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Originally published In Press as doi:10.1074/jbc.M509071200 on September 9, 2005

J. Biol. Chem., Vol. 280, Issue 45, 37565-37571, November 11, 2005
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Identification of a Functional Phosphatidylinositol 3,4,5-Trisphosphate Binding Site in the Epithelial Na+ Channel*

Oleh Pochynyuk, Alexander Staruschenko, Qiusheng Tong, Jorge Medina, and James D. Stockand1

From the Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229-3900

Received for publication, August 17, 2005 , and in revised form, September 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na+ channel (ENaC). Whereas PI(3,4,5)P3 directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of {gamma}-mENaC just following the second trans-membrane domain (residues 569–583) important to PI(3,4,5)P3 binding and regulation. Deletion of this track decreases activity of ENaC heterologously expressed in Chinese hamster ovary cells. K-Ras and its first effector phosphoinositide 3-OH kinase (PI3-K), as well as RhoA and its effector phosphatidylinositol 4-phosphate 5-kinase increase ENaC activity. Whereas the former, via generation of PI(3,4,5)P3, increases ENaC open probability, the latter increases activity by increasing membrane levels of the channel. Deletion of the region just distal to the second trans-membrane domain disrupted regulation by K-Ras and PI3-K but not RhoA and phosphatidylinositol 4-phosphate 5-kinase. Moreover, PI(3,4,5)P3 binds ENaC with deletion of the region following the second transmembrane domain disrupting this interaction and disrupting direct activation of the channel by PI(3,4,5)P3. Mutation analysis revealed the importance of conserved positive and negative charged residues as well as bulky amino acids within this region to modulation of ENaC by PI3-K. The current results identify the region just distal to the second trans-membrane domain within {gamma}-mENaC as being part of a functional PI(3,4,5)P3 binding site that directly impacts ENaC activity. Phospholipid binding to this site is probably mediated by the positively charged amino acids within this track, with negatively charged and bulky residues also influencing specificity of interactions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Direct regulation of ion channel activity by phospholipid signaling molecules is becoming widely appreciated (reviewed in Ref. 1). This mechanism for ion channel modulation is recognized to be physiologically important for its disruption, in some instances, leads to disease (e.g. Bartter and Andersen syndromes) (2–5). Diverse types of ion channels, including those composed of subunits having only two trans-membrane domains, such as ENaC2 and inwardly rectifying K+ channels (Kir), as well as those with subunits having multiple (four or six) membrane-spanning domains, such as KCNQ, HERG, and two pore domain K+ channels, and transient receptor potential and cyclic nucleotide-gated channels, are modulated by phospholipids (6–13). Phospholipids, such as PI(4,5)P2 and PI(3,4,5)P3, directly interact with these channels to modulate gating (6, 9, 14).

ENaC is a heteromeric channel composed of three distinct but similar subunits: {alpha}, {beta}, and {gamma} (15, 16). Each subunit has an amino- and carboxyl-terminal cytosolic domain separated by two trans-membrane domains and a large extracellular region. ENaC serves an essential physiological function, since its activity is limiting for Na+ absorption across many epithelia, including that in the distal renal nephron (reviewed in Refs. 17–19). Thus, ENaC is well positioned to influence system Na+ balance and blood pressure. Indeed, gain and loss of function mutations in ENaC lead to inheritable forms of hypertension and hypotension, respectively, associated with inappropriate salt conservation and wasting at the kidney (20, 21). In addition, ENaC plays a critical role in hydration and fluid reabsorption across many mucosal membranes. Deletion and overexpression of ENaC correspondingly lead to excessively wet and dry air spaces with associated disease (21, 22).

The mineralocorticoid aldosterone increases ENaC activity. Several lines of evidence suggest a role for PI(3,4,5)P3 signaling in regulation of ENaC by aldosterone and other hormones, such as insulin (23–30). Aldosterone increases PI3-K activity and production of its phospholipid products (29). In some preparations, aldosterone activates PI3-K to augment ENaC activity via transcriptional control of the upstream PI3-K-regulator, K-Ras (31–33). Both K-Ras and PI3-K increase ENaC open probability (31–33). Moreover, we recently demonstrated a direct effect of PI(3,4,5)P3 on ENaC to increase channel open probability (33).

The phospholipid binding site in several types of ion channels, particularly that for PI(4,5)P2 binding to Kir and transient receptor potential channels, is becoming clear (2, 6–9, 34–39). Channel-phospolipid interactions are electrostatic in nature, where the negatively charged phosphate groups of anionic phospholipids interact with positively charged residues within the cytosolic domains of the channel. Moreover, the typical phospholipid binding site includes several regions/residues spread across the cystosolic domain(s) of the polypeptide (6, 8, 36–38). Importantly, the cystosolic domains just following trans-membrane domains, which often are rich in positively charged residues, appear to be particularly involved in phospholipid binding (6, 8–10, 36, 39). Once bound, phospholipids exert either an allosteric effect, changing the conformation of the channel, or change the free energy requirement for open and/or closed state transition to affect channel gating/open probability.

Compared with PI(4,5)P2 binding sites, much less is known about putative PI(3,4,5)P3 binding sites. This becomes particularly important when considering issues of specificity, because the former phospholipid is much more abundant in the plasma membrane than the latter. Recall, however, that aldosterone increases PI(3,4,5)P3 levels (29). Since ENaC is directly modulated by PI(3,4,5)P3 with this phospholipid increasing channel open probability, it serves as a good model to define domains and residues involved in PI(3,4,5)P3 binding and regulation of ion channels. We demonstrate here that the region just distal to the second trans-membrane domain in {gamma}-ENaC is involved in regulation of the channel by signaling cascades which activate ENaC via PI(3,4,5)P3. Moreover, deletion of this region disrupted ENaC association with PI(3,4,5)P3 and direct activation of the channel by PI(3,4,5)P3. Thus, the current results argue strongly that the conserved, positively charged residues immediately following the second membrane-spanning domain in {gamma}-ENaC play a functional role in mediating PI(3,4,5)P3 actions on ENaC activity and are involved in binding of this phospholipid to the channel.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All chemicals were of reagent grade and were purchased from either BioMol or Sigma unless noted otherwise. The mammalian expression vectors encoding Myc-tagged mouse ENaC subunits ({alpha}, {beta}, and {gamma}) have been described previously (32, 33, 40–44). Similarly, the deletion mutants {gamma}{Delta}573Q-600P, {gamma}{Delta}573Q-583R, and {gamma}{Delta}584T-600P (deletions of Gln573–Pro600,Gln573–Arg583, and Thr584–Pro600, respectively), which remove the region following the second trans-membrane domain in {gamma}-mENaC, have been described (43, 45). Site-directed mutagenesis of {gamma}-mENaC was performed using the QuikChange site-directed mutagenesis kit (Stratagene). All ENaC constructs were sequenced to ensure proper sequence, orientation, reading frame, and incorporation of substitution and deletion mutations. Expression vectors encoding K-Ras, PI3-K, RhoA, and PI(4)P5-K also have been described previously (32, 33, 42). All materials used in Western blot analysis were from Bio-Rad. PIP beads (PI(3,4)P2- and PI(3,4,5)P3-agarose as well as control agarose beads) were from Echelon Biosciences Inc. The mouse monoclonal anti-Myc antibody was from Clontech. Anti-mouse horseradish peroxidase-conjugated second antibody was from Kirkegaard-Perry Laboratories. ECL reagents were from PerkinElmer Life Sciences. CHO cells were from ATCC and maintained with standard culture conditions (DMEM + 10% FBS, 37 °C, 5% CO2) and transfected using the Polyfect reagent (Qiagen) as described previously (32, 33, 40–44).

Electrophysiology—Whole cell macroscopic and excised outside-out current recordings of mENaC expressed in CHO cells were made under voltage clamp conditions using standard methods (32, 33, 40–44). In brief, current through ENaC was the inward, amiloride-sensitive Na+ current with a bath solution of 160 mM NaCl, 1 mM CaCl2, 2 mM MgCl2, and 10 mM HEPES (pH 7.4) and a pipette solution of 120 mM CsCl, 5 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES (pH 7.4), 2.0 mM ATP, and 0.1 mM GTP. In some experiments, 2 mM GDP{beta}S replaced the 0.1 mM GTP in the pipette solution. Current recordings were acquired with either an Axopatch 200B (Axon Instruments) or a PC-505B (Warner Instruments) patch clamp amplifier interfaced via a Digidata 1322A (Axon Instruments) to a PC running the pClamp 9.2 suite of software (Axon Instruments). For whole cell recording, cells were held at 40 mV with voltage ramps (500 ms) from 60 down to –100 mV used to elicit current. ENaC activity is reported as the amiloride-sensitive current density at –80 mV. Whole cell capacitance was routinely compensated and was ~9 pF for CHO cells. Series resistances, on average 2–4 megohms, were also compensated.

For excised patches, gap-free current recordings were made at 0 mV with inward Na+ current downwards. Data were digitized at 500 Hz and filtered at 100 Hz. Displayed data were subsequently software-filtered at 20 Hz. Channel activity was expressed as NPo = I/i, where I represents mean current in a patch, and i is unitary current as determined by all point histograms. Open probability (Po) was established by normalizing NPo for channel number. Water-soluble, short-chain, dioctanoyl phospholipids were from Echelon Biosciences Inc. (Salt Lake City, UT). Aqueous phospholipid stocks were prepared at 1 mM by sonication for 30 min and stored at –70 °C. For outside-out experiments, stock phospholipids were mixed just prior to use with an equal volume of a carrier solution containing histone H1 (0.2 mM; Echelon Biosciences Inc.) and sonicated again for 10 min.

Biochemistry—Experiments testing precipitation of ENaC with phospholipids were performed using standard biochemical methods (40, 42, 44, 46–48). In brief, CHO cells expressing all three Myc-tagged ENaC subunits were extracted with gentle lysis buffer (76 mM NaCl, 50 mM Tris-HCl (pH 7.4), and 2 mM EGTA plus 1% Nonidet P-40 and 10% glycerol) supplemented with protease (1 mM phenylmethylsulfonyl fluoride) and phosphatase (0.1 mM NaPPi, 0.5 mM NaF, 0.1 mM Na2MoO4, 0.1 mM ZnCl2, and 0.04 mM Na3VO4) inhibitors. After standardizing total protein concentration, 40 µl of 10 nM PIP beads were added to 400 µg of whole cell lysate with a final volume of 400 µl and incubated together overnight at 4 °C. Precipitants were then washed three times with 400 µl of a 0.5 M NaCl, 25 mM NaH2PO4 (pH 8.0) solution, resuspended in sample buffer (0.005% bromphenol blue, 10% glycerol, 3% SDS, 1 mM EDTA, 77 mM Tris-HCl, and 20 mM dithiothreitol), and heated at 95 °C for 10 min. Precipitated proteins were separated by standard SDS-PAGE (7.5% gel), transferred to nitrocellulose, and probed with anti-Myc antibody in Tris-buffered saline supplemented with 5% dried milk and 0.1% Tween 20. Negative controls for these experiments included transfecting cells with only enhanced green fluorescence protein and incubating lysate containing ENaC with agarose beads lacking conjugated PIPs. Specific precipitation of Akt, which is known to interact with PI(3,4,5)P3 and PI(3,4)P2, by PI(3,4,5)P3/PI(3,4)P2 beads served as a positive control.

Statistics—All patch clamp data are presented as mean ± S.E. Data were compared using Student's t test, with p ≤ 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Deletion of the Region following the Second Trans-membrane Domain of {gamma}-ENaC Decreases Channel Activity—In earlier work (43, 45), we identified the region just distal to the second trans-membrane domain in {gamma}-ENaC as interacting with the plasma membrane or a membrane protein and playing a role in modulation of ENaC open probability and activity. This region is similar to that found in many other intrinsic membrane proteins, particularly to the portion of the polypeptide exiting the plasma membrane at the intracellular face of the lipid bilayer. Fig. 1A shows a sequence alignment of the region just distal to the second trans-membrane domain in {gamma}-ENaC from several different mammalian species. Investigation of this alignment reveals several well conserved positive charged residues. This conserved, polar region within {gamma}-ENaC, similar to that found in other phospholipid-sensitive ion channels (6, 8, 9, 39), separates the hydrophobic trans-membrane domain from the more hydrophilic cytosolic domain specifically standing at the cystosolic side of the {alpha}-helix spanning the membrane. Thus, this region is well positioned to influence channel gating.

To further explore the function of the region just distal to the second trans-membrane domain in {gamma}-ENaC, we created ENaC mutants lacking these residues and assessed the activity of the channel when heterologously expressed in CHO cells. Fig. 1B shows overlays of typical currents from CHO cells expressing wild-type ENaC and channels containing the {gamma}-ENaC deletion mutants, {gamma}{Delta}573Q-600P, {gamma}{Delta}573Q-583R, and {gamma}{Delta}584T-600P, before (arrow) and after the addition of the ENaC blocker, amiloride, to the bathing solution. Currents were elicited by voltage ramping from 60 to –100 mV. As summarized in Fig. 1C, channels containing the deletion mutants {gamma}{Delta}573Q-600P and {gamma}{Delta}573Q-583R had significantly decreased activity (101 ± 15 and 80 ± 13 pA/pF) compared with wild-type ENaC (213 ± 23 pA/pF) and ENaC containing {gamma}{Delta}584T-600P (180 ± 38 pA/pF). Since deletion of the region just following the second trans-membrane domain in {gamma}-ENaC does not influence oligomerization and delivery of the channel to the plasma membrane (43), these results are consistent with it playing a role in modulating channel gating.



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  		FIGURE 1.
Deletion of residues just distal to the second trans-membrane domain in {gamma}-ENaC decrease channel activity. A, sequence alignment for{gamma}-ENaC from mammalian species showing the region following the second trans-membrane domain. B, overlays of typical macroscopic currents from voltage-clamped CHO cells expressing wild-type ENaC and channels containing the {Delta}573Q-600P, {Delta}573Q-583R, and {Delta}584T-600P deletions of {gamma}-ENaC before (noted with an arrow) and after amiloride. Currents were evoked with a voltage ramp. C, summary graph of ENaC activity for wild-type and {gamma}{Delta}573Q-600P, {gamma}{Delta}573Q-583R, and {gamma}{Delta}584T-600P channels. *, versus wild type.

 
ENaC Lacking the Region following the Second Membrane-spanning Domain in {gamma}-ENaC Does Not Respond to PI3-K Signaling—We demonstrated previously that small, monomeric G proteins in both the Ras and Rho families increase ENaC activity (23, 32, 33, 42, 49). Co-expression of K-RasA and RhoA with ENaC significantly increased activity from 174 ± 18 (n = 14) to 430 ± 30 (n = 21) and 437 ± 82 pA/pF (n = 11), respectively (not shown; see Refs. 23, 32, 33, 42, and 49 and Fig. 4). The phospholipid kinases, PI3-K and PI(4)P5-K, are recognized downstream effectors of Ras and RhoA, respectively, and, when co-expressed with ENaC, similarly increase channel activity (23, 32, 33, 42, 49). Co-expression of PI3-K (461 ± 82 pA/pF, n = 18) and PI(4)P5-K (453 ± 50 pA/pF, n = 15) with ENaC significantly increased channel activity as expected (not shown; see Refs. 23, 32, 33, 42, 49 and Fig. 4). In CHO cells, K-Ras activates ENaC via PI3-K with its phospholipid products PI(3,4)P2 and PI(3,4,5)P3 increasing channel open probability (32, 33). In contrast, the primary effect of RhoA is to increase the number of ENaC in CHO cell plasma membranes via PI(4)P5-K and chronic increases in its product PI(4,5)P2 (42).

We took advantage of the different mechanisms by which Ras-PI3-K and RhoA-PI(4)P5-K activate ENaC in CHO cells to test whether the region following the second trans-membrane domain in {gamma}-ENaC plays a role in mediating PI(3,4)P2 and PI(3,4,5)P3 actions on the channel. Fig. 2 summarizes the effects of PI3-K, K-Ras, PI(4)P5-K, and RhoA on channels containing the {gamma}{Delta}573R-600P deletion mutant. Whereas both RhoA (188 ± 30 pA/pF) and PI(4)P5-K (289 ± 59 pA/pF) significantly increased the activity of these channels, K-Ras (88 ± 18 pA/pF) and its first effector PI3-K (102 ± 16 pA/pF) did not. As shown in Fig. 3, RhoA (165 ± 38 pA/pF) and PI(4)P5-K (404 ± 69 pA/pF) signaling also significantly increased activity of channels containing the shorter deletion mutant {gamma}{Delta}573Q-583R. Again, K-Ras (102 ± 12 pA/pF) and PI3-K (60 ± 12 pA/pF) did not. In contrast to the full deletion (Gln573–Pro600) and deletion of residues spanning Gln573–Arg583, which is a track that contains several conserved, positive charged amino acids, deletion of Thr584–Pro600, as shown in Fig. 4, had no effect on regulation by K-Ras (429 ± 85 pA/pF), PI3-K (375 ± 67 pA/pF), RhoA (467 ± 72 pA/pF), and PI(4)P5-K (369 ± 80 pA/pF). This latter portion of {gamma}-ENaC contains no conserved positively charged residues.



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  		FIGURE 2.
RhoA and PI5-K but not K-RasA and PI3-K increase the activity of channels containing the {Delta}573Q-600P deletion. Shown is a summary of the activity of {gamma}{Delta}573Q-600P channels in the absence and presence of co-expression of PI3-K, K-Ras, PI5-K, and RhoA. *, versus mutant ENaC alone.

 
ENaC activity in CHO cells is closely tied to cytoplasmic GTP levels, reflecting, in part, regulation of the channel by K-Ras and PI3-K (32, 33). Consistent with results in Figs. 2 and 3, dialysis of intracellular GTP with GDP{beta}S had a significantly greater effect on the relative activity of wild-type channels compared with channels containing the {gamma}{Delta}573Q-583R deletion mutant decreasing activity of the prior and latter to 32 ± 8% (n = 5) and 62 ± 5% of starting values (n = 8; after 2 min; data not shown), respectively. These results, as well as those in Figs. 2, 3, 4 strongly argue that the region following the second trans-membrane domain in {gamma}-ENaC plays a role in K-Ras-PI3-K and by extension PI(3,4)P2 and PI(3,4,5)P3 modulation of ENaC.

ENaC Lacking the Region following the Second Trans-membrane Domain in {gamma}-ENaC Does Not Respond to PI(3,4,5)P3—The results in Fig. 5 are from experiments directly testing the idea that the region just following the second trans-membrane domain in {gamma}-ENaC is critical to PI(3,4,5)P3 regulation of the channel. Shown in Fig. 5, A and B, respectively, are representative current traces in excised, outside-out patches for wild type ENaC and channels containing {gamma}{Delta}573Q-583R before (top) and after (bottom) the addition of exogenous PI(3,4,5)P3. Changes in activity for wild type and mutant ENaC upon the addition of PI(3,4,5)P3 are summarized in Fig. 5C, with phospholipid significantly increasing activity of wild type (from 1.94 ± 0.47 to 3.0 ± 0.37, n = 5) but not mutant channels (from 0.90 ± 0.29 to 0.84 ± 0.27, n = 5). Summarized in Fig. 5D is the relative increase in activity (NPo) and open probability (Po) in response to PI(3,4,5)P3 for wild type and mutant ENaC. ENaC containing {gamma}{Delta}573Q-583R had significantly smaller changes in relative activity and Po (1.0 ± 0.09 and 1.0 ± 0.08, n = 5, respectively) in response to PI(3,4,5)P3 compared with wild type channels (1.8 ± 0.26 and 1.8 ± 0.26, n = 5, respectively). Indeed, the addition PI(3,4,5)P3 failed to affect either variable for mutant channels but increased both for wild type channels. Thus, as expected and similar to earlier findings (see Refs. 33 and 43), channels containing {gamma}{Delta}573Q-583R had less activity compared with wild type channels, and PI(3,4,5)P3 increased activity of wild type channels. Importantly, however, this is the first observation that {gamma}{Delta}573Q-583R channels fail to respond to PI(3,4,5)P3. This finding is consistent with the idea that the 11 residues following the second transmembrane domain in {gamma}-ENaC are part of a PI(3,4,5)P3 binding site within the channel.



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  		FIGURE 3.
RhoA and PI5-K but not K-Ras and PI3-K increase the activity of channels containing the {Delta}573Q-583R deletion. Shown is a summary of the activity of {gamma}{Delta}573Q-583R channels in the absence and presence of co-expression of PI3-K, K-Ras, PI5-K, and RhoA. *, versus mutant alone.

 



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  		FIGURE 4.
K-Ras, PI3-K, RhoA, and PI5-K increase the activity of channels containing the {Delta}584T-600P deletion. Shown is a summary of the activity of {gamma}{Delta}584T-600P channels in the absence and presence of co-expression of PI3-K, K-Ras, PI5-K, and RhoA. *, versus mutant alone.

 
Deletion of the 11 Amino Acids following the Second Trans-membrane Domain in {gamma}-ENaC Disrupts PI(3,4)P2 and PI(3,4,5)P3 Binding to the Channel—We next tested whether ENaC directly interacts with PI(3,4)P2 and PI(3,4,5)P3 and what the effect of deleting the area just following the second trans-membrane domain in {gamma}-ENaC is on this possible interaction. Fig. 6A shows representative Western blots (n = 4) containing whole cell lysate and PI(3,4,5)P3 and PI(3,4)P2 precipitants from cells expressing wild-type ({alpha}, {beta}, and {gamma}) ENaC and channels containing the {gamma}{Delta}573Q-600P and {gamma}{Delta}573Q-583R deletion mutants. Expressed ENaC is Myc-tagged and identified in these blots with anti-Myc antibody. Negative controls for these phospholipid-ENaC interaction experiments included co-transfection with only control enhanced green fluorescence protein plasmid and precipitation with beads not conjugated with phospholipids (see also Fig. 6D). As a positive control, we asked with the experiments in Fig. 6B whether PI(3,4,5)P3 and PI(3,4)P2 but not PI(4,5)P2 specifically precipitated endogenous Akt. Shown in Fig. 6B is a Western blot probed with anti-Akt antibody containing whole cell lysate and PI(3,4,5)P3-, PI(3,4)P2-, and PI(4,5)P2 precipitants from CHO cells (lysate the same as in lane 1; Fig. 6A).

As summarized in Fig. 6C, ENaC interacts with phospholipids with deletion of residues Gln573–Pro600 and Gln573–Arg583 in {gamma}-ENaC disrupting this interaction. Deletion of Gln573–Pro600 significantly decreased relative PI(3,4)P2 and PI(3,4,5)P3 binding to 0.39 ± 0.12 and 0.15 ± 0.06 (n = 4), respectively. Similarly, deletion of Gln573–Arg583 significantly decreased relative PI(3,4)P2 and PI(3,4,5)P3 binding to 0.18 ± 0.02 and 0.14 ± 0.05 (n = 4), respectively. In contrast, channels containing {gamma}{Delta}584T-600P had normal interactions with phospholipids (not shown).

To test whether {gamma}-ENaC could directly interact with phospholipids in the absence of {alpha}- and {beta}-subunits, we overexpressed Myc-tagged {gamma}-ENaC alone in CHO cells. The left micrograph in Fig. 6D is of a representative (n = 3) Western blot containing the PI(3,4,5)P3 precipitants of control lysates (cells transfected with enhanced green fluorescence protein) and of lysates prepared from cells expressing wild type {gamma}-ENaC alone and the {Delta}573Q-600P and {Delta}573Q-583R deletion mutants alone. Also contained in the second lane of this blot is wild-type {gamma}-ENaC alone precipitated with control beads (beads not conjugated with phospholipids). This blot was probed with anti-Myc antibody. The right blot in Fig. 6D, which was also probed with anti-Myc antibody, is of whole cell lysates from cells expressing {gamma}-ENaC alone and the {Delta}573Q-600P and {Delta}573Q-583R deletion mutants alone, as well as cells only transfected with enhanced green fluorescent protein. Precipitants in the left blot were from these lysates.

Positively and Negatively Charged Residues in the Area following the Second Trans-membrane Domain in {gamma}-ENaC Modulate PI3-K Regulation of the Channel—To more fully explore the putative PI(3,4,5)P3-binding site immediately following the second trans-membrane domain in {gamma}-ENaC, we made targeted substitutions of the conserved positive charged residues in this region. Fig. 7A summarizes the effects on ENaC activity of alanine substitution mutations of conserved arginines and lysines within residues 569–583 in {gamma}-ENaC. For wild type ({alpha}-, {beta}-, and {gamma}-ENaC) channels, as well as those containing substituted arginines and lysines in the {gamma}-subunit, basal activity and activity in the presence of co-expression of PI3-K is reported. Simultaneous substitution of Arg569 and Arg570 had minimal effect on basal activity (213.0 ± 22.7 pA/pF (n = 14) versus 199.0 ± 30.0 pA/pF (n = 12)) but markedly attenuated activity in response to co-expression of PI3-K (438.8 ± 53.1 pA/pF (n = 13) versus 323.0 ± 43.0 pA/pF (n = 12)). In combination with substituting Lys574 and Lys576, this double arginine mutation significantly decreased ENaC activity in the presence of PI3-K to 294.1 ± 29.5 pA/pF (n = 12) but again had little effect on basal activity (210.3 ± 22.2 pA/pF, n = 12). In contrast, mutation of Lys574 and Lys576 in the absence of Arg569 and Arg570 had no effect on basal (223.5 ± 24.5 pA/pF, n = 12) or PI3-K-sensitive activity (482.0 ± 68.9 pA/pF, n = 12). Substitution of the last three arginines (Arg581, Arg582, and Arg583), however, had the single greatest effect, markedly decreasing basal activity to 173.0 ± 33.0 pA/pF (n = 12) and significantly decreasing activity in the presence of PI3-K to 202.0 ± 25.0 pA/pF (n = 12). Further substitution of Lys574 and Lys576 along with these three arginines resulted in basal and PI3-K-sensitive activities of 182.0 ± 27.0 (n = 12) and 213.0 ± 33.0 pA/pF (n = 12), respectively, with the latter again being significantly less than wild type in the presence of PI3-K. Similarly, substitution of all seven positive residues in the putative PI(3,4,5)P3 binding site resulted in markedly less basal activity (172.0 ± 20.0 pA/pF, n = 12) and significantly less activity in the presence of PI3-K (166.0 ± 25.0, n = 12). Thus, substitution of the three distal arginines within the putative PI(3,4,5)P3 binding domain had a greater relative effect on PI3-K-sensitive activity compared with mutation of the two proximal arginines, with mutation of these latter two arginines having a greater relative effect compared with substitution of the middle two lysines. These experiments then suggest that the conserved arginines play a greater role in PI(3,4,5)P3 binding compared with the conserved lysines. The contribution of the lysines to PI(3,4,5)P3 binding may be tempered by nearby negatively charged and bulky residues (see below).



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  		FIGURE 5.
PI(3,4,5)P3 directly activates wild type but not {Delta}573Q-583R ENaC in excised patches. A, representative ENaC current traces in an excised, outside-out patch before (top trace) and after (bottom trace) the addition of 20 µM exogenous PI(3,4,5)P3, patch clamped to 0 mV with inward current down. B, representative current traces for ENaC containing {gamma}{Delta}573Q-583R before (top trace) and after (bottom trace) the addition of PI(3,4,5)P3. All other conditions are the same as in A. C, summary graph (n = 5 for wild type and {Delta}573Q-583R) of ENaC activity before and after the addition of PI(3,4,5)P3. Both individual data points and means are shown for wild-type (black) and {Delta}573Q-583R (gray) channels with lines connecting paired data points. *, significant increase. D, summary graph of the relative increase in NPo and Po in response to 20 µM PI(3,4,5)P3 added to the bathing solution of patches containing wild type (black) and {Delta}573Q-583R (gray) channels. *, significantly lower compared with increases for wild type channels.

 



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  		FIGURE 6.
Deletion of the region just distal to the second trans-membrane domain in {gamma}-ENaC disrupts ENaC interactions with PI(3,4,5)P3 and PI(3,4)P2. A, representative Western blots (n = 4) from CHO cells expressing Myc-tagged wild-type {alpha}-, {beta}-, and {gamma}-ENaC and tagged channels having the {gamma}{Delta}573Q-600P and {gamma}{Delta}573Q-583R deletions. These blots were probed with anti-Myc antibody and contained whole cell lysate (top) and the PI(3,4,5)P3 precipitant (middle) and PI(3,4)P2 precipitant (bottom) of this lysate. Equal amounts of whole cell lysate (400 µg) were used for each precipitation. B, control blot probed with anti-Akt antibody. This blot contains whole cell lysate (left lane) from cells expressing ENaC in A, as well as the PI(3,4,5)P3, PI(3,4)P2, and PI(4,5)P2 precipitants from this lysate. C, summary graph quantifying disruption of PI(3,4,5)P3 and PI(3,4)P2 binding to ENaC by deletion of 573Q-600P and 573Q-583R in {gamma}-ENaC (n = 4). *, significant decrease. D, representative Western blots (n = 3) probed with anti-Myc antibody containing whole cell lysate (right) and the PI(3,4,5)P3 precipitant (left) from control cells expressing enhanced green fluorescence protein and cells expressing only Myc-tagged {gamma}-ENaC and tagged, deletion mutants of {gamma}-ENaC. The {gamma}-ENaC control lane in the left blot contains precipitant from control beads lacking conjugated PI(3,4,5)P3 and is from the same lysate as the first lane.

 
We also investigated the role of conserved negatively charged and bulky hydrophobic residues within the putative PI(3,4,5)P3-binding site. Shown in Fig. 7B is a summary graph of ENaC activity in the absence and presence of co-expression of PI3-K for wild type channels and channels containing {gamma}-subunits with conserved aspartic acid and tryptophan residues immediately following the second trans-membrane substituted with alanine. Channels containing {gamma}D577A compared with those containing wild type subunits had significantly greater basal activity (337.0 ± 37.4 (n = 12) versus 213.0 ± 22.7 (n = 14)) and activity in the presence of co-expressed PI3-K (668.5 ± 50.5 (n = 12) versus 438.8 ± 53.1 (n = 13)). Substitution of the two conserved bulky residues just distal to Asp577 in combination with the D577A mutation further augmented basal ENaC activity (563 ± 89, n = 12) and activity in the presence of PI3-K (895 ± 95, n = 12).



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  		FIGURE 7.
Conserved positively and negatively charged residues, as well as bulky aromatic amino acids in the region just following TM2 in {gamma}-ENaC, modulate PI3-K regulation of ENaC. A, summary graph showing ENaC activity in the absence (black bars) and presence of co-expression of constitutively active PI3-K (gray bars). Cells expressed either wild type {alpha}-, {beta}-, and {gamma}-ENaC or wild type {alpha}- and {beta}-ENaC plus {gamma}-ENaC containing the indicated (in boldface type) alanine-substituted amino acids just following TM2. *, versus activity in the absence of PI3-K; **, mutant ENaC + PI3-K significantly less then wild-type ENaC + PI3-K. B, summary graph showing ENaC activity in the absence (black bars) and presence of co-expression of constitutively active PI3-K (gray bars). Cells expressed wild type {alpha}-, {beta}-, and {gamma}-ENaC or wild type {alpha}- and {beta}-ENaC plus {gamma}-ENaC containing the indicated alanine-substituted aspartic acid and tryptophan residues just following TM2. *, activity of mutant ENaC in the absence and presence of PI3-K versus wild-type ENaC in the absence and presence of PI3-K, respectively. All groups had significantly more current in the presence of PI3-K versus in its absence.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The current results identify the region just following the second trans-membrane domain in {gamma}-ENaC as necessary for PI(3,4)P2 and PI(3,4,5)P3 binding to the channel and necessary for K-Ras, PI3-K, and PI(3,4,5)P3 regulation of ENaC. In CHO cells, K-Ras via PI3-K and production of its phospholipid products increases ENaC activity, with PI(3,4)P2 and PI(3,4,5)P3 directly increasing channel open probability (32, 33). RhoA via PI(4)P5-K, in comparison, increases ENaC activity in CHO cells by increasing the number of active channels in the membrane (42). In contrast to affecting regulation by K-Ras, PI3-K, and PI(3,4,5)P3, the region following the second trans-membrane domain in {gamma}-ENaC did not play a role in regulation of ENaC by RhoA and PI(4)P5-K. These results argue that the residues just following the second trans-membrane domain in {gamma}-ENaC are part of a functional PI(3,4)P2 and PI(3,4,5)P3 binding site that impacts ENaC gating.

Several other laboratories have also demonstrated regulation of ENaC and/or Na+ transport by PI3-K and its phospholipid product, PI(3,4,5)P3 (28–30, 50). Indeed, PI3-K and its phospholipid products play a central role in activation of ENaC in renal epithelial cells in response to both aldosterone and insulin. One effect of PI3-K and PI(3,4,5)P3 signaling on ENaC is to increase the membrane levels of the channel (26, 51). Increases in membrane levels of ENaC are mediated by direct phospholipid binding to the channel and/or activation of the PI3-K downstream effector, serum and glucocorticoid-regulated kinase (Sgk), which disrupts channel interactions with the ubiquitin ligase, Nedd4(-2) (24, 52, 53). With either of these mechanisms, PI(3,4,5)P3 signaling increases ENaC residency in the membrane. We demonstrated previously that PI(3,4,5)P3, in addition, has a more direct effect on ENaC, increasing channel open probability (33). This more direct effect on gating is similar to the actions of anionic phospholipids, such as PI(4,5)P2, on ENaC and other ion channels, including Kir (6, 14, 50, 54, 55). We emphasize that the functional PI(3,4,5)P3 binding site described in the current study plays a role in modulating ENaC gating. The current results provide no evidence for or against the idea that this site also plays a role in setting plasma membrane levels of ENaC.

Considering the direct effect of phospholipids on ENaC gating, it is important to define the domains within the channel responsible for coordinating interactions with phospholipids and for transducing phospholipid binding into changes in open probability. The current results demonstrate that {gamma}-ENaC in isolation from the other channel subunits can directly interact with PI(3,4,5)P3. Similar to the PI(4,5)P2 binding site in Kir and transient receptor potential channels (6, 8, 9, 36, 39), the PI(3,4,5)P3 and PI(3,4)P2 binding site in the COOH-terminal tail of {gamma}-ENaC contains several well conserved positively charged arginine and lysine residues. Specific substitution of these positively charged residues disrupted modulation by PI3-K. Thus, PI(3,4,5)P3 and PI(3,4)P2 binding to ENaC probably involves electrostatic interactions between positively charged residues and the negatively charged head groups of the phospholipids. In this respect, the PI(3,4,5)P3 and PI(3,4)P2 binding site in ENaC then is similar to the PI(4,5)P2 binding site found in Kir and transient receptor potential channels. Another shared feature with Kir and transient receptor potential is that the phospholipid binding site in ENaC includes, in part, residues just distal to the end of the second trans-membrane domain. We believe that the region just following the second trans-membrane domain in {gamma}-ENaC, in some manner, communicates with the channel gate to influence open probability. The experiments investigating PI(3,4,5)P3 actions on wild-type and mutant ENaC included in the current study are consistent with this idea. We believe, however, that the tract just following the second transmembrane domain in {gamma}-ENaC probably represents only a partial binding site. Our rationale is that other residues/regions within ENaC, namely positively charged, conserved residues in the amino terminus of {beta}- and {gamma}-ENaC, have also been implicated in phospholipid binding to the channel (54, 55). In this regard, then, ENaC-phospholipid interactions may continue to parallel Kir-phospholipid interactions, with phospholipid binding to both channel types probably involving residues in both the cytosolic amino and carboxyl termini of these channels.

The current results demonstrate that at least five of the seven positively charged residues immediately following the second trans-membrane domain in {gamma}-mENaC must be present for PI3-K regulation. Indeed, we find that both the proximal and distal arginines within the putative PI(3,4,5)P3 binding site, but not the middle two lysines, must be present for a complete response to PI3-K. Of these five arginines, the latter three (Arg581, Arg582, and Arg583) in mENaC are the most important. Interestingly, only five of the seven positively charged residues (Arg569, Arg570, Lys574, Lys576, and Arg582/Lys582) are absolutely conserved across all vertebrates, with only one of the critical three arginines (Lys582/Arg582) in mENaC being conserved. We speculate that this residue is the single most important with respect to PI(3,4,5)P3 binding, with the more proximal arginines and possible lysines contributing to binding to a lesser degree. Our results support the idea that the negatively charged and bulky residues near Lys574 and Lys576 probably temper the contribution of the conserved positively charged residues. Thus, the PI(3,4,5)P3 binding site identified by this study has a proximal and distal region with net positive charges separated by a more neutral bulky region. We propose that the conserved positively charged residues play a role in phospholipid binding, with the binding site actually being an amino acid track containing a diffuse net positive charge or two positive charged pockets separated by a more neutral bulky region that resides near the inner leaflet of the plasma membrane.

An unexpected but exciting finding was that alanine substitution of the conserved negatively charged aspartic acid residue and the bulky tryptophans within the region just following the second trans-membrane domain in {gamma}-ENaC increased basal activity and activity in response to PI3-K. Whereas the exact role of these residues with respect to phospholipid regulation remains to be determined, we believe that they, in a yet to be determined manner, influence the specificity of phospholipid-ENaC interactions and/or the maximum response to phospholipids. This speculation arises from parallel findings regarding phospholipid regulation of Kir channels, where mutation of neutral and negatively charged residues within the latter's phospholipid binding sites change the specificity and maximum response (6, 34).


   FOOTNOTES
 
* This research was supported by NIDDK, National Institutes of Health, Grant RO1-DK-59594 and American Heart Association, Texas Affiliate, Grant 0355012Y (to J. D. S.) and a National Kidney Foundation Research Fellowship (to A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: University of Texas Health Science Center at San Antonio, Dept. of Physiology 7756, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-4360; E-mail: stockand{at}uthscsa.edu.

2 The abbreviations used are: ENaC, epithelial Na+ channel; mENaC, mouse ENaC; Kir, inwardly rectifying K+ channel; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI3-K, phosphoinositide 3-OH kinase; CHO, Chinese hamster ovary; GDP{beta}S, guanyl-5'-yl thiophosphate; pF, picofarads; PI(4)P5-K, phosphatidylinositol 4-phosphate 5-kinase.. Back


   ACKNOWLEDGMENTS
 
We thank Pravina Patel for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hilgemann, D., Feng, S., and Nasuhoglu, C. (2001) Sci. STKE http://stke.sciencemag.org/cgi/content/full/oc_sigtrans;2001/111/re19
  2. Lopes, C., Zhang, H., Rohacs, T., Jin, T., and Logothetis, D. (2002) Neuron 34, 933–944[CrossRef][Medline] [Order article via Infotrieve]
  3. Schulte, U., Hahn, H., Konrad, M., Jeck, N., Derst, C., Wild, K., Weidemann, S., Ruppersberg, J. P., Fakler, B., and Ludwig, J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15298–15303[Abstract/Free Full Text]
  4. Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., Clark, J., Deymeer, F., George, A. L., Jr., Fish, F. A., Hahn, A., Nitu, A., Ozdemir, C., Serdaroglu, P., Subramony, S. H., Wolfe, G., Fu, Y. H., and Ptacek, L. J. (2001) Cell 105, 511–519[CrossRef][Medline] [Order article via Infotrieve]
  5. Donaldson, M. R., Jensen, J. L., Tristani-Firouzi, M., Tawil, R., Bendahhou, S., Suarez, W. A., Cobo, A. M., Poza, J. J., Behr, E., Wagstaff, J., Szepetowski, P., Pereira, S., Mozaffar, T., Escolar, D. M., Fu, Y. H., and Ptacek, L. J. (2003) Neurology 60, 1811–1816[Abstract/Free Full Text]
  6. Zhang, H., He, C., Yan, X., Mirshahj, T., and Logothetis, D. E. (1999) Nat. Cell Biol. 1, 183–188[CrossRef][Medline] [Order article via Infotrieve]
  7. Lopes, C. M., Rohacs, T., Czirjak, G., Balla, T., Enyedi, P., and Logothetis, D. E. (2005) J. Physiol. 564, 117–129[Abstract/Free Full Text]
  8. Dong, K., Tang, L., Macgregor, G. G., and Hebert, S. C. (2002) J. Biol. Chem. 277, 49366–49373[Abstract/Free Full Text]
  9. Prescott, E., and Julius, D. (2003) Science 300, 1284–1288[Abstract/Free Full Text]
  10. Bian, J. S., Kagan, A., and McDonald, T. V. (2004) Am. J. Physiol. 287, H2154–H2163
  11. Suh, B. C., and Hille, B. (2002) Neuron 35, 507–520[CrossRef][Medline] [Order article via Infotrieve]
  12. Spehr, M., Wetzel, C. H., Hatt, H., and Ache, B. W. (2002) Neuron 33, 731–739[CrossRef][Medline] [Order article via Infotrieve]
  13. Zhainazarov, A. B., Spehr, M., Wetzel, C. H., Hatt, H., and Ache, B. W. (2004) J. Membr. Biol. 201, 51–57[CrossRef][Medline] [Order article via Infotrieve]
  14. Xiao, J., Zhen, X. G., and Yang, J. (2003) Nat. Neurosci. 6, 811–818[CrossRef][Medline] [Order article via Infotrieve]
  15. Canessa, C. M., Horisberger, J. D., and Rossier, B. C. (1993) Nature 361, 467–470[CrossRef][Medline] [Order article via Infotrieve]
  16. Canessa, C. M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J. D., and Rossier, B. C. (1994) Nature 367, 463–467[CrossRef][Medline] [Order article via Infotrieve]
  17. Garty, H., and Palmer, L. G. (1997) Physiol. Rev. 77, 359–396[Abstract/Free Full Text]
  18. Snyder, P. M. (2002) Endocr. Rev. 23, 258–275[Abstract/Free Full Text]
  19. Kellenberger, S., and Schild, L. (2002) Physiol. Rev. 82, 735–767[Abstract/Free Full Text]
  20. Lifton, R. P., Gharavi, A. G., and Geller, D. S. (2001) Cell 104, 545–556[CrossRef][Medline] [Order article via Infotrieve]
  21. Hummler, E., and Horisberger, J. D. (1999) Am. J. Physiol. 276, G567–G571[Medline] [Order article via Infotrieve]
  22. Mall, M., Grubb, B., Harkema, J., O'Neal, W., and Boucher, R. (2004) Nat. Med. 10, 487–493[CrossRef][Medline] [Order article via Infotrieve]
  23. Tong, Q., Booth, R. E., Worrell, R. T., and Stockand, J. D. (2004) Am. J. Physiol. 286, F1232–F1238
  24. Bhargava, A., Fullerton, M. J., Myles, K., Purdy, T. M., Funder, J. W., Pearce, D., and Cole, T. J. (2001) Endocrinology 142, 1587–1594[Abstract/Free Full Text]
  25. Faletti, C. J., Perrotti, N., Taylor, S. I., and Blazer-Yost, B. L. (2002) Am. J. Physiol. 282, C494–C500
  26. Blazer-Yost, B. L., Esterman, M. A., and Vlahos, C. J. (2003) Am. J. Physiol. 284, C1645–C1653
  27. Paunescu, T. G., Blazer-Yost, B. L., Vlahos, C. J., and Helman, S. I. (2000) Am. J. Physiol. 279, C236–C247
  28. Blazer-Yost, B. L., Liu, X., and Helman, S. I. (1998) Am. J. Physiol. 274, C1373–C1379[Medline] [Order article via Infotrieve]
  29. Blazer-Yost, B. L., Paunescu, T. G., Helman, S. I., Lee, K. D., and Vlahos, C. J. (1999) Am. J. Physiol. 277, C531–C536[Medline] [Order article via Infotrieve]
  30. Record, R. D., Froelich, L. L., Vlahos, C. J., and Blazer-Yost, B. L. (1998) Am. J. Physiol. 274, E611–E617[Medline] [Order article via Infotrieve]
  31. Stockand, J. D., Spier, B. J., Worrell, R. T., Yue, G., Al-Baldawi, N., and Eaton, D. C. (1999) J. Biol. Chem. 274, 35449–35454[Abstract/Free Full Text]
  32. Staruschenko, A., Patel, P., Tong, Q., Medina, J. L., and Stockand, J. D. (2004) J. Biol. Chem. 279, 37771–37778[Abstract/Free Full Text]
  33. Tong, Q., Gamper, N., Medina, J. L., Shapiro, M. S., and Stockand, J. D. (2004) J. Biol. Chem. 279, 22654–22663[Abstract/Free Full Text]
  34. Rohacs, T., Lopes, C., Jin, T., Ramdya, P., Molnar, Z., and Logothetis, D. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 745–750[Abstract/Free Full Text]
  35. Du, X., Zhang, H., Lopes, C., Mirshahi, T., Rohacs, T., and Logothetis, D. E. (2004) J. Biol. Chem. 279, 37271–37281[Abstract/Free Full Text]
  36. Soom, M., Schonherr, R., Kubo, Y., Kirsch, C., Klinger, R., and Heinemann, S. H. (2001) FEBS Lett. 490, 49–53[CrossRef][Medline] [Order article via Infotrieve]
  37. Cukras, C. A., Jeliazkova, I., and Nichols, C. G. (2002) J. Gen. Physiol. 120, 437–446[Abstract/Free Full Text]
  38. Cukras, C. A., Jeliazkova, I., and Nichols, C. G. (2002) J. Gen. Physiol. 119, 581–591[Abstract/Free Full Text]
  39. Shyng, S. L., Cukras, C. A., Harwood, J., and Nichols, C. G. (2000) J. Gen. Physiol. 116, 599–608[Abstract/Free Full Text]
  40. Tong, Q., and Stockand, J. D. (2005) Am. J. Physiol. 288, F150–F161
  41. Staruschenko, A., Adams, E., Booth, R. E., and Stockand, J. D. (2005) Biophys. J. 88, 3966–3975[Abstract/Free Full Text]
  42. Staruschenko, A., Nichols, A., Medina, J. L., Camacho, P., Zheleznova, N. N., and Stockand, J. D. (2004) J. Biol. Chem. 279, 49989–49994[Abstract/Free Full Text]
  43. Booth, R. E., Tong, Q., Medina, J., Snyder, P. M., Patel, P., and Stockand, J. D. (2003) J. Biol. Chem. 278, 41367–41379[Abstract/Free Full Text]
  44. Booth, R. E., and Stockand, J. D. (2003) Am. J. Physiol. 284, F938–F947
  45. Hendron, E., Patel, P., Hausenfluke, M., Gamper, N., Shapiro, M. S., Booth, R. E., and Stockand, J. D. (2002) J. Biol. Chem. 277, 34480–34488[Abstract/Free Full Text]
  46. Casamayor, A., and Snyder, M. (2003) Mol. Cell. Biol. 23, 2762–2777[Abstract/Free Full Text]
  47. Gupta, S., Fanzo, J. C., Hu, C., Cox, D., Jang, S. Y., Lee, A. E., Greenberg, S., and Pernis, A. B. (2003) J. Biol. Chem. 278, 43541–43549[Abstract/Free Full Text]
  48. Hiromura, M., Okada, F., Obata, T., Auguin, D., Shibata, T., Roumestand, C., and Noguchi, M. (2004) J. Biol. Chem. 279, 53407–53418[Abstract/Free Full Text]
  49. Staruschenko, A., Pochynyuk, O. M., Tong, Q., and Stockand, J. D. (2005) Biochim. Biophys. Acta 1669, 108–115[Medline] [Order article via Infotrieve]
  50. Ma, H. P., Saxena, S., and Warnock, D. G. (2002) J. Biol. Chem. 277, 7641–7644[Abstract/Free Full Text]
  51. Blazer-Yost, B. L., Vahle, J. C., Byars, J. M., and Bacallao, R. L. (2004) Am. J. Physiol. 287, C1569–C1576[CrossRef]
  52. Snyder, P. M., Olson, D. R., and Thomas, B. C. (2002) J. Biol. Chem. 277, 5–8[Abstract/Free Full Text]
  53. Debonneville, C., Flores, S. Y., Kamynina, E., Plant, P. J., Tauxe, C., Thomas, M. A., Munster, C., Chraibi, A., Pratt, J. H., Horisberger, J. D., Pearce, D., Loffing, J., and Staub, O. (2001) EMBO J. 20, 7052–7059[CrossRef][Medline] [Order article via Infotrieve]
  54. Yue, G., Malik, B., Yue, G., and Eaton, D. C. (2003) J. Biol. Chem. 277, 11965–11969
  55. Kunzelmann, K., Bachhuber, T., Regeer, R., Markovich, D., Sun, J., and Schreiber, R. (2005) FASEB J. 19, 142–143[Abstract/Free Full Text]

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