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. 2014 May 16;289(20):14351-9.
doi: 10.1074/jbc.M113.526020. Epub 2014 Apr 1.

Cysteine palmitoylation of the γ subunit has a dominant role in modulating activity of the epithelial sodium channel

Anindit Mukherjee  1 , Gunhild M Mueller  1 , Carol L Kinlough  1 , Nan Sheng  1 , Zhijian Wang  1 , S Atif Mustafa  1 , Ossama B Kashlan  1 , Thomas R Kleyman  2 , Rebecca P Hughey  3
Affiliations

Cysteine palmitoylation of the γ subunit has a dominant role in modulating activity of the epithelial sodium channel

Anindit Mukherjee et al. J Biol Chem. .

Abstract

The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, β, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the β subunit, βCys-43 and βCys-557, are Cys-palmitoylated. ENaCs with mutant βC43A/C557A exhibit normal surface expression but enhanced Na(+) self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ subunit-activating cleavage. These mutant channels have significantly enhanced Na(+) self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the γ subunit places γCys-33 within an α-helix and γCys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the β and γ subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that γ subunit palmitoylation has a dominant role over β subunit palmitoylation in modulating ENaC gating.

Keywords: Acid-sensing Ion Channel (ASIC); ENaC; Gating; Ion Channels; Protein Palmitoylation.

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Figures

FIGURE 1.
FIGURE 1.
Two γ subunit cytoplasmic Cys residues are palmitoylated. α and β subunits were co-expressed with a wild type or mutant γ subunit (γWT, γC33A, γC41A, or γC33A/C41A with N-terminal HA and C-terminal V5 tags) in MDCK cells. Cys palmitoylation of the γ subunit in anti-V5 IPs was assessed with fatty acid-exchange chemistry where palmitate is removed with hydroxylamine treatment (+), using Tris treatment as a negative control (−), and replaced with biotin. A fraction was reserved to assess total γ subunit in an initial IP (10%), and biotinylated γ subunit was recovered with avidin-conjugated beads (90% biotin) for immunoblotting with anti-V5 antibodies. Note that the percentage of cleaved HA-γ-V5 subunit (revealed as the C-terminal 75-kDa V5-tagged fragment) is similar between the total IP and that eluted from the avidin-conjugated beads. This is consistent with efficient recovery of biotin-labeled cleaved N-terminal HA-tagged 18-kDa fragment (co-precipitating with the C-terminal V5-tagged 75 kDa fragment), as well as biotin-labeled full-length HA-γ-V5 subunit. The percentage palmitoylated is calculated from the difference in γ subunit biotinylation after treatment with hydroxylamine or Tris, relative to the γ subunit recovered in the total IP. A, representative immunoblots are shown with the mobility of the noncleaved (γ93) and cleaved (γ75) γ subunit on the left, and Bio-Rad molecular mass markers on the right. A control experiment was carried out to ensure that all biotinylated γ subunit was recovered by precipitation with avidin-conjugated beads (Av1), as a second incubation with avidin-conjugated beads (Av2) revealed no additional γ subunit. B, data from multiple experiments are presented as mean ± S.E. (error bars, n = 4–5) with levels of palmitoylation normalized to channels with a wild type γ subunit (1.0) in each experiment (3.3 ± 0.9% palmitoylated, n = 5). Only mutation of both Cys residues (γC33A/C41A) significantly reduced γ subunit palmitoylation (*, p < 0.05).
FIGURE 2.
FIGURE 2.
Channels with a γC33A, γC41A, or γC33A/C41A mutant exhibit reduced activity. Whole cell amiloride-sensitive Na+ currents were measured in Xenopus oocytes expressing nontagged wild type ((αβγ) WT) or channels with mutant γ subunits (n = 21–27). Data obtained 24 h after cRNA injections were normalized to wild type channels measured in the same batch of oocytes, and statistically significant differences for the mutants compared with wild type channels are indicated (***, p < 0.001 by one-way analysis of variance with Tukey's post hoc test). Data are presented as mean ± S.E. (error bars).
FIGURE 3.
FIGURE 3.
Surface expression and activating cleavage of ENaCs are not altered by the γC33A or γC41A mutation. Oocytes expressing channels with wild type or mutant γ subunits (γC33A or γC41A) were treated with membrane-impermeant sulfo-NHS-SS-biotin. Nontagged β and γ subunits were co-expressed with a double epitope-tagged α subunit (N-terminal HA and C-terminal V5) to assess α subunit processing. Nontagged α and β subunits were co-expressed with a double epitope-tagged γ subunit (N-terminal HA and C-terminal V5) to assess γ subunit processing. Oocytes were solubilized, 2% of the extract was incubated with anti-V5 antibodies conjugated to beads to measure total α or γ subunit, and surface biotinylated proteins were recovered with streptavidin-conjugated beads. Eluted samples were subjected to SDS-PAGE and immunoblotting (IB) with anti-V5 antibodies. Bands on scanned films were analyzed with Bio-Rad Quantity One Software. A, representative profiles are shown from one of three experiments. B, surface expression of ENaCs with mutant subunits was compared with ENaC with wild type subunits. The levels of surface expression in each experiment were normalized to wild type ENaC. Data are presented as mean ± S.E. (error bars; n = 3). No statistically significant differences were found (NS). C, the percentages of mature, cleaved α or γ subunits of surface (i.e. biotinylated) ENaCs were determined using the levels of immature noncleaved full-length (α95 kDa and γ93 kDa) and mature cleaved forms (α65 kDa and γ75 kDa) of the subunits. Percentage cleaved = (mature × 100)/(mature + immature). Data are presented as mean and S.E. (n = 3). No statistically significant differences were found where indicated (NS).
FIGURE 4.
FIGURE 4.
Channels with the γC33A or γC41A mutant exhibit enhanced Na+ self-inhibition. A, whole cell currents were measured in oocytes expressing ENaCs bathed in a low (1 mm) [Na+] bath and subsequently with high (110 mm) [Na+] bath as indicated by the bar in the representative tracings. The Ipeak and the Iss were recorded. ENaCs with γWT, γC33A, and γC41A had N-terminal HA and C-terminal V5 tags, whereas ENaC with γC33A/C41A had no epitope tag. B, the ratio of the Iss to the Ipeak is presented for ENaCs with wild type or mutant subunits as mean ± S.E. (error bars) (n = 16–51). Statistically significant differences for the mutants compared with wild type channels are indicated (***, p < 0.001).
FIGURE 5.
FIGURE 5.
Channels with the mutant γC33A exhibit a reduced Po. A, cell-attached patch clamp recordings were obtained from oocytes expressing wild type (WT) channels or channels with the γC33A or γC41A mutant. Patches were clamped as indicated. Representative recordings are shown for αβγ and αβγC33A. B, recordings of single channels lasting at least 5 min were selected for analysis. C, statistical analyses of Po values for ENaCs with mutant subunits were compared with wild type (*, p < 0.05, n = 9–17; error bars, S.E.).
FIGURE 6.
FIGURE 6.
γ subunit palmitoylation has a dominant effect over β subunit palmitoylation on ENaC activity. Whole cell amiloride-sensitive Na+ currents were measured in Xenopus oocytes expressing wild type ENaC or mutant channels lacking sites for β (αβC43A/C557Aγ) or γ (αβγC33A/C41A) subunit palmitoylation, or lacking palmitoylation sites on both subunits (αβC43A/C557AγC33A/C41A) (n = 25–43). All subunits lacked epitope tags. Data obtained 24 h after cRNA injection were normalized to channels with wild type subunits each day, and statistically significant differences between mutant and wild type channels were determined by analysis of variance followed by a Tukey's post hoc test for pairwise comparisons as indicated (***, p < 0.001; error bars, S.E.). There was also a statistically significant difference in activity between αβC43A/C557Aγ and αβγC33A/C41A, and between αβC43A/C557Aγ and αβC43A/C557AγC33A/C41A (###, p < 0.001). There was no statistical (NS) difference between αβγC33A/C41A and αβC43A/C557AγC33A/C41A.
FIGURE 7.
FIGURE 7.
ENaC activity is enhanced by co-expression of a palmitoyltransferase. A, whole cell amiloride-sensitive Na+ currents were measured in Xenopus oocytes expressing nontagged ENaC with either wild type subunits, mutant β subunit (αβC43A/C557Aγ), mutant γ subunit (αβγC33A/C41A), or both mutant β and γ subunits (αβC43A/C557AγC33A/C41A) (n = 14–51), with or without DHHC2. Amiloride-sensitive Na+ currents obtained 48 h after injection of cRNAs were normalized to currents measured in oocytes expressing wild type channels in the same batch. Data are presented as mean ± S.E. (error bars). Statistically significant differences with and without DHHC2 expression were observed for wild type αβγ (***, p < 0.0001), αβC43A/C557Aγ (***, p < 0.0001), and αβC43A/C557AγC33A/C41A (*, p < 0.05). DHHC2 did not alter αβγC33A/C41A currents (NS, not significant). Statistically significant differences were also observed for wild type αβγ + DHHC2 versus αβC43A/C557Aγ + DHHC2 (p < 0.001), αβγ + DHHC2 versus αβγC33A/C41A + DHHC2 (p < 0.001), and αβγ + DHHC2 versus αβC43A/C557AγC33A/C41A + DHHC2 (p < 0.0001) (not illustrated in the figure). Statistically significant differences were observed for αβC43A/C557Aγ versus αβγC33A/C41A (*, p < 0.05) and αβC43A/C557Aγ + DHHC2 versus αβγC33A/C41A + DHHC2 (***, p < 0.0001) but not for αβγC33A/C41A versus αβC43A/C557AγC33A/C41A (NS). Statistically significant differences were also observed between αβC43A/C557Aγ + DHHC2 versus αβC43A/C557AγC33A/C41A + DHHC2 (p < 0.0001), and αβγC33A/C41A + DHHC2 versus αβC43A/C557AγC33A/C41A + DHHC2 (p < 0.001) (not illustrated in the figure). B, whole cell amiloride-sensitive Na+ currents were measured in Xenopus oocytes expressing nontagged wild type ENaC subunits alone or ENaC subunits co-expressed with either DHHC2 or the mutant DHHC (C156S) (n = 28–32). Amiloride-sensitive Na+ currents obtained 48 h after injection of cRNAs were normalized to currents measured in oocytes expressing only wild type channels in the same batch. Statistically significant differences were observed when αβγ was expressed with DHHC2 (**, p < 0.001), but not with mutant DHHC2 (NS, not significant). Error bars, S.E.
FIGURE 8.
FIGURE 8.
DHHC2 co-immunoprecipitates with ENaC. Extracts of MDCK cells transfected with enhanced GFP-tagged DHHC2 (FW 71,350) and αβγENaC (all three subunits had C-terminal V5 epitope tags) were incubated with anti-V5-conjugated beads (V5) or control beads with no antibody (NA), and immunoprecipitates were immunoblotted with anti-GFP antibodies (Ab; IB: GFP). An aliquot of each cell extract (5%) was retained as “total.” Mobility of Bio-Rad molecular mass markers are on the right of the gel.
FIGURE 9.
FIGURE 9.
Model of sites of γ subunit palmitoylation. A, the rotation, angle, and length of the transmembrane domains 1 ((TM1) γLeu-55 to Ile-102) and 2 ((TM2) γAsn-528 to Phe-560) are based on the resolved structure of ASIC1 (5, 6). These domains are represented as rectangles (M1 and M2, respectively). The N-terminal His-Gly (HG) and C-terminal Pro-Tyr (PY) motifs are located at the indicated positions. Palmitate is indicated within the inner leaflet of the membrane as a wavy line. The presence of α-helices in the cytoplasmic domains, also represented as rectangles, was predicted using the PROF program (online). Two α-helix structures are predicted in the γ subunit cytoplasmic N terminus that include γGly-4 to Lys-13 and γThr-24 to Cys-33, placing γCys-33 at the terminus of an α-helix. γCys-41 is located between this α-helix and TM1. An α-helix is also predicted immediately following TM2 for the γ subunit (γIle-561 to Ala-580). It may represent a continuation of TM2 into the intracellular space, or exist as a distinct α-helix as depicted. B, alignment of mouse α, β, and γ subunit sequences within the N-terminal cytoplasmic domains, noting the HG motif (bolded) and surrounding residues. Palmitoylated βCys-43, γCys-33, and γCys-41 are underlined.
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