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

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

doi:10.7554/elife.39340

Cited by 158 publications

(208 citation statements)

References 72 publications

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“…There are no known heme binding sites on ENaC. Therefore, to identify these potential sites of ENaC, we performed computer modeling using a known cryo-electron microscopy structure of ENaC (37). The software predicted 22 potential docking sites of heme molecule on ENaC with the energy of binding ranging from 86 to1563 kJ/mol.…”

Section: Discussionmentioning

confidence: 99%

“…For this purpose, we used a recently developed cryo-electron microscopy structure of ENaC (Protein Data Bank: 6BQN) (37). The ion channel has large extracellular domains and a narrow transmembrane pore and the α :β:γ subunits are arranged in a counter-clockwise manner in a 1:1:1 stoichiometry (37). The software predicted 22 potential docking sites of heme on ENaC with the energy of binding ranging from 86 to1563 kJ/mol.…”

Section: Heme Impairs Enac Activity and Na + Transport Across Lung Epmentioning

confidence: 99%

“…To determine potential mechanisms by which heme exposure impairs ENAC activity within few seconds, we performed computer modeling using YASARA software (35,36) to simulate heme docking and binding to the known cryo-electron microscopy structure of ENaC (Protein Data Bank: 6BQN) (37). The docking of heme molecule to the ENaC structure was performed using AutoDock program developed at the Scripps Research Institute using the default docking parameters and point charges initially assigned according to the AMBER03 force field (76), and then damped to mimic the less polar Gasteiger charges used to optimize the AutoDock scoring function.…”

Section: Molecular Modeling and Heme Dockingmentioning

confidence: 99%

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Heme Impairs Alveolar Epithelial Sodium Channels Post Toxic Gas Inhalation

Aggarwal

Lazrak

Ahmad

et al. 2020

Preprint

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Fax: (205) 934-7476; Tel: (205) 975-9673 Conflict of interest statement: The authors have declared that no conflict of interest exists.Authors Contribution: SA and IA designed and performed studies assessing RBC lysis measurements of heme and heme induced oxidative injury; AL and ZY designed and performed the ion transport experiments; JAM designed, conducted and analyzed the mass spectrometry studies; AB, DF and SM completed the necessary forms to obtain permissions from the UAB and St. Louis University IRBs to perform measurements in human samples; DAF designed and performed all measurements to assess brominated lipids in humans and mice; SA performed the modelling studies; SA, AL and SM wrote all drafts of this manuscript; SM conceived the study, designed and organized experiments, analyzed data, and inspected all primary data. ABSTRACTWe previously reported that cell-free heme (CFH) is increased in the plasma of patients with acute and chronic lung injury and causes pulmonary edema in animal model of acute respiratory distress syndrome (ARDS) post inhalation of halogen gas. However, the mechanisms by which CFH causes pulmonary edema are unclear. Herein we report for the first time the presence of CFH and chlorinated lipids (formed by the interaction of halogen gas, Cl 2 , with plasmalogens) in the plasma of patients and mice exposed to Cl 2 gas. Ex vivo incubation of red blood cells (RBC) with halogenated lipids caused oxidative damage to RBC cytoskeletal protein spectrin, resulting in hemolysis and release of CFH. A single intramuscular injection of the heme-scavenging protein hemopexin (4 µg/kg body weight) in mice, one hour post halogen exposure, reversed RBC fragility and decreased CFH levels to those of air controls. Patch clamp and short circuit current measurements revealed that CFH inhibited the activity of amiloridesensitive (ENaC) and cation sodium (Na + ) channels in mouse alveolar cells and transepithelial Na + transport across human airway cells with EC 50 of 125 nM and 500 nM, respectively. Molecular modeling identified 22 putative heme-docking sites on ENaC (energy of binding range: 86-1563 kJ/mol) with at least 2 sites within its narrow transmembrane pore, potentially capable of blocking Na + transport across the channel. In conclusion, results suggested that CFH mediated inhibition of ENaC activity may be responsible for pulmonary edema post inhalation injury.

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Section: Discussionmentioning

confidence: 99%

Section: Heme Impairs Enac Activity and Na + Transport Across Lung Epmentioning

confidence: 99%

Section: Molecular Modeling and Heme Dockingmentioning

confidence: 99%

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Heme Impairs Alveolar Epithelial Sodium Channels Post Toxic Gas Inhalation

Aggarwal

Lazrak

Ahmad

et al. 2020

Preprint

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“…To address the hypothesis that the identified 16 sites may be comprise of cleavage centres spatially in 3D structure (Figure 9a), we labeled three deletion mutants in key domain organization scheme (Figure 9b) and GRIP domain (Figure 9c) based on the cryo-EM model (Noreng, Bharadwaj, Posert, Yoshioka & Baconguis, 2018). Apparently, one center was made up of six cleavage sites at the end of a1 helix and another with cleavage sites located in antiparallel P1 and P2 strands.…”

Section: Identified Cleavage Sites For Plasmin Compose Two Proteolytimentioning

confidence: 99%

“…On the other hand, we employed protein docking approach to substantiate this notion and potential interactions between individual amino acid residue. We generated a homology model of human γENaC with the I-TASSER server ( Figure S8a) (Noreng, Bharadwaj, Posert, Yoshioka & Baconguis, 2018;Roy, Kucukural & Zhang, 2010). There were three highly accessible clusters of positively charged residues in the GRIP domain of the γ ENaC ( Figure S8b).…”

Section: Identified Cleavage Sites For Plasmin Compose Two Proteolytimentioning

confidence: 99%

Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels

Zhao¹,

Ali²,

Nie³

et al. 2020

Preprint

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Background and Purpose: Lung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. We aimed to test whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid.Experimental Approaches: Human lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, protein docking, and alveolar fluid clearance were combined for identifying plasmin specific cleavage sites and benefits.Key Results: Plasmin led to a marked increment in lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin specifically activated abgENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts (aD432-444, gD131-138, gD178-193, and gD410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human gENaC proteins. In addition to confirming the 7 cleavage sites, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin with mass spectrometry. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both a and g subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal. Conclusion and Implications:We demonstrate that plasmin could cleave ENaC to benefit the blood-gas exchange by resolving oedema fluid as a potent fibrinolytic therapy for oedematous pulmonary diseases.

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Aldosterone: Renal Action and Physiological Effects

Johnston

Welch

Cain

et al. 2023

Comprehensive Physiology

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Aldosterone exerts profound effects on renal and cardiovascular physiology. In the kidney, aldosterone acts to preserve electrolyte and acid-base balance in response to changes in dietary sodium (Na + ) or potassium (K + ) intake. These physiological actions, principally through activation of mineralocorticoid receptors (MRs), have important effects particularly in patients with renal and cardiovascular disease as demonstrated by multiple clinical trials. Multiple factors, be they genetic, humoral, dietary, or otherwise, can play a role in influencing the rate of aldosterone synthesis and secretion from the adrenal cortex. Normally, aldosterone secretion and action respond to dietary Na + intake. In the kidney, the distal nephron and collecting duct are the main targets of aldosterone and MR action, which stimulates Na + absorption in part via the epithelial Na + channel (ENaC), the principal channel responsible for the fine-tuning of Na + balance. Our understanding of the regulatory factors that allow aldosterone, via multiple signaling pathways, to function properly clearly implicates this hormone as central to many pathophysiological effects that become dysfunctional in disease states. Numerous pathologies that affect blood pressure (BP), electrolyte balance, and overall cardiovascular health are due to abnormal secretion of aldosterone, mutations in MR, ENaC, or effectors and modulators of their action. Study of the mechanisms of these pathologies has allowed researchers and clinicians to create novel dietary and pharmacological targets to improve human health. This article covers the regulation of aldosterone synthesis and secretion, receptors, effector molecules, and signaling pathways that modulate its action in the kidney. We also consider the role of aldosterone in disease and the benefit of mineralocorticoid antagonists.

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

Cited by 158 publications

References 72 publications

Heme Impairs Alveolar Epithelial Sodium Channels Post Toxic Gas Inhalation

Heme Impairs Alveolar Epithelial Sodium Channels Post Toxic Gas Inhalation

Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels

Aldosterone: Renal Action and Physiological Effects

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