scholarly journals A Functionally Conserved Mechanism of Modulation via a Vestibule Site in Pentameric Ligand-Gated Ion Channels

2019 ◽  
Author(s):  
Marijke Brams ◽  
Cedric Govaerts ◽  
Kumiko Kambara ◽  
Kerry Price ◽  
Radovan Spurny ◽  
...  
Keyword(s):  
Ion Channels ◽  
Binding Site ◽  
Binding Sites ◽  
Allosteric Modulators ◽  
Channel Opening ◽  
Single Domain Antibodies ◽  
Allosteric Binding Sites ◽  
Target Binding ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

ABSTRACTPentameric ligand-gated ion channels (pLGICs) belong to a class of ion channels involved in fast synaptic signaling in the central and peripheral nervous systems. Molecules acting as allosteric modulators target binding sites that are remote from the neurotransmitter binding site, but functionally affect coupling of ligand binding to channel opening. Here, we investigated an allosteric binding site in the ion channel vestibule, which has converged from a series of studies on prokaryote and eukaryote channel homologs. We discovered single domain antibodies, called nanobodies, which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote channel ELIC bound either to a positive (PAM) or a negative (NAM) allosteric modulator. We extrapolate the functional importance of the vestibule binding site to eukaryote ion channels, suggesting a conserved mechanism of allosteric modulation. This work identifies key elements of allosteric binding sites and extends drug design possibilities in pLGICs using nanobodies.

scholarly journals Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Marijke Brams ◽  
Cedric Govaerts ◽  
Kumiko Kambara ◽  
Kerry L Price ◽  
Radovan Spurny ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Common Mechanism ◽  
Allosteric Modulators ◽  
Functional Importance ◽  
Channel Opening ◽  
Negative Allosteric Modulator ◽  
Single Domain Antibodies ◽  
Allosteric Binding Sites ◽  
Small Molecule Modulators

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.

Structural elements involved in activation of the γ-aminobutyric acid type A (GABAA) receptor

2004 ◽  
Vol 32 (3) ◽  
pp. 540-546 ◽  
Author(s):  
T.L. Kash ◽  
J.R. Trudell ◽  
N.L. Harrison
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Binding Site ◽  
Type A ◽  
Aminobutyric Acid ◽  
Structural Elements ◽  
Agonist Binding ◽  
Acid Type ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

Ligand-gated ion channels function as rapid signal transducers, converting chemical signals (in the form of neurotransmitters) into electrical signals in the postsynaptic neuron. This is achieved by the recognition of neurotransmitter at its specific-binding sites, which then triggers the opening of an ion channel (‘gating’). For this to occur rapidly (<1 ms), there must be an efficient coupling between the agonist-binding site and the gate, located more than 30 Å (1 Å=0.1 nm) away. Whereas a great deal of progress has been made in elucidating the structure and function of both the agonist-binding site and the ion permeation pathway in ligand-gated ion channels, our knowledge of the coupling mechanism between these domains has been limited. In this review, we summarize recent studies of the agonist-binding site and the ion channel in the γ-aminobutyric acid type A receptor, and discuss those structural elements that may mediate coupling between them. We will also consider some possible molecular mechanisms of receptor activation.

scholarly journals Probing Pentameric Ligand-Gated Ion Channels with Bromoform Reveals Many Interconnected Anesthetic Binding Sites

2014 ◽  
Vol 106 (2) ◽  
pp. 342a
Author(s):  
Benoist Laurent ◽  
Samuel Murail ◽  
Ludovic Sauguet ◽  
Marc Delarue ◽  
Marc Baaden
Keyword(s):  
Ion Channels ◽  
Binding Sites ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

The Cys-loop superfamily of ligand-gated ion channels: the impact of receptor structure on function

2004 ◽  
Vol 32 (3) ◽  
pp. 529-534 ◽  
Author(s):  
C.N. Connolly ◽  
K.A. Wafford
Keyword(s):  
Ion Channels ◽  
Conformational Changes ◽  
Channel Structure ◽  
Channel Function ◽  
Receptor Structure ◽  
Channel Opening ◽  
The Impact ◽  
Ion Pore ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

The Cys-loop receptors constitute an important superfamily of LGICs (ligand-gated ion channels) comprising receptors for acetylcholine, 5-HT3 (5-hydroxytryptamine; 5-HT3 receptors), glycine and GABA (γ-aminobutyric acid; GABAA receptors). A vast knowledge of the structure of the Cys-loop superfamily and its impact on channel function have been accrued over the last few years, leading to exciting new proposals on how ion channels open and close in response to agonist binding. Channel opening is initiated by the extracellular association of agonists to discrete binding pockets, leading to dramatic conformational changes, culminating in the opening of a central ion pore. The importance of channel structure is exemplified in the allosteric modulation of channel function by the binding of other molecules to distinct sites on the channel, which exerts an additional level of control on their function. The subsequent conformational changes (gating) lead to channel opening and ion transport. Following channel pore opening, ion selectivity is determined by receptor structure in, and around, the ion pore. As a final level of control, cytoplasmic determinants control the magnitude (conductance) of ion flow into the cell. Thus the Cys-loop receptors are complex molecular motors, with moving parts, which can transduce extracellular signals across the plasma membrane. Once the full mechanical motions involved are understood, it may be possible to design sophisticated therapeutic agents to modulate their activity, or at least be able to throw a molecular spanner into the works!

scholarly journals Disrupting inter-subunit interactions favors channel opening in P2X2 receptors

2021 ◽  
Author(s):  
Federica Gasparri ◽  
Sarune Bielickaite ◽  
Mette Homann Poulsen ◽  
Stephan Alexander Pless
Keyword(s):  
Ion Channels ◽  
Subunit Interactions ◽  
Cellular Protection ◽  
Protection Mechanism ◽  
Subunit Interface ◽  
Channel Opening ◽  
Close Proximity ◽  
Low Threshold ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding to their large extracellular domain (ECD). The seven known P2XR subtypes typically assemble as homo- or heterotrimeric complexes and they contribute to numerous physiological functions, including nociception, inflammation and hearing. Both the overall structure of P2XRs and the details of how ATP is coordinated at the subunit interface are well established. By contrast, little is known about how inter-subunit interactions in the ECD contribute to channel function. Here we investigate both single and double mutants at the subunit interface of rP2X2Rs using electrophysiological and biochemical approaches. Our data demonstrate that the vast majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity and that double mutants at the subunit interface show significant energetic coupling, especially if the mutations are located in close proximity. Overall, we show that inter-subunit interactions, as well as possibly interactions in other parts of the receptor, stabilize WT rP2X2Rs in the closed state. This suggests that, unlike other ligand-gated ion channels, P2X2 receptors have not evolved for an intrinsically low threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

scholarly journals Allosteric Modulation of Ligand Gated Ion Channels by Ivermectin

2014 ◽  
pp. S215-S224 ◽  
Author(s):  
H. ZEMKOVA ◽  
V. TVRDONOVA ◽  
A. BHATTACHARYA ◽  
M. JINDRICHOVA
Keyword(s):  
Ion Channels ◽  
Binding Site ◽  
Glycine Receptor ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Determinants ◽  
P2x4 Receptor ◽  
Electrophysiological Recordings ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

Ivermectin acts as a positive allosteric regulator of several ligand-gated channels including the glutamate-gated chloride channel (GluCl),  aminobutyric acid type-A receptor, glycine receptor, neuronal α7-nicotinic receptor and purinergic P2X4 receptor. In most of the ivermectin-sensitive channels, the effects of ivermectin include the potentiation of agonist-induced currents at low concentrations and channel opening at higher concentrations. Based on mutagenesis, electrophysiological recordings and functional analysis of chimeras between ivermectin-sensitive and ivermectin-insensitive receptors, it has been concluded that ivermectin acts by insertion between transmembrane helices. The three-dimensional structure of C. elegans GluCl complexed with ivermectin has revealed the details of the ivermectin-binding site, however, no generic motif of amino acids could accurately predict ivermectin binding site for other ligand gated channels. Here, we will review what is currently known about ivermectin binding and modulation of Cys-loop receptor family of ligand-gated ion channels and what are the critical structural determinants underlying potentiation of the P2X4 receptor channel.

scholarly journals Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels

2016 ◽  
Vol 124 (3) ◽  
pp. 664-673 ◽  
Author(s):  
Monica N. Kinde ◽  
Weiming Bu ◽  
Qiang Chen ◽  
Yan Xu ◽  
Roderic G. Eckenhoff ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Ion Channels ◽  
Magnetic Resonance ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Functional Responses ◽  
Nuclear Magnetic ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

Abstract Background Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. Methods We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1β3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations. Results Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. Conclusions The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.

scholarly journals In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 453
Author(s):  
Jiayi Yuan ◽  
Chen Jiang ◽  
Junmei Wang ◽  
Chih-Jung Chen ◽  
Yixuan Hao ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Cannabinoid Receptors ◽  
Md Simulations ◽  
Docking Studies ◽  
Allosteric Modulators ◽  
3D Structures ◽  
Potential Binding ◽  
Allosteric Sites ◽  
Allosteric Binding Sites

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

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