Mechanisms and Consequences of Casein Kinase II regulation of the Epithelial Sodium Channel

Abstract

Activity of the Epithelial Na+ Channel, ENaC, in the distal nephron fine-tunes renal sodium excretion. Appropriate renal sodium excretion is a key factor in the normal regulation of blood pressure. Consequently, dysregulation of ENaC causes hypertension. Casein Kinase II phosphorylates ENaC. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. The binding of Ank-3 facilitates the proper membrane localization of ENaC increasing its activity. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and influence channel trafficking in a manner that increases activity. Herein, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for ankyrin-3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion and blood pressure. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching to follow movement of ENaC toward the plasma membrane in living cells and electrophysiology of ENaC activity in split-open collecting ducts from principal cell-specific CKII and Ank3 knockout mice. We also used whole animal physiological studies of sodium excretion in knockout mice to understand the physiological consequences of CKII and Ank3 regulation of ENaC. In addition, scanning mutagenesis was used to identify key residues in the overlapping CKII and Ank3 sites in ENaC. Findings showed that disrupting CKII signaling and abrogation of the CKII phosphorylation and Ank3 binding site within ENaC decreases channel trafficking toward the plasma membrane and activity, and promotes improper sodium excretion. These results showed that the CKII phosphorylation of ENaC functions as a “switch” that favors Ank-3 binding to increase channel activity.