Mice lacking gamma ENaC palmitoylation sites maintain amiloride/benzamil-sensitive Na+ transport in vivo, despite markedly reduced open probability of typical ENaC-like channels.
Authors: Nickerson Andrew , Sheng Shaohu , Ray Evan , Kashlan Ossama , Mutchler Stephanie , Carattino Marcelo , Marciszyn Allison , Hughey Rebecca , Kleyman Thomas 
Published: May 2023

Background: Epithelial Na+ channels (ENaCs) play a major role in controlling extracellular fluid volume and electrolyte homeostasis by facilitating absorption of Na+ across transporting epithelia, such as the kidney and distal colon. ENaCs are heterotrimeric channels comprised of homologous α, β and γ subunits that are each regulated by various transcriptional and post-translational mechanisms. Studies using heterologous expression systems have shown that β andγ subunits undergo cys-palmitoylation in vitro, and that mutation of specific palmitoylation sites – particularly within the γ subunit – results in significantly reduced channel open probability. This study was designed to test whether γ-ENaC palmitoylation is necessary for channel function in vivo. We hypothesized that mice lacking γ subunit palmitoylation sites would have decreased channel activity and an impaired ability to maintain Na+ and fluid homeostasis during salt deprivation. Methods: 129SV mice lacking key palmitoylation sites (ENaCγC33A, C41A) were generated using CRISPR-Cas9 technology. We assessed ENaC function in dissected kidney tubules and colonic epithelia from WT and ENaCγC33A, C41A mice via patch clamp and Ussing chamber electrophysiology, respectively. We also used metabolic cages and quantitative magnetic resonance (QMR) analyses to evaluate the effects of ENaC palmitoylation in terms of systemic salt and fluid balance. Experiments were performed under either normal or Na+-restricted dietary conditions. Results: No differences in 1) body weight, 2) body water composition, 3) blood and urine parameters, or 4) ENaC subunit expression were observed between WT and ENaCγC33A, C41A mice. Both WT and ENaCγC33A, C41A mice also displayed robust ENaC activity in the distal colon following Na+ restriction, as assessed in Ussing chambers. However, ENaC-like channel activity (8 pS with Li+ as the charge carrier, assessed by cell attached patch clamp) was significantly lower in cortical collecting ducts (CCDs) of ENaCγC33A, C41A versus WT mice. Plasma aldosterone levels were also higher in ENaCγC33A, C41A versus WT male mice following Na+ restriction, while this was not observed in females. Surprisingly, ENaC-dependent Na+ transport at the whole kidney level (benzamil-sensitive natriuresis) was more robust in ENaCγC33A, C41A versus WT mice, suggesting the presence of transport mechanism(s) not detected by initial patch clamp analyses. We identified a second, larger conductance (20 pS) channel in the CCD with similar kinetics to canonical ENaCs. The activity of this channel was higher in Na+-restricted ENaCγC33A, C41A versus WT mice, and preliminary studies suggest that neither channel is observed in the presence of amiloride, providing a possible compensatory mechanism for diminished activity of typical ENaCs. Conclusion: γ-ENaC palmitoylation plays a role in regulating typical ENaC activity in vivo but is not an absolute requirement for Na+ (re)absorption in the kidney or distal colon. 5T32DK061296-20 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.