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Front Physiol
2019 Jan 01;10:7. doi: 10.3389/fphys.2019.00007.
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Annexin II Light Chain p11 Interacts With ENaC to Increase Functional Activity at the Membrane.
Cheung TT
,
Ismail NAS
,
Moir R
,
Arora N
,
McDonald FJ
,
Condliffe SB
.
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The epithelial Na+ channel (ENaC) provides for Na+ absorption in various types of epithelia including the kidney, lung, and colon where ENaC is localized to the apical membrane to enable Na+ entry into the cell. The degree of Na+ entry via ENaC largely depends on the number of active channels localized to the cell membrane, and is tightly controlled by interactions with ubiquitin ligases, kinases, and G-proteins. While regulation of ENaC endocytosis has been well-studied, relatively little is understood of the proteins that govern ENaC exocytosis. We hypothesized that the annexin II light chain, p11, could participate in the transport of ENaC along the exocytic pathway. Our results demonstrate that all three ENaC channel subunits interacted with p11 in an in vitro binding assay. Furthermore, p11 was able to immunoprecipitate ENaC in epithelial cells. Quantitative mass spectrometry of affinity-purified ENaC-p11 complexes recovered several other trafficking proteins including HSP-90 and annexin A6. We also report that p11 exhibits a robust protein expression in cortical collecting duct epithelial cells. However, the expression of p11 in these cells was not influenced by either short-term or long-term exposure to aldosterone. To determine whether the p11 interaction affected ENaC function, we measured amiloride sensitive Na+ currents in Xenopus oocytes or mammalian epithelia co-expressing ENaC and p11 or a siRNA to p11. Results from these experiments showed that p11 significantly augmented ENaC current, whereas knockdown of p11 decreased current. Further, knockdown of p11 reduced ENaC cell surface population suggesting p11 promotes membrane insertion of ENaC. Overall, our findings reveal a novel protein interaction that controls the number of ENaC channels inserted at the membrane via the exocytic pathway.
FIGURE 1. p11 interacts with all three epithelial Na+ channel (ENaC) subunits. HA-tagged ENaC subunits were transiently expressed alone or together as an αβHAγ channel complex in COS-7 cells. Cells were then lysed, pre-cleared with GST bound to glutathione-agarose beads and then incubated with 50 μg of GST alone or GST-p11. After washing, bound proteins were eluted and analyzed by SDS-PAGE and immnoblotting (IB) with anti-HA antibody. The top panel labeled ‘Input’ shows the successful expression of each subunit (Mr 85 kDa). The bottom lane labeled ‘Pulldown’ shows binding of each ENaC subunit and the channel complex to GST-p11. The GST lane indicates that no ENaC subunits interacted with GST alone. Data shown are representative of three independent experiments with similar results.
FIGURE 2. p11 co-immunoprecipitates with ENaC. HEK293 cells were transfected with p11-myc-DDK and αβHAγ ENaC. After 24 h, cells were lysed and immunoprecipitated with anti-p11 (ab89438) and protein G-agarose beads. Precipitates were analyzed by SDS-PAGE and immunoblotted (IB) with anti-HA to detect βHA ENaC (Mr ∼ 72 kDa) and p11 (Mr ∼ 11 kDa). ENaC was co-immunoprecipitated in the presence of anti-p11 but not when an unrelated antibody was used. Data shown are representative of three independent experiments with similar results.
FIGURE 3. Identification of novel p11-ENaC interacting partners by LC–MS/MS analysis. (A) Schematic representation of the proteomics approach used to identify proteins interacting with the p11-ENaC complex. HEK293 cell lysates were co-transfected with p11-myc-DDK and αβ-HAγ ENaC. After 24 h expression, cells were lysed and co-immunoprecipitated sequentially with anti-myc-DDK and anti-HA with additional protein G-agarose beads in each tube. The eluent of the complex was then collected and subjected to SDS-PAGE 1D gel. The gel was carefully excised into three fragments and processed through in-gel trypsinization for LC–MS/MS analysis to determine potential proteins interacting with ENaC and p11. (B) MS spectrum recorded during analysis of purified protein complexes. Red text denotes specific peptides that identified proteins listed in Table 1. The list of identified peptides was used to tag the corresponding peptide-related ion spectra based on m/z differences, deviations from the predicted elution times, and the match between the theoretical and observed isotopic envelopes. The maximum deviation accepted in m/z and the retention time was established separately for each of the processed LC–MS spectra to account for possible variations in mass measurement accuracy and chromatographic separation between runs.
FIGURE 4. Effect of aldosterone on the endogenous expression of p11. M1 CCD or mCCDcl1 cells were grown to 80% confluence in full medium (F) then 24 h in dexamethasone and serum-free (SF) medium before treatment with 10 nM aldosterone or ethanol (veh) for 1, 3, or 24 h. Cells were then lysed and immunoblotted (IB) for p11 subunit (Mr ∼ 11 kDa) and annexin II (Anx II or annexin a2) subunit (38 kDa), or used for qRT-PCR. (A,C) Representative western blot showing the presence of endogenous p11 and annexin II proteins in M1-CCD cells and the effects of aldosterone versus vehicle treatment. (B,D) Quantification of p11 protein level normalized to annexin II in M1 CCD or mCCDcl1 cells in culture conditions described above. (E)
p11, Annexin II, and Sgk1 expression is shown relative to expression of 18S following 1 h of 10 nM aldosterone treatment. The threshold value was automatically set by the CFX Manager software and the Ct value was calculated from the average of three technical replicates. Data are expressed as mean ± SEM (n = 3–4 independent experiments) by one-way ANOVA followed by Bonferroni’s post-test where ∗ indicates a significant difference to control at a level of p < 0.05.
FIGURE 5. Epithelial Na+ channel current is increased by p11 co-expression. Oocytes were injected with α-, β-, and γ ENaC subunit cRNAs with or without p11 cRNA before recording whole cell Na+ currents using two electrode voltage clamp. (A) Representative whole cell Na+ currents from oocytes expressing ENaC alone (black line) or together with p11 (gray line) and the effect of perfusing 10 μM amiloride. (B) Relative amiloride-sensitive currents in oocytes injected with ENaC alone (n = 17) or together with p11 (n = 18). Oocytes were obtained from four different batches/animals. Amiloride-sensitive currents were measured as the difference before and after addition of 10 μM amiloride at a holding potential of –60 mV. Data are expressed as the mean (± SEM) fraction of amiloride-sensitive current from each oocyte relative to the ENaC control mean where ∗∗ indicates a significant difference to control at a level of p < 0.01 by unpaired t-test.
FIGURE 6. Knockdown of p11 reduces ENaC current. FRT epithelia were grown on SnapwellTM filters, and co-transfected with plasmids encoding α-, β-, and γ ENaC (0.067 μg each) together with either 20 pmol of control siRNA or p11 siRNA. Amiloride-sensitive short-circuit current (Isc-amiloride) was measured 72 h after transfection. (A) Representative traces of Isc-amiloride for control and p11 knockdown epithelia. (B) Pooled results of Isc-amiloride for control and p11 knockdown epithelia, where ∗∗ indicates a significant difference to control at a level of p < 0.01 or ∗∗∗p < 0.001 by one sample t-test, n = 5–7.
FIGURE 7. Knockdown of p11 reduces ENaC cell surface levels. FRT epithelia were cotransfected with plasmids encoding α-, βHA-, and γ ENaC (1 μg each) together with either control siRNA or increasing amounts of p11 siRNA (20 and 40 pmol). Cell surface proteins were labeled with Sulfo-NHS-LC-biotin and captured on Neutravidin beads after cell lysis. βHA ENaC was detected in both the cell surface and whole cell lysate fractions. (A) Representative immunoblot of surface βHA ENaC. (B) Pooled results show a significant decrease in the cell surface population of βHA ENaC with 40 pmol sip11, n = 3, ∗∗p < 0.01 by one-way ANOVA followed by Bonferroni’s post-test.
FIGURE 8. Schematic for p11 in ENaC trafficking. ENaC is translated at the endoplasmic reticulum (ER) and is modified by glycosylation, folded and assembled in the ER. ENaC is then moved to the cis-Golgi and emerges from the trans-Golgi network (TGN) in vesicles or tubules. The Annexin II-p11 tetramer may associate directly with vesicular ENaC and assist with apical targeting and tethering. At the apical membrane rab, v-SNARE, and t-SNARE molecules in the vesicle and apical membrane respectively will promote vesicle fusion, delivering ENaC to the apical cell surface. Not to scale.
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