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Department of Medicine and Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 
-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.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
· A · ([Na]i [Na]o · e
)/(1 e)
where = 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).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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).
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6 to 0.47 × 106 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 × 106 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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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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 of60 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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; n = 9) and
0.25 M (
; 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.
; n = 5), indicating that
amiloride-insensitive current does not appreciably change during
experiment. Control oocytes were also insensitive to
Na2SO4
injection (
; n = 3) was without effect on
ENaC currents, and so was injection of isotonic
K2SO4
made hypertonic with sucrose (
