Cys palmitoylation of the beta subunit modulates gating of the epithelial sodium channel.
The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na(+) self-inhibition, and reduced single channel P(o) when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.
Mueller
Gunhild M
GM
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Maarouf
Ahmad B
AB
Kinlough
Carol L
CL
Sheng
Nan
N
Kashlan
Ossama B
OB
Okumura
Sora
S
Luthy
Sarah
S
Kleyman
Thomas R
TR
Hughey
Rebecca P
RP
D000584:Amiloride / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003198:Computer Simulation; D004285:Dogs; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology*; D054878:Lipoylation / Q000502:physiology*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D020125:Mutation, Missense; D009865:Oocytes; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D014982:Xenopus laevis
D053503:Epithelial Sodium Channels; D026941:Sodium Channel Blockers; D000584:Amiloride; D012964:Sodium