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High blood pressure is a significant risk factor for cardiovascular disease. Many details about the molecular mechanisms that regulate blood pressure are unknown. The Epithelial Na+ Channel (ENaC) is the final arbiter of Na+ excretion in the kidneys. Appropriate renal sodium excretion is a key factor in the regulation of blood pressure. Abnormalities in ENaC function have been directly linked to several human diseases of blood pressure. The kinase, casein kinase II (CKII), phosphorylates ENaC but the physiological importance of this is obscure. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. Phosphorylation of Nav and KCNQ channels by CKII acts as a molecular “switch” favoring the binding of ankyrin-3 (Ank-3). The binding of Ank-3 facilitates the proper membrane localization of these channels increasing their activity. Here, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for Ank-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. We tested this hypothesis using endpoint measurements including analyses of ENaC activity and trafficking to the plasma membrane, and quantification of CKII-dependent ENaC-Ank3 binding. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching (TIRF/FRAP) 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 and blood pressure 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. Furthermore, we tested whether CKII influences Ank-3 binding to β-ENaC subunit using fluorescence resonance energy transfer (FRET). 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, promotes improper sodium excretion, and decreases blood pressure in KO mice. These results showed how and when CKII phosphorylation of ENaC functions as a “switch” to favor Ank-3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation. This work was supported by National Institutes of Health (NIH) grants R01 HL139841 and R01 DK113816. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cellular release of nucleotides is of physiological importance to regulate and maintain cell function and integrity. Also in the tubular and collecting duct system of the kidney, nucleotides are released in response to changes in cell volume or luminal flow rate and act in a paracrine and autocrine way on basolateral and luminal P2Y receptors. Recent studies using gene knockout mice assigned a prominent role to G protein-coupled P2Y(2) receptors, which are activated by both ATP and UTP. The antidiuretic hormone, arginine-vasopressin (AVP), and possibly an increase in collecting duct cell volume induce ATP release. The subsequent activation of P2Y(2) receptors inhibits AVP-induced cAMP formation and water reabsorption, which stabilizes cell volume and facilitates water excretion. An increase in NaCl intake enhances luminal release of ATP and UTP in the aldosterone-sensitive distal nephron which by activating apical P2Y(2) receptors and phospholipase C lowers the open probability of the epithelial sodium channel ENaC, thereby facilitating sodium excretion. Thus, the renal ATP/UTP/P2Y(2) receptor system not only serves to preserve cell volume and integrity but is also regulated by stimuli that derive from body NaCl homeostasis. The system also inhibits ENaC activity during aldosterone escape, i.e. when sodium reabsorption via ENaC is inappropriately high. The P2Y(2) receptor tone inhibits the expression and activity of the Na-K-2Cl cotransporter NKCC2 in the thick ascending limb and mediates vasodilation. While the role of other P2Y receptors in the kidney is less clear, the ATP/UTP/P2Y(2) receptor system regulates NaCl and water homeostasis and blood pressure.