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

Sigrid Noreng,Isabelle Baconguis,Richard Posert,Arpita Bharadwaj,Craig Yoshioka

doi:10.7554/elife.39340

Sigrid Noreng, Isabelle Baconguis + Show 3 more

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https://doi.org/10.7554/elife.39340

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Journal: eLife	Publication Date: Sep 25, 2018
Citations: 145	License type: CC BY 4.0

Affiliation: Oregon Health & Science University

Abstract

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.

Highlights

The fine-tuning of Na+ homeostasis is largely mediated by epithelial sodium channels (ENaC) that are related in amino acid sequence to acid-sensing ion channels (ASIC) found in eukaryotes, degenerin channels (DEG) of Caenorhabditis elegans, and the FMRF-amide peptide-gated channels (FaNaCh) of mollusk (Driscoll and Chalfie, 1991; Chalfie and Wolinsky, 1990; Kellenberger and Schild, 2002; Waldmann et al, 1997a; Waldmann et al, 1997b; Krishtal and Pidoplichko, 1981; Chelur et al, 2002; Garty and Palmer, 1997; Cottrell et al, 1990; Lingueglia et al, 1995)
We first assessed the expression of full-length (FL) ENaC by small-scale expression in adherent HEK293S GnTI- cells and fluorescence-detection size-exclusion chromatography (FSEC) (Kawate and Gouaux, 2006)
We screened a number of deletions and mutations in each ENaC subunit, harnessing information derived from previous biochemical and functional experiments gauging the propensity for heterotrimeric formation of ENaC and its susceptibility to proteolytic processing (Canessa et al, 1994; Orce et al, 1980; Vallet et al, 1997; Vallet et al, 2002; Vuagniaux et al, 2002; Hughey et al, 2004; Hughey et al, 2003; Caldwell et al, 2004; Passero et al, 2010), before arriving at the construct referred to here as DENaC (Figure 1a–c, Figure 1—figure supplement 1, Figure 1—figure supplement 2)

Summary

Introduction

The fine-tuning of Na+ homeostasis is largely mediated by epithelial sodium channels (ENaC) that are related in amino acid sequence to acid-sensing ion channels (ASIC) found in eukaryotes, degenerin channels (DEG) of Caenorhabditis elegans, and the FMRF-amide peptide-gated channels (FaNaCh) of mollusk (Driscoll and Chalfie, 1991; Chalfie and Wolinsky, 1990; Kellenberger and Schild, 2002; Waldmann et al, 1997a; Waldmann et al, 1997b; Krishtal and Pidoplichko, 1981; Chelur et al, 2002; Garty and Palmer, 1997; Cottrell et al, 1990; Lingueglia et al, 1995) These ion channels belong to the voltage-independent, Na+-selective, and amiloride-sensitive ENaC/DEG superfamily which together perform diverse cellular functions in different organisms. The number of subunits in an active channel remains unclear (Shobair et al, 2016)

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