Molecular principles of assembly, activation, and inhibition in epithelial sodium channel
- Department of Biochemistry and Molecular Biology, Oregon Health & Science University, United States
- Vollum Institute, Oregon Health & Science University, United States
- Neuroscience Graduate Program, Oregon Health & Science University, United States
Figures
Figure 1 with 6 supplements
The unique molecular interactions at the subunit interface define heteromeric assembly of ENaC.
(a) Top-down cartoon schematic illustration of ENaC with α-β-γ counterclockwise as resolved by cryo-EM (top left) and three possible assemblies of ENaC based on the defined purification scheme (see …
Figure 1—figure supplement 1
Biochemical and functional characterization of ENaCFL.
(a) Schematic illustration of the ENaCFL subunit constructs. (b) Size-exclusion chromatogram of purified ENaCFL in complex with 7B1 and 10D4 Fabs (c) Representative SDS-PAGE of the purified protein. …
Figure 1—figure supplement 2
Cryo-EM initial data processing workflow.
CryoSPARC Blob Picker was used for automated particle picking, to select an initial set of 1,787,887 particles. Multiple rounds of 2D classification were performed to select true-positive 2D classes …
Figure 1—figure supplement 3
Cryo-EM data processing for the final map.
To remove false-positive particles, three rounds of 3D classification was performed using cryoSPARC heterogeneous refinement. For each round, only the best class was selected, and the other two …
Figure 1—figure supplement 4
Cryo-EM analysis of ENaCFL dataset.
(a) Representative 2D class averages of ENaCFL. (b) Fourier Shell Correlation plot of model vs masked map. (c) Angular particle distribution. (d) Local resolution estimate (e) Gold-standard Fourier …
Figure 1—figure supplement 5
Cryo-EM potential maps of different regions in ENaCFL map.
The α, β, and γ subunit maps are represented in blue, red and magenta colored mesh, respectively. ENaCFL coordinates are represented in grey stick superposed with the colored mesh maps.
Figure 1—figure supplement 6
Stereoview of cryo-EM potential maps of the GRIP domain in ENaCFL map.
The α, β, and γ subunit maps are colored in blue, red and magenta colored mesh, respectively. The ENaCFL coordinates are represented in grey stick superposed with the colored mesh maps.
Figure 2 with 1 supplement
Human ENaC is a heteromeric channel with three different subunits.
(a) Generated model of homomeric α ENaC using coordinates of the α subunit from the ENaCFL structure. The two additional α subunits that complete the trimer were generated around the three-fold axis …
Figure 2—figure supplement 1
The domains within the ENaC subunits favor a heteromeric assembly.
(a-c) Superpositions of the three subunits from the ENaCFL structure at the upper palm domain show that the α subunit is distinct from the β and γ subunits. There is a rigid-body shift of the α …
Figure 3
A cation binding site is located in the β6-β7 loop of the α subunit finger domain.
(a) Cartoon representation of ENaC perpendicular to the membrane. α, β and γ are colored blue, red and magenta, respectively. The orange box shows the region of the cation site that is speculated to …
Figure 4
The inhibitory peptides in α and γ interact distinctly with the gating domains.
(a) Cartoon representation of ENaC perpendicular to the membrane where the GRIP domains in all three subunits are shown as surface representation. α, β and γ are colored blue, red and magenta, …
Figure 5 with 3 supplements
3D focused classification of the inhibitory peptide pocket in the α subunit reveals important site for ENaC regulation.
(a) 3D classification of the GRIP domain in the α subunit revealed four major classes. Two classes clearly demonstrate the uncleaved and cleaved states of the α subunit representing 48% and 10% of …
Figure 5—figure supplement 1
Cryo-EM data analysis of all α-GRIP classes in cryoSPARC.
(a) Illustration of the region in which focused classification was performed. (b–e) Local resolution estimate, angular distribution and gold standard FSC of the resulting four maps from focused …
Figure 5—figure supplement 2
Cryo-EM data analysis of all γ-GRIP classes in cryoSPARC.
(a) Illustration of the region in which focused classification was performed. (b–f) Local resolution estimate, angular distribution and gold standard FSC of the resulting four maps from focused …
Figure 5—figure supplement 3
3D focused classification of the inhibitory peptide pocket in the γ subunit demonstrates heterogeneity in the inhibitory peptide cleavage states.
(a) 3D classification of the GRIP domain in the γ subunit revealed five major classes. The major class representing 36% of the particles clearly demonstrate the uncleaved state of the γ subunit. As …
Figure 6
The 7B1 Fab binds to the α subunit making contacts with residues in the finger and thumb domains.
(a) Top-down view of the ENaCFL in complex with 7B1 and 10D4. The subunits and Fabs are colored as in Figure 1. (b) View of the interaction between the 7B1 Fab and the α2/α3 helices of the finger …
Figure 7 with 1 supplement
7B1 binds to the α subunit independent of the cleavage state of the α subunit.
(a) Whole-cell patch clamp measurements of ENaC-mediated Na+ current indicate the 7B1 Fab does not alter current amplitude and shape. (b) Similar to the control current, 7B1 does not mediate acute …
Figure 7—figure supplement 1
7B1 binds to both uncleaved and cleaved ENaCFL.
(a, b) Biochemical characterization by Fluorescence size-exclusion chromatography (FSEC) shows that the 7B1 Fab binds to purified uncleaved (a) and cleaved ENaCFL (b) in the presence of 150 mM NaCl …
Author response image 1
Schematic illustration of human ENaC-α subunit (hENaC-α).
The two furin sites are illustrated as white dotted lines. The location of the epitope for the polyclonal antibody SC-210112 is illustrated in the schematic.
Tables
Table 1
IC50 values of ENaC for three different blockers (amiloride, phenamil mesylate and benzamil).
IC50 values (mean ± S.E.M) determined from dose-response curves for three different blockers (amiloride, phenamil mesylate and benzamil) at different holding voltages (-60 mV, -40 mV, -20 mV, 0 mV).
| IC50 values (nM) | |||
|---|---|---|---|
| Amiloride | Phenamil | Benzamil | |
| 0 mV -20 mV -40 mV -60 mV | 97.14 ± 21.62 80.05 ± 8.78 80.25 ± 11.37 86.34 ± 27.04 | 51.37 ± 10.42 49.97 ± 11.18 43.37 ± 11.86 51.01 ± 14.12 | 36.74 ± 13.25 29.41 ± 6.47 27.72 ± 6.65 32.90 ± 12.66 |
Table 2
Statistics of data collection, three-dimensional reconstruction, and model refinement.
| ENaCFL | |||
|---|---|---|---|
| Pre-merge dataset | 1 | 2 | 3 |
| Material Source | Membrane | Whole cell | Whole cell |
| Detergent | Digitonin | Digitonin | Digitonin |
| Fab | 7B1 and 10D4 | 7B1 and 10D4 | 7B1 and 10D4 |
| Microscope | FEI Krios | FEI Krios | FEI Krios |
| Voltage (kV) | 300 | 300 | 300 |
| Detector | Gatan K2 Summit | Gatan K2 Summit | Gatan K2 Summit |
| Defocus range (µm) | −0.8 – −2.2 | −0.8 – −2.2 | −0.8 – −2.2 |
| Exposure time (s) | 3 | 3 | 3 |
| Dose rate (e−/Å2/frame) | 1.0 | 1.0 | 1.0 |
| Frames per movie | 60 | 60 | 60 |
| Pixel size (Å) | 0.415 | 0.415 | 0.415 |
| Total dose (e−/Å2) | 60 | 60 | 60 |
| Motion correction | UCSF MotionCor2 | UCSF MotionCor2 | UCSF MotionCor2 |
| CTF estimation | CTFFIND 4 | CTFFIND 4 | CTFFIND 4 |
| Particle picking | cryoSPARC blob | cryoSPARC blob | cryoSPARC blob |
| 2D/3D classification | cryoSPARC 2.11 | cryoSPARC 2.11 | cryoSPARC 2.11 |
| 3D classification and refinement | Relion 3.0, | Relion 3.0, | Relion 3.0, |
| cryoSPARC 2.11, | cryoSPARC 2.11, | cryoSPARC 2.11, | |
| cisTEM 1.0 | cisTEM 1.0 | cisTEM 1.0 | |
| Symmetry | C1 | C1 | C1 |
| Particles processed | 172 954 | 218 428 | 71 549 |
| Resolution masked (Å) | 3.57 | 3.05 | 3.96 |
| Map Sharpening B-factor (Å2) | 91.8 | 87.3 | 97.9 |
| cryoSPARC 2.11 merged map | |||
| Merged Symmetry | C1 | ||
| Merged particle count | 252 071 | ||
| Merged resolution masked (Å) | 3.06 | ||
| cisTEM 1.0.0 merged map | |||
| Merged Symmetry | C1 | ||
| Merged particle count | 248 079 | ||
| Merged resolution masked (Å) | 3.11 | ||
| Initial model | 6BQN | ||
| Non-hydrogen atoms | 11 740 | ||
| Protein residues | 1 594 | ||
| Ligands (Na+, NAG) | 1, 10 | ||
| Resolution (FSC = 0.143, Å) | 3.06 | ||
| Molprobity score | 1.37 | ||
| Cβ deviations | 0 | ||
| Poor rotamers | 0.84% | ||
| Ramachandran outliers | 0 | ||
| Ramachandran allowed | 2.7% | ||
| Ramachandran favored | 97.3% | ||
| Bond length rmsd (Å) | 0.002 | ||
| Bond angle rmsd (°) | 0.390 | ||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | Amiloride-sensitive sodium channel subunit alpha isoform 1 | Synthetic | NCBI Reference Sequence: NP_001029.1 | Gene synthesized by BioBasic |
| Gene (Homo sapiens) | Amiloride-sensitive sodium channel subunit beta | Synthetic | NCBI Reference Sequence: NP_000327.2 | Gene synthesized by BioBasic |
| Gene (Homo sapiens) | Amiloride-sensitive sodium channel subunit gamma | Synthetic | NCBI Reference Sequence: NP_001030.2 | Gene synthesized by BioBasic |
| Cell line (Homo sapiens) | HEK293T/17 | ATCC | Cat #ATCC CRL-11268 | |
| Cell line (Homo-sapiens) | HEK293S GnTI- | ATCC | Cat #ATCC CRL-3022 | |
| Antibody | 7B1 mouse monoclonal | OHSU VGTI, Monoclonal Antibody Core | AB_2744525 | Isotype IgG2a, kappa, 1:2 molar ratio |
| Antibody | 10D4 mouse monoclonal | OHSU VGTI, Monoclonal Antibody Core | AB_2744526 | Isotype IgG1, kappa. 1:2 molar ratio |
| Recombinant DNA reagent | pEG BacMam | Gift from Eric Gouaux | Doi: 10.1038/nprot.2014.173 | |
| Chemical compound, drug | Amiloride hydrochloride hydrate | Sigma | Cat#: A7410 | |
| Chemical compound, drug | Phenamil Mesylate | Tocris | Cat#: 3379 | |
| Chemical compound, drug | Benzamil hydrochloride hydrate | Sigma | Cat#: B2417 | |
| Other | TRITC | ThermoFischer | Cat#: 46112 | |
| Software algorithm | HOTSPUR | Doi: 10.1017/s1431927619006792 | ||
| Software algorithm | MotionCor2 | Doi:10.1038/nmeth.4193 | SCR_016499 | https://emcore.ucsf.edu/ucsf-motioncor2 |
| Software algorithm | Ctffind4 | Doi: 10.1016/j.jsb.2015.08.008 | RRID:SCR_016732 | https://grigoriefflab.umassmed.edu/ctffind4 |
| Software algorithm | CryoSPARC | Doi:10.1038/nmeth.4169 | SCR_016501 | https://cryosparc.com/ |
| Software algorithm | cisTEM1.0.0 | Doi: 10.7554/eLife.35383 | SCR_016502 | https://cistem.org/ |
| Software algorithm | pyem | Doi: 10.5281/zenodo.3576633 | https://github.com/asarnow/pyem | |
| Software algorithm | Pymol | Pymol Molecular Graphics System, Schrodinger, LLC | RRID:SCR_000305 | http://www.pymol.org/ |
| Software algorithm | UCSF Chimera | Doi: 10.1002/jcc.20084 | RRID:SCR_004097 | http://plato.cgl.ucsf.edu/chimera/ |
| Software, algorithm | UCSF ChimeraX | Doi: 10.1002/pro.3235 | RRID:SCR_015872 | https://www.cgl.ucsf.edu/chimerax/ |
| Software, algorithm | Coot | Doi: 10.1107/S0907444910007493 | RRID:SCR_014222 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ |
| Software, algorithm | Phenix | Doi:10.1107/S2059798318006551 | RRID:SCR_014224 | https://www.phenix-online.org/ |
| Software, algorithm | MolProbity | Doi:10.1107/S0907444909042073 | RRID:SCR_014226 | http://molprobity.biochem.duke.edu |
