Structural insights into subunit-dependent functional regulation in epithelial sodium channels

Abstract

Epithelial sodium channels (ENaCs) play a crucial role in Na⁺ reabsorption in mammals. To date, four subunits have been identified-α, β, γ, and δ-believed to form different heteromeric complexes. Currently, only the structure of the αβγ complex is known. To investigate the formation of channels with different subunit compositions and to determine how each subunit contributes to distinct channel properties, we co-expressed human δ, β, and γ. Using single-particle cryoelectron microscopy, we observed three distinct ENaC complexes. The structures unveil a pattern in which β and γ positions are conserved among the different complexes while the α position in αβγ trimer is occupied by either δ or another β. The δ subunit induces structural rearrangements in the γ subunit, which may contribute to the differences in channel activity between αβγ and δβγ channels. These structural changes provide molecular insights into how ENaC subunit composition modulates channel function.

Keywords: ENaC; Zn(2+) modulation; channel assembly; cryo-EM; heteromeric complex; ion channel; structural biology; subunit stoichiometry; δ subunit.

Figure 1.. Functional and biochemical characterization of the δβγ complex.

A, B. Representative traces of δβγ (A) and αβγ (B) with and without 100 μM Zn²⁺. The red and blue lines indicate application of 100 μM amiloride and 100 μM Zn²⁺, respectively. C. Current-voltage experiment in HEK293 cells demonstrating that δβγ_WT is Na⁺-selective, permeable to Li⁺ and impermeable to K⁺. Voltage potential range used for the experiment is −80 mV to 80 mV in 20 mV increments. The external solutions contained equimolar concentration of Na⁺, Li⁺, and K⁺. Internal solution contained K⁺. Each point is represented as mean ± SEM (n = 7). D. Normalized FSEC traces of purified δβγWT, δβγR138A, and δβγ_CYS monitored on the tryptophan fluorescence channel. Traces were normalized to the height of the peak at 14 mL. E. Representative current traces of δβγWT, δβγR138A, and δβγ_CYS expressed in HEK cells and in whole-cell patch-clamp experiments. Holding potential at −60 mV. F, G. Current-voltage experiments of δβγ_R138A (F) and δβγ_CYS (G) using the same conditions in C. Data are represented as mean SEM (n = 5 for δβγ_R138A and n=5 for δβγCYS). See also Figure S1.

Figure 2.. Cryo-EM analysis reveal two different heteromeric complexes.

A. Cryo-EM map of the extracellular domain of δβγ_CYS viewed parallel and perpendicular to the membrane plane viewed from the extracellular side, and in surface representation. The subunits δ, β, and γ are colored teal, red, and magenta respectively. The 10d4 Fab bound to the β subunit and other unmodeled densities are colored yellow. B. Cryo- EM map of ββγ_CYS viewed similarly as in A. The β¹ subunit is colored salmon. Only a small segment of the 10d4 Fab, colored yellow, is resolved after local refinement. C, D. The models of the extracellular domains of δβγ_CYS (A) and ββγ_CYS (B) viewed from the extracellular side and down the pore axis. They are in cartoon representation and colored as in Figure 2A and B E. Schematic illustration viewed from the extracellular side and looking down the pore axis of the three positions of the subunits in ENaC denoted as positions 1, 2, and 3. F. Inset: An overall view of the δβγ_CYS extracellular domain in cartoon representation. The close-up view of δ is rotated 180° relative to the view in the inset. The δ subunit follows a similar architecture as other ENaC subunits consisting of domains arranged like a hand grasping a ball. The unique domain first characterized in the human αβγ structure, the GRIP domain colored blue, is conserved in δ. The knuckle, finger, thumb, palm, and β-ball are colored cyan, purple, green, yellow, and orange, respectively. See also Figure S2.

Figure 3.. Position 1 subunit mediates changes in the extracellular domain

A, B. Cryo-EM maps of δβγ_CYS (A) and ββγ_CYS (B) showing the position and local map resolution of α2 helices, which include the tryptophans forming the TriTrp triangle. Only the α2 helix map features are shown and colored according to the estimated local resolution. The rest of the map features are colored as in Figure 2. For clarity, the knuckle and α1 helices are removed in the views looking down the pore axis. C-E. Comparison of the extracellular domains of δβγ_CYS (C), ββγ_CYS (D), and αβγ (E, PDB:6WTH). The Cα positions of the tryptophans belonging to the TriTrp are shown as spheres. The distances between the Cα atoms are shown in Å. See also Figure S3.

Figure 4.. Unique conformational features in δ contribute to changes in the extracellular domains.

A. Superposition of all position 1 subunits show differences in the finger and β-ball domains. The δ, β¹, and α are colored teal, salmon, and blue. The subunits are shown in cartoon representation. B-D. Close-up view of boxed regions in A belonging to δ (B), β¹ (C), and α (D). The sidechains of β6-β7 and β7-β8 loop residues are shown in sticks representation. A 7-residue segment of the β7-β8 loop is shown along with the corresponding sequence. E. View looking down the pore axis to show relative positions of the finger domains in positions 1–3. The close-up views of the boxed area in position 1 are displayed. The sidechains of residues at corresponding positions in δ, β1, and α are depicted in stick form to illustrate the orientation of their side chains. The black dot marks the relative direction of the pore axis position. F. Close-up views of the boxed regions in E of positions 2 and 3 in the δβγ_CYS structure. The views are rotated ~120° clockwise (yellow) or counterclockwise (green) relative to the panels in E highlighted with a red box. G. Illustration of the position 1/position 3 interface focusing on the knuckle/finger domain contacts. Only the sidechain of the conserved tryptophan in the knuckle domain is displayed in stick form while the rest of the region is shown in cartoon. H. Equivalent interfaces in position 1/position 2 and position 2/position 3 in δβγ_CYS are shown and represented similarly as in G. See also Figure S4.

Figure 5.. The γ finger domain is altered in the presence of δ

A. Superposition of all γ subunits in position 3 from the three trimers. The subunits are displayed in cartoon representation and colored magenta (δβγ_CYS), light pink (ββγ_CYS), and gray (αβγ_6WTH). B-D. Close-up views of the boxed region in A. The sidechains of residues in β6-β7 loop in the finger domain are shown in stick form to demonstrate the positions of the side chains. E, F. Surface representation of the δβγ_CYS (E) and ββγ_CYS (F) models is shown. The positions of the γ-α2 helices in δβγ_CYS and ββγ_CYS relative to those of αβγ_6WTH are displayed. See also Figure S5.

Figure 6.. Trimer superpositions using the β subunit in position 2 as reference reveal global rearrangements in position 1.

A. View looking down the pore axis from the extracellular side. The δβγ_CYS and αβγ_6WTH trimers are shown in cartoon and subunits are colored as in figures 4 and 5. The helices are shown as cylinders. The β subunit occupying position 2 is colored red. The knuckle, finger, and thumb are opaque while the rest of the extracellular domains are transparent for clarity. B-D. Comparison of the finger and thumb domains of position 1 and 3 subunits in δβγ_CYS vs ββγ_CYS (B), δβγ_CYS vs αβγ_6WTH (C), and ββγ_CYS vs αβγ_6WTH (D). The β subunit in position 2 is omitted for clarity. The centers of mass of the finger and thumb domain helices, calculated using Pymol, are represented as spheres. The solid bars indicate the distances between the centers of mass. Angle between corresponding helices of the finger and thumb domains are shown. E. Overlay of β¹ and β² subunits in cartoon representation. F. Close-up views of the finger and thumb domains shown in E shown as cylinders. G, H. Cryo-EM maps of ββγ_CYS (G) and δβγ_CYS (H) showing a peptide-like map feature in the palm domain that is not observed in αβγ. Position 1 subunits are removed for clarity (right image). The molecules and other unmodeled densities in ββγ_CYS (G) and δβγ_CYS (H) are colored yellow and blue, respectively. I. Close-up view and superposition of the features observed in the lower palm domain shown in G and H. See also Figure S6.

Figure 7.. Heteromeric ENaC assembly intermediate shows β and γ assemble as dimers.

A. Cryo-EM map of the βγ-dimer with subunits and Fab colored as in figure 2. The second γ subunit is colored in light pink for clarity. Solid bars indicate the direction of the putative membrane plane. B. Schematic illustration of the dimer-dimer interaction shown in A with the missing position 1 subunit shown as dashed circle. C. Close-up view of the α2 helices, which include the tryptophans forming the TriTrp triangle, in the βγ complex. The α2 helix map features are shown as a surface representation and are colored according to the estimated local resolution. D. Cartoon representation of the βγ dimer with distances of the Cα atoms of conserved tryptophans in the α2 helices. The highlighted region in green is the γ-GRIP portion that is not resolved in ENaC trimers. This region mediates extensive interdimer contacts. E. Inset: An overall view of the δβγ_CYS extracellular domain in cartoon representation. The region highlighted with a yellow rectangle includes the finger and thumb domains of γ. Close-up views of the boxed region showing comparison of the α1, α2, α4, and α5 positions in the finger and thumb domains of γ from trimers to the γ from βγ-dimer. The γ from trimers are colored as in figures 4 and 5, while γ from the dimer is colored yellow. All helices are shown in cylinders. See also Figure S7.