Atomic force microscopy reveals the architecture of the epithelial sodium channel (ENaC)

Abstract

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (α, β, and γ); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the α-, β- and γ-subunits. We show that for α-, β- and γ-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that αβγ-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.

FIGURE 1.

Epitope-tagged ENaCs are functional. A, representative whole-cell current traces from X. laevis oocytes expressing either wild type αβγ-ENaC (WT; left) or αHA/FLAG-ENaC, βHA/His₆-ENaC, and γHA/V5-ENaC (3xTag; right), at a holding potential of −60 mV. Amiloride (2 μm) was present in the bath to specifically inhibit ENaC as indicated by the black bars. Washout of amiloride revealed an inward current corresponding to the ENaC-mediated sodium current; reapplication of amiloride returned the whole-cell current toward the initial baseline level. B, summary of results of experiments obtained from oocytes expressing various combinations of tagged and untagged ENaC subunits. Only the C-terminal tag is indicated; all constructs also had an N-terminal HA tag. Numbers inside of the columns indicate the number of individual oocytes measured. Four different batches of oocytes were used. ***, p < 0.001, unpaired t test. C, representative single-channel current traces at different holding potentials (V_hold) from an outside-out patch of an oocyte expressing αHA/FLAG-, βHA/His₆-, and γHA/V5-ENaC. The current level at which all channels are closed was determined in the presence of amiloride (2 μm). D, average single-channel I/V plots calculated from recordings (four patches from two batches of oocytes) similar to those shown in (C). Binned current amplitude histograms (not shown for clarity) were used to determine the single-channel current amplitude (i) at each holding potential. The dashed line represents a Goldman-Hodgkin-Katz fit of the data for a Na⁺-selective channel with a predicted Na⁺ equilibrium potential (E_Na+) of 74.4 mV. Single-channel conductance (g) was calculated from the fit of the average I/V plots. E, representative single-channel current recording obtained at V_hold = −70 mV from an outside-out patch from an oocyte expressing αHA/FLAG-ENaC, βHA/His₆-ENaC, and γHA/V5-ENaC. Amiloride (ami; 2 μm), NaCl, KCl, or LiCl were present in the bath solution as indicated by the bars. The single-channel current amplitudes for Na⁺ and Li⁺ (i_Na+ and i_Li+, respectively) were determined as described in D. Note that no single-channel events were detected in the presence of KCl in the bath.

FIGURE 2.

Expression of ENaCs and isolation from tsA 201 cells. A, cells were transiently transfected with DNA encoding HA/V5-tagged α-, β-, or γ-ENaC. Cells were fixed, permeabilized, and incubated with either mouse monoclonal anti-HA or mouse monoclonal anti-V5 antibodies, followed by Cy3-conjugated goat anti-mouse secondary antibody. Cells were imaged by confocal laser scanning microscopy. Scale bar, 50 μm. B, cells were singly transfected with αHA/FLAG-ENaC, βHA/His₆-ENaC, or γHA/V5-ENaC. Cells were fixed, permeabilized, and incubated with mouse monoclonal anti-FLAG, rabbit polyclonal anti-His₆, or mouse monoclonal anti-V5 antibodies, as appropriate, followed by Cy3-conjugated goat secondary antibodies. C, detection of HA/V5-tagged α-, β-, and γ-ENaC in samples isolated from singly transfected cells by anti-HA affinity chromatography. Samples were analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody, followed by horseradish peroxidase-conjugated goat secondary antibodies, with enhanced chemiluminescence detection. Arrowheads indicate molecular mass markers (kDa). D, detection of αHA/FLAG-ENaC, βHA/His₆-ENaC, and γHA/V5-ENaC in samples isolated from triply transfected cells by anti-FLAG affinity chromatography. α-, β-, and γ-ENaC were visualized using anti-FLAG, anti-His₆, and anti-V5 antibodies, respectively.

FIGURE 3.

Determination of the assembly state of ENaC homomers. A, low magnification AFM image of αHA/V5-ENaC. Note that the sample contains particles of various sizes. Scale bar, 200 nm; shade-height scale, 0–4 nm. B, low magnification image of αHA/V5-ENaC that had been incubated with anti-V5 antibodies. Arrowheads indicate singly decorated particles; arrows indicate doubly decorated particles. Scale bar, 200 nm; shade-height scale, 0–4 nm. C, gallery of zoomed images of αHA/V5-ENaC (top), βHA/V5-ENaC (middle), and γHA/V5-ENaC (bottom) particles that are decorated by two peripheral particles (anti-V5 antibodies). Scale bar, 50 nm; shade-height scale, 0–2.5 nm. D–F, frequency distributions of molecular volumes of αHA/V5-ENaC (D), βHA/V5-ENaC (E), and γHA/V5-ENaC (F) particles that were decorated by two anti-V5 antibodies. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated. Note that the antibody-decorated particles shown in C are from the lowest volume peaks in D–F. G–I, frequency distributions of angles between pairs of anti-V5 antibodies bound to αHA/V5-ENaC (G), βHA/V5-ENaC (H), and γHA/V5-ENaC (I). The curves indicate the fitted Gaussian functions. The peaks of the distributions are indicated.

FIGURE 4.

Determination of the assembly state of ENaC heteromers. A, low magnification image of proteins isolated from cells expressing αHA/FLAG-ENaC, βHA/His₆-ENaC, and γHA/V5-ENaC. Note that the sample contains particles of various sizes. Scale bar, 200 nm; shade-height scale, 0–4 nm. B, low magnification image of a sample of isolated αβγ-ENaC that had been incubated with anti-His₆ antibodies and anti-V5 Fab fragments. The arrowhead indicates an ENaC particle that is singly decorated by an anti-His₆ antibody; the arrow indicates an ENaC particle that is singly decorated by an anti-V5 Fab fragment; an ENaC particle that is decorated by both an anti-His₆ antibody and an anti-V5 Fab fragment is circled. Scale bar, 200 nm; shade-height scale, 0–4 nm. C, frequency distribution of molecular volumes of ENaC particles that were decorated by both an anti-His₆ antibody and an anti-V5 Fab fragment. The curves indicate the fitted Gaussian functions. The means of the distribution are indicated. D, gallery of zoomed images of ENaC particles from the lowest volume peak in (C) that are decorated by both an anti-His₆ antibody and an anti-V5 Fab fragment. Scale bar, 50 nm; shade-height scale, 0–2.5 nm. E, schematic illustration of the alternative antibody/Fab decoration patterns seen in D. F, frequency distribution of angles between bound anti-His₆ antibodies and anti-V5 Fab fragments. The curve indicates the fitted Gaussian function. The peak of the distribution is indicated.

FIGURE 5.

Analysis of the higher-order structure of ENaC. A, low magnification image of proteins isolated from cells expressing αHA/FLAG-ENaC, βHA/His₆-ENaC, and γHA/V5-ENaC. Representative particles belonging to each of the three volume peaks in Fig. 4C are indicated as follows: circle, peak 1; box, peak 2; arrowhead, peak 3. The arrow indicates a triple particle. Scale bar, 200 nm; shade-height scale, 0–4 nm. B, gallery of zoomed images of triple particles isolated from cells expressing α-, β-, γ-, or αβγ-ENaC. Scale bar, 50 nm; shade-height scale, 0–2.5 nm. C, low magnification images of samples of isolated αβγ-ENaC that had been incubated with either anti-His₆ (left panel) or anti-V5 antibodies (right panel). Arrowheads indicate singly decorated ENaC particles; arrows indicate doubly decorated particles. Scale bar, 200 nm; shade-height scale, 0–4 nm. D, gallery of zoomed images of ENaC particles from volume peaks 2 and 3, isolated from cells expressing α-, β-, γ-, or αβγ-ENaC, that are decorated by two antibodies. Scale bar, 50 nm; shade-height scale, 0–2.5 nm. E and F, frequency distributions of molecular volumes of αβγ-ENaC particles that were decorated by two anti-His₆ (E) or anti-V5 (F) antibodies. The curves indicate the fitted Gaussian functions. The means of the distribution are indicated. Note the absence of the lowest volume peak seen in Fig. 3, D–F, and Fig. 4C.