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

Abstract

Kidneys play a pivotal role in long-term blood pressure regulation by controlling extracellular fluid volume through renal salt reabsorption. Discretionary control of the Epithelial Na⁺ Channel, ENaC, is the final step in fine-tuning renal Na⁺ excretion. Consequently, gain-of-function mutations in ENaC result in hypertension caused by inappropriate retention of Na⁺, whereas loss-of-function of this channel causes abnormal renal salt wasting. Thus, understanding how ENaC activity is regulated is important for a complete understanding of the molecular origins of hypertension. Our data show that casein kinase 2 (CK2) phosphorylates β-ENaC at a conserved motif homologous to the CK2 site in Nav1.2 and KCNQ channels, where phosphorylation controls ankyrin-3 (Ank-3) binding and channel membrane localization. In addition, CK2-dependent phosphorylation of β-ENaC is required for Ank-3 binding, which stabilizes ENaC at the apical membrane, sustains channel activity, and thereby ensures appropriate renal Na⁺ excretion. Using structure-guided mutagenesis of the β-ENaC C-terminus combined with advanced live-cell imaging (TIRF-FRAP, FRET) and patch-clamp electrophysiology, we found that mutation of the CK2 consensus site or disruption of the Ank-3-binding motif abolishes ENaC surface mobility and suppresses macroscopic current density. Principal cell-specific deletion of CK2 or Ank-3, or pharmacologic CK2 inhibition, eliminates ENaC activity in native collecting ducts and drives markedly accelerated urinary sodium loss in vivo. These results identify CK2-dependent Ank-3 scaffolding as an essential post-translational mechanism that maintains ENaC at the apical membrane and sustains renal Na⁺ reabsorption, thereby presenting a potential new targetable regulatory pathway with implications for sodium homeostasis and blood pressure control.