Mechanisms and consequences of casein kinase II and ankyrin-3 regulation of the epithelial Na+ channel

Abstract

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 electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. 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.

Stimulation of the Epithelial Na+ Channel in Renal Principal Cells by Gs-Coupled Designer Receptors Exclusively Activated by Designer Drugs

“…Total internal reflection fluorescence (TIRF) microscopy followed standard procedures ( Staruschenko et al, 2005 ; Abd El-Aziz et al, 2021 ). In brief, fluorescence emissions from membrane eYFP-tagged ENaC were collected in COS-7 cells at room temperature using TIRF microscopy to selectively illuminate the plasma membrane.…”

Lessons learned about epithelial sodium channels from transgenic mouse models

“…El-Aziz and coworkers now proposed that casein kinase II-phosphorylated bENaC promotes Ank-3-mediated trafficking to the membrane. Principal cell-specific Ank-3 knockout mice showed decreased ENaC activity and increased sodium excretion supporting evidence that Ank-3 binding increased channel activity [52]. The ankyrin repeat domain 36 (ANKRD36) mediates a variety of protein-protein interactions and was found to be significantly lower expressed in hypertension [53 && ].…”

Regulation of Epithelial Sodium Transport by SARS-CoV-2 Is Closely Related with Fibrinolytic System-Associated Proteins

“…The subcellular localization of ENaC on the apical side of epithelial cells determines either competition for the furin site by the SARS-CoV-2 spike protein in the Golgi apparatus or the failure of cleaved one to be smoothly transported to the apical side, which results in the functional loss of ENaC in pulmonary fluid regulation. It has been demonstrated that by interfering with casein kinase II (CK II) signaling or knocking down CK II phosphorylation and ankyrin-3 binding sites, the translocation and activity of ENaC are reduced, proving that CK II is essential for the function of ENaC and proper localization to the apical side [ 42 , 43 , 44 ]. A recent quantitative mass spectrometry-based phosphorylation proteomics study showed that two subunits of CK II, CSNK2B, and CSNK2A2, interacted with SARS-CoV-2 N proteins to drive changes by affecting the subcellular localization of CK II or sterically blocking the entry of kinases, which may interfere with the phosphorylation of β-ENaC and its subcellular localization ( Figure 1 II) [ 45 ].…”