances of the currents in the absence and presence of 10 µM amiloride, estimated from the width of the fitted Gaussian functions. This relationship assumes that fluctuations in currents through the channel ({sigma}Na2) and through leak pathways ({sigma}A2) are independent. Po was then estimated from the relationship (10, 14)

(2)


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Previous patch-clamp measurements of Na channel activity in the collecting tubule were done in rat kidneys (9, 22). We therefore carried out pilot experiments using the commercial mouse strain (C57BL/6) used to produce the mutant mice for the later studies to see whether similar results were obtained. Figure 1 shows whole cell currents from a cell of a CCT isolated from a mouse that had been treated with aldosterone by an osmotic minipump for 7 days. Currents were obtained in the absence and presence of 10 µM amiloride. Because the pipette contained no Na, there is a large Na gradient across the membrane. Under the conditions used, amiloride reduced the inward current in the absence of a transmembrane voltage and blocked a large fraction of the total inward conductance. The amiloride-sensitive currents approached zero at large positive potentials, consistent with blockade of Na-selective channels with low intracellular Na. At a cell potential of –100 mV relative to the bath, INa was 246 ± 58 pA (n = 16 cells). In CCTs from animals not treated with aldosterone, only 1 of 13 cells had a clear amiloride-sensitive current and the mean INa was 13 ± 13 pA. These data are similar to those obtained in rat CCT (7, 9, 20). Na channel function in these cells, at least as evaluated in vitro, was dependent on an elevation of plasma mineralocorticoid activity.



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Fig. 1. Current-voltage (I-V) relationships in mouse cortical collecting tubule (CCT). Data were obtained from a wild-type C57BL/6 mouse infused with aldosterone for 7 days. A: currents were measured under whole cell voltage-clamp conditions. The holding potential was 0, and the test potentials were stepped from –120 to +60 mV in 10-mV increments. a: Traces obtained in the absence of amiloride; b: traces set in the presence of 10 µM amiloride. B: I-V curves were generated by measuring currents at the end of each voltage pulse in the absence ({blacksquare}) and presence ({circ}) of amiloride. The {square}represent the difference currents (INa).

 

We next examined the L/L mice and their wild-type (WT) littermates backcrossed into this same mouse strain. Because human patients with Liddle's syndrome have high blood pressure, presumably reflecting increased Na reabsorption, together with low levels of aldosterone, we hypothesized that the CCTs from the untreated L/L animals would escape from tight mineralocorticoid control and would therefore have high channel activity under control conditions. Contrary to this expectation, INa was very low in both the L/L and WT mice under these control conditions (Fig. 2), and there was no statistically significant difference between the two groups (Fig. 3).



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Fig. 2. I-V relationships for wild-type (WT; normal; left) and Liddle (L/L; right) mouse CCT. Animals were fed either standard chow (bottom) or a low-Na diet (top). In each case, I-V curves were obtained in the absence ({square}) and presence ({circ}) of amiloride and INa-V curves ({blacksquare}) calculated from the difference in current at each voltage.

 


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Fig. 3. Mean values of INa measured at Vm = –100 mV under different in vivo and in vitro conditions. Open bars represent WT mice. Filled bars represent Liddle mice. Control = standard chow, no infusion. + Aldo = standard chow, aldosterone infusion by osmotic minipump for 6–8 days. Low Na = low-Na diet for 2 or 7 days (d). High K = high-K diet for 6–8 days. cAMP = CCTs from mice fed standard chow, without aldosterone infusion treated in vitro with 105 M 8-cpt-cAMP for 5 to 30 min. Data are shown as means ± SE for 11 to 23 different cells. Statistical tests for Liddle vs. WT littermates: *P < 0.05, **P < 0.01, ***P < 0.001.

 

We next tested whether a difference could be detected after activation of channels with aldosterone. Figure 2 shows representative current-voltage (I-V) curves from aldosterone-infused mice. The WT animals had Na currents that were similar to those from the standard mouse strain described above. In contrast, the currents from tubules taken from L/L mice were considerably larger. In the cell shown, currents could not even be measured at voltages more negative than –60 mV due to saturation of the amplifier. In addition, the reversal potential of the amiloride-sensitive current was shifted toward zero. The pipette solution contained no Na, so the most likely interpretation is that Na entered the cells through the channels so rapidly that the submembrane space contained a significant Na concentration leading to a finite reversal potential. Mean values for INa under these conditions are given in Fig. 3. Values for the L/L cells are more than five times larger than those from the WT cells, and the differences are highly significant. Thus, although the basal currents in the two groups were not different, the L/L animals responded much more vigorously to elevation of plasma aldosterone than did controls.

Reduction in dietary Na intake is a more physiological way of elevating mineralocorticoid status. WT mice that were fed a low-Na diet for 6–8 days also had easily measurable INas that were comparable to those obtained with aldosterone infusion (Fig. 3). Again, the L/L animals had much larger currents than did the WT animals, the average values being increased by a factor of 7. We also investigated animals after 2 days of Na depletion. This is sufficient to activate INa but not to the presumably maximal levels seen after 1 wk of the low-Na diet. The response of the L/L animals was again significantly larger than that of the WT (Fig. 3).

To see whether channels regulated through other physiological pathways were also hyperactivated in the L/L mice, we fed mice a high-K diet for 6–8 days. Although plasma aldosterone can be elevated under these conditions, we observed enhanced Na channel activity even in adrenalectomized rats, suggesting a process that is at least in part aldosterone independent (21). The high-K diet increased INa in both WT and L/L mice, although to a lesser extent than did the low-Na diet or aldosterone infusion. The mean currents in the L/L animals were significantly larger, although the ratio of L/L to WT was only 2.3, less than that for the other maneuvers (Fig. 3).

Na channels in the CCT can be activated in vitro by raising cytoplasmic cAMP (8). We compared the response to an application of 8-CTP-cAMP in CCTs from WT and L/L mice. As shown in Fig. 3, 8-CTP-cAMP increased INa in both sets of animals, and there was no difference in the mean levels of the two groups. We conclude that the effects of the Liddle mutation on Na channels depend on the pathway (i.e., mineralocorticoids vs. cAMP) through which the channels are activated.

To examine the single-channel properties of the WT and L/L channels, we first examined cell-attached patches. The results were disappointing. Seals good enough for single-channel analysis were difficult to make regularly, and in those patches where the seal was satisfactory, the channels were largely absent. In many cases, channels were seen briefly after seal formation but inactivated quickly. Thus the mouse CCT is not as advantageous as that of the rat for investigating single-channel events.

Because good whole cell recordings could be made relatively routinely, we chose to further characterize the channels using noise analysis. Two types of experiments were done. First, we used analysis of amiloride-induced fluctuations (14, 15) to estimate the single-channel current. We then analyzed noise from spontaneous fluctuations in channel currents to estimate the Po of the channels.

Spectra from a representative whole cell recording in the absence of amiloride and in the presence of submaximal (1 µM) and maximal (10 µM) amiloride concentrations are illustrated in Fig. 4. In the absence of amiloride, significant power densities were seen over a large range of frequencies. The shape of the power density spectrum is complex, however, and was not analyzed further. With 1 µM amiloride, a clear Lorentzian component of noise emerged with a plateau in the frequency range of 0.1 to 1 Hz. The entire spectrum from 0.1 to 100 Hz could be well described by a single Lorentzian component with a plateau of 3.06 pA2s and a corner frequency of 3.8 Hz. Finally, in the presence of 10 µM amiloride, the noise in the range of 0.1 to 10 Hz was greatly reduced. The spectrum between 1 and 300 Hz could also be reasonably well described by a Lorentzian with a much reduced plateau of 0.06 pA2s and an increased corner frequency of 42 Hz.



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Fig. 4. Power density spectra of currents in mouse CCT. The examples shown are from a L/L mouse infused with aldosterone. The cell membrane was clamped to 0 mV and currents were recorded for 10–20 s with 0 (A), 1 (B), and 10 µM amiloride (C) in the bath. Power density spectra were obtained by fast Fourier transform. The line through the spectrum with 1 µM amiloride is the best fit to a Lorentzian function with So = 3.06 pA2s and fc = 3.8 Hz.

 

The corner frequency of the amiloride-induced noise is expected to increase with amiloride concentration. This is shown in Fig. 5, which shows data from both WT and L/L animals. The results from the two genotypes are identical, implying that the kinetics of amiloride block are unchanged by the mutation. The slope of the plot gives the on-rate for block, which was 56 µM/s. The intercept with the frequency axis gives the off-rate, but this was too close to zero to be accurately determined.



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Fig. 5. Kinetics of amiloride block. A: dose-response curves for amiloride-sensitive currents. INa measured in the presence of submaximal amiloride concentrations is plotted as a fraction of the current in the absence of amiloride for Liddle ({bullet}) and normal ({circ}) mice. All animals were infused with aldosterone. The line represents the best fit of the data with Liddle's mice to a simple blocking scheme with Ki = 0.25 µM. B: corner frequency as a function of amiloride concentration. Data were obtained by fitting power density spectra with Lorentzian functions for Liddle ({bullet}) or normal ({circ}) mice. The line is a best fit to the equation with kon = 56 µM/s and koff = 5.6/s.

 

These data can be used to estimate single-channel currents from Eq. 1. For each cell studied, we calculated i (at a membrane potential of 0) for one to three submaximal amiloride concentrations. Although there was some variation from cell to cell, values obtained using different concentrations on the same cell were usually in good agreement. The mean values for i were 0.33 pA for WT and 0.24 pA for L/L (Table 1). The reduced values for the mutant channels can be at least partially attributed to higher intracellular Na concentrations as shown in Fig. 2 (see DISCUSSION). In any case, the higher whole cell currents in the L/L cells are not due to an increased single-channel current.


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Table 1. Effect of Liddle mutation on single-channel parameters estimated from fluctuation analysis

 

Channel Po can be estimated assuming that the noise in the current traces in the absence of amiloride arises from spontaneous transitions of channels between open and closed states. As shown in Fig. 6, distributions of currents about the mean could be described by Gaussian functions both in the absence of amiloride and in the presence of a maximal amiloride dose. This permits the variance of the currents to be estimated from the width of the Gaussian fit. Assuming that the noise measured in the presence of amiloride represents current fluctuations unrelated to Na channels, the variance of INa can be calculated from the relationship {sigma}Na2 = {sigma}T2{sigma}A2 as described in the METHODS section.



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Fig. 6. Spontaneous current fluctuations. Current histograms were obtained from the same cell used for Fig. 4 in the absence (A) and presence (B)of10 µM amiloride. Histograms were fitted with Gaussian functions Without amiloride Î = 470 pA, {sigma} = 3.73 pA. With amiloride, Î = 122 pA, {sigma} = 2.45 pA.

 

Equation 2 shows the relationship between the variance of the current around the mean and the single-channel parameters. The mean values for Po calculated in this way were quite high: 0.77 for WT and 0.80 for L/L (Table 1). Thus the higher currents in the L/L cells appear to be the result of an increase in the number of conducting channels (N) rather than to a change in Po or i. Limitations to this interpretation are discussed below.


    DISCUSSION
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Na channels in mouse CCT. The results shown in Figs. 1 and 2 indicate that in the mouse CCT control of Na channels by aldosterone is very tight. In most cases, no clear amiloride-sensitive current could be observed in control animals fed a standard rodent chow. Typically, the sensitivity of the assay was such that a current of 10–20 pA at –100 mV would have been detectable. In contrast, most cells had measurable Na currents after elevation of plasma aldosterone either directly through infusion or indirectly by decreasing dietary Na intake. In this sense, the mouse is similar to the rat (7, 20, 25, 31) but is unlike the rabbit. In the latter case, there appears to be a constitutive channel activity that gives rise to an amiloride-sensitive Na conductance and active Na transport even when the mineralocorticoid status is low (19, 27).

Augmented channel activity in CCTs from Liddle mice. Under conditions in which Na channels are activated in the CCT in vivo, particularly by Na depletion or aldosterone infusion, the amiloride-sensitive currents were five to seven times higher in the L/L animals compared with their WT controls. This degree of stimulation is similar to that observed in the oocyte system, in which injection of comparable amounts of cRNA for the WT and truncated {beta}-subunit along with normal {alpha}- and {gamma}-ENaC generate amiloride-sensitive currents that are generally two to five times larger in the case of the mutant channels (1, 6, 26, 29).

The channel activity in the L/L cells is quantitatively rather than qualitatively altered. The single-channel currents at a membrane potential of zero were slightly smaller for the L/L channels, but this can be explained at least in part by a smaller Na gradient across the membrane. It appears (Fig. 2) that when the inward Na currents are very high, as in the case of the L/L cells, that the intracellular Na concentration at the inside of the cell membrane can be fairly high even though the pipette solution in communication with the cytoplasm is Na free. The typical reversal potential of the amiloride-sensitive I-V relationship was about –35 mV. Assuming that the channels remain highly selective for Na, this corresponds to a submembrane Na concentration of ~38 mM. Given the very large fluxes across the apical membrane under these conditions, this value seems plausible, although we cannot completely rule out the possibility that the change in reversal potential could reflect a reduction in Na/K or Na/Cs selectivity of the channel. Thus the gradient for Na across the membrane could be reduced from 140 to ~100 mM, which could account for the lower single-channel currents. This finding is in agreement with single-channel recordings of channels with Liddle mutations in oocytes (6, 26, 29).

The open probability, again estimated from noise analysis, was also similar for WT and L/L channels. This analysis assumes a single population of channels with the same Po that may not be the case (23). Because channels with very low Po would contribute little to either the noise or the current, conversion of such channels to a high Po form would be indistinguishable from a simple increase in channel number at a constant Po. However, the results rule out a uniform increase in Po as a major mechanism for the effects of the mutation.

In the oocyte system, Firsov et al. (6) found that the increased INa could be only partially accounted for by an increase in the amount of protein expressed at the cell surface. Po, measured as the ratio of the macroscopic current to the product of the channel protein and the single-channel currents, was quite low (< 0.01) and was increased in the Liddle channels. Our data would be consistent with activation of channels already resident in the cell membrane provided that the channels initially had a Po close to zero.

The inhibition constant for the interaction of the channels with amiloride estimated from dose-response curves was ~0.25 µM at Vm = 0 (Fig. 5A). The on-rate for amiloride block, calculated from the slope of the plot of corner frequency vs. concentration, was 56 µM/s. In principle, the off-rate can be determined as the intercept of this plot with the frequency axis. The best fit gives an estimate of 5.6/s, but this is not very reliable because small errors in the estimate of the slope would have large effects on the intercept that is close to the origin. A better estimate of the off-rate can be obtained by dividing the on-rate by the inhibition constant, giving a value of 14/s. There was no difference in the kinetics of amiloride block of WT and L/L channels.

Unfortunately, we could not routinely record single-channel events from the apical membrane of the mouse CCT. Several problems appeared to contribute to this failure. First, many seals, although sufficiently tight to achieve good whole cell clamps, had resistances that were too low to resolve single-channel events. Second, patches for which the seal was adequate were often unstable when a voltage was applied to the pipette. Third, in a number of patches in which activity was visible immediately after seal formation, the channels inactivated spontaneously before a recording could be made. Finally, patches with high resistance and stable seals tended to have no channel activity at all. Although we could occasionally obtain data similar to those seen in the rat CCT, we could not be sure how representative such recordings were and therefore have not reported them here.

Requirement for activation. The most surprising finding to us was the requirement for conditions in which channels are normally activated, e.g., high plasma aldosterone, to observe the increased currents associated with the Liddle mutation. Indeed, our hypothesis when we began the work was that one consequence of the mutation would be a loss of mineralocorticoid control of the channels. We therefore expected that currents under control conditions would be larger in the L/L animals than in WT. Furthermore, if the main effect of the mutation was to bypass this control mechanism, it is possible that the currents under conditions of maximal stimulation might not be very different. In this sense, the channel activity would reflect the state of pseudohyperaldosteronism, which characterizes patients with Liddle's syndrome (13, 32). The observations were opposite to these expectations. Under control conditions, channel activity remained suppressed, and they increased to a much greater extent in response to stimulation. They also differ from results obtained from primary cultures derived from CCTs isolated from WT and L/L mice, in which a significant amiloride-sensitive short-circuit current could be measured in the absence of aldosterone, and this current was larger in L/L than in WT mice (Pradervand S, Vandewalle A, Bens M, Gautschi I, Loffing J, Hummler E, Schild L, and Rossier BC, unpublished observations). Thus the ability of the cell to suppress channel activity when aldosterone levels are low appears to be stronger in vivo than in vitro.

Not every mode of stimulation resulted in larger currents in the L/L mice. The largest differences from WT were observed with high plasma aldosterone, achieved either with a low-Na diet or with direct infusion of the hormone. Significantly higher currents were also observed when the animals were given a high-K diet. Although increased K intake can also stimulate aldosterone secretion (3), there is also evidence that at least some of the effects of high K are independent of aldosterone (18, 30, 33). In particular, the stimulation of Na channels in the CCTs of rats on a high-K diet was unaffected by adrenalectomy (21). Thus the Na currents measured under these conditions may be only partly induced by aldosterone. This could explain the smaller degree of hyperactivation in the L/L animals, but experiments with adrenalectomized WT and L/L mice would be necessary to test this point rigorously.

In contrast, stimulation of INa in vitro using cAMP evoked similar currents in WT and L/L genotypes. Increased intracellular cAMP is believed to underlie the activation of channels by ADH and perhaps by other hormones using this second messenger system (12). With the use of an epithelial exogenous expression system, Snyder (28) found that cAMP-activated currents were actually lower in Liddle mutant channels than with WT channels. Although we did not find an inhibition in the cAMP response in the native kidney cells, we did confirm a striking difference in the effects of the Liddle mutation on cAMP-stimulated channels compared with those activated through other pathways.

The increase in activity of Liddle channels in oocytes was dependent on the rate of Na influx across the plasma membrane, suggesting that the mutant channels had escaped from downregulation due to high intracellular Na concentration (11). Such a mechanism may also be involved in the experiments reported here in the CCT. Such an effect could not involve an acute response to high cell Na because under the conditions of measurement, the concentrations are very low, at least in the control cells. A more chronic effect is more likely. However, similar results were obtained in animals on a low-Na diet, where Na delivery to the CCT (and Na influx into the cells) is low, and in animals infused with aldosterone in which Na delivery is presumably high. Thus the effects of the mutation do not appear to depend entirely on an elevation of intracellular Na in vivo. Nevertheless, a different response to increased cell Na could contribute to the findings.

Implications for hypertension. Liddle's syndrome is a form of pseudohyperaldosteronism. Patients have symptoms of primary aldosteronism, including hypertension, hypokalemia, and metabolic alkalosis, but actual plasma levels of the hormone are low (32). If low-aldosterone levels can suppress the activity of Na channels even with a Liddle mutation, as appears to be the case at least in the mouse CCT, how can high blood pressure develop?

One possibility is that channels are suppressed more effectively in the rodent than in the human. As mentioned above, the rabbit CCT has a significant constitutive (aldosterone independent) rate of Na transport. If this were also the case in the human kidney, the constitutive channels might also be hyperactive in Liddle's syndrome. According to this explanation, hypertension should be less pronounced in the L/L mouse than in the human disease. This appears to be the case as blood pressure in these animals was only mildly elevated and only when dietary Na was high (24).

A second possibility is that Na channels in other nephron segments are less completely suppressed by low aldosterone in both mice and men. One candidate for such a segment is the connecting tubule (CNT), where at least in the rabbit Na transport rates were higher than in the CCT and less sensitive to mineralocorticoid status (2). In this scenario, hyperreabsorption of Na would take place in the CNT rather than the CCT. No data are available for mouse or rat CNT in vitro. However, a substantial amiloride-sensitive transport rate was observed in the late distal convoluted tubule of control rats by micropuncture techniques (4).

Third, it is possible that channels in any of the distal segments could be active in vivo due to the presence of hormones that are eliminated when the tubules are studied in vitro. The effects of such hormones could be exaggerated in Liddle's syndrome, leading to overreabsorption of Na. An obvious candidate is ADH, whose action is rapidly reversed and therefore not "remembered" by the in vitro tissue. However, activation by cAMP, the second messenger for ADH, was not elevated in the L/L mouse CCT. We cannot, however, rule out a role for other hormone-second messenger systems.

Implications for aldosterone action. A clear result of these studies is that the channels with the Liddle mutation can be regulated by aldosterone. This finding indicates that an intact COOH terminus of the {beta}-ENaC subunit, and its PPPxY motif presumed to be an internalization signal, is not required for the long-term effects of mineralocorticoids. Recently, Staub and colleagues (1) proposed a mechanism of action of aldosterone involving a phosphorylation of the COOH-terminal binding protein Nedd-4–2 by serum and glucocorticoid activated kinase (SGK) with a subsequent impairment of channel ubiquitination and retrieval from the apical membrane. This model predicts that mineralocorticoid control of the channels should be diminished in the absence of the internalization signal, which we did not observe. Two caveats should be mentioned. First, Abriel et al. (1) used channels in which the PPPxY motif was eliminated from both the {beta}- and {gamma}-subunits, whereas in the Liddle mice only the {beta}-subunit was altered. Second, the SGK-mediated pathway may be important in the rapid (1–3 h) effects of the hormone, whereas our experiments involved chronic (>2 days) exposure to mineralocorticoids and/or salt depletion.

Because the long-term effects of aldosterone appear to involve a redistribution of channel protein from cytoplasmic to plasma membrane sites (16, 17), one possibility is that aldosterone could stimulate the rate of insertion of channels into the apical membrane. This would increase the steady-state density of channels available for transport, and the effect would be exaggerated if the rate of channel retrieval were at the same time diminished, as may occur in Liddle's syndrome.


    DISCLOSURES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59659, a grant-in-aid from the American Heart Association Heritage Affiliate, and a Fellowship Grant Da536/1–1 from the Deutsche Forschungsgemeinschaft (to A. Dahlmann).


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. G. Palmer, Dept. of Physiology and Biophysics, Weill Medical College of Cornell Univ., 1300 York Ave., New York, NY 10021 (E-mail: lgpalm{at}med.cornell.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 METHODS
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 DISCUSSION
 REFERENCES
 

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