ENaC phosphorylation facilitates ankyrin-3 interaction critical for renal sodium balance

Figure 1. CK2-dependent phosphorylation and Ank-3 binding clusters in the β-ENaC C-terminal region. A) Schematic of the β-ENaC subunit showing the C-terminal region containing Ank-3 binding sites (blue) and CK2 phosphorylation sites (red), indicated by the dashed box. B) β-ENaC C-terminal sequences numbered according to the full-length protein, highlighting an Ank-3 binding cluster and a nearby CK2 phosphorylation cluster. Alanine substitutions introduced into the CK2 phosphorylation cluster beginning at residue 631 (β_CK2), the Ank-3 binding cluster beginning at residue 625 (β_Ank-3), or both overlapping clusters (β_CK2+Ank-3) are highlighted in red and blue, respectively.

Figure 2. Phosphorylation of β-ENaC by CK2 within the anchoring motif is required for Ank-3-dependent regulation of channel trafficking and activity. A) Representative TIRF-FRAP images of COS-7 cells expressing eYFP-ENaC variants, including WT β-ENaC (top row), the CK2-site mutant (second row), the Ank-3-site mutant (third row), and the double-cluster mutant (fourth row). Images show prebleach, 10 s after bleach, and 10 min after bleach. B) Time-dependent FRAP curves at the plasma membrane for cells expressing WT eYFP-ENaC (black), the CK2-site mutant (red), the Ank-3-site mutant (blue), and the CK2+Ank-3 double mutant (green). Data represent mean recovery values from 8 to 10 cells obtained across 2–3 independent transfections corresponding to the imaging conditions shown in (A). C) Summary of FRAP values at 10 min after bleach for cells expressing WT and mutant eYFP-ENaC constructs. Individual data points are shown for WT (black), CK2-site mutants (red), Ank-3-site mutants (blue), and CK2+Ank-3 double mutants (green). Data are from the same experiments as in (A). D) Representative whole-cell current recordings from CHO cells expressing WT mENaC, the CK2-site mutant, the Ank-3-site mutant,…

Figure 3. Ank-3 binds to β-ENaC subunit, and if so, whether CK2 influences this binding. A) Representative FRET images from COS-7 cells coexpressing eYFP-βENaC and eCFP-Ank-3. Left panels show YFP and CFP fluorescence before bleaching; right panels show YFP and FRET images after bleaching. Top row: untreated cells. Middle row: cells treated with the CK2 inhibitor TBB. Bottom row: cells cotransfected with CK2. B) Quantification of FRET efficiency for cells expressing eYFP-βENaC and eCFP-Ank-3 under the conditions shown in (A): untreated (black), TBB-treated (red), and CK2-overexpressing cells (blue). Data are from 11 to 20 cells and are presented as mean ± SEM. C) FRET imaging of COS-7 cells coexpressing the βENaC mutant lacking CK2/Ank-3 phosphorylation and binding sites (eYFP-mt βENaC) together with eCFP-Ank-3. Cells are shown under three conditions: untreated (top row), TBB treatment (middle row), and CK2 overexpression (bottom row). YFP and CFP channels are displayed before (left columns) and after (right columns) YFP bleaching. D) Quantification of FRET efficiency for COS-7 cells expressing eYFP-mt βENaC together with eCFP-Ank-3 under the conditions shown in (C): untreated (black),…

Figure 4. Expression of CK2 in principal cells is necessary for ENaC activity in the mouse collecting duct. A) Left, schematic of the breeding strategy used to generate PC-CK2 KO mice by crossing CSNK2A1fl/fl animals with Aqp2-Cre transgenic mice. Right, representative gel containing PCR products from genotyping reactions for breeders (lanes 1 and 2) and PC-specific CK2 KO mice (lane 3). Bands corresponding to the Aqp2-Cre transgene and the floxed CK2 allele are indicated with arrowheads. The gel image was inverted (black to white) to enhance visual clarity without altering the underlying data. bp, base pairs. B, C) Representative gap-free ENaC single-channel current recordings obtained in the cell-attached configuration from the apical membrane of principal cells in freshly isolated collecting duct tubules from WT littermates and PC-CK2 KO mice. Traces are shown under baseline conditions (top) and after pretreatment of isolated tubules with TBB (200 nM, 30 min; bottom). Inward Na+ current is downwards with dashed lines indicating the closed state. D–F) Summary graph of ENaC activity (NPo), open probability (Po), and channel number (N) obtained from cell-attached recordings of WT…

Figure 5. Principal-cell CK2 deficiency alters systemic sodium excretion and urinary electrolyte composition. A) Cumulative Na+ excretion over a 9-h period in WT mice (black filled circles, n = 7), WT mice treated with TBB (open black circles, n = 8), PC-CK2 KO mice (red filled circles, n = 8), and PC-CK2 KO mice treated with TBB (open red circles, n = 9). Mice received an intraperitoneal Na+ load (100 µL, 0.9% NaCl) at time 0, and cumulative excretion was measured every 3 h. B) Cumulative Na+ excretion at the 9-h endpoint for WT and PC-CK2 KO mice with or without TBB pretreatment. Data are plotted as mean ± SEM. C–F) Summary graphs showing 24 h urine volume (mL), Osmolality (mmol/kg), urinary Na+ excretion (UNaV), and urinary K+ excretion (UKV) for WT littermate (black circles, n = 4) and PC-specific CK2 KO mice (red squares, n = 4). Data are displayed as mean ± SEM. Both male and female mice were used in similar proportions. Statistical significance for all data was assessed using two-tailed unpaired Student's t tests (ns, P ≥ 0.05; *P < 0.05).

Figure 6. Loss of CK2 or Ank-3 abolishes benzamil-sensitive Na+ reabsorption. A) Experimental protocol. WT littermate, PC-CK2 KO, and PC-Ank-3 KO mice were placed in metabolic cages and maintained on a low-Na+ diet. Urine samples were collected at 0, 24, 48, and 72 h. Benzamil (1.4 mg/kg, i.p.) was administered at 24 h. B) UNaV during low-Na+ diet in WT littermate, PC-CK2 KO, and PC-Ank-3 KO mice. Benzamil (1.4 mg/kg, i.p.) was administered at 24 h (red arrow). Data are presented as mean ± SEM.

Figure 7. CK2- and Ankyrin-3–dependent regulation of ENaC in principal cells of the cortical collecting duct. CK2 phosphorylates on a regulatory cluster within the β-ENaC C-terminal domain, comprising a CK2-associated cluster (red) positioned adjacent to an Ank-3-interacting cluster (blue), thereby enabling effective association with Ank-3. This interaction is thought to link ENaC to the ankyrin-spectrin cytoskeletal scaffold, promoting efficient trafficking and stable retention of the channel at the apical membrane. Elevated ENaC abundance and activity at the luminal surface facilitate Na+ entry, which is complemented by Na+ extrusion through the basolateral Na+/K+-ATPase, thereby sustaining transepithelial Na+ transport and contributing to renal salt homeostasis.