Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical “anchor” ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electro physiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice.
Fig: CKII activity is necessary for ENaC trafficking to the plasma membrane and channel activity. (A) Fluorescence micrographs of COS-7 cells expressing eYFP-ENaC in the absence (top) and presence (bottom) of TBB treatment prior to photobleaching (left) and 10 s (middle) and 10 min (right) after photobleaching. Cells were treated with 200 nM TBB for 30 min. Images were collected with TIRF microscopy. (B) Time course of relative FRAP at the plasma membrane for cells expressing eYFP-ENaC in the absence (black circles) and presence of TBB (black squares). Summary data from experiments (n = 8–10 cells from 2–3 distinct transfections) identical to those shown in (A). (C) Summary graph of relative FRAP 10 min after photobleaching in cells expressing eYFP-ENaC in the absence (black circles, gray bar) and presence (black squares, white bar) of TBB. Summary data from experiments identical to those shown in (A). *P < 0.05 vs. absence of TBB. (D) Overlays of typical macroscopic current traces from representative CHO cells expressing mENaC in the absence (top) and presence of TBB (bottom) before and after 10 µM amiloride (dotted lines). Cells were treated with 200 nM TBB for 30 min prior to the recording. Currents elicited by voltage ramps stepped from a holding potential of 40 mV to 60 mV and ramped to − 100 mV. (E) Summary graph of ENaC activity (amiloride-sensitive current density at -100 mV) quantified in whole-cell voltage clamped CHO cells transfected with mENaC in the absence (black circles, gray bar) and presence (black squares, white bar) of TBB. Summary data from experiments (n = 8–9 cells from 3–5 distinct transfections) identical to those shown in (D). *P < 0.05 vs. absence of TBB.
Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in β-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a “switch” that favors Ank-3 binding to increase channel activity.
Abd El-Aziz, T.M., Soares, A.G., Mironova, E. et al. Mechanisms and consequences of casein kinase II and ankyrin-3 regulation of the epithelial Na+ channel. Sci Rep 11, 14600 (2021). https://doi.org/10.1038/s41598-021-94118-3