Regulation of the epithelial Na+ channel by intracellular Na+

Mouhamed S. Awayda

Department of Medicine and Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hypothesis that the intracellular Na+ concentration ([Na+]i) is a regulator of the epithelial Na+ channel (ENaC) was tested with the Xenopus oocyte expression system by utilizing a dual-electrode voltage clamp. [Na+]i averaged 48.1 ± 2.2 meq (n = 27) and was estimated from the amiloride-sensitive reversal potential. [Na+]i was increased by direct injection of 27.6 nl of 0.25 or 0.5 M Na2SO4. Within minutes of injection, [Na+]i stabilized and remained elevated at 97.8 ± 6.5 meq (n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after the initial injection of 0.5 and 0.25 M Na2SO4, respectively. This increase of [Na+]i caused a biphasic inhibition of ENaC currents. In oocytes injected with 0.5 M Na2SO4 (n = 9), a rapid decrease of inward amiloride-sensitive slope conductance (gNa) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slower decrease to 0.304 ± 0.043 of control at 30 min were observed. Similar but smaller inhibitions were also observed with the injection of 0.25 M Na2SO4. Injection of isotonic K2SO4 (70 mM) or isotonic K2SO4 made hypertonic with sucrose (70 mM K2SO4-1.2 M sucrose) was without effect. Injection of a 0.5 M concentration of either K2SO4, N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thus increases of [Na+]i have multiple specific inhibitory effects on ENaC that can be temporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.

epithelial sodium channel; Xenopus oocytes; inhibition; autoregulation; intracellular sodium concentration


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT HAS LONG BEEN RECOGNIZED that macroscopic rates of Na+ transport across epithelia display saturation kinetics with increasing extracellular Na+ concentration ([Na+]o) and that inhibition of Na+ entry by various means causes a stimulation of channel activity (12, 22). Because the single-channel current saturates at much higher [Na+] than the macroscopic current, it is inferred that intrinsic regulatory processes that cause inhibition of the open probability and/or channel density must exist (29, 33). Lindemann (19) classified these types of intrinsic regulation of the epithelial Na+ channel into self-inhibition and feedback inhibition. Self-inhibition is thought to reflect direct interaction between the channel and external Na+. On the other hand, feedback inhibition or autoregulation may be mediated via the indirect actions of Na+ and second messengers on the Na+ channel (32).

Fuchs et al. (9) provided the initial evidence for intrinsic regulation of the Na+ channel by luminal [Na+] in the short-circuited, K+-depolarized epithelium of frog skin. They found that the apical membrane Na+ permeability (PNa) was inhibited within seconds after increasing apical [Na+]. These effects were observed in the absence of detectable changes of membrane voltage (Vm) and presumably intracellular Na+ concentration ([Na+]i) and were attributed to self-inhibition of the apical Na+ channel by [Na+]o. Kroll et al. (17) arrived at a similar conclusion for the epithelial Na+ channel expressed in Xenopus oocytes, where PNa was found to inversely vary with [Na+]o, with no apparent correlation with [Na+]i.

Palmer et al. (24) arrived at a similar conclusion by utilizing a whole cell patch clamp of rat cortical collecting tubules (CCT). These investigators found that decreasing [Na+]i by decreasing pipette [Na+] by substitution with K+ did not affect the whole cell currents. However, changes of [Na+]o were found to be accompanied by changes of channel activity, indicating that extracellular Na+ is responsible for inhibiting the Na+ channel.

Another intrinsic regulatory process of feedback inhibition or autoregulation is observed after inhibition of apical membrane Na+ entry (1, 6, 7, 11, 20, 27, 28). This process exhibits a longer time course than self-inhibition and is thought to be mediated via second messengers that may involve protein kinase C (PKC) (10, 20) and potential interactions with the actin cytoskeleton (6). Data from Komwatana et al. (16) also indicate that [Na+]i inhibited channel activity via an indirect mechanism that involves G proteins. Unfortunately a time course of the effect of [Na+]i on channel activity could not be obtained because these studies were carried out by using the whole cell patch clamp mode on cells dialyzed with the pipette contents. In a follow-up study these authors (4) concluded that the regulation of the Na+ channel in salivary glands by [Na+]i also involves the ubiquitin ligase protein Nedd4.

Data indicating that the cloned epithelial Na+ channel (ENaC) is also regulated by Na+ have been recently accumulating. Ishikawa et al. (14) have reported that ENaC transfected into Madin-Darby canine kidney cells is inhibited by increasing [Na+]i in excised inside-out membrane patches, indicating a likely direct effect of [Na+]i on this channel. Kellenberger et al. (15) have also reported that ENaC expressed in Xenopus oocytes is inhibited by increasing [Na+]i. These investigators increased [Na+]i by increasing [Na+]o and estimated the magnitude of [Na+]i from the membrane reversal potentials. However, their methods could not completely differentiate the effects of [Na+]o and [Na+]i on gNa. Nevertheless, these reports indicate that ENaC is inhibited by Na+ and that this is likely a property of the cloned channel itself.

In this study the regulation of ENaC expressed in Xenopus oocytes by [Na+]i was examined to determine whether changes of [Na+]i in the absence of changes of [Na+]o can affect this channel in intact cells. The effect of a general increase of intracellular ions on ENaC activity was also assessed. Experiments were carried out with the alpha beta gamma -subunit of rat ENaC-expressing oocytes voltage clamped to 0 mV to eliminate the effects of changing Vm subsequent to altering [Na+]i. [Na+]i was increased by direct injection of Na+ into intact oocytes, thus circumventing issues of loss of cell signaling by cell dialysis and further avoiding changes of [Na+]o.

Increases of [Na+]i caused a biphasic inhibition of ENaC. A rapid initial phase was observed within seconds and is consistent with a direct inhibition of ENaC by [Na+]i. A secondary and slower inhibition that continued to 30 min was also observed during a phase in which the [Na+]i was elevated but constant. Injection of hypertonic nonionic solutions did not affect ENaC activity, whereas injection of ionic solutions that do not contain Na+ (cations or anions) caused a much smaller rapid inhibition in the absence of an appreciable secondary inhibition at 30 min.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Oocyte isolation and injection. Toads were obtained from Xenopus Express (Beverly Hills, FL) and were kept in dechlorinated tap water at 18°C. Conditions for oocyte removal, processing, injection, and cRNA synthesis were as previously described (3). Injected oocytes were incubated at 18°C for 1-3 days until the recordings were made. All recordings were performed at 19-21°C.

Two procedures were utilized for the direct injection of solutions. In the first, oocytes were impaled with the injecting electrode at the beginning of the experiment and the injections were followed by impalement of the oocytes with the two recording microelectrodes as previously described (3). A small air bubble was initially drawn into the tip of the electrode to minimize leakage of its contents into the oocyte cytoplasm. In the second, oocytes were impaled with the injecting electrode immediately before the injection of its contents. No differences between the results of these two procedures were found. All injections were limited to 27.6 nl to avoid membrane disruption.

Some oocytes that were injected with the 0.5 M salt swelled and lysed within the 30-min experimental period. This lysis phenomenon was previously described in connection with volume injections of ~180 nl into ENaC-expressing oocytes and a >33% decrease of external solution osmolarity (3). To circumvent this problem, the external perfusion solution was changed during the initial 5 min after injection to one that also contained 50 or 75 mM sucrose. Furthermore, each oocyte was visually inspected to determine its volume status. A small degree of cell swelling was preferred because it assured that there was no small but undetectable cell shrinking. This was an important criterion because cell swelling is without immediate or long-term effects on ENaC in contrast to cell shrinking, which causes a slow inhibition of gNa (3).

Solutions and chemicals. All solutions and chemicals were as described by Awayda and Subramanyam (3). Amiloride was a gift from Merck Sharp & Dohme (Rahway, NJ). All other chemicals were of the highest grade and were obtained from Sigma Chemical (St. Louis, MO). Solution Na+ content was measured with a flame photometer (model 443; Instrumentation Laboratory, Watertown, MA).

Dual-electrode clamp. Whole cell currents were recorded and analyzed as described by Awayda and Subramanyam (3) with a TEV-200 two-electrode voltage clamp (Dagan Instrument, Minneapolis, MN). In most experiments the bath was perfused with solution at the rate of 6 ml/min, or ~4 chamber volumes/min. In some experiments a smaller volume chamber that allowed an exchange rate of ~12 chamber volumes/min was used. No differences between the results of experiments in either chamber were detected. Values for gNa were calculated from the amiloride-sensitive current-voltage (I-V) relationship between -100 and -80 mV as described by Awayda et al. (2). By convention the inward flow of cations is designated as inward current (negative current), and all voltages are reported with respect to ground or bath. Except where noted, all data are reported as means ± SE.

Intracellular Na+ activity was estimated from the fit of the I-V relationship to the Goldman equation (13): INa = PNa · F · beta  · A · ([Na]i - [Na]o · ebeta )/(1 - ebeta ) where beta  = Vm · F/R · T, and F, R, T and A are Faraday's number, gas constant, absolute temperature, and area, respectively.

An activity coefficient for Na+ of 0.778 was used (18). Data were fit in the voltage range of -100 to -20 or -40 mV. Data were fit by using the least-squares minimization fitting subroutine in SigmaPlot (Jandel Scientific, San Rafael, CA); an oocyte membrane area of 0.15 cm2 was assumed (16).

Statistical analysis was carried out by using the paired Student t-test where appropriate. Significance was determined at the 95% confidence level (P < 0.05).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of increasing [Na+]i. ENaC-expressing oocytes were incubated in media that contained 81 mM [Na+]o, and the whole cell currents were recorded 1-3 days later in Ringer containing 100 mM Na+. [Na+]i, current at -100 mV, gNa, and PNa averaged 48.1 ± 2.2 meq, -2,583 ± 204 nA, 27.6 ± 2.3 µS, and 0.446 ± 0.038 × 10-6 cm/s (n = 27), respectively. PNa was calculated from the amiloride-sensitive I-V relationship as described in MATERIALS AND METHODS (also see below).

A representative effect of injecting 0.5 M Na2SO4 on the whole cell current is shown in Fig. 1. Within 2 min, an inhibition of current was observed and gNa decreased from 45.2 to 37.1 µS (Fig. 1, A and B). This rapid inhibition was followed by a secondary inhibition that caused a decrease of the gNa to 21.7 µS at 30 min (Fig. 1C). The remaining amiloride-insensitive current is shown in Fig. 1D and exhibited a conductance of 1.0 µS.


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Fig. 1.   Whole cell currents (Im) in an epithelial Na+ channel (ENaC)-expressing oocyte and changes of Im after a rapid increase of intracellular Na+ concentration ([Na+]i). A: Im in an oocyte preincubated in 81 mM solution [0.5× L-15 (Sigma)], and recorded in high Na+ (100 mM [Na+]o). B: rapid inhibition is observed within 2 min of injection of 27.6 nl of 0.5 M Na2SO4. (C) Im were further inhibited 30 min after increase of [Na+]i. The amiloride (10 µM)-insensitive Im are shown in D. Note the shift in reversal potential after increase of [Na+]i.

The effect of 0.5 M Na2SO4 injection on the amiloride-sensitive I-V relationship is shown in Fig. 2. The current data were summarized from the average of five points at the end of each voltage episode. The solid line represents the fit to the Goldman equation as described in MATERIALS AND METHODS. As expected with Goldman-type rectification (13), the I-V relationship is inwardly rectified under conditions of higher [Na+]o than [Na+]i. The injection of Na2SO4 increased [Na+]i from 52 to 88 meq and decreased PNa from 0.69 × 10-6 to 0.47 × 10-6 cm/s (Fig. 2, A and B). The [Na+]i at 30 min was essentially the same as that at 2 min; however PNa decreased to 0.30 × 10-6 cm/s (Fig. 2C). Consistent with Goldman-type rectification, the increase of [Na+]i beyond [Na+]o was accompanied by a change of the I-V relationship from one that exhibited inward rectification to one that exhibited outward rectification.


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Fig. 2.   Amiloride-sensitive current-voltage (I-V) relationship for an ENaC-expressing oocyte, and changes of this relationship after rapid increase of [Na+]i. Amiloride-sensitive currents were obtained after subtraction of amiloride-insensitive components obtained at either beginning or end of experiment (no appreciable differences in these amiloride-insensitive currents were detected). The I-V values were fit to the Goldman equation as described in MATERIALS AND METHODS. A: [Na+]i and membrane Na+ permeability (PNa) were 52 meq and 0.69 × 10-6 cm/s, respectively, under control high-Na+ solution-bathed conditions. Injection of 27.6 nl of 0.5 M Na2SO4 decreased PNa to 0.47 × 10-6 cm/s within 2 min (B) and to 0.30 × 10-6 cm/s at 30 min (C). These changes of PNa were accompanied by an increase of [Na+]i from 52 to 88 meq at 2 min and to 90 mM at 30 min. Data were fit in voltage range of -100 to -20 mV.

Summarized in Table 1 are the effects of intracellular Na+ injection on ENaC. Within 2 min of injecting 0.5 M Na2SO4, [Na+]i was elevated from 52.1 ± 2.6 to 98.9 ± 4.5 meq (n = 9). This increase of [Na+]i was accompanied by a 29.5 ± 3.8% decrease of gNa and a 34.8 ± 3.1% decrease of PNa. The [Na+]i at 30 min remained elevated at 97.8 ± 6.5 meq and was not different from the value observed at 2 min. Despite the absence of further increases of [Na+]i, gNa and PNa decreased by 67.3 and 66.9% of control, respectively, at 30 min. This indicated the presence of an additional slower effect of elevating [Na+]i on ENaC. Two inhibitory phases were also observed with the injection of 27.6 nl of 0.25 M Na2SO4. However, as expected this injection resulted in smaller increases of [Na+]i and was accompanied by smaller initial and secondary decreases of gNa and PNa (see Table 1).

                              
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Table 1.   Changes of [Na+]i and ENaC activity subsequent to intracellular Na2SO4 injection in oocytes in 100 mM [Na+]o

To determine the dynamic effects of increasing [Na+]i on ENaC, the time course of changes of gNa with Na+ injection was examined. As shown in Fig. 3, the response of gNa was biphasic and consisted of a rapid inhibition by ~31.9% within 3 min, followed by a slower secondary inhibition by ~69.6% at 30 min. The initial rapid inhibition was nearly complete within 2-5 min, and the second inhibitory phase began thereafter. The effects of injecting 27.6 nl of 0.25 M Na2SO4 were qualitatively similar, and gNa decreased by a smaller amount at both 3 and 30 min. It should be noted that ENaC is not directly sensitive to mechanical perturbations in oocytes, and therefore these effects cannot be attributed to the injected volume. This volume (27.6 nl) is well below that which was found to cause mechanical membrane disruption, and, additionally, the injection of isotonic KCl at volumes <180 nl does not cause time-dependent changes of ENaC conductance (3).


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Fig. 3.   Time courses of inhibition of slope conductance (gNa) by intracellular Na+. Data are summarized as changes of gNa (calculated between -100 and -80 mV) and are normalized to value of gNa immediately before injection of Na2SO4. Two concentrations of Na2SO4 were injected: 0.5 M (; n = 9) and 0.25 M (open circle ; n = 5). In both cases, rapid inhibition, which reached a relative plateau within ~2-5 min, was observed. This was followed by a secondary and much slower inhibitory phase, which continued to 30 min. All data points with exception of 10- and 20-s points in 0.5 M group and 0.5-min point in 0.25 M group were significantly different from control. E/C, experimental value divided by control value; [Na+]o, extracellular Na+ concentration.

The changes of currents observed in these experiments are specifically due to changes of the gNa because there were no effects of injecting 0.5 M Na2SO4 in ENaC-expressing oocytes treated with a saturating concentration of amiloride (10 µM) or in control oocytes (see Fig. 4). Thus this injection procedure does not cause any appreciable changes of endogenous or amiloride-insensitive currents. Moreover, the injection of isotonic K2SO4 (70 mM) did not affect ENaC's conductance (Fig. 4), and gNa was 101.1 ± 1.5 and 99.1 ± 2.8% of control (n = 3) at 3 and 30 min, respectively. The injection of isotonic K2SO4 made hypertonic with sucrose (70 mM K2SO4-1.2 M sucrose) was also without effect, and gNa was 103.3 ± 2.0 and 103.2 ± 4.8% of control (n = 6) at 3 and 30 min, respectively (Fig. 4). Thus these data rule out any nonspecific-injection- or time-dependent changes of gNa.


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Fig. 4.   Lack of nonspecific effects of Na2SO4 injection on ENaC currents. Time courses of changes of whole cell gNa after injection of Na2SO4, isotonic K2SO4, or hypertonic sucrose are shown. Data are summarized as changes of gNa from control or time 0 point. ENaC-expressing oocytes treated with 10 µM amiloride were not affected by injection of Na2SO4 (; n = 5), indicating that amiloride-insensitive current does not appreciably change during experiment. Control oocytes were also insensitive to Na2SO4 injection (open circle ; n = 10), indicating lack of effect on background currents. Injection of isotonic K2SO4 (; n = 3) was without effect on ENaC currents, and so was injection of isotonic K2SO4 made hypertonic with sucrose (; n = 6).

The time course of changes of [Na+]i in high-[Na+]i oocytes is summarized in Fig. 5. As shown, the [Na+]i in oocytes injected with 0.5 M Na2SO4 began changing within 10 s and continued to increase to a maximal value in 2-5 min. These values were essentially stable between 5 and 30 min. A small, statistically insignificant decrease of [Na+]i between the 5- and 30-min time points (P > 0.185) was observed. Similar results for the time course of changes of [Na+]i in oocytes injected with 0.25 M Na2SO4 were observed.


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Fig. 5.   Time courses of changes of [Na+]i in oocytes injected with 0.5 M () and 0.25 M (open circle ) Na2SO4. Data are summarized as changes of [Na+]i from control values immediately before injection of Na2SO4. All data were calculated from Goldman equation as described in MATERIALS AND METHODS, by using same oocytes used for Fig. 3. With both concentrations of Na2SO4, a rapid increase of [Na+]i, which reached a relative plateau within ~5 min, is observed. Values were unchanged up to 30 min (n = 8). See Fig. 3 legend for more detail.

As seen from Figs. 3 and 5, the time course of the changes of [Na+]i and that of changes of gNa correlate well within the first 5 min when the rapid inhibition is observed. This inhibition was followed by a secondary, slower inhibition that continued to 30 min in the absence of changes of [Na+]i. This secondary decrease and the initial inhibition exhibited a concentration dependence because they were larger in oocytes injected with 0.5 M Na2SO4. These results provide strong evidence for immediate rapid and delayed long-term concentration-dependent inhibitions of ENaC by intracellular Na+.

The finding that injection of 0.5 M Na2SO4 causes larger inhibition than injection of 0.25 M Na2SO4 during both the rapid and slow inhibitory phases is consistent with a dose dependency between the [Na+]i and ENaC activity. Thus it is expected that smaller increases of [Na+]i may also produce inhibition. To better understand this relationship, the conductance and permeability data obtained in the first 3 min were plotted against the changes of [Na+]i. As shown in Fig. 6A, an inverse linear relationship between the changes of gNa and the changes of [Na+]i is observed. The regression line exhibited a slope of -5.56 and a y-intercept of 1.016, indicating no self-inhibition of the gNa in the absence of changes of [Na+]i. This is similar to the relationship observed when plotting the average change of [Na+]i and the corresponding percent change of gNa within the first 5 min of injecting 0.5 or 0.25 M Na2SO4 (Fig. 6B; y-intercept of 1.025 and slope of -6.27). Moreover, as shown in Fig. 6C, an inverse relationship for the PNa was also observed, indicating decreased permeability with increased [Na+]i. This relationship exhibited a slope of -7.36 and also indicated no feedback inhibition in the absence of changes of [Na+]i because the y-intercept was 1.034. Thus these data provide evidence for a dose dependence between gNa and [Na+]i during the initial rapid phase and indicate that small changes of [Na+]i could result in rapid inhibition of ENaC. It is important to point out that it is not necessary to observe large changes of ENaC activity in native tissues such as the CCT for this phenomenon to be an important regulator of Na+ transport, because a change of just a few percentage points in ENaC activity can have significant effects on body Na+ homeostasis.


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Fig. 6.   Relationship between changes of [Na+]i in oocytes in high [Na+]o (100 mM) and observed self-inhibition-induced changes of gNa and PNa. Data are obtained from first 3 min after injection of Na2SO4 from experiments summarized in legend for Fig. 3. A: inverse relationship between changes of [Na+]i and gNa. B: similar relationship between mean changes of [Na+]i and gNa in oocytes injected with 0.5 or 0.25 M Na2SO4. C: inverse relationship for changes of [Na+]i and PNa. Solid line, linear regression fit. See text for more detail.

Effects of increasing the intracellular concentration of poorly permeant ions. To determine whether the above intrinsic regulatory processes are selective for ions that permeate ENaC, oocytes were injected with 27.6 nl of 0.5 M K2SO4 or N-methyl-D-glucamine (NMDG) sulfate. As expected from a membrane that predominantly contains Na+ channels that are also highly selective for Na+ over K+ and NMDG+, there was little effect of this procedure on the membrane reversal potential (data not shown). The time courses of the effect on gNa in these two groups of oocytes are shown in Fig. 7. Injection of either solution caused a rapid inhibition of gNa similar to that observed with the injection of Na2SO4. However, this inhibition was clearly much smaller than that observed with the injection of 0.5 M Na2SO4. Moreover, the gNa was essentially constant after 5 min, and the primary response was not followed by any significant secondary response. Thus injection of Na+ causes additional inhibition that is not observed with injection of other salts.


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Fig. 7.   Time courses of effects of increasing intracellular concentration of impermeant ions on gNa. Injection of 0.5 M K2SO4 (n = 8), 0.5 M NMDG sulfate (n = 8), or 0.75 M NMDG gluconate (n = 4) caused a small initial inhibition similar in time course to that observed with injection of Na2SO4. In all 3 groups, secondary inhibition at 30 min was either slightly smaller or statistically not different from that at 5 min. Data from oocytes injected with 0.5 M Na2SO4 are same as those summarized in Fig. 3 and are shown for comparison purposes.

Injection of solution containing NMDG gluconate resulted in an inhibition similar to that observed with NMDG sulfate, indicating the lack of any appreciable effects of the injected anion on ENaC activity. In combination with the observation that injection of hypertonic sucrose does not cause inhibition (see above), these results indicate a small but significant effect of increasing the intracellular concentration of impermeant ions. The reason for this finding is unknown but may be attributed to a crowding effect whereby these ions shield the Na+ from interacting with ENaC.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Xenopus oocyte expression system was utilized for its ability to reproduce the electrophysiological properties of the epithelial Na+ channel to study the regulation of ENaC by [Na+]i. In this system the changes of [Na+]i are separated from the effects on Vm as oocytes are voltage clamped. A holding voltage of 0 mV was used, and this voltage is close to that observed across the apical membranes of many open-circuited Na+-transporting epithelia. In this system the [Na+]i could also be changed by direct injection of solutions in the absence of changes of [Na+]o. I report that rapid increases of [Na+]i by direct intracellular injection of Na2SO4 caused a biphasic inhibition of ENaC consisting of rapid and slow phases. The rapid phase was completed within 2-3 min. During this phase the gNa changed almost instantaneously with changes of [Na+]i. This observation is consistent with a direct interaction of intracellular Na+ with ENaC. The second phase was slower in its time course and continued to 30 min after the increase of [Na+]i. This phase is consistent with an indirect mechanism because the gNa was found to decrease in the absence of additional changes of [Na+]i.

Relationship to physiological levels of [Na+]i. The spontaneous intracellular Na+ activity in the bulk cytoplasm in many Na+-transporting epithelia is in the range of 10-30 meq (21, 26, 30). Thus the baseline [Na+]i encountered in the present study may represent a supraphysiological concentration. Moreover, the largest increase of [Na+]i in this study (~47 meq) may also represent a supraphysiological increase. However, it is important to point out that inhibition was also observed in the group injected with 0.25 M Na2SO4, in which [Na+]i increased by <20 meq. Moreover, the inverse relationships observed in Fig. 6 indicate that smaller changes of [Na+]i will likely result in small but significant changes of gNa or PNa. This was also observed for both groups injected with Na2SO4 at time points shorter than 3 min, in which the [Na+]i was increased by small amounts but was nevertheless accompanied by significant changes of gNa.

It is important to recognize that the activities measured by intracellular microelectrodes represent those found in the bulk cytoplasm and are not those present in the subapical membrane space at the inner mouth of the channel, whereas those calculated from reversal potentials are more representative of the activities in the submembrane space and may not reflect the bulk cytoplasmic concentrations. It is also important to consider that although microelectrode studies report a low bulk [Na+]i, these values are dependent on the activity of transporters in both the apical and basolateral membranes. For example, an [Na+]i of 14 meq in frog skin is reported to increase to 66 meq after inhibition of the basolateral pump by ouabain (29).

An additional caution against a direct comparison between the activities in the present study and those obtained from cytoplasmic measurements in epithelia is the presence of a standing voltage across the apical membranes (Va) of these ENaC-containing epithelia. This Va is dependent not only on the apical membrane permeability to Na+ but also on whether the epithelium is studied in the open circuit or short circuit modes and in high- or low-[Na+]o Ringer solution. In the short circuit mode, taking account of the example of frog skin epithelia, the apical membrane is clamped to a Va in the range of its intracellular voltage of -60 to -80 mV (8). In combination with an [Na+]o of ~110 meq, it is clear that the equilibrium activity at the inner mouth of the channel could exceed 1,100 meq! Because the membrane is not at equilibrium, as evident from the presence of a short circuit current, the [Na+]i at the subapical space is not 1,100 meq; however, it is also unlikely to be 10 meq. A similar situation applies in the open circuit mode, in which Va is in the range of 0 to -20 mV (5, 30, 31) and is very close to the Thevenin equilibrium potential. At an [Na+]o of 110 meq, the subapical [Na+]i near the inner mouth of the channel is expected to be ~50 meq or higher.

Possible origin of these inhibitory phases. The initial inhibitory phase is a rapid process and is consistent with a direct effect of [Na+]i on ENaC. Indeed the onset of this response (within 10-20 s) is in the range of the delay expected if one assumed a free-diffusion rate of ~50 µM/s. The effects of injecting second messenger proteins such as protein kinase A or PKC into oocytes expressing ENaC or cystic fibrosis transmembrane conductance regulator are delayed by 1-2 min and are also prolonged, such that a plateau is not observed until 30-60 min after injection (M. S. Awayda, unpublished observation). Moreover, at any one instant during the initial response the changes of reversal potential, and presumably the increases of [Na+]i, correlate well with the decrease of gNa. Although the involvement of a rapid second messenger system cannot be ruled out, the observed changes during this phase are consistent with a direct effect of Na+ on ENaC (see Figs. 3, 5, and 6).

On the other hand, the observed secondary inhibition is unlikely to be directly caused by Na+, because changes of gNa and PNa are observed during this phase in the absence of further changes of [Na+]i. Thus this phase may be consistent with one that involves a second messenger system such as PKC (10, 20) or Nedd4 (4). It should be emphasized that Na+-dependent feedback regulation of ENaC by ubiquitination and that by PKC are not necessarily mutually exclusive because Nedd4, which interacts with ENaC, also possesses Ca2+ and phospholipid binding sites in addition to its ubiquitin ligase site (25) and is likely activated by Ca2+ and phospholipids, similar to PKC.

Intracellular vs. extracellular Na+. The present experiments were designed to increase [Na+]i. Although small changes of [Na+]o due to Na+ exit through ENaCs cannot be completely ruled out, there are two observations that favor the conclusion that these changes of gNa are the result of changes of [Na+]i rather than [Na+]o. First, the time course of the changes of gNa mimics that of changes of [Na+]i during the initial rapid phase. These changes occur within seconds, thereby eliminating the contribution of Na+ exit. Second, the reversal potentials after the injection of Na+ increase and reach a plateau during this initial phase and then remain constant through the remainder of this phase and throughout the entire secondary phase (see Fig. 5).

Similarly, it is also unlikely that Na+ leak through ENaC is causing the secondary slower inhibition because the [Na+]i remained essentially constant during this period, indicating little or no Na+ loss. It should be noted that, because the intracellular volume is much smaller than the extracellular volume, any Na+ loss resulting in appreciable change of [Na+]o would be accompanied by a much larger and exaggerated decrease of [Na+]i.

Na+ vs. other ions. The lack of effect of injecting 70 mM K2SO4-1.2 M sucrose indicates that both of these inhibitory processes are independent of intracellular osmolarity. Additionally, the observation that the injection of various anions or cations other than Na+ results in a much smaller nonspecific initial inhibition with no appreciable secondary inhibition indicates that Na+ selectively causes these two inhibitory processes.

It is not known whether the initial small inhibition observed with injection of K2SO4, NMDG sulfate, or NMDG gluconate is due to the injection of cations or anions or a combination of both. However, it is clear that the effect of a general increase of intracellular ionic strength is distinct from that of a specific increase of [Na+]i for both the initial and delayed phases of inhibition. If the effect of increased ionic strength is attributed to cations, one can speculate that these cations may possess a finite but smaller affinity to a Na+ binding site on ENaC. Alternatively, if this effect is due to a general increase of ionic strength, one can speculate that this may be due to a crowding effect whereby these ions shield the Na+ from interacting with ENaC.

Conclusions. [Na+]i was found to regulate ENaC activity in a dose-dependent manner. This regulation can be divided into rapid and slow phases. These two phases would be expected to control tonic and phasic channel activities, respectively. These two modes of regulation are likely to correspond to a direct and indirect effect of [Na+]i on ENaC and are not observed with the injection of various other cations or anions.


    ACKNOWLEDGEMENTS

I thank Dr. Bernard Rossier (University of Lausanne, Lausanne, Switzerland) for the gift of rat ENaC subunits and Dr. Lawrence Palmer (Cornell University) and Roxanne Reger for reading the manuscript.


    FOOTNOTES

This work was supported by a Grant-In-Aid from the Louisiana American Heart Association and by a Louisiana Education Quality Support Fund grant from the Louisiana Board of Regents.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. S. Awayda, Dept. of Medicine, SL 35, Tulane Univ. School of Medicine, New Orleans, LA 70112 (E-mail: mawayda{at}mailhost.tcs.tulane.edu).

Received 2 March 1999; accepted in final form 8 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 277(2):C216-C224
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