Characterizing Ligand-Gated Ion Channel Receptors with Genetically Encoded Ca++ Sensors

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca2+ permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT3A serotonin receptors and a chimera of human α7/mouse 5-HT3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters

Towards an understanding of human alpha-7 nicotinic acetylcholine receptor selectivity : the creation and characterization of a soluble ligand binding domain template

Receptors play a vital role in the transduction of cell to cell signaling. They are important proteins with recognition capacity in signal transduction, particularly in neurons. Cholinergic neurons in the central and peripheral nervous systems are defined by storage, release, and stimulation of the neurotransmitter acetylcholine. At the synaptic cleft, the release of acetylcholine from presynaptic neurons triggers activation through channel opening of the nicotinic acetylcholine receptors (nAChRs) in the postsynaptic membrane. Previous research implicates the nAChRs in diseases including schizophrenia, drug addiction (specifically nicotine addiction), Alzheimer's and Parkinson's diseases. The work presented here uses a comprehensive approach including structural studies, protein engineering, functional studies, and protein characterization to gain insight into the distinguishing characteristics that may contribute to ligand selectivity and structural components that contribute to receptor function. The main aim of this work was to create a soluble template of the ligand binding domain of human alpha-7-nAChRs to understand pharmacological selectivity, while also creating an efficient tool for therapeutic development. In order to validate this study, methods to test the functional activity of nAChRs were developed and in the process a novel calcium FRET sensor based assay was developed as a cost-effective tool for drug screening and functional ligand characterization on nAChRs and other ligand-gated, calcium-permeable, ion channels. To process the information generated by the developed assay, a novel data storage system in the form of a database was created. The creation of a soluble template fully identical to the ligand binding domain of human alpha-7-nAChR was not successful, but the protein generated served to identify structurally a glycosylation site on the receptor as well as proved to be a better binding homologue for alpha-7- nAChRs than any other proteins currently available. Overall, the mutational conversion of a soluble homologue to the human alpha-7-nAChR gave insight into the structural understanding of the extracellular domain of nAChRs. This template provides a better model to create lead compounds as therapeutics for diseases associated with the nAChR

Emerging Molecular Mechanisms of Signal Transduction in Pentameric Ligand-Gated Ion Channels

International audienceNicotinic acetylcholine, serotonin type 3, γ-amminobutyric acid type A, and glycine receptors are major players of human neuronal communication. They belong to the family of pentameric ligand-gated ion channels, sharing a highly conserved modular 3D structure. Recently, high-resolution structures of both open- and closed-pore conformations have been solved for a bacterial, an invertebrate, and a vertebrate receptor in this family. These data suggest that a common gating mechanism occurs, coupling neurotransmitter binding to pore opening, but they also pinpoint significant differences among subtypes. In this Review, we summarize the structural and functional data in light of these gating models and speculate about their mechanistic consequences on ion permeation, pathological mutations, as well as functional regulation by orthosteric and allosteric effectors

Fumarate as positive modulator of allosteric transitions in the pentameric ligand-gated ion channel GLIC: Requirement of an intact vestibular pocket.: Fumarate as positive allosteric modulator of GLIC.

International audienceGLIC is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain di-carboxylate compounds are positive modulators of pHo 5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue-dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati et al. 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both di- carboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore gating modulation

Full mutational mapping of titratable residues helps to identify proton-sensors involved in the control of channel gating in the Gloeobacter violaceus pentameric ligand-gated ion channel.

The Gloeobacter violaceus ligand-gated ion channel (GLIC) has been extensively studied by X-ray crystallography and other biophysical techniques. This provided key insights into the general gating mechanism of pentameric ligand-gated ion channel (pLGIC) signal transduction. However, the GLIC is activated by lowering the pH and the location of its putative proton activation site(s) still remain(s) unknown. To this end, every Asp, Glu, and His residue was mutated individually or in combination and investigated by electrophysiology. In addition to the mutational analysis, key mutations were structurally resolved to address whether particular residues contribute to proton sensing, or alternatively to GLIC-gating, independently of the side chain protonation. The data show that multiple residues located below the orthosteric site, notably E26, D32, E35, and D122 in the lower part of the extracellular domain (ECD), along with E222, H235, E243, and H277 in the transmembrane domain (TMD), alter GLIC activation. D122 and H235 were found to also alter GLIC expression. E35 is identified as a key proton-sensing residue, whereby neutralization of its side chain carboxylate stabilizes the active state. Thus, proton activation occurs allosterically to the orthosteric site, at the level of multiple loci with a key contribution of the coupling interface between the ECD and TMD

Crystal structures of a pentameric ion channel gated by alkaline pH show a widely open pore and identify a cavity for modulation

International audiencePentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonists in the extracellular domain, the transmembrane pore opens, allowing ions to go through, via a gating mechanism that can be modulated by a number of drugs. Even though high-resolution structural information on pLGICs has increased in a spectacular way in recent years, both in bacterial and in eukaryotic systems, the structure of the open channel conformation of some intensively studied receptors whose structures are known in a nonactive (closed) form, such as Erwinia chrysanthemi pLGIC (ELIC), is still lacking. Here we describe a gammaproteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is closely related to the pLGIC from ELIC with 28% identity. We provide an X-ray crystallographic structure at 2.3 Å in an active conformation, where the pore is found to be more open than any current conformation found for pLGICs. In addition, two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions, such as tetramethylammonium. Additionally, we found that sTeLIC is allosterically potentiated by aromatic amino acids Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC

Protein expression of nonfunctional mutants.

<p>Rabbit anti-HA tag Alexa Fluor-695 immunostaining results of the mutants that produced no detectable current in functional tests, compared to Wt and GFP-alone injected oocytes. Left, colored merge of GFP and Alexa-695 and right, the Alexa-695 imaging alone. Both D122N and H235A show no expression. GLIC, <i>G</i>. <i>violaceus</i> ligand-gated ion channel; GFP, green fluorescent protein; HA, human influenza hemagglutinin; Wt, wild-type.</p

Protein expression of nonfunctional mutants.

<p>Rabbit anti-HA tag Alexa Fluor-695 immunostaining results of the mutants that produced no detectable current in functional tests, compared to Wt and GFP-alone injected oocytes. Left, colored merge of GFP and Alexa-695 and right, the Alexa-695 imaging alone. Both D122N and H235A show no expression. GLIC, <i>G</i>. <i>violaceus</i> ligand-gated ion channel; GFP, green fluorescent protein; HA, human influenza hemagglutinin; Wt, wild-type.</p

TMD residues decrease sensitivity.

<p>Stick representation of the evaluated Glu and His residues are labeled. Glu residues are color coded based upon effect, with residues in red producing a significant decrease in pH₅₀, and in yellow, an insignificant or weak effect, whereas the dark purple and magenta for His residues are synonymous to the red of Glu residues. The main subunit is shown in black, with ribbon and cartoon representation with the α-helices labeled, whereas neighboring subunits are represented in white ribbon. The M2 α-helix of the neighboring subunit (labeled M2ʹ) is also highlighted in cyan and in cartoon representation. A table of the pH₅₀ and ΔpH₅₀ is shown below with mean values ± standard deviation for evaluated mutants with recorded values noted in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004470#pbio.2004470.s001" target="_blank">S1 Table</a>. Light red and green zones with dashed lines represent the respective ½ log decrease and increase from Wt. Values outside this region are color coded for ease of interpretation, with red and darker red as significant decreases and a decrease of greater significance, respectively. TMD, transmembrane domain; Wt, wild-type.</p