Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker

Charged residues in the β10–M1 linker region (“pre-M1”) are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational “wave.” We examined the effects of mutations to all five residues in pre-M1 (positions M207–P211) plus E45 in loop 2 in the mouse α1-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (Keq), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in Keq (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Φ values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Φ values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only −0.33 kcal/mol (for both α subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.

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Modular Design of Cys-loop Ligand-gated Ion Channels: Functional 5-HT3 and GABA ρ1 Receptors Lacking the Large Cytoplasmic M3M4 Loop

The Journal of General Physiology ◽

10.1085/jgp.200709896 ◽

2008 ◽

Vol 131 (2) ◽

pp. 137-146 ◽

Cited By ~ 85

Author(s):

Michaela Jansen ◽

Moez Bali ◽

Myles H. Akabas

Keyword(s):

Ion Channels ◽

Acetylcholine Receptors ◽

Modular Design ◽

Transmembrane Domain ◽

Single Channel ◽

Cytoplasmic Domain ◽

Wild Type ◽

Channel Assembly ◽

And Function ◽

Gated Ion Channels

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the α-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT3A) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT3A-glvM3M4 receptors were comparable to 5-HT3A wild type. The channels remained cation-selective but the 5-HT3A-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT3 and acetylcholine receptors, significantly attenuated 5-HT–induced currents with wild-type 5-HT3A but not 5-HT3A-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-ρ1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.

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Formation and Function of Formate in the Side-Chain Alkylation of Toluene with Methanol

CHINESE JOURNAL OF CATALYSIS (CHINESE VERSION) ◽

10.3724/sp.j.1088.2012.11243 ◽

2013 ◽

Vol 33 (6) ◽

pp. 1041-1047

Author(s):

Dan LIN ◽

Huimin ZHAO ◽

Xiaoyue ZHANG ◽

Dongxue LAN ◽

Yuan CHUN

Keyword(s):

Side Chain ◽

And Function

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Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic acetylcholine receptors.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32248-2 ◽

1994 ◽

Vol 269 (29) ◽

pp. 18870-18876

Author(s):

K. Blüml ◽

E. Mutschler ◽

J. Wess

Keyword(s):

Ligand Binding ◽

Acetylcholine Receptors ◽

Functional Role ◽

Transmembrane Domain ◽

Receptor Activation ◽

Muscarinic Acetylcholine Receptors ◽

Muscarinic Acetylcholine ◽

Asparagine Residue

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New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

Scientific Reports ◽

10.1038/s41598-021-83240-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sara Bitam ◽

Ahmad Elbahnsi ◽

Geordie Creste ◽

Iwona Pranke ◽

Benoit Chevalier ◽

...

Keyword(s):

Phosphinic Acid ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Transmembrane Conductance Regulator ◽

Intracellular Loop ◽

Respiratory Cells ◽

Cftr Protein ◽

Dynamics Simulations ◽

And Function ◽

Molecular Mode

AbstractC407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

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Cryo-EM structure of the human histamine H1 receptor/Gq complex

Nature Communications ◽

10.1038/s41467-021-22427-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ruixue Xia ◽

Na Wang ◽

Zhenmei Xu ◽

Yang Lu ◽

Jing Song ◽

...

Keyword(s):

Transmembrane Domain ◽

Binding Pocket ◽

Receptor Activation ◽

Intracellular Loop ◽

Cryo Electron Microscopy ◽

Allergy Treatment ◽

Domain 3 ◽

Extracellular Side ◽

Key Residues ◽

Loop 2

AbstractHistamine receptors play important roles in various pathophysiological conditions and are effective targets for anti-allergy treatment, however the mechanism of receptor activation remain elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human H1R in complex with a Gq protein in an active conformation via a NanoBiT tethering strategy. The structure reveals that histamine activates receptor via interacting with the key residues of both transmembrane domain 3 (TM3) and TM6 to squash the binding pocket on the extracellular side and to open the cavity on the intracellular side for Gq engagement in a model of “squash to activate and expand to deactivate”. The structure also reveals features for Gq coupling, including the interaction between intracellular loop 2 (ICL2) and the αN-β junction of Gq/11 protein. The detailed analysis of our structure will provide a framework for understanding G-protein coupling selectivity and clues for designing novel antihistamines.

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Anti-Peptide Specific Antibodies for the Characterization of Different α Subunits of α-Bungarotoxin Binding Acetylcholine Receptors Present in Chick Optic Lobe

Journal of Receptor Research ◽

10.3109/10799899309073672 ◽

1993 ◽

Vol 13 (1-4) ◽

pp. 453-465 ◽

Cited By ~ 7

Author(s):

Cecilia Gotti ◽

Milena Moretti ◽

Renato Longhi ◽

Luca Briscini ◽

Ernesto Manera ◽

...

Keyword(s):

Acetylcholine Receptors ◽

Optic Lobe ◽

Specific Antibodies ◽

Chick Optic Lobe ◽

Α Subunits

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Gating Dynamics of the Acetylcholine Receptor Extracellular Domain

The Journal of General Physiology ◽

10.1085/jgp.200309004 ◽

2004 ◽

Vol 123 (4) ◽

pp. 341-356 ◽

Cited By ~ 109

Author(s):

Sudha Chakrapani ◽

Timothy D. Bailey ◽

Anthony Auerbach

Keyword(s):

Conformational Change ◽

Acetylcholine Receptors ◽

Single Channel ◽

Extracellular Domain ◽

Mouse Muscle ◽

Α Subunit ◽

Acetylcholine Binding Protein ◽

Almost All ◽

Loop 2 ◽

Sandwich Core

We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the α-subunit of mouse muscle–type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a β-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the β-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the α-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Φ = 0.93) and is followed by the loop 2/loop 7 domain (Φ = 0.80). These movements precede that of the extracellular linker (Φ = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.

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Acetylcholine Receptors

The Journal of General Physiology ◽

10.1085/jgp.60.3.248 ◽

1972 ◽

Vol 60 (3) ◽

pp. 248-262 ◽

Cited By ~ 174

Author(s):

H. Criss Hartzell ◽

Douglas M. Fambrough

Keyword(s):

Acetylcholine Receptors ◽

Time Course ◽

Plasma Membranes ◽

Scintillation Counting ◽

Receptor Density ◽

Rat Diaphragm ◽

Leg Muscles ◽

And Function ◽

Quantitative Radioautography ◽

Ach Receptors

Using 125iodine-labeled α-bungarotoxin (α-BGT-125I) and quantitative radioautography, we have studied the time-course of the change in acetylcholine (ACh) receptor distribution and density occurring in rat diaphragm after denervation. In innervated fibers, ACh receptors are localized at the neuromuscular junction and the extrajunctional receptor density is less than five receptors per square micrometer. The extrajunctional receptor density begins to increase between 2 and 3 days after denervation and increases approximately linearly to 1695 receptors/µm2 at 14 days, subsequently decreasing to 529 receptors/µm2 at 45 days. We have isolated plasma membranes from rat leg muscles at various times after denervation and find that the change in concentration of ACh receptors in the membranes measured by α-BGT-125I binding and scintillation counting follows a time-course similar to the change in ACh receptor density measured radioautographically. Furthermore, we have correlated extrajunctional ACh receptor density measured by radioautography with extrajunctional ACh sensitivity measured by iontophoretic application of ACh and intracellular recording and find that the log of ACh receptor density is related to 0.53 times the log of ACh sensitivity. These results are discussed in terms of the electrophysiological experiments on the ACh receptor and the recent, more biochemical approaches to the study of ACh receptor control and function.

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Dynamics and mechanism of ultrafast water–protein interactions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1602916113 ◽

2016 ◽

Vol 113 (30) ◽

pp. 8424-8429 ◽

Cited By ~ 77

Author(s):

Yangzhong Qin ◽

Lijuan Wang ◽

Dongping Zhong

Keyword(s):

Protein Interactions ◽

Hydration Shell ◽

Water Dynamics ◽

Side Chain ◽

Water Molecules ◽

Ultrafast Dynamics ◽

Hydration Water ◽

Water Side ◽

Dynamics And Function ◽

And Function

Protein hydration is essential to its structure, dynamics, and function, but water–protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/side-chain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein side-chain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship.

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