Session: 727 APS Renal ion transport, transporter regulation, and trafficking Poster Session
(727.4) Cellular and Molecular Mechanisms Regulating the Normal Physiological Function of the Epithelial Sodium Channel
Monday, April 4, 2022
10:15 AM – 12:15 PM
Location: Exhibit/Poster Hall A-B - Pennsylvania Convention Center
Poster Board Number: E197
Tarek Abd El-Aziz (University of Texas Health Science Center San Antonio, Minia University), Antonio Soares (University of Texas Health Science Center at Houston), Elena Mironova (University of Texas Health Science Center San Antonio), James Stockand (University of Texas Health Science Center San Antonio)
Postdoctoral Fellow University of Texas Health Science Center San Antonio San Antonio, Texas
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 (see attached Figure). 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 (see attached Figure). 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.
Support or Funding Information
This work was supported by National Institutes of Health (NIH) grants R01 HL139841 and R01 DK113816.
lt;section data-title="Acknowledgements" data-gtm-vis-recent-on-screen-10482319_401="6637" data-gtm-vis-first-on-screen-10482319_401="6637" data-gtm-vis-total-visible-time-10482319_401="10000" data-gtm-vis-recent-on-screen-10482319_393="6637" data-gtm-vis-first-on-screen-10482319_393="6637" data-gtm-vis-total-visible-time-10482319_393="10000" data-gtm-vis-has-fired-10482319_401="1" data-gtm-vis-has-fired-10482319_393="1"gt;This work was supported by National Institutes of Health (NIH) grants R01 HL139841 and R01 DK113816.
The figure shows the mechanisms and consequences of CKII and Ank-3 regulation of ENaC. CKII increases ENaC activity by phosphorylating β-ENaC within its anchor motif to promote Ank-3 mediated trafficking to the membrane. Our hypothesis is that phosphorylation by CKII of a key serine residue within the COOH-terminal of β-ENaC is necessary for ENaC to bind Ank-3 with the latter being necessary for appropriate channel locale and activity, which is required for the physiological function of ENaC.
