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

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sigrid Noreng ◽  
Arpita Bharadwaj ◽  
Richard Posert ◽  
Craig Yoshioka ◽  
Isabelle Baconguis
Keyword(s):  
Electron Microscopy ◽  
Ion Channel ◽  
Sodium Channel ◽  
Conformational Changes ◽  
Single Particle ◽  
Transmembrane Domain ◽  
Epithelial Sodium Channel ◽  
Cryo Electron Microscopy ◽  
Inhibitory Domain ◽  
Epithelial Sodium Channel Enac

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.

scholarly journals Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel

2010 ◽  
Vol 299 (4) ◽  
pp. F854-F861 ◽  
Author(s):  
Christopher J. Passero ◽  
Marcelo D. Carattino ◽  
Ossama B. Kashlan ◽  
Mike M. Myerburg ◽  
Rebecca P. Hughey ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Xenopus Laevis Oocytes ◽  
Airway Epithelia ◽  
Gamma Subunit ◽  
Γ Subunit ◽  
Inhibitory Region ◽  
Inhibitory Domain ◽  
Complete Deletion ◽  
Epithelial Sodium Channel Enac

Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the γ-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the γ-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the γ inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel.

scholarly journals Author response: Structure of the human epithelial sodium channel by cryo-electron microscopy

2018 ◽  
Author(s):  
Sigrid Noreng ◽  
Arpita Bharadwaj ◽  
Richard Posert ◽  
Craig Yoshioka ◽  
Isabelle Baconguis
Keyword(s):  
Electron Microscopy ◽  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Author Response ◽  
Cryo Electron Microscopy

scholarly journals ATP and Substrate Binding Regulates Conformational Changes of Human Peroxisomal ABC Transporter ALDP

2021 ◽  
Author(s):  
Lin Tang ◽  
Chao Xiong ◽  
Lina Jia ◽  
Ming-He Shen ◽  
Wei-Xi Xiong ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fatty Acids ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Long Chain Fatty Acids ◽  
Variable Size ◽  
Binding Domains ◽  
Cryo Electron Microscopy ◽  
Peroxisome Membrane ◽  
Long Chain

Abstract The malfunction of ABCD1 causes X-linked adrenoleukodystrophy (X-ALD), a rare neurodegenerative disease that affect all tissues in human. Residing in the peroxisome membrane, ABCD1 plays a role in the translocation of very long chain fatty acids (VLCFA) for their damage by β-oxidation. Here, we present five Cryo-Electron microscopy structures of ABCD1 in four conformational states. Combined with functional analysis, we found that substrate and ATP trigger the closing of two nucleotide binding domains (NBDs) over a distance of 40 Å and the rearrangement of the transmembrane domains. Each of the three inward-facing structure of ABCD1 has a vestibule opens to cytosol with variable size. Furthermore, the structure of ABCD1 in the outward-facing state supports that ATP molecules pull the two NBDs together and open the transmembrane domain to the peroxisomal lumen for substrate release. The five structures provide a snapshot of substrate transporting cycle and mechanistic implications for disease-causing mutations.

scholarly journals Cryo-EM reveals a previously unrecognized structural protein of a dsRNA virus implicated in its extracellular transmission

2021 ◽  
Vol 17 (3) ◽  
pp. e1009396
Author(s):  
Qianqian Shao ◽  
Xudong Jia ◽  
Yuanzhu Gao ◽  
Zhe Liu ◽  
Huan Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Life Cycle ◽  
High Resolution ◽  
Conformational Changes ◽  
Single Particle ◽  
Life Cycle Stage ◽  
Dsrna Virus ◽  
Structural Protein ◽  
Cryo Electron Microscopy

Mosquito viruses cause unpredictable outbreaks of disease. Recently, several unassigned viruses isolated from mosquitoes, including the Omono River virus (OmRV), were identified as totivirus-like viruses, with features similar to those of the Totiviridae family. Most reported members of this family infect fungi or protozoans and lack an extracellular life cycle stage. Here, we identified a new strain of OmRV and determined high-resolution structures for this virus using single-particle cryo-electron microscopy. The structures feature an unexpected protrusion at the five-fold vertex of the capsid. Disassociation of the protrusion could result in several conformational changes in the major capsid. All these structures, together with some biological results, suggest the protrusions’ associations with the extracellular transmission of OmRV.

scholarly journals ATP and Substrate Binding Regulates Conformational Changes of Human Peroxisomal ABC Transporter ALDP

2021 ◽  
Author(s):  
chao xiong ◽  
Li-Na Jia ◽  
Ming-He Shen ◽  
Wei-Xi Xiong ◽  
Liu-Lin Xiong ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fatty Acids ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Long Chain Fatty Acids ◽  
Variable Size ◽  
Binding Domains ◽  
Cryo Electron Microscopy ◽  
Peroxisome Membrane ◽  
Long Chain

The malfunction of ABCD1 causes X-linked adrenoleukodystrophy (X-ALD), a rare neurodegenerative disease that affect all tissues in human. Residing in the peroxisome membrane, ABCD1 plays a role in the translocation of very long chain fatty acids (VLCFA) for their damage by β-oxidation. Here, we present five Cryo-Electron microscopy structures of ABCD1 in four conformational states. Combined with functional analysis, we found that substrate and ATP trigger the closing of two nucleotide binding domains (NBDs) over a distance of 40 &Aring and the rearrangement of the transmembrane domains. Each of the three inward-facing structure of ABCD1 has a vestibule opens to cytosol with variable size. Furthermore, the structure of ABCD1 in the outward-facing state supports that ATP molecules pull the two NBDs together and open the transmembrane domain to the peroxisomal lumen for substrate release. The five structures provide a snapshot of substrate transporting cycle and mechanistic implications for disease-causing mutations.

scholarly journals Single-particle cryo-EM structure of a voltage-activated potassium channel in lipid nanodiscs

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Doreen Matthies ◽  
Chanhyung Bae ◽  
Gilman ES Toombes ◽  
Tara Fox ◽  
Alberto Bartesaghi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lipid Bilayer ◽  
Single Particle ◽  
Transmembrane Domain ◽  
X Ray ◽  
Kv Channels ◽  
Cryo Electron Microscopy ◽  
Transmembrane Voltage ◽  
Structural Mechanisms ◽  
Lipid Nanodiscs

Voltage-activated potassium (Kv) channels open to conduct K+ ions in response to membrane depolarization, and subsequently enter non-conducting states through distinct mechanisms of inactivation. X-ray structures of detergent-solubilized Kv channels appear to have captured an open state even though a non-conducting C-type inactivated state would predominate in membranes in the absence of a transmembrane voltage. However, structures for a voltage-activated ion channel in a lipid bilayer environment have not yet been reported. Here we report the structure of the Kv1.2–2.1 paddle chimera channel reconstituted into lipid nanodiscs using single-particle cryo-electron microscopy. At a resolution of ~3 Å for the cytosolic domain and ~4 Å for the transmembrane domain, the structure determined in nanodiscs is similar to the previously determined X-ray structure. Our findings show that large differences in structure between detergent and lipid bilayer environments are unlikely, and enable us to propose possible structural mechanisms for C-type inactivation.

scholarly journals Ankyrin G Expression Regulates Apical Delivery of the Epithelial Sodium Channel (ENaC)

2016 ◽  
Vol 292 (1) ◽  
pp. 375-385 ◽  
Author(s):  
Christine A. Klemens ◽  
Robert S. Edinger ◽  
Lindsay Kightlinger ◽  
Xiaoning Liu ◽  
Michael B. Butterworth
Keyword(s):  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Ankyrin G ◽  
Epithelial Sodium Channel Enac
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