Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na<sup>+</sup> transport despite reduced ENaC activity

Andrew J. Nickerson; Stephanie M. Mutchler; Shaohu Sheng; Natalie A. Cox; Evan C. Ray; Ossama B. Kashlan; Marcelo D. Carattino; Allison L. Marciszyn; Aaliyah Winfrey; Sebastien Gingras; Annet Kirabo; Rebecca P. Hughey; Thomas R. Kleyman

doi:10.1172/jci.insight.172051

Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na⁺ transport despite reduced ENaC activity

Andrew J. Nickerson,¹ Stephanie M. Mutchler,¹ Shaohu Sheng,¹ Natalie A. Cox,¹ Evan C. Ray,¹ Ossama B. Kashlan,¹ Marcelo D. Carattino,¹ Allison L. Marciszyn,¹ Aaliyah Winfrey,¹ Sebastien Gingras,² Annet Kirabo,³ Rebecca P. Hughey,¹ and Thomas R. Kleyman¹

Published September 14, 2023 - More info

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Abstract

Epithelial Na⁺ channels (ENaCs) control extracellular fluid volume by facilitating Na⁺ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γ ENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γ^C33A,C41A) using CRISPR-Cas9 technology. ENaCs in dissected kidney tubules from γ^C33A,C41Amice had reduced open probability compared to wild type (WT) littermates maintained on either standard or Na⁺-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na⁺ restriction. However, γ^C33A,C41A mice did not have reduced amiloride-sensitive Na⁺ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na⁺-restricted γ^C33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo, but are not necessary for amiloride/benzamil-sensitive Na⁺ transport in the distal nephron or colon.

Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na⁺ transport despite reduced ENaC activity

Andrew J. Nickerson,¹ Stephanie M. Mutchler,¹ Shaohu Sheng,¹ Natalie A. Cox,¹ Evan C. Ray,¹ Ossama B. Kashlan,¹ Marcelo D. Carattino,¹ Allison L. Marciszyn,¹ Aaliyah Winfrey,¹ Sebastien Gingras,² Annet Kirabo,³ Rebecca P. Hughey,¹ and Thomas R. Kleyman¹

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Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na+ transport despite reduced ENaC activity

Andrew J. Nickerson,1 Stephanie M. Mutchler,1 Shaohu Sheng,1 Natalie A. Cox,1 Evan C. Ray,1 Ossama B. Kashlan,1 Marcelo D. Carattino,1 Allison L. Marciszyn,1 Aaliyah Winfrey,1 Sebastien Gingras,2 Annet Kirabo,3 Rebecca P. Hughey,1 and Thomas R. Kleyman1

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Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na⁺ transport despite reduced ENaC activity

Andrew J. Nickerson,¹ Stephanie M. Mutchler,¹ Shaohu Sheng,¹ Natalie A. Cox,¹ Evan C. Ray,¹ Ossama B. Kashlan,¹ Marcelo D. Carattino,¹ Allison L. Marciszyn,¹ Aaliyah Winfrey,¹ Sebastien Gingras,² Annet Kirabo,³ Rebecca P. Hughey,¹ and Thomas R. Kleyman¹