Structure of the human epithelial sodium channel by cryo-electron microscopy

The epithelial sodium channel (ENaC), a member of the ENaC/DEG superfamily, regulates Na+ and water homeostasis. ENaCs assemble as heterotrimeric channels that harbor protease-sensitive domains critical for gating the channel. Here, we present the structure of human ENaC in the uncleaved state determined by single-particle cryo-electron microscopy. The ion channel is composed of a large extracellular domain and a narrow transmembrane domain. The structure reveals that ENaC assembles with a 1:1:1 stoichiometry of α:β:γ subunits arranged in a counter-clockwise manner. The shape of each subunit is reminiscent of a hand with key gating domains of a ‘finger’ and a ‘thumb.’ Wedged between these domains is the elusive protease-sensitive inhibitory domain poised to regulate conformational changes of the ‘finger’ and ‘thumb’; thus, the structure provides the first view of the architecture of inhibition of ENaC.


Sample-size estimation
• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: No power analysis was used to determine sample size. Sample size for cryo-EM experiments was determined based on access to the Krios microscope at the Multiscale Microscopy Core (OHSU). For two-electrode voltage clamp electrophysiology experiments, sample size was dependent on the number of oocytes that were in optimal condition for RNA injection, as well as reproducibility of the recordings. Conditions were based on color and shape of the oocytes. The sample size for the cryo-EM experiments is in Figure  Biological replicates are defined as protein obtained following independent infections of HEK293S cells. Micrographs with ice contamination and aggregates were excluded from particle picking. At the 3D classification stage, classes that gave rise to maps that resemble a channel with Fabs bound were selected for final reconstruction and refinement. Details are provided in the cryo-EM flowchart in Figure 3 -figure supplement 1.
For electrophysiology experiments, discolored oocytes post-RNA injections were excluded from the experiments.

Statistical reporting • Statistical analysis methods should be described and justified
• Raw data should be presented in figures whenever informative to do so (typically when N per group is less than 10) • For each experiment, you should identify the statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures (e.g., mean, median, SD, SEM, confidence intervals; and, for the major substantive results, a measure of effect size (e.g., Pearson's r, Cohen's d) • Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.
Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage files, such as numerical data that are represented as a graph in you to upload relevant additional data a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" No statistical tests were used.
For Cryo-EM experiments, particles in micrographs were picked using the program DoGPicker, and Relion was used for 2D and 3D classification to remove ice contamination, micelles and denatured protein. For structure refinement using cryoSPARC, particles were split into two groups to generate half maps and then used to calculate gold standard FSC. More details provided in the Methods section. For Electrophysiology experiments, we characterized ion channel properties with different subunit composition/mutant combination. We measured activity of oocytes that demonstrated currents blocked by amiloride. Details provided in pages 7 & 8, legends of Figure  We have also provided numerical data as a source data corresponding to the graph shown in Figure 2 -figure supplement 3