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

Sigrid Noreng,Richard Posert,Isabelle Baconguis,Alexandra Houser,Arpita Bharadwaj

doi:10.7554/elife.59038

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.

Highlights

The ability to balance the amount of water inside and outside cells is absolutely essential for life
ENaC belongs to the ENaC/degenerin family, defined by Na+-selectivity, voltage independence, and amiloride sensitivity (Kellenberger and Schild, 2002). Members of this family, including the well-studied relative Acid-Sensing Ion Channel (ASIC), have subunits that consist of short intracellular N- and C-termini, two membrane-spanning helices, and a large cysteine-rich extracellular domain (ECD) that can form homo- or heterotrimeric ion channels (Jasti et al, 2007; Noreng et al, 2018)
To investigate the structural source of ENaC trimer assembly, we exploited a set of constructs, deemed ENaCFL, which comprises wild-type a and b, and N-terminally eGFP-tagged g, and behaves like wild-type ENaC as measured by electrophysiology (Figure 1—figure supplement 1 and Table 1)

Summary

Introduction

The ability to balance the amount of water inside and outside cells is absolutely essential for life. ENaC belongs to the ENaC/degenerin family, defined by Na+-selectivity, voltage independence, and amiloride sensitivity (Kellenberger and Schild, 2002). Members of this family, including the well-studied relative Acid-Sensing Ion Channel (ASIC), have subunits that consist of short intracellular N- and C-termini, two membrane-spanning helices, and a large cysteine-rich extracellular domain (ECD) that can form homo- or heterotrimeric ion channels (Jasti et al, 2007; Noreng et al, 2018).

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