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The epithelial sodium channel, ENaC, present in kidney epithelia, plays an integral role in sodium homeostasis. Stimulated by the steroid hormone aldosterone, ENaC in the kidney reabsorbs sodium into the blood, thus increasing fluid retention, causing an increase in blood volume and mean blood pressure. ENaC, a hetero-trimeric protein, consists of three subunits, α, β, and γ. One mechanism of altering ENaC activity involves the ENaC recycling pathway that retrieves functional channels from the surface of cells into an intracellular cytosolic compartment. Once within the intra-cellular compartment, ENaC has two alternative fates: it may be recycled to the surface membrane where it can continue to transport sodium or it may be targeted for degradation by the proteasome, a subcellular organelle that recognizes monomeric proteins and degrades them. Since ENaC is a trimeric protein, it cannot be degraded by the proteasome unless the channel is separated into its component subunits. Preliminary data suggests that P97 ATPase, an unusual chaperone protein, may play an integral role in the degradative pathway of ENaC. The current study aims to elucidate the role of P97 in ENaC recycling by confirming physical interaction between the two proteins and the impact of P97 protein on ENaC function and composition. Interaction between P97 protein and ENaC was confirmed by co-immunoprecipitation of P97 protein with ENaC subunits; however, apical membrane-resident ENaC appears not to be associated with P97 protein as determined by a strepavidin pull-down assay of biotin-labeled apical membrane-resident proteins, including ENaC apical membrane-resident subunits. ENaC subunit composition in whole-cell lysate was altered in cells with over-expressed P97 protein; these cells express endogenous, native ENaC subunit proteins.
To maintain homeostatic blood pressure in the body, the output of sodium and water must be closely regulated. The kidney is a major regulator for maintaining proper sodium and water balance because the nephron, a functional unit of kidney, filters a large amount of water and salt from the blood and then regulates blood sodium concentration by reabsorbing varying amounts of filtered sodium back into the blood; non-reabsorbed filtered sodium is finally excreted by the kidney. Low blood pressure is interpreted by the kidney as a state of low blood volume (i.e., reduced total body sodium and water). This is a signal for the kidney to excrete less but reabsorb more sodium and water, causing an increase in blood volume, and thereby restoring blood pressure, whereas high blood pressure results in reduced reabsorption and increased excretion of sodium and water, causing blood volume to fall, thus restoring blood pressure to its proper state.
Sodium re-absorption is controlled by the hormone aldosterone through regulation of the epithelial sodium channel, ENaC. ENaC is a hetero-trimeric protein consisting of three subunits, α, β, and γ. ENaC activity, and thus salt re-absorption, is controlled through either the channel-open probability or the number of functional ENaC at the cell’s apical membrane. The number of functional ENaC at the apical membrane is determined by the balance between the rate of ENaC insertion and the rate of the channel retrieval from the apical membrane. The internalized channel can be either recycled backed to the apical membrane or degraded.
Covalent coupling of a small peptide, ubiquitin, to ENaC promotes ENaC internalization and is a signal for degradation of the channel. However, once within the cell, the ubiquitin tag can be removed by de-ubiquinating enzymes, and the untagged ENaC then recycles back to the plasma membrane. If the ubiquitin is not removed, ENaC is degraded by the proteasomal complex. Because only monomeric proteins can be degraded through the proteasomal complex, the hetero-trimer ENaC must be separated into individual subunits. Since the association of the subunits in the hetero-trimer is energetically favorable, energy is required to separate the channels into individual subunits. This energy can be provided by an enzyme that can hydrolyse ATP, an ATPase. Since the chaperone protein P97 has the unusual ability to hydrolyze ATP, it could supply energy to separate the ENaC into individual subunits. In this study, we investigated the role of P97 in ENaC degradation and recycling pathways.
1st aim: Immunoprecipitation will be used to confirm that P97 associates with ENaC.
2nd aim: Immunoprecipitation and biotynlation, a small-molecule tagging technique that labels membrane proteins, will be used to determine if P97 associates with ENaC derived from the membrane.
3rd aim: Cells will be transfected with P97 cDNA to determine if over-expression of P97 protein is possible and the effects of P97 protein on ENaC composition.
Cell culture and harvesting.
2F3 cells, a clone of the Xenopus laevis distal nephron epithelial cell line, from passage numbers 99 to 104 were grown to confluent monolayers on 0.02mm Anapore membrane permeable support (Nalge NUNC) in media containing 1.5 µM aldosterone. After ten days of growth, cells were harvested at 4° C by washing twice in PBS buffer containing Ca2+ and Mg2+ and then scraped in PBS buffer containing a protease inhibitor cocktail (100 μM leupeptin, 100 μM antipain, 1 mM phenylmethylsulfonyl fluoride, 100 μM 1-chloro-3-tosylamido-7-amino-2-heptanone, and 100 μM L-1-tosylamido-2-phenylethyl chloromethyl ketone).
Cell lysis.
Harvested cells were lysed in one of three conditions: in Buffer D (0.25 M sucrose, 1 mM EDTA, 20 mM Tricine-NaOH, pH= 7.8), a non-denaturing lysis solution that retains native protein structure and protein-protein interactions; in Buffer D containing 1% digitonin, a detergent that solubilizes membranes but retains ENaC structure and, therefore, should retain the ENaC protein complexes; or in denaturing buffer, RIPA, containing detergents that solubilize membranes and disrupt protein-protein interactions. A protease inhibitor cocktail (100 μM leupeptin, 100 μM antipain, 1 mM phenylmethylsulfonyl fluoride, 100 μM 1-chloro-3-tosylamido-7-amino-2-heptanone, and 100 μM L-1-tosylamido-2-phenylethyl chloromethyl ketone) was added to all three lysis buffers. When lysing with Solution D or Solution D with 1% digitonin, the lysis solution was added to the harvested cell pellet and then dounced with a glass douncer followed by passing through a syringe (25 G 5/8). Cells lysed in RIPA were not not dounced but were incubated at 4° C for 1 hour. After cells were lysed, whole-cell lysate was separated from nuclei and unlysed cells by centrifugation at 1,200 g for 15 minutes.
Immunoprecipitation.
Whole-cell lysate was immunoprecipitated with either anti- α-,β-, or γ-ENaC antibodies at a of 1:20 dilution and incubated at 4° C for overnight. The next morning, the lysate was incubated with pre-washed Protein-A beads at room temperature for two hours. The beads were washed with gentle lysis buffer (1% NaPO4, 10% Glycerol, 2 mM EGTA) and then eluted with buffer containing 100 mM DDT and 5% SDS. The volume of the eluted sample was reduced in a speed vacuum.
Biotinylation and isolation of biotinylated proteins.
Confluent 2F3 cells grown on permeable support were washed twice with PBS buffer containing Ca2+ and Mg2+. The cells’ apical membranes were labeled with borrate buffer (85 mM NaCl, 4 mM KCl, 15 mM Na2B4O7· 10H2O ) containing 0.5 mg/mL biotin (Thermo Scientific) and incubated for 20 minutes at 4° C; the procedure was repeated. The apical side was washed first with media containing 10% serum and then three times with PBS containing 100 mM lysine and finally twice with PBS. Cells were then scraped into PBS containing Ca2+, Mg2+, and the protease inhibitor cocktail (100 μM leupeptin, 100 μM antipain, 1 mM phenylmethylsulfonyl fluoride, 100 μM 1-chloro-3-tosylamido-7-amino-2-heptanone, and 100 μM L-1-tosylamido-2-phenylethyl chloromethyl ketone). Cells were lysed with buffer D as mentioned before. Protein concentration of the lysate was determined using the BCA assay (Pierce, MA). Equal amounts of lysate proteins were incubated with pre-washed streptavidin coated beads overnight at 4° C to isolate biotinylated proteins. Beads were then washed 5 times with gentle lysis buffer and eluted in buffer containing 100 mM DDT and 5% SDS.
Transfection.
Cells were plated 24 hours prior to transfection and then transiently transfected with Vector PCDNA 3.1 containing P97 cDNA lipofectamine PLUS (Invitrogen) according to manufacturer’s instructions. Mock-transfected control cells were incubated with the lipofectamine PLUS reagents without the vector. Cells were grown for two days after transfection and then harvested. Prior to harvesting, trans-epithelial current was measured (EVOM, World Precision Instruments). Cells were harvested using PBS containing Ca2+, Mg2+, and the protease inhibitor cocktail (100 μM leupeptin, 100 μM antipain, 1 mM phenylmethylsulfonyl fluoride, 100 μM 1-chloro-3-tosylamido-7-amino-2-heptanone, and 100 μM L-1-tosylamido-2-phenylethyl chloromethyl ketone) and lysed with RIPA as mentioned before.
Immunoblotting.
Precipitated proteins were separated on a linear 7.5% SDS-PAGE and then transferred onto a nitrocellulose membrane. The membrane was blocked with TBS buffer containing 5% milk and 0.1% Tween-20. The blots were incubated overnight at 4° C with either rabbit anti-ENaC subunit antibodies or mouse anti-P97 antibodies at a 1:100 dilution followed by secondary antibody incubation, rabbit IgG or mouse IgG (KPL), at a 1:5000 dilution for two hours at room temperature. The secondary antibody was conjugated to alkaline phosphate. The antigen-antibody complex was imaged using the CDP-star (Tropix, MA) chemiluminescent detection system and a Kodak 2000M camera (Eastman Kodak Company, NY).
Figure 1: Confluent monolayers of 2F3 cells were grown on permeable supports and subsequently scraped. To ensure that protein-protein interactions were preserved, cells were lysed in non-denaturing buffer. Cell lysate was immunoprecipitated with either anti-α-,β-, or γ-ENaC antibodies and incubated with Protein-A beads to isolate the antibody-ENaC protein complex. The isolated proteins were resolved on a 7.5% SDS-PAGE gel, transferred onto nitrocellulose membranes, and probed with anti-P97 antibodies. A band was observed at approximately 90 kDa, corresponding to the predicted molecular weight of the P97 protein; the same band at 90 kDa was also observed with 2F3 whole-cell lysate. A second band at 60 kDa corresponding to the heavy chain of immunoprecipitating antibodies was observed in the immunoprecipitate. A band of the correct size was observed with anti-P97 antibody in the α-,β-, and γ-ENaC immuno-precipitates, which suggests that P97 protein associates with ENaC subunits in whole-cell lysate.
Figure 2: 2F3 cells were grown on permeable support to confluence and then harvested. Cells were lysed in non-denaturing buffer containing 1% digitonin, a detergent that solubilizes cell membranes without disrupting ENaC protein structure and, therefore, preserves the ENaC protein complex. The lysate was immunoprecipitated with either anti-α-,β-, or γ-ENaC antibodies, and the antigen-antibody protein complexes were isolated with Protein-A coated beads. The isolated proteins were resolved using a 7.5% SDS-PAGE gel, transferred onto nitrocellulose membranes, and probed with anti-P97 antibodies. Bands were observed at 90 kDa and 60 kDa weights, corresponding to P97 protein and the heavy chain of the immunoprecipitating antibodies, respectively. The presence of P97 protein in whole-cell lysate containing digitonin further confirms our previous conclusion that P97 protein is associated with ENaC subunits in 2F3 whole-cell lysate.
Figure 3: 2F3 cells were grown on permeable supports until they were confluent. Apical membrane-resident proteins were labeled covalently with biotin, then harvested, and lysed in non-denaturing conditions. Since biotin has a very high affinity for streptavidin, the lysate was incubated with streptavidin-coated beads to isolate biotinylated proteins complexes. The isolated proteins were resolved in a 7.5% SDS-PAGE gel, transferred onto nitrocellulose membranes, and then probed with either anti-P97 antibody or anti-γ-ENaC antibody. Although biotin-labeled lysate contained a band corresponding to γ-ENaC, no band was observed corresponding to P97 protein. However, bands corresponding to both P97 (90 KDa) and γ-ENaC (approximately 70 KDa) were observed in whole-cell lysate blots that were resolved and probed at the same time as the strepavidin pull-down blots. Because P97 protein is absent in the biotinylated protein complex, we conclude that P97 does not associate with apical membrane-resident ENaC protein complexes.
Figure 4: 2F3 cells were plated on permeable support and then transiently transfected with vector containing P97 cDNA. Control cells were mock transfected with the transfection lipofectamine reagents without vector. Cells were grown for 2 days, harvested, and lysed in RIPA buffer. Equal amounts of protein were resolved on a 7.5% SDS-PAGE gel and then transferred onto nitrocellulose membranes. The membranes were probed with anti-P97, anti-α-,β-, and γ-ENaC antibodies. Bands of correct sizes were observed with P97 protein, α-, β-, and γ- ENaC antibodies. A comparison of P97 protein band intensities between transfected and control mock-transfected cells suggests that transfected cells contained significantly larger amount of P97 protein; therefore, the P97 protein was successfully over-expressed in transfected cells. Composition of ENaC appears to alter in the transfected cells since the α-subunit’s band intensity increased, suggesting an increase in amount of α-ENaC, and a shift in the γ-subunit from higher to lower molecular weight bands, indicating an increase in the cleaved form of γ-ENaC in cells with P97 over-expressed.
P97 is co-localized and physically interacts with ENaC subunits in intra-cellular vesicles.
P97 is not co-localized with apical membrane-resident ENaC.
Over-expression of P97 protein increases amount of α-ENaC and cleaved form of γ-ENaC
Detailed schematic of trafficking of functional, heterotrimeric ENaC. α, β, and γ ENaC heterotrimers are assembled and leave the ER to travel through the Golgi. They then traffic through a post Golgi compartment (step 1) before entering the apical membrane in phosphatidyl-inositol-rich lipid rafts (step 2). These channels are functional and responsible for most, if not all, Na+ transport (step 3). The subunit are stabilized by interaction with inositol phospholipids produced by phospatidylinositol kinases and GAP43 (step 4). The functional heterotrimers in lipid rafts are associated wit and ubiquitinated by an ENaC-specific ubiquitin ligase, Nedd4. Ubiquitination promotes internalization of the α, β, and γ trimers into a subapical, sorting compartment (step 5). ENaC can suffer two fates within this compartment. One fate is to be de-ubiquitinated by the enzyme, UCH-L3, and returned to a post-Golgi recycling compartment (step 9) from which they can return to the surface membrane (step 10). The other fate is to be captured by a chaperone molecule, P-97, that disassembles the trimer into individual, ubiquitinated subunits (step 6). Some of the ubiquitinated subunits will be recognized by the 26S proteasome and degraded (step 7) while others will be de-ubiquitinated by ubiquitin-specific proteases (like USP-45) and trafficked back to the Golgi (step 8).
Our data supports our model for ENaC trafficking:
P97 is an important chaperone in the endocytic ENaC trafficking pathway
P97 associates with ENaC within an intracellular compartment
P97 appears to alter ENaC subunit composition
To determine the effects of P97 on ENaC function, trans-epithelial current will be measured from either P97 over-expressing or P97 knockout 2F3 cells.
Patch-clamp analysis of P97 over-expression and P97 knockout cells will determine if the change in ENaC function is due to a change in either Po (channel-open probability) or N (the number of functional channels), or both Po and N.
Co-localization of P97 and ENaC within endosomal vesicles will be determined using confocal microscopy and also by co-immunoprecipitation of P97 and ENaC subunits in isolated intracellular compartments.
Thanks to the members of Dr. Douglas Eaton’s lab for their support.
This work was supported in part by the Howard Hughes Medical Institute and NIH grant, DK037963.
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