scholarly journals β11-12 linker isomerization governs Acid-sensing ion channel desensitization and recovery

2019 ◽  
Author(s):  
Matthew Rook ◽  
Abby Williamson ◽  
John D. Lueck ◽  
Maria Musgaard ◽  
David M. MacLean
Keyword(s):  
Functional Data ◽  
Resting State ◽  
Low Ph ◽  
Cross Linking ◽  
State Dependent ◽  
Acid Sensing Ion Channels ◽  
Canonical Amino Acid ◽  
Functional States ◽  
Dynamics Simulations ◽  
Fast Perfusion

AbstractAcid-sensing ion channels (ASICs) are neuronal sodium-selective channels activated by reductions in extracellular pH. Structures of the three presumptive functional states, high-pH resting, low-pH desensitized, and toxin-stabilized open, have all been solved for chicken ASIC1. These structures, along with prior functional data, suggest that the isomerization or flipping of the β11-12 linker in the extracellular, ligand-binding domain is an integral component of the desensitization process. To test this, we combined fast perfusion electrophysiology, molecular dynamics simulations and state-dependent non-canonical amino acid cross-linking. We find that both desensitization and recovery can be accelerated by orders of magnitude by mutating resides in this linker or the surrounding region. Furthermore, desensitization can be suppressed by trapping the linker in the resting state, indicating that isomerization of the β11-12 linker is not merely a consequence of, but a necessity for the desensitization process in ASICs.

scholarly journals β11-12 linker isomerization governs acid-sensing ion channel desensitization and recovery

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Matthew L Rook ◽  
Abby Williamson ◽  
John D Lueck ◽  
Maria Musgaard ◽  
David M Maclean
Keyword(s):  
Functional Data ◽  
Resting State ◽  
Low Ph ◽  
Cross Linking ◽  
State Dependent ◽  
Acid Sensing Ion Channels ◽  
Canonical Amino Acid ◽  
Functional States ◽  
Dynamics Simulations ◽  
Fast Perfusion

Acid-sensing ion channels (ASICs) are neuronal sodium-selective channels activated by reductions in extracellular pH. Structures of the three presumptive functional states, high-pH resting, low-pH desensitized, and toxin-stabilized open, have all been solved for chicken ASIC1. These structures, along with prior functional data, suggest that the isomerization or flipping of the β11–12 linker in the extracellular, ligand-binding domain is an integral component of the desensitization process. To test this, we combined fast perfusion electrophysiology, molecular dynamics simulations and state-dependent non-canonical amino acid cross-linking. We find that both desensitization and recovery can be accelerated by orders of magnitude by mutating resides in this linker or the surrounding region. Furthermore, desensitization can be suppressed by trapping the linker in the resting state, indicating that isomerization of the β11–12 linker is not merely a consequence of, but a necessity for the desensitization process in ASICs.

scholarly journals Characterization of the dynamic resting state of a pentameric ligand-gated ion channel by cryo-electron microscopy and simulations

2020 ◽  
Author(s):  
U Rovšnik ◽  
Y Zhuang ◽  
L Axelsson ◽  
BO Forsberg ◽  
V Lim ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ion Channel ◽  
Resting State ◽  
Structural Data ◽  
Cryo Electron Microscopy ◽  
Electrochemical Signal ◽  
Moderate Resolution ◽  
Functional States ◽  
Dynamics Simulations

AbstractLigand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological development in this family relies on high-quality structural data in multiple, subtly distinct functional states. However, structural data remain limited, particularly for the unliganded or resting state. Here we report cryo-electron microscopy structures of the Gloeobacter violaceus ligand-gated ion channel (GLIC) under resting and activating conditions (neutral and low pH). Parallel models were built either manually or using recently developed density-guided molecular simulations. The moderate resolution of resting-state reconstructions, particularly in the extracellular domain, was improved under activating conditions, enabling the visualization of residues at key subunit interfaces including loops B, C, F, and M2–M3. Combined with molecular dynamics simulations, the cryo-electron microscopy structures at different pH describe a heterogeneous population of closed channels, with activating conditions condensing the closed-channel energy landscape on a pathway towards gating.

scholarly journals Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

1984 ◽  
Vol 99 (2) ◽  
pp. 734-741 ◽  
Author(s):  
W A Braell ◽  
D M Schlossman ◽  
S L Schmid ◽  
J E Rothman
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Low Ph ◽  
Coated Vesicles ◽  
Magnesium Concentration ◽  
Cross Linking ◽  
Dependent Atpase ◽  
Event Triggering ◽  
Hydrolysis Of

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

State-Dependent Disulfide Cross-Linking in Rhodopsin†

Biochemistry ◽  
1999 ◽  
Vol 38 (37) ◽  
pp. 12028-12032 ◽  
Author(s):  
Hongbo Yu ◽  
Masahiro Kono ◽  
Daniel D. Oprian
Keyword(s):  
Cross Linking ◽  
State Dependent

Swelling of PVA/Chitosan/TiO2 Nanofibers Membrane in Different PH

2020 ◽  
Vol 990 ◽  
pp. 220-224
Author(s):  
Ari Dwi Nugraheni ◽  
Diki Purnawati ◽  
Ani Rohmatillah ◽  
Dian Nur Mahardika ◽  
Ahmad Kusumaatmaja
Keyword(s):  
Time Constant ◽  
Weight Ratio ◽  
Low Ph ◽  
Membrane Stability ◽  
Swelling Behavior ◽  
Cross Linking ◽  
High Ph ◽  
Chitosan Concentration ◽  
Nanofiber Membranes ◽  
Water Swelling

The nanofiber PVA/chitosan have been successfully fabricated by the addition of TiO2 or without TiO2. Nanofiber membranes of PVA/chitosan/TiO2 were fabricated with weight ratio of PVA/chitosan (w/w) 90/10, 85/15, and 80/20. Glutaraldehyde cross-linking was added to increase membrane stability in water. Swelling behavior was tested in three different pH to investigate the swelling behavior of nanofiber membranes of PVA/chitosan with and without TiO2. The result indicated that the addition of chitosan concentration would decrease the swelling index, and increase the time constant (τ). The swelling index will increase in high pH (pH 10.01) compare to low pH (pH 4.01).

scholarly journals High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase

2020 ◽  
Vol 117 (8) ◽  
pp. 4071-4077 ◽  
Author(s):  
Yohta Fukuda ◽  
Yu Hirano ◽  
Katsuhiro Kusaka ◽  
Tsuyoshi Inoue ◽  
Taro Tamada
Keyword(s):  
Computational Chemistry ◽  
Structural Biology ◽  
Resting State ◽  
Catalytic Mechanism ◽  
Low Ph ◽  
Intramolecular Electron Transfer ◽  
Hydrogen Atoms ◽  
X Ray ◽  
Electron Transfer Pathway ◽  
Neutron Structure

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Å resolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

TSH receptor structure

1987 ◽  
Vol 116 (1_Suppl) ◽  
pp. S166-S172 ◽  
Author(s):  
John Chan ◽  
Pilar Santisteban ◽  
Michele De Luca ◽  
Osamu Isozaki ◽  
Evelyn Grollman ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Low Ph ◽  
The Other ◽  
Tsh Receptor ◽  
Cross Linking ◽  
Thyroid Cells ◽  
Autoimmune Thyroid ◽  
Receptor Structure ◽  
Rat Thyroid

Abstract. When solubilized, radiolabelled membrane preparations from FRTL-5 rat thyroid cells are applied to TSH affinity columns, two separate peaks of protein can be eluted by high salts/high pH and low pH buffers, respectively. Immunoprecipitation with monoclonal antibodies to the TSH receptor shows that both peaks contain proteins related to the TSH receptor. If extracts were from cells grown without TSH, one peak has a ~ 300 K and the other a ~ 70 K protein the 70 K protein can be derived from the purified 300 K protein in vitro. A 50 and 20 K protein can be derived from the 70 K protein. If extracts are from cells grown with TSH, the peaks contain a multiplicity of additional immunoprecipitable bands of ~ 200, 175, 130, 90, 50, 20 K etc. These bands are shown to result from the ability of TSH to increase the synthesis (3–4-fold) and degradation (2–3-fold) of the 300 and 70 K proteins. The 300/70 K protein fractions are reactive with monoclonal autoimmune thyroid stimulating antibodies and contain a specific disialo ganglioside. The ganglioside migrates near GM2, i.e., like a lower order ganglioside, and contains fucose. In translation experiments, the monoclonal antibodies to the TSH receptor identify a single mRNA component which produces a protein of ~ 220 K. This protein is not present in thyroid cells which have no functional TSH receptor and which cannot be surface labelled with monoclonal antibodies to the TSH receptor. The data thus indicate that the multiplicity of TSH binding proteins demonstrated in many labs may be breakdown products of a receptor which is synthesized by a single message but has both 330 and 70 K forms and is tightly complexed with a specific thyroid ganglioside. The 70 K form is composed of ~ 50 and ~ 20 K fragments seen in TSH cross-linking studies.

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