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Am J Physiol Renal Physiol 294: F38-F46, 2008. First published October 3, 2007; doi:10.1152/ajprenal.00403.2007
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Aldosterone and Epithelial Na+ Channels

Purinergic control of apical plasma membrane PI(4,5)P2 levels sets ENaC activity in principal cells

Oleh Pochynyuk,1 Vladislav Bugaj,1 Alain Vandewalle,2,3 and James D. Stockand1

1Department of Physiology, University of Texas Health Science Center, San Antonio, Texas; and 2INSERM U773, Centre de Recherche Biomedicale Bichat-Beaujon, and 3Universite Paris, Paris, France

Submitted 29 August 2007 ; accepted in final form 26 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activity of the epithelial sodium channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] modulate the activity of this channel. Activation of purinergic receptors triggers multiple events, including activation of PKC and PLC, with the latter depleting plasma membrane PI(4,5)P2. Here, we investigate regulation of ENaC in renal principal cells by purinergic receptors via PLC and PI(4,5)P2. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)P2 levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor U73122 [GenBank] , but not U73343 [GenBank] , its inactive analog, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting either purinergic receptors or PLC in resting cells dramatically increased ENaC activity above basal levels, indicating tonic activation of purinergic signaling in these polarized renal epithelial cells. Increased ENaC activity was associated with elevation of apical membrane PI(4,5)P2 levels. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)P2 levels. Dwell-time analysis reveals that depletion of PI(4,5)P2 forces ENaC toward a closed state. In contrast, increasing PI(4,5)P2 levels above basal values locks the channel in an open state interrupted by brief closings. Thus our results suggest that purinergic control of apical membrane PI(4,5)P2 levels is a major regulator of ENaC activity in renal epithelial cells.

phosphoinositides; PLC signaling; P2Y receptors; sodium reabsorption; hypertension

THE EPITHELIAL NA+ CHANNEL (ENaC) is localized to the apical plasma membrane of many epithelia, including that in the aldosterone-sensitive distal renal nephron. The activity of this channel is limiting for Na+ (re)absorption across these epithelia (12, 15, 31, 33, 45). ENaC is a critical end-effector of the renin-angiotensin-aldosterone system. Thus ENaC is well positioned to regulate sodium homeostasis and, consequently, blood pressure. The physiological importance of ENaC to negative-feedback regulation of blood pressure is emphasized by inheritable forms of severe hypertension resulting from gain-of-function mutations in the channel (13, 21, 29, 30, 32). In contrast, loss-of-function mutations lead to improper salt wasting and low blood pressure (29, 30).

ENaC is a highly Na+-selective, non-voltage-gated, noninactivating ion channel in the ENaC/Deg superfamily (3, 12, 15). This channel is heteromeric, comprising three distinct but similar subunits: {alpha}, β, and {gamma} (7). Each subunit has NH2- and COOH-terminal cytosolic domains separated by two transmembrane domains and a large extracellular loop (3, 10, 12). The stoichiometry of ENaC remains controversial (9, 16, 34, 35), but all three subunits are required to form a functional channel with maximal activity and normal regulation.

The activity of ENaC is controlled by both systemic endocrine inputs, such as aldosterone (12, 21, 39, 45), and local paracrine factors, such as ATP (17, 18, 22). ATP is released from distal renal epithelial cells and lung epithelial cells in response to various stimuli, including mechanical stretch (28, 46). Several recent reports suggest that extracellular ATP decreases ENaC activity (8, 20). It has been suggested that activation of purinergic receptors localized to cells also containing the channel underpins this regulation (17, 19, 42). The metabotropic G protein-coupled P2Y receptors could inhibit ENaC activity via PLC signaling either through stimulating PKC or depleting PI(4,5)P2 levels. PKC activation causes long-term decreases in ENaC surface expression via ERK1/2 signaling (5). Alternatively, a role for PLC via PI(4,5)P2 hydrolysis during purinergic regulation of ENaC was recently demonstrated by Kunzelmann and colleagues (17). The primary effect of PI(4,5)P2 is not related to changes in ENaC plasma membrane levels but rather effects on channel open probability (17, 23, 44, 48). Direct interaction is required for normal channel gating since exogenous PI(4,5)P2 reverses rapid run-down in ENaC activity in excised patches (23). Moreover, depletion of plasma membrane PI(4,5)P2 levels by activation of receptor tyrosine kinases and Gq/11-coupled receptors decreases ENaC activity (44). Therefore, rapid changes in apical plasma membrane PI(4,5)P2 levels in response to purinergic stimulation may serve as a signaling mechanism to adjust the rate of Na+ reabsorption via ENaC across epithelial barriers. However, the physiological importance of such regulation remains to be fully tested. Moreover, the mechanism of PI(4,5)P2 action on ENaC open probability and gating has not been completely probed.

Here, we investigate the physiological mechanisms and consequences of regulation of ENaC by acute changes in apical plasma membrane PI(4,5)P2 levels in response to purinergic stimulation. Our results are consistent with ENaC activity being dynamically regulated by PLC via control of apical plasma membrane PI(4,5)P2 levels. Moreover, resting PI(4,5)P2 levels in renal epithelial cells set basal ENaC activity. Fluctuations in apical PI(4,5)P2 allow ENaC to range between a state with brief channel openings to one with prolonged channel openings.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and cDNA constructs. All chemicals and materials were from Fisher Scientific, Sigma, BioMol, or Calbiochem unless noted otherwise. The PI(4,5)P2 reporter green fluorescent protein (GFP)-PLC-{delta}1-PH is a chimeric protein consisting of the PI(4,5)P2-binding plekstrin homology (PH) domain of PLC-{delta}1 fused to GFP (14). The cDNA encoding this reporter was a kind gift from the T. Meyer laboratory.

Cell culture. Immortalized mouse cortical collecting duct (mpkCCDc14) principal cells were grown in defined medium on permeable supports (Costar Transwells, 0.4-µm pore, 24-mm diameter) as described previously (4, 36). Cells were maintained with FBS and corticosteroids until they polarized and formed monolayers with high resistances and avid Na+ transport. When used, the PKC inhibitor Ro 31-8220 was added to both the apical and basolateral membranes at a final concentration of 160 nM for 24 h. All other reagents were added during an experiment.

Exogenous expression of protein. Plasmid cDNA encoding the fluorescent PI(4,5)P2 reporter was introduced into mpkCCDc14 principal cells within a confluent monolayer with the biolistic particle delivery system (Biolistic PDS-1000/He Particle Delivery System, Bio-Rad). Use of this system has been described previously (11, 36). We closely followed established protocols. In brief, mpkCCDc14 cells were grown to confluence on permeable supports. After forming high-resistance monolayers avidly transporting Na+, cells were washed with physiological saline, aspirated, and quickly bombarded (at the apical membrane) under vacuum with microcarriers coated with reporter cDNA. The medium was immediately returned to the cells, which were then placed within a tissue culture incubator for 2–3 days to allow expression of the PI(4,5)P2 reporter. Bombardment had little disruptive effect on cellular and monolayer integrity, as established by maintenance of Na+ transport and a high transepithelial resistance.

Electrophysiology. Transepithelial Na+ current across mpkCCDc14 cell monolayers was calculated as described previously (5, 36). In brief, current was calculated using Ohm's law as the quotient of transepithelial voltage to transepithelial resistance corrected for surface area under open-circuit conditions using the Millicel Electrical Resistance System with dual Ag/AgCl pellet electrodes (Millipore) to measure voltage and resistance.

For cell-attached patches made on the apical membrane of mpkCCDc14 cells, bath and pipette solutions were (in mM) 155 NaCl, 1 CaCl2, 2 MgCl2, 5 glucose, and 10 HEPES (pH 7.4) and 140 LiCl, 2 MgCl2 and 10 HEPES (pH 7.4), respectively. Current recordings were made using an Axopatch 200B. Patched membranes were clamped (–Vp) to –40 or –60 mV. Currents were low-pass filtered at 100 Hz by an eight-pole Bessel filter (Warner Instruments) and digitized and stored on a PC using the Digidata 1322A interface. Current data were analyzed using pClamp 9.2. Channel activity, defined as NPo, was calculated as NPo = {Sigma}(t1 + 2t2 +...iti), where ti is the fractional open time spent at each of the observed current levels. Only patches containing five channels or fewer were used to calculate NPo. For dwell-time analysis, patches containing only a single channel were used. Extremely fast events, beyond reliable resolution (≤5 ms), were excluded from analysis.

Total internal reflection fluorescence microscopy. Fluorescence emissions from the PI(4,5)P2 reporter at the apical membrane of mpkCCDc14 cells within a confluent monolayer were collected using total internal reflection fluorescence TIRF (also called evanescent-field) microscopy. TIRF generates an evanescent field that declines exponentially with increasing distance from the interface between the cover glass and plasma membrane, illuminating only a thin section (~100 nm) of the cell in contact with the cover glass (2, 38). For these experiments, GFP-PLC-{delta}1-PH was introduced into polarized monolayers of mpkCCDc14 cells grown on permeable supports with the particle delivery system described above. On expression of the reporter, 5 x 5-mm sections of the support were excised, inverted, and placed onto cover glass coated with poly-L-lysine. This arrangement made it possible to visualize dynamic changes in the level of the PI(4,5)P2 reporter at the apical membrane in real time in living cells. All TIRF experiments were performed in the TIRF microscopy core facility housed within the Department of Physiology at the University Texas Health Sciences Center (http://physiology.uthscsa.edu/tirf). We have previously described imaging the GFP-PLC-{delta}1-PH reporter and other fluorophore-tagged proteins using this core facility (24, 25, 35, 36, 43, 44). The methods used here closely follow these published protocols. In brief, fluorescence emissions from the GFP-PLC-{delta}1-PH reporter were collected using an inverted TE2000 microscope with through-the-lens (prismless) TIRF imaging (Nikon). Samples were viewed through a plain Apo TIRF x60 oil-immersion, high-aperture (1.45 NA) objective. Fluorescence emissions were collected through a 535 ± 25-nm band-pass filter (Chroma Technology) by exciting GFP with an argon-ion laser with an acoustic optic tunable filter (Prairie Technology) used to restrict excitation wavelength to 488 nm. Fluorescence images were collected and processed with a 16-bit, cooled charge-coupled device camera (Cascade 512F, Roper Scientific) interfaced to a PC running Metamorph software. This camera uses a front-illuminated EMCCD with on-chip multiplication gain. Images were collected once a minute with a 100-ms exposure time. Images were not binned or filtered, with pixel size corresponding to a square of 122 x 122 nm.

Statistics and data treatment. All summarized data are reported as means ± SE. Summarized data were compared with either Student's (2-tailed) t-test or a one-way ANOVA in conjunction with the Dunnett or Student-Newman-Keuls posttest where appropriate. P ≤ 0.05 was considered significant. Emissions from GFP-PLC-{delta}1-PH were normalized to starting levels. Vehicle treatment was used to quantify spontaneous decreases in fluorescence emissions (bleaching) over time. All fluorescence data were corrected to this value. For presentation, current data from some cell-attached patches were subsequently software filtered at 50 Hz, and spontaneous baseline drifts were corrected.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Extracellular ATP acutely decreases ENaC activity via activation of purinergic receptors. Here, we tested the hypothesis that ENaC activity and open probability are dynamically regulated by ATP via purinergic signaling control of apical plasma membrane PI(4,5)P2 levels. For these experiments, we used polarized epithelial monolayers of mpkCCDc14 cells with robust transepithelial transport (5.2 ± 0.3 µA/cm2, n = 25) and resistances (1.51 ± 0.01 M{Omega}, n = 25). ENaC activity (NPo) was continuously monitored in paired cell-attached experiments. Extracellular application of 100 µM ATP rapidly decreases ENaC activity within a couple of minutes. A representative current trace from a patch containing a single ENaC showing such inhibition is reported in Fig. 1A. As summarized in Fig. 1B, ATP acutely decreases ENaC NPo within 5 min, from 0.47 ± 0.08 to 0.04 ± 0.02 (n = 11). We next asked whether such decreases in ENaC activity in response to ATP are mediated by activation of purinergic receptors. The current trace monitoring changes in ENaC activity in Fig. 1C is representative of such experiments. As apparent in this representative experiment and summarized in Fig. 1D, inhibiting purinergic receptors with 100 µM suramin completely restores ENaC activity, even above basal levels, following inhibition by ATP. ENaC NPo was 0.58 ± 0.13 before and 0.08 ± 0.04 after application of 100 µM of ATP and 1.01 ± 0.11 on subsequent inhibition of purinergic receptors with suramin in the continued presence of ATP (n = 7). These results are consistent with extracellular ATP acutely decreasing ENaC activity via activation of purinergic receptors.


Figure 1
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  		Fig. 1. Exogenous ATP rapidly decreases epithelial Na+ channel (ENaC) activity via activation of purinergic receptors in polarized mpkCCDc14 cells. A: representative continuous current trace from a cell-attached patch containing a single ENaC before and after treatment with ATP. Areas before (1) and after (2) treatment are shown below at an expanded time scale. Inward Li+ current is downward. c and o, Closed and open states, respectively. This patch was held at a test potential of Vh = –Vp = –40 mV. B: summary graph of ENaC open probability (NPo) before and after ATP. *P ≤ 0.05 vs. before. C: continuous current trace from a representative cell-attached experiment containing a single ENaC before and after ATP treatment followed by subsequent inhibition of purinergic receptors with suramin in the continued presence of ATP. Areas 1, 2, and 3 are shown below the top trace at an expanded time scale. All other conditions are the same as in A. D: summary graph of ENaC NPo before and after ATP and following suramin treatment. *P < 0.05 vs. before. **P < 0.05 vs. +ATP.

 
Activation of PLC is necessary for regulation of ENaC activity by purinergic signaling. We next tested possible involvement of PLC signaling in regulation of ENaC activity by extracellular ATP. The representative current recording in Fig. 2A documents the time course of change in ENaC activity on inhibiting PLC with 10 µM U73122. [GenBank] U73343, its inactive analog (Fig. 2B), was used as a negative control. As apparent from these representative experiments, the PLC inhibitor U73122 [GenBank] , but not its inactive analog U73343 [GenBank] , completely restores ENaC activity following inhibition by ATP. Moreover, U73122 [GenBank] restored ENaC activity even after its inactive analog U73343 [GenBank] failed to act. Figure 2C summarizes the effects of U73122 [GenBank] on ENaC in paired experiments. Mean NPo was 0.42 ± 0.10 before and 0.07 ± 0.03 after ATP and 1.09 ± 0.30 following subsequent inhibition of PLC with U73122 [GenBank] (n = 6). In contrast, as summarized in Fig. 2D, NPo was 0.78 ± 0.08, 0.09 ± 0.02, and 0.16 ± 0.04 before and after ATP and ATP followed by U73343 [GenBank] (n = 6), respectively.


Figure 2
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  		Fig. 2. ATP inhibits ENaC activity via activation of PLC. A: representative continuous current trace from a cell-attached patch containing 2 channels before and after treatment with ATP in the absence and presence of PLC inhibition with U73122. Areas 1, 2, and 3 are shown below the top trace at an expanded time scale. c, o1, and o2: closed and first and second open current levels, respectively. All other conditions are the same as in Fig. 1. B: representative continuous current trace from a cell-attached patch containing 2 channels before, after treatment with ATP, and following addition of the inactive PLC inhibitor analog U73343 and following inhibition of PLC with active U73122 analog. Areas 1, 2, 3, and 4 are shown below the top trace at an expanded time scale. All other conditions are the same as in A. C: summary graph of ENaC NPo before and after ATP with and without U73122. *P < 0.05 vs. before. **P < 0.05 vs. +ATP. D: summary graph of ENaC NPo before and after ATP followed by addition of the inactive and active PLC inhibitors U73343 and U73122. *P < 0.05 vs. before.

 
These experiments demonstrate that inhibiting PLC with U73122 [GenBank] but not its inactive analog U73343 [GenBank] recapitulates the effects of inhibiting purinergic receptors with suramin. Thus activation of PLC following stimulation of purinergic receptors plays a critical role in regulation of ENaC by ATP.

PKC signaling does not play a role in acute regulation of ENaC by ATP. It is well established that activation of PLC in response to purinergic receptor stimulation triggers activation of PKC in addition to hydrolysis of PI(4,5)P2. We and others have shown that PKC modulates ENaC activity by long-term control of plasma membrane levels of the channel (1, 5, 40). However, a possible role for PKC during acute inhibition of ENaC also exists. Thus we next tested involvement of PKC in the rapid decrease in ENaC activity in response to extracellular ATP. For this purpose, mpkCCDc14 cell monolayers were pretreated with 160 nM Ro 31-8220, the pan-specific PKC inhibitor. Inhibition of PKC did not affect acute decreases in ENaC activity in response to ATP and subsequent recovery from ATP-dependent inhibition on inhibiting PLC with U73122. [GenBank] A representative current trace of ENaC from this set of experiments is shown in Fig. 3A. Figure 3B shows summary data. In the presence of Ro 31-8220, mean NPo was 0.55 ± 0.24 before and 0.04 ± 0.01 after ATP and 0.41 ± 0.10 on subsequent inhibition of PLC with U73122 [GenBank] (n = 5).


Figure 3
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  		Fig. 3. PKC signaling does not play a role in acute regulation of ENaC activity by ATP. A: representative continuous current trace from a cell-attached patch containing 3 ENaC before and after ATP and ATP plus U73122. For these experiments, mpkCCDc14 monolayers were pretreated with the PKC inhibitor Ro 31-8220 for 24 h. Areas 1, 2, and 3 are shown below the top trace at an expanded time scale. This patch was held at a –60-mV test potential (–Vp). All other conditions are the same as in Fig. 1. B: summary graph of ENaC NPo for cells pretreated with the PKC inhibitor Ro 31-8220, before and after ATP, and ATP plus U73122. *P < 0.05 vs. before. **P < 0.05 vs. +ATP. C: representative continuous current trace from a cell-attached patch containing 2 ENaC before and after addition of the PKC activator PMA followed by treatment with ATP and the PLC inhibitor U73122. Areas 1, 2, 3, and 4 are shown below the top trace at an expanded time scale. This patch was held at a test potential (–Vp) of –60 mV. All other conditions are the same as in Fig. 1. D: summary graph of ENaC NPo before and after PMA followed by ATP and U73122. *P < 0.05 vs. before.

 
To further investigate a possible role for PKC signaling in regulation of ENaC by ATP, we next treated cells with 200 nM PMA, the PKC activator. A representative current trace of ENaC from this set of experiments is shown in Fig. 3C. Surprisingly, we observed a small increase in ENaC activity following addition of PMA from 0.31 ± 0.10 to 0.53 ± 0.15 (n = 5). However, this value did not reach significance. Nevertheless, even in the presence of PMA, ATP rapidly decreases ENaC activity to 0.03 ± 0.01. Moreover, subsequent inhibition of PLC with U73122 [GenBank] rescues ENaC activity in a manner similar to that reported above in the absence of PKC activation (see Fig. 2). These results are summarized in Fig. 3D. Thus inhibiting and activating PKC did not affect acute regulation of ENaC activity by extracellular ATP.

Apical plasma membrane PI(4,5)P2 levels are dynamically controlled by PLC in response to activation of purinergic receptors. Our results are consistent with the idea that PLC plays a dominant role in acute regulation of ENaC activity by extracellular ATP. This raises the possibility that ENaC activity could be acutely controlled by changes in apical plasma membrane PI(4,5)P2 levels in response to PLC signaling. To test this hypothesis, we next used TIRF microscopy to directly monitor the changes in apical plasma PI(4,5)P2 levels in confluent mpkCCDc14 cell monolayers in response to ATP stimulation. The reporter GFP-PLC-{delta}1-PH was used to follow apical PI(4,5)P2 levels. The representative fluorescence micrographs in Fig. 4A show GFP emissions from the apical membrane of mpkCCDc14 principal cells before (1), 5 min after (2), and 15 min (3) after addition of vehicle (top row), 100 µM ATP (middle row), and 10 µM U73122 [GenBank] (bottom row). As apparent from these representative micrographs, addition of vehicle has no effect on the relative levels of PI(4,5)P2 in the apical plasma membrane. In contrast, addition of extracellular ATP rapidly decreases PI(4,5)P2 levels within 5 min. The time course of inhibition is presented in Fig. 4B. Surprisingly, inhibiting PLC with U73122 [GenBank] significantly increased PI(4,5)P2 levels above basal values even in the absence of exogenous stimulation of purinergic signaling. Moreover, ATP failed to decrease PI(4,5)P2 levels when coapplied with U73122. [GenBank] These results are consistent with apical plasma membrane PI(4,5)P2 levels, being acutely controlled by changes in PLC activity during activation of purinergic receptor signaling.


Figure 4
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  		Fig. 4. Apical plasma membrane phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] levels are dynamically controlled by exogenous ATP via activation of PLC. A: fluorescence micrographs of emission from the GFP-PLC-{delta}1-PH PI(4,5)P2 reporter in the apical plasma membrane of mpkCCDc14 cells within a confluent monolayer before (1) and 5 min (2) and 15 (3) min after treatment with vehicle (top), ATP (middle), and the PLC inhibitor U73122 (bottom). Emissions were collected in a paired manner using total internal reflection fluorescence (TIRF) microscopy. B: time course of change for relative PI(4,5)P2 levels in the apical plasma membrane after addition of ATP ({blacksquare}; n = 9), U73122 ({blacktriangleup}; n = 8), and ATP+U73122 (bullet; n = 7). All reagents were added at the beginning of the experiments. All emissions were normalized to those at t = 0. Moreover, all values were corrected for a small decay in signal over time resulting from modest photobleaching, which was established by following changes in emission in response to vehicle.

 
Resting levels of apical plasma membrane PI(4,5)P2 set basal ENaC activity. The similarity in time courses for decreases in ENaC activity and apical plasma membrane PI(4,5)P2 levels in response to extracellular ATP suggests tight spatial-temporal coupling between the channel and this second messenger. This gives rise to the idea that the activity of ENaC is dynamically controlled by PI(4,5)P2 abundance in the apical membrane, with decreases in PI(4,5)P2 causing dramatic decreases in ENaC open probability. Moreover, inhibiting purinergic receptors with suramin (Fig. 1B) and PLC with U73122 [GenBank] (Fig. 2) restores ENaC activity after ATP action to above basal levels (Figs. 1D and 2C). Finally, inhibiting PLC increases apical plasma membrane PI(4,5)P2 levels above basal levels even in the absence of ATP stimulation. Thus we hypothesize that ENaC in polarized principal cells is tonically inhibited by basal purinergic signaling. To test this possibility, we treated cells with the purinergic receptor inhibitor suramin before exposing them to any other stimuli (Fig. 5A). Suramin alone increased ENaC activity from basal levels of 0.32 ± 0.13 to 2.26 ± 0.40 (Fig. 5B, n = 8). Interestingly, in these experiments we observed an increase in the number of active channels from 1.50 ± 0.32 to 2.88 ± 0.54. Thus the increase in activity may be related to increasing open probability and also involve activation of previously inactive/quiescent channels or channels with activity too low to be readily detectable. Importantly, subsequent application of ATP in the presence of suramin failed to decrease ENaC activity. ENaC NPo after addition of ATP in the presence of suramin was 2.19 ± 0.34 (Fig. 5B).


Figure 5
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  		Fig. 5. ENaC activity is set to basal levels in resting cells by paracrine purinergic signaling via PLC. A: representative continuous current trace from a cell-attached patch containing 3 ENaC before and after inhibiting purinergic receptors with suramin followed by ATP. Areas 1, 2, and 3 are shown below the top trace at an expanded time scale. This patch was held at a test potential (–Vp) of –60 mV. All other conditions are the same as in Fig. 1. B: summary graphs of ENaC NPo before and after suramin and plus ATP (left) and the number of observable, active channels in the same patch before and after treatment with suramin (right). *P < 0.05 vs. before. C: representative continuous current trace from a cell-attached patch containing 5 ENaC before and after inhibiting PLC with U73122 and subsequent treatment with ATP. Areas 1, 2, and 3 are shown below the top trace at an expanded time scale. This patch was held at a test potential (–Vp) of –40 mV. All other conditions are the same as in Fig. 1. D: summary graphs of ENaC NPo before and after U73122 and subsequent ATP treatment (left) and the number of observable, active channels in the same patch before and after treatment with U73122 (right). *P < 0.05 vs. before.

 
We next tested whether inhibiting PLC also affected resting ENaC activity. The current trace of ENaC in Fig. 5C is representative of such experiments. Inhibiting PLC with U73122 [GenBank] in the absence of all other stimuli dramatically enhances ENaC NPo from 0.33 ± 0.09 to 2.02 ± 0.046 (n = 8), as summarized in Fig. 5D. Similar to suramin, we observed an increase in the number of active channels that were identifiable with U73122 [GenBank] from 1.60 ± 0.27 to 3.10 ± 0.60. Again, subsequent application of ATP failed to decrease ENaC activity following prior inhibition of PLC. ENaC NPo in this case was 2.04 ± 0.65 (Fig. 5D). Thus inhibiting PLC with U73122 [GenBank] recapitulates the effect of suramin. Together, these results show that ENaC activity is not only dynamically controlled by acute changes in apical PI(4,5)P2 levels but also preset by resting levels of PI(4,5)P2.

Changes in apical plasma membrane PI(4,5)P2 levels affect both ENaC closed and open times. To learn more about the mechanism of PI(4,5)P2 action on ENaC in polarized epithelia, we next performed dwell-time analysis of ENaC closed and open states following manipulation of apical plasma membrane PI(4,5)P2 levels. Data from patches containing only a single channel were used. Figure 6A shows the distribution of closed time durations for ENaC with basal activity (n = 10). The average closed time was 1,249 ± 90 ms (Fig. 6B). Decreases in apical plasma membrane PI(4,5)P2 levels following treatment with ATP increased ENaC closed time durations. ENaC closed time durations in the presence of ATP are shown in Fig. 6C. As apparent, decreases in PI(4,5)P2 levels by ATP shift the peak of the histogram to the right. The average closed time in the presence of ATP of 4,643 ± 463 ms (n = 9) is significantly greater compared with control mean closed time (Fig. 6B). In contrast, inhibiting purinergic receptors with suramin shortened closed time durations, shifting the peak of the histogram to the left (Fig. 6C). The mean closed time of 174 ± 23 ms (n = 6) in the presence of suramin is significantly shorter compared with that in untreated cells. As expected, inhibiting PLC with U73122 [GenBank] yielded results similar to suramin (Fig. 6B). The average closed time with U73122 [GenBank] was 363 ± 68 ms (n = 5). Together, this dwell-time analysis suggests that decreasing apical plasma membrane PI(4,5)P2 levels drives the channel toward the closed state by increasing closed time durations, whereas, increasing PI(4,5)P2 levels opens the channel by shortening closed times.


Figure 6
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  		Fig. 6. Changes in apical plasma PI(4,5)P2 levels affect both ENaC open and closed times. A: histogram of single-channel closed state durations for ENaC with basal activity (in the absence of stimulation) from mpkCCDc14 cells. Events from 10 different patches containing a single ENaC were used. B: summary graph of mean closed time for ENaC with basal activity and following treatment with ATP, U73122 and suramin. *P < 0.05 significant increase vs. basal. **P < 0.05 significant decrease vs. basal. C: merged histograms of single-channel closed state durations for ENaC from cells treated with ATP (dark gray bars) and suramin (light gray bars), respectively. White bars represent overlap between the 2 histograms. Events from 9 and 6 different patches containing a single ENaC were used, respectively. D: summary graph of ENaC mean open time for channels with basal activity and after treatment with ATP, U73122, and suramin. *P < 0.05 significant increase vs. basal. **P < 0.05 significant decrease vs. basal.

 
PI(4,5)P2 has a parallel effect on ENaC open state durations. Figure 6D summarizes mean open time durations in response to manipulation of apical plasma membrane PI(4,5)P2 levels. As apparent in this graph, drops in PI(4,5)P2 levels following ATP significantly decrease ENaC open times from 540 ± 45 (n = 10) to 129 ± 19 ms (n = 9). In contrast, elevating apical plasma membrane PI(4,5)P2 levels with either suramin or U73122 [GenBank] markedly increases ENaC mean open time to 1,349 ± 182 (n = 6) and 1,544 ± 289 ms (n = 5), respectively. Together, these results suggest that apical plasma membrane PI(4,5)P2 availability directly controls ENaC gating in polarized renal principal cells. A lack of PI(4,5)P2 forces ENaC to its closed state by shortening open times and prolonging closed times. In contrast, a PI(4,5)P2 abundance locks the channel in its open state by increasing open times and shortening closed times.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We demonstrate here that a decrease in apical plasma membrane PI(4,5)P2 levels following purinergic stimulation is a major regulator of ENaC activity. Moreover, basal ENaC activity is set by resting apical PI(4,5)P2 levels and changes in PI(4,5)P2 levels affect ENaC gating.

Our results are most consistent with ATP acting on ENaC via a signal transduction cascade. We report that inhibiting purinergic receptors with suramin completely reverses ATP action on ENaC. Moreover, inhibiting PLC signaling recapitulates the effects of suramin. These findings strongly suggest involvement of metabotropic P2Y receptors in ATP-mediated inhibition of ENaC. Metabotropic P2Y receptors, similar to many other G protein-coupled receptors, are coupled to PLC by Gq/11. There is overwhelming evidence that activation of such G protein-coupled receptors leads to a pronounced decrease in the plasma membrane PI(4,5)P2 levels (37). Indeed, purinergic inhibition of transepithelial sodium transport via activation of P2Y receptors and subsequent hydrolysis of PI(4,5)P2 were recently demonstrated in tracheal epithelia and M1 collecting duct cells (17). Here, we define the mechanism of such regulation at the single-channel level. A different study suggests possible involvement of ionotropic P2X receptors in regulation of ENaC activity and trafficking (47). However, we find no role for P2X receptors in acute regulation of ENaC, particularly since ATP fails to affect ENaC activity when PLC is inhibited. Importantly, the potential physiological significance of P2Y receptors in the regulation of ENaC and other proteins involved in renal salt handling has recently been revealed. Mice lacking P2Y2 receptors have hypertension with facilitated renal sodium and water reabsorption (27).

Activation of PLC signaling decreases ENaC activity either by activating PKC or hydrolysis of PI(4,5)P2. Activation of PKC is known to cause long-term downregulation of ENaC in the plasma membrane via a pathway involving ERK1/2 signaling (5, 40). However, we find no role for PKC in acute regulation of ENaC activity in response to purinergic signaling.

The finding that apical plasma membrane PI(4,5)P2 levels rapidly decrease on purinergic stimulation led to the idea that hydrolysis of PI(4,5)P2 could be a crucial factor affecting ENaC activity in renal principal cells. The observation that inhibiting PLC with U73122 [GenBank] prevented both apical plasma membrane PI(4,5)P2 depletion and decreases in ENaC activity by ATP further supports this hypothesis. We also find that recovery of ENaC activity following PI(4,5)P2 depletion in cells with inhibited PLC involves PI(4,5)P2 resynthesis. This observation is similar to that reported previously for recovery of KCNQ K+ channel activity following inhibition by G protein-coupled muscarinic receptors in sympathetic neurons (reviewed in Refs. 6 and 41).

The similar time courses for decreases in apical PI(4,5)P2 levels and ENaC activity following purinergic stimulation suggest tight spatiotemporal coupling between the channel and this signaling molecule. We previously reported that changes in ENaC activity also parallel decreases in PI(4,5)P2 levels on activation of receptor tyrosine kinases (44). These observations are consistent with ENaC directly interacting with PI(4,5)P2. Findings that direct application of PI(4,5)P2 to excised patches prevents spontaneous rundown of ENaC activity (48) further support this idea. Moreover, putative binding sites for PI(4,5)P2 within the intracellular domains of β- and {gamma}-ENaC subunits have recently been proposed (17, 48).

An important finding of this study is that resting apical PI(4,5)P2 levels set basal ENaC activity. Our TIRF experiments show that inhibition of PLC in the absence of purinergic stimulation clamps PI(4,5)P2 to a new higher level. This is associated with dramatic increases in ENaC activity. In contrast, depletion of apical PI(4,5)P2 with ATP causes significant decreases in ENaC activity. This leads to the possibility that ENaC has low affinity for PI(4,5)P2 since resting PI(4,5)P2 levels are not saturated with respect to modulating ENaC. The high level of basal PLC activity suggests that paracrine release of ATP tonically inhibits ENaC via P2Y receptors in principal cell monolayers. Similar paracrine regulation of ENaC by endogenous ATP via P2Y receptors was reported for rabbit airway epithelia (26). However, release of intracellular Ca2+ but not PI(4,5)P2 depletion was identified to be the major cause of this latter inhibition.

Thus ENaC is sensitive to apical plasma membrane PI(4,5)P2 levels. This allows for rapid adjustment of the rate of sodium reabsorption in principal cell monolayers in response to different inputs, including purinergic signaling, utilizing this second messenger.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-59594 and R01-DK-070571 and American Heart Association Grant EIA 0640054N (to J. D. Stockand).


   ACKNOWLEDGMENTS
 
We thank Jorge Medina for excellent technical assistance. We also thank Dr. A. Staruschenko for a critical reading of this manuscript and for making helpful comments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Pochynyuk, Dept. of Physiology, Univ. of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio TX 78229-3900 (e-mail: pochynyuk{at}uthscsa.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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