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

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 γENaC 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,C41A mice had reduced open probability compared with 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.

Proteolytic Cleavage of the ENaC γ Subunit – Impact Upon Na+and K+Handling

“…This likely reflects our use of 129/Sv as a background strain and is consistent with our other ENaC single channel recordings in CNTs/CCDs from this mouse strain (55). Recent evidence showed that impaired ENaC activity due to a  subunit mutation that prevents its palmitoylation was associated with increased activity of a higher conductance (20 pS), non-selective cation channel that is blocked by a high (50 M) concentration of benzamil (55). We determined NP O , N and NP O of the 20 pS channel in CNTs/CCDs from WT and  Q4/Q4 mice on a low Na + diet.…”

“…NP O did not differ in patches from  WT/WT (0.10 ± 0.06, N = 5) and  Q4/Q4 (0.21 ± 0.24, N = 7) mice (p = NS). We also observed a previously described 20 pS channel of unclear identity (55). This channel is non-cation selective and is inhibited by high (50 M) concentrations of benzamil (55).…”

“…We also observed a previously described 20 pS channel of unclear identity (55). This channel is non-cation selective and is inhibited by high (50 M) concentrations of benzamil (55).…”

“…Connecting tubules/cortical collecting ducts (CNTs/CCDs) were isolated manually with dissecting needle and tweezers and transferred to a cover glass coated with poly-lysine (Sigma-Aldrich, Inc., St. Louis, MO) for patch clamp studies, as previously described (55). CNTs/CCDs were opened with sharp pipettes under an inverted microscope, and principal cells were identified by morphology.…”