Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

Abstract

The epithelial Na ؉ channel (ENaC) mediates the rate-limiting step in transepithelial Na ؉ transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the ␣ subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the fingerthumb domain interface.The epithelial Na ϩ channel (ENaC) 2 is expressed at the apical surface of Na ϩ -transporting epithelia such as the distal nephron of the kidney, distal colon, and lung alveoli and airway. In conjunction with the Na ϩ /K ϩ -ATPase, ENaC transfers Na ϩ from the luminal to the interstitial space. This transfer is crucial in regulating blood pressure through its role in renal Na ϩ absorption and in regulating airway surface liquid volume and mucociliary clearance through its role in airway Na ϩ absorption. In accord with its role in these processes, improper ENaC function is implicated in several disorders. There is a growing body of evidence that enhanced ENaC activity in the airways of individuals with cystic fibrosis contributes to depletion of airway surface liquids resulting in poor mucociliary clearance (1-3). In the kidney, increased levels of aldosterone activate ENaC and increase the reabsorption of filtered Na ϩ (4). In both instances, increases in channel activity reflect, in part, enhanced channel proteolysis. Proteinuric states, characterized by excessive protein in the urine, are often accompanied by renal Na ϩ retention, volume expansion, and hypertension. Recent work indicates that volume expansion in proteinuric states reflects proteolytic activation of ENaC (5-7).ENaC is a trimer composed of three homologous subunits, ␣, ␤, and ␥ (8, 9). ENaC subunits are members of the much larger ENaC/Degenerin family of ion channel proteins. These channels share a few salient features as follows: 1) most are gated by ligands and/or mechanical forces; 2) they are Na ϩ -permeable and blocked by amiloride, a potassium-sparing diuretic; and 3) each subunit has two transmembrane helices (six transmembrane helices for the full channel), short intracellular N and C termini, and a large extracellular region comprised of sever...

Inhibitory Tract Traps the Epithelial Na+ Channel in a Low Activity Conformation

“…We reported recently that mutations in the thumb near the outer mouth of the pore modulated the efficacy of multiple extracellular stimuli, suggesting mechanistic convergence (18). The remarkably similar kinetics of peptide, Cl Ϫ , and Na ϩ inhibition hint at a common rate-limiting step (10,16,19,20). As ENaC/degenerin channels have evolved to respond to numerous extracellular stimuli, the basic architecture of the thumb domain seems to have been preserved.…”

“…Consequently, peptides that correspond to the released fragments are inhibitory (3, 6 -9). We recently reported that introduction of Trp mutations at a large number of sites in the ␣ subunit reduced the inhibition of channel activity by the ␣ subunit-derived 8-mer peptide, Ac-LPHPLQRL-amide (10). A structural model of the ␣ subunit extracellular region was constructed based on these data and on homology to ASIC1 (11), the only member of the ENaC/degenerin family whose structure has been resolved (12,13).…”

Conserved cysteines in the finger domain of the epithelial Na+ channel α and γ subunits are proximal to the dynamic finger–thumb domain interface

“…A peptide derived from the furinreleased autoinhibitory fragment of the a subunit decreased channel currents by binding at the finger-thumb domain interface of the a subunit (19). Previously, we recapitulated this effect in the absence of the inhibitory peptide by constraining the distance between the finger domain (using residue 171 in helix 1, red circle in Fig.…”

“…Synthetic peptides corresponding to each of the liberated tracts inhibit the channel (17,18). We have shown that the a subunit-derived inhibitory peptide and the autoinhibitory tract from which it is derived likely bind the a subunit at overlapping sites (10,19). One key feature of these sites is involvement of the finger-thumb domain interface.…”

TMPRSS4-dependent activation of the epithelial sodium channel requires cleavage of the γ-subunit distal to the furin cleavage site

“…Modeling of the ␣-subunit based on the crystal structure of the related acid-sensing ion channel 1 (19) allowed us to characterize the binding site for the ␣-subunit-derived 8-mer inhibitory peptide between the "finger" and "thumb" subdomains of the extracellular loop. We proposed that the ␣-subunit inhibitory peptide affects channel gating by constraining motions within these two major subdomains (20,21).…”