The Relative Orientation of the TM3 and TM4 Domains Varies between α1 and α3 Glycine Receptors

Glycine receptors (GlyRs) are anion-conducting members of the pentameric ligand-gated ion channel family. We previously showed that the dramatic difference in glycine efficacies of α1 and α3 GlyRs is largely attributable to their nonconserved TM4 domains. Because mutation of individual nonconserved TM4 residues had little effect, we concluded that the efficacy difference was a distributed effect of all nonconserved TM4 residues. We therefore hypothesized that the TM4 domains of α1 and α3 GlyRs differ in structure, membrane orientation, and/or molecular dynamic properties. Here we employed voltage-clamp fluorometry to test whether their TM4 domains interact differently with their respective TM3 domains. We found a rhodamine fluorophore covalently attached to a homologous TM4 residue in each receptor interacts differentially with a conserved TM3 residue. We conclude that the α1 and α3 GlyR TM4 domains are orientated differently relative to their TM3 domains. This may underlie their differential ability to influence glycine efficacy

Development of the assay for single cell-based functional imaging.

<p><b>A.</b> Determination of optimal assay conditions. HEK293 cells YFP_I152L and α1 GlyRs were seeded into 384-well plates. The culture medium was replaced by 20 µl control NaCl solution and cells were imaged once to record cellular fluorescence in unquenched state (grey dot at 0 µM glycine). This step was conducted to optimise auto-focussing and detection of fluorescent objects in recorded fluomicrographs. Cells were perfused with NaI solution containing indicated lycine concentrations, whereupon a series of 5 images was recorded every 1.5 s. The lowest and highest fluorescence intensity, recorded in the first and fourth/fifth image is colored blue and red, respectively, with the intensities in intermediate images colored grey. <b>B.</b> Data filtering. Images of fluorescent cells always contained a substantial amount of debris (e.g. dead cells, cells expressing YFP_I152L but not GlyRs or cells detaching during perfusion). To discriminate functionally relevant fluorescence signals from artefactual data, dose responses were filtered after automatic curve fitting. The histogram shows the average number (mean ± SD) of objects per image, averaged from 10 experiments, with 20 randomly selected wells per experiment before (black, cells & debris) and after filtering (striped, mainly cells) using either of the parameters EC₅₀, slope (nH), R² and ΔF as described in Methods. To achieve a maximal number of unbiased concentration responses all four parameters were combined for filtering (grey), resulting in approx. 50% of data points considered acceptable. <b>C.</b> Representation of normalized concentration-responses measured in a single well after filtering. In this example a total of 186 (grey) and 95 (black) cells were accepted and rejected, respectively. <b>D.</b> Scatter plot of EC₅₀ and slope values derived from curves shown in panel C. ***P<0.0001 relative to untreated control, unpaired t-test.</p

Clustering functional GlyR phenotypes in pure and heterogeneous cell populations.

<p><b>A.</b> Color map of mean glycine EC₅₀ (µM) in presence and absence of the drugs strychnine, picrotoxin and lindane, as indicated. Warm and cold colors represent low and high EC₅₀ values indicating no effect and inhibition, respectively. The black rectangle highlights experiments shown in B and C. <b>B.</b> Dot plot of glycine EC₅₀ and slope values measured in presence of 10 and 100 µM lindane in pooled pure (α2, blue; α2β, orange) and mixed (α2+α2βgreen, 1∶1 ratio) cell populations from the experiment in A. Values measured in pure populations were used for training a J48 decision tree classification algorithm. <b>C.</b> Functional phenotyping of lindane-exposed GlyRs in pure (α2, blue; α2β, orange) and mixed (α2+α2β green) cell populations. Cells were classified according to their descriptors with a training set of 402 cells distributed in 2 classes: α2 (black), α2β (grey). Bars represent the percentage of cells assigned to either of the two classes and reflect the initial 1∶1 mixing ratio (green).</p

Work flow of experiment and data analysis.

<p>HEK293 cells were transiently co-transfected with YFP_I152L and GlyR cDNA (). Approximately 48 h later, cells were seeded into the wells of 384-well plates at defined density and are cultured for another 24 h (). Functional analysis of GlyRs is carried out by progressive receptor activation and iterative fluorescence imaging using an in house-built automated screening device with integrated liquid-handling robotics (). Recorded images are segmented and fluorescence dose-responses calculated () are fitted (). Finally, functional parameters measured in single cells, such as R², ΔF, slope and EC₅₀ are filtered to discriminate functional from non-functional data ().</p

Effects of the lindane on α2, α3, α2β and α3β GlyRs as determined by fluorescence assay.

<p><b>A, B, D, E.</b> Dot plots of glycine EC₅₀ and slope values in absence (black) and presence (grey) of 10 µM lindane measured in single cells expressing α2 and α2β (A–B) and α3 and α3β (D–E) GlyR. <b>C, F.</b> Histogram of median glycine EC₅₀ (±SD) calculated from data shown in panels A, B (C) and D,E (F). These results provide evidence for lindane as a pharmacological tool for identifying the presence of β subunits in α2β and α3β heteromeric GlyRs. ***P<0.0001 relative to untreated control, unpaired t-test.</p

Quality control and proof-of-principle of the screening assay.

<p><b>A.</b> Dot plot of glycine EC₅₀ and slope values obtained from dose-response experiments with cells expressing α1, α2 and α3 GlyRs conducted in individual wells. <b>B.</b> Averaged and normalized concentration-responses from the experiment depicted in A. <b>C.</b> Comparison of EC₅₀ values from the experiment shown in panels A and B to data from whole-cell patch-clamp electrophysiology (Islam and Lynch, 2011). <b>D.</b> Box-plot of glycine EC₅₀ in α2β GlyRs measured in 64 wells of a 384-well plate demonstrating the stability and usability of the assay for operation in high-throughput mode. Boxes and whiskers display the 25–75% and 5–95% data ranges, respectively, of all accepted values in each well. <b>E.</b> Drug-effects of 10 µM lindane (grey) and 30 µM strychnine (light grey) on α1 and α1β GlyRs, validating our assay for identification and characterization of chemicals modulating GlyRs. ***P<0.0001 relative to untreated control, unpaired t-test.</p

Stoichiometry and Subunit Arrangement of α1β Glycine Receptors As Determined by Atomic Force Microscopy

The glycine receptor is an anion-permeable member of the Cys-loop ion channel receptor family. Synaptic glycine receptors predominantly comprise pentameric α1β subunit heteromers. To date, attempts to define the subunit stoichiometry and arrangement of these receptors have not yielded consistent results. Here we introduced FLAG and six-His epitopes into α1 and β subunits, respectively, and imaged single antibody-bound α1β receptors using atomic force microscopy. This permitted us to infer the number and relative locations of the respective subunits in functional pentamers. Our results indicate an invariant 2α1:3β stoichiometry with a β–α–β–α–β subunit arrangement

Phosphorylation of α3 Glycine Receptors Induces a Conformational Change in the Glycine-Binding Site

Inflammatory pain sensitization is initiated by prostaglandin-induced phosphorylation of α3 glycine receptors (GlyRs) that are specifically located in inhibitory synapses on spinal pain sensory neurons. Phosphorylation reduces the magnitude of glycinergic synaptic currents, thereby disinhibiting nociceptive neurons. Although α1 and α3 subunits are both expressed on spinal nociceptive neurons, α3 is a more promising therapeutic target as its sparse expression elsewhere implies a reduced risk of side-effects. Here we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences that might be exploited in designing α3-specific analgesics. Using voltage-clamp fluorometry, we show that glycine-mediated conformational changes in the extracellular M2-M3 domain were significantly different between the two GlyR isoforms. Using a chimeric approach, we found that structural variations in the intracellular M3-M4 domain were responsible for this difference. This prompted us to test the hypothesis that phosphorylation of S346 in α3 GlyR might also induce extracellular conformation changes. We show using both voltage-clamp fluorometry and pharmacology that Ser346 phosphorylation elicits structural changes in the α3 glycine-binding site. These results provide the first direct evidence for phosphorylation-mediated extracellular conformational changes in pentameric ligand-gated ion channels, and thus suggest new loci for investigating how phosphorylation modulates structure and function in this receptor family. More importantly, by demonstrating that phosphorylation alters α3 GlyR glycine-binding site structure, they raise the possibility of developing analgesics that selectively target inflammation-modulated GlyRs

Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode <i>H</i>. <i>contortus</i>

<div><p>Ivermectin (IVM) is a widely-used anthelmintic that works by binding to and activating glutamate-gated chloride channel receptors (GluClRs) in nematodes. The resulting chloride flux inhibits the pharyngeal muscle cells and motor neurons of nematodes, causing death by paralysis or starvation. IVM resistance is an emerging problem in many pest species, necessitating the development of novel drugs. However, drug optimisation requires a quantitative understanding of GluClR activation and modulation mechanisms. Here we investigated the biophysical properties of homomeric α (avr-14b) GluClRs from the parasitic nematode, <i>H</i>. <i>contortus</i>, in the presence of glutamate and IVM. The receptor proved to be highly responsive to low nanomolar concentrations of both compounds. Analysis of single receptor activations demonstrated that the GluClR oscillates between multiple functional states upon the binding of either ligand. The G36’A mutation in the third transmembrane domain, which was previously thought to hinder access of IVM to its binding site, was found to decrease the duration of active periods and increase receptor desensitisation. On an ensemble macropatch level the mutation gave rise to enhanced current decay and desensitisation rates. Because these responses were common to both glutamate and IVM, and were observed under conditions where agonist binding sites were likely saturated, we infer that G36’A affects the intrinsic properties of the receptor with no specific effect on IVM binding mechanisms. These unexpected results provide new insights into the activation and modulatory mechanisms of the <i>H</i>. <i>contortus</i> GluClRs and provide a mechanistic framework upon which the actions of drugs can be reliably interpreted.</p></div

Comparison of the effect of 1 mM glutamate on wild-type and G36’A mutant GluClRs.

<p><b>A)</b> Examples of continuous single channel activity recorded from G36’A mutant GluClRs. Note the emergence of a ‘spiky’ activation mode (red boxes) that is not observed in wild-type GluClRs. Wild-type-like activations are termed ‘mode 1’ or ‘high Po’, whereas spiky activations are termed ‘mode 2’ or ‘low Po’. <b>B)</b> Examples of continuous single channel activity recorded from wild-type GluClRs included for comparison. <b>C)</b> Comparison of mean active durations (upper panel) and Po (lower panel) of low (LP_O) and high (HP_O) P_O events recorded from G36’A mutant GluClRs (n = 6 patches). <b>D)</b> Examples of activations demarcated by a grey bar in A and B. These activations are of the high P_O mode for the G36’A mutant (above) and normal mode for wild-type (below). The comparison indicates that there are more numerous open-shut events within the activations of G36’A compared to wild-type. <b>E)</b> Shut and open dwell histograms for data obtained from G36’A mutant GluClRs at 1 mM glutamate. This plot combined LP_O and HP_O activations of G36’A receptors at 1 mM glutamate. The histograms show that the mutant receptors have two shut and three open components. <b>F)</b> Shut and open dwell histograms for data obtained from wild-type GluClRs at 1 mM glutamate, revealing two shut and three open components.</p