Molecular principles of assembly, activation, and inhibition in epithelial sodium channel

Abstract

The molecular bases of heteromeric assembly and link between Na+ self-inhibition and protease-sensitivity in epithelial sodium channels (ENaCs) are not fully understood. Previously, we demonstrated that ENaC subunits – α, β, and γ – assemble in a counterclockwise configuration when viewed from outside the cell with the protease-sensitive GRIP domains in the periphery (Noreng et al., 2018). Here we describe the structure of ENaC resolved by cryo-electron microscopy at 3 Å. We find that a combination of precise domain arrangement and complementary hydrogen bonding network defines the subunit arrangement. Furthermore, we determined that the α subunit has a primary functional module consisting of the finger and GRIP domains. The module is bifurcated by the α2 helix dividing two distinct regulatory sites: Na+ and the inhibitory peptide. Removal of the inhibitory peptide perturbs the Na+ site via the α2 helix highlighting the critical role of the α2 helix in regulating ENaC function.

The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs

“…Recent advances in structural biology have resulted in the publication of multiple structures of chick ASIC1 and hENaC [15,16,21,22,35,36]. However, structures of open, full-length…”

“…However, structures of open, full-length ASICs have not been obtained and especially the conformation of the lower pore varies considerably among ASIC structures [15, 21, 22, 36]. Similarly, the resolution of the pore-forming M1 and M2 segments in the available ENaC structures is comparatively low, giving rise to uncertainty regarding their absolute and relative positions, also with respect to the (unresolved) N-and C-termini [16, 35]. It is therefore difficult to reconcile the stark differences in ion selectivity among these channel types solely based on available structural data.…”

“…Although structural information on the ENaC pore is of relatively low resolution [16, 35], there is strong functional evidence that the G/SXS motif in the middle of the pore is critical for ion selectivity in ENaCs [28, 31, 33, 41]. Additionally, and in contrast to the findings for ASICs, the negatively charged side chains in the M2-18’ and M2-21’ positions do not appear to play a crucial role in ion selectivity [28].…”

“…Recent advances in structural biology have resulted in the publication of multiple structures of chick ASIC1 and hENaC [15,16,21,22,40,41]. However, structures of open, full-length ASICs have not been obtained and especially the conformation of the lower pore varies considerably among ASIC structures [15,21,22,41].…”

Enhanced Shear Force Responsiveness of Epithelial Na+ Channel’s (ENaC) δ Subunit Following the Insertion of N-Glycosylation Motifs Relies on the Extracellular Matrix

“…The full-length extracellular domain of human ENaC at 3 Å (PDB: 6WTH [ 35 ]) was chosen as a template for creating predicted 3D models of δ wt and δ N292/487 ENaC. Therefore, the amino acid sequence of the extracellular domain of human δ ENaC (UniProt identifier Q09HT1) was used as input and aligned to 6WTH_A (human α ENaC) using UCSF Chimera 1.15rc [ 36 ] with the BLOSUM62 scoring matrix.…”