Next-gen antivenoms bite back: explorations in antibodies, de novo proteins, snake toxins and ion channels

Melisa Benard Valle, Technical University of Denmark, DTU, Copenhagen, Denmark; Susana Vazquez, Torres University of Washington, Seattle, USA; Damian Bell, Sophion, Copenhagen, Denmark; Kim Boddum, Sophion Bioscience, Copenhagen, Denmark; David Baker, University of Washington, Seattle, USA; Aneesh Karatt-Vellatt Maxion Therapeutics, Cambridge, United Kingdom; Anna Damsbo Technical University of Denmark, DTU, Copenhagen, Denmark; Andreas Laustsen-Kiel Technical University of Denmark, DTU, Copenhagen, Denmark; Timothy Jenkins Technical University of Denmark, DTU, Copenhagen, Denmark.

There is an urgent need for more effective snakebite envenoming treatments, a neglected global health issue responsible for high mortality and morbidity rates. Traditional antivenoms, typically derived from animal plasma, have limitations in safety, efficacy and cost, especially against the diverse toxin components found in snake venoms.

Recent advances in therapeutic design, including the use of phage display technology, have enabled the discovery and optimization of broadly-neutralizing human monoclonal antibodies and oligoclonal nanobody mixtures. These biotherapeutics demonstrate significant neutralization capacity against various neurotoxins and venom compositions, including those of coral snakes and other elapids, with high efficacy in murine models. Additionally, deep learning-driven de novo protein design has produced stable, high-affinity proteins that effectively neutralize multiple 3FTx sub-families,offering promising and accessible alternatives to traditional antivenoms.

Collectively, these strategies underscore a shift towards safer, cost-effective, and multi-target antivenom solutions, providing a foundation for next-generation therapeutics against snakebite and other complex pathologies.

_______________________________________________

Functional characterization of GRIN2B variants using automated patchclamp technology

Mehwish Akram,  Sygnature Discovery; Glasgow, United Kingdom; Christina Kadi, Sygnature Discovery, Glasgow, United Kingdom; Eleanor Parker Sygnature Discovery, Glasgow, United Kingdom; Claire Brown, Sygnature Discovery, Glasgow, United Kingdom; Laura Pisarek, Sygnature Discovery, Glasgow, United Kingdom; Simon Rice, Sygnature Discovery, Glasgow, United Kingdom; Rosario Finocchiaro, Sygnature Discovery, Glasgow, United Kingdom; Rachel Long, United Kingdom; Sian Morrison, Sygnature Discovery, Glasgow, United Kingdom; David Dalrymple, Sygnature Discovery, Glasgow, United Kingdom; Ian McPhee, Sygnature Discovery, Glasgow, United Kingdom; Davide Pau, Sygnature Discovery, Glasgow, United Kingdom; Laura Hutchison, Sygnature Discovery, Glasgow, United Kingdom. Clark, Metrion, Cambridge, UK.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors composed of two Glycine-binding NR1subunits (encoded by GRIN1 gene) in combination with two Glutamate-binding NR2 subunits (encoded by GRIN2A,GRIN2B, GRIN2C or GRIN2D genes). Rare de novo variants of the GRIN genes have been associated with neurodevelopmental disorders (NDD) and epileptic encephalopathy resulting in seizures, behavioural symptoms and movement disorders. Current investigations are focusing on functional and pharmacological analyses to understand the properties of these variants and potentially lead to a more rapid classification of GRIN variants as gain-of-function (GoF) orloss-of-function (LoF). The classification of these variants from patients may provide diagnostic advantages and together with precision medicine could enable the development of a personalised therapy.

The functional analysis of these variants includes at least six different assays which aim to investigate the receptor’s sensitivity to the agonists Glutamate and Glycine, extracellular Mg²⁺ inhibition, alterations in response time-course (e.g. kinetics), channel open probability and trafficking to the plasma membrane. The results of these assays are required to determine the variant classification (Myers et al., 2023).

Until now, the functional analysis of the GRIN variants has been restricted to the conventional manual patch-clamp and two electrode voltage techniques. Although this approach has successfully been used to classify variants, the low throughput slows the rapid screening that may be required for target-directed pharmacological treatments. Here, using automated patch-clamp technology, we describe the functional and pharmacological characterization of human embryonic kidney (HEK) cells transfected with GRIN1A (wild-type) or GRIN1-A652C in combination with the GoF and LoF GRIN variants.

The aim of this work is to demonstrate the capability of the automated patch-clamp system to characterise variants as LoFor GoF. In the future, the establishment of a higher throughput assay will enable a faster evaluation of the GRIN variants, where multiple variants can be assessed simultaneously, while obtaining robust data. Furthermore, it is expected that this time efficient approach can be applied more generally in a wide range of ion channels for the functional classification of other missense variants related to neurological disorders.

[1] Myers, S.J, Yuan, H., Perszyk, R.E., Zhang, J., Kim, S., Nocilla, K.A., Allen, J.P., Bain, J.M., Lemke, J.R., Lal, D., Benke,T.A., Traynelis, S.F. (2015) NMDA receptor subunit mutations in neurodevelopmental disorders. Curr. Opin. Pharmacol.,20, 73–82.

_______________________________________________

De novo variants in the KCNC4 potassium channel gene disrupt Kv3.4ion channel function and neuronal excitability in a novelneuro developmental disorder

Mariana Vargas-Caballero, University of Southampton, Southampton, United Kingdom; Jenny Lord, University of Sheffield, Sheffield, United Kingdom; Annie Godwin, University of Portsmouth, Portsmouth, United Kingdom; Cerys Roberts, University of Southampton, Southampton, United Kingdom; Tia Fletcher, University of Portsmouth, Portsmouth, United Kingdom; Midhun Krishnan, University of Southampton, Southampton, United Kingdom; Alexander Lowe, University of Southampton, Southampton, United Kingdom; Callum Ives, Atlantic Technologic University, Sligo, Ireland; Katrin Deinhardt, University of Southampton, Southampton, United Kingdom; Nichola Foulds, University of Southampton, Southampton, United Kingdom; Meriel Mcentagart St George's University Hospital, NHS, London, United Kingdom; Elisa Fadda, University of Southampton, Southampton, United Kingdom; Sarah Ennis, University of Southampton, Southampton, United Kingdom; Matthew Guille, University of Portsmouth, Portsmouth, United Kingdom; Diana Baralle, University of Southampton, Southampton, United Kingdom.

Introduction

Voltage-gated potassium (Kv) channels are essential for regulating neuronal excitability, with mutations in KCNC1–3associated with various neurological disorders. However, mutations in KCNC4, which encodes the Kv3.4 potassium channel subunit [1], have not previously been linked to disease. In this study, we identifed variants in KCNC4 in 4 patients presenting with developmental delay, ataxia, microcephaly, cerebellar atrophy, and hypomyelination. We hypothesised that these variants would result in ion channel dysfunction, impacting excitability and neurodevelopment.

Method

We used patch clamp electrophysiology to test KCNC4 variants in HEK293 cells. We found three loss-of-function mutations (p.Ser268Gly, p.Arg362Cys, p.Leu481Pro) and one gain-of-function mutation (p.Arg404His). Computational models using NEURON simulation environment suggested profound changes in neuronal excitability on human Purkinje neurons [2].

To further investigate loss of function in vivo, we developed a CRISPR-induced KCNC4 Xenopus tropicalis model [3],which recapitulated key neurodevelopmental defects, including growth delay and microcephaly. Neuronal firing analysis in this model showed impaired excitability, consistent with the role of Kv3.4 in high-frequency neuronal firing. These findings establish KCNC4 as a novel disease gene, highlighting the role of potassium channel mutations in neuro developmental disorders and broadening the spectrum of potassium channelopathies. Our results have important implications for both clinical diagnostics and the development of targeted therapies.

[1] Kaczmarek LK, Zhang Y. Kv3 Channels: Enablers of Rapid Firing, Neurotransmitter Release, and Neuronal Endurance. Physiological Reviews. 2017;97(4):1431-1468. doi: 10.1152/physrev.00002.2017
[2] Masoli S, Sanchez-Ponce D, Vrieler N, et al. Human Purkinje cells outperform mouse Purkinje cells in dendritic complexity and computational capacity. Commun Biol . 2024;7(1):5. doi: 10.1038/s42003-023-05689-y
[3] Ismail V, Zachariassen LG, Godwin A, et al. Identification and functional evaluation of GRIA1 missense and truncation variants in individuals with ID: An emerging neurodevelopmental syndrome. Am J Hum Genet. 2022;109(7):1217-1241.doi: 10.1016/j.ajhg.2022.05.009

_______________________________________________

Glutamatergic pharmacology and receptor modification by EndosomalSorting Complex Required for Transport proteins

Mohamed Shalaby, University of Bradford, Bradford, United Kingdom; Harsha Kimtameneni, University of Bradford, Bradford, United Kingdom; Samantha McLean, University of Bradford, Bradford, United Kingdom.

Introduction

Growing evidence supporting the NMDA receptor hypofunction theory of schizophrenia highlights the importance ofrestoring glutamatergic signaling as a potential strategy for developing novel antipsychotic therapies. The endosomal sorting complexes required for transport (ESCRT) proteins are critical in sorting ubiquitinated membrane receptors to lysosomes, a key process for reducing cell surface receptor signaling. This study investigates the impact of Tsg101 andVps4a gene knockdown on the expression and function of ionotropic glutamate receptors.

Methods

HEK293T cells were transfected with NR1a/NR2a (NMDA receptors) and GluK2 (kainate receptors) and treated with shRNA targeting Tsg101 and dominant-negative Vps4a to inhibit ESCRT function. Expression of mutant receptors was confirmed by western blot analysis. The surface expression of receptors was assessed using live-cell biotinylation and immunocytochemistry. To evaluate receptor functionality, calcium influx was measured using Rhod-2 AM fluorescence imaging, and whole-cell patch-clamp recordings were performed to assess channel currents and agonist response.

Results

Knockdown of Vps4a and Tsg101 resulted in increased epidermal growth factor receptor (EGFR) accumulation, indicating impaired receptor trafficking and potential disruption of receptor recycling pathways. Functional assays showed that NMDA and kainate agonists significantly increased calcium influx and channel currents in cells expressing mutant receptors. Specifically, Tsg101 knockdown resulted in elevated NMDA receptor-mediated calcium signaling (405.64 ± 34.12 RFU vs. control 297.23 ± 19.8, P < 0.05), which was sufficient to counteract the inhibitory effects of phencyclidine (PCP, an NMDAR antagonist), as evidenced by an increased IC₅₀ for PCP (73 ± 6.1 μM vs. 19.6 ± 1.6 μM, n = 3, P < 0.01).Additionally, immunocytochemistry and biotinylation assays revealed increased surface expression of the NR2a subunit(38.1% ± 2.8, P < 0.05) and enhanced trafficking of receptors to the plasma membrane (12.68% ± 1.08, P < 0.05).

Interestingly, the overexpression of GluK2 receptors (in contrast to NMDA receptors) showed a neuroprotective effect ,reducing calcium signaling at higher kainate concentrations, suggesting that GluK2 receptors may buffer excessive calcium influx and protect against excitotoxicity. This finding highlights the distinct role of kainate receptors in modulating cellular calcium dynamics compared to NMDA receptors.

Conclusions

These findings identify Tsg101 as a novel regulator of glutamate receptor trafficking, influencing NMDA and kainate receptor surface expression and function. The results suggest that modulating ESCRT-mediated receptor trafficking could be a promising strategy to enhance excitatory signaling and stabilize neuronal circuits, with potential applications in neurodegenerative and psychiatric diseases such as schizophrenia. Targeting ESCRT pathways may provide novel therapeutic approaches aimed at correcting glutamatergic dysregulation and improving synaptic plasticity.

_______________________________________________

The MIDAS motif and glycosylation of CACHD1 are important for expression and function as a CaV3.1 voltage-gated calcium channel modulator

Maria Roznovcova, University of Reading, Reading, United Kingdom; Tyler C. Deutsch, University of Virginia, Charlottersville, USA; Caitlin Lamb-Jordan, University of Reading, Reading, United Kingdom; Muna Ibrahim, University of Reading, Reading, United Kingdom; Manoj K. Patel, University of Virginia, Charlottersville, USA; Graeme S. Cottrell, University of Reading, Reading, United Kingdom; Gary J. Stephens, University of Reading, Reading, United Kingdom.

Voltage-gated calcium channels (VGCCs) regulate intracellular calcium, and their dysfunction contributes to diseases including pain. CACHD1 (calcium channel and chemotaxis receptor domain-containing protein 1), an α2δ-like protein, has been identified as a Ca_V3 modulator and, similar to α2δ auxiliary subunits, contains a MIDAS (metal ion-dependent adhesion site) motif within the von Willebrand Factor A (VWA) domain, previously shown to be essential for the trafficking and synaptic function of HVA VGCCs. Additionally, glycosylation plays a crucial role in α2δ function, stability, and surface expression. Specifically, glycosylation of Asn136 and Asn184 in the α2δ subunit is necessary for its function, likely affecting its interaction with other channel subunits and its role in modulating calcium channel activity. Therefore, understanding the roles of the MIDAS motif and CACHD1 glycosylation could reveal new regulatory mechanisms and has potential therapeutic implications.

We investigated the roles of MIDAS motif and glycosylation in CACHD1 on its expression and function as a modulator of Ca_V3.1 VGCCs in HEK293 cells. MIDAS motif mutants; CACHD1-AAA (3 key residues mutated to Ala, D 234 xGxS to AxAxA) and CACHD1-G236S (mimicking α2δ (DxSxS) MIDAS motif); and glycosylation mutants(N145/329/373/587/940/985Q, NΔ6Q) were generated by PCR. Sub-cellular localisation of CACHD1 was assessed by immunocytochemistry, using live and fixed cell labelling (n=5). Expression levels of CACHD1 were characterised by western blotting and densitometry (n=5); statistical significance was determined using ANOVA (Tukey’s post hoc test).CACHD1 modulation of Ca_V3.1 was characterised by patch clamp electrophysiology; statistical significance was determined using ANOVA (Bonferroni post hoc test) and two-tailed unpaired t-test.

CACHD1-wt and CACHD1-G236S were localised to the cell surface and intracellular vesicles, whereas CACHD1-AAA was only localised to intracellular vesicles. In stable expression studies, a significant reduction in total CACHD1 levels was seen for CACHD1-AAA (22±6.8% vs CACHD1-wt, mean±SEM, p<0.05) with no significant change forCACHD1-G236S (93±4.2% vs CACHD1-wt, mean±SEM). CACHD1-wt and CACHD1-G236S significantly increased CaV3.1 current density and maximal conductance (1.46- and1.49-fold), while CACHD1-AAA caused significant reduction (0.62-fold). All glycosylation mutants were expressed at the cell surface, and all mutations caused a decrease inmolecular mass (except N940Q; n=3), suggesting CACHD1 has five N-linked glycosylation sites.

Our findings highlight the essential role of the MIDAS motif in CACHD1, suggesting that, like the α2δ MIDAS motif, theCACHD1 MIDAS motif contributes to protein expression and CACHD1-associated modulation of Ca_V3.1 VGCCs. Modulation of Ca_V3 expression might have therapeutic utility for hyperexcitability diseases, including pain.

_______________________________________________

Computational Insights into KV10.1 Channels in Breast Cancer:Electrophysiological Mechanisms and Therapeutic Prospects

Chitaranjan Mahapatra, Indian Institute of Technology Bombay, Mumbai, India; Arshdeep Kaur, University of California San Francisco, San Francisco, USA.

Background

KV10.1 (Eag1) channels are frequently overexpressed in various malignancies, including breast cancer, where they contribute to critical cellular processes such as motility, cell cycle progression, and the inhibition of apoptosis. Notably, KV10.1 channels have been implicated in facilitating epithelial-to-mesenchymal transition (EMT), a key mechanism underlying cancer metastasis. Recent insights from immuno-oncology propose that KV10.1 may also modulate immune cell dynamics within the tumor microenvironment, potentially influencing immune surveillance and evasion. Despite their recognized significance, the detailed electrophysiological mechanisms and oncogenic potential ofKV10.1 in breast cancer remain inadequately characterized.

Methods

This study integrates computational modeling simulations to elucidate the electrophysiological properties ofKV10.1 channels. The models were designed to replicate channel dynamics under diverse physiological conditions, focusing on membrane potential modulation, ion flux, and resultant cellular responses in breast cancer cells. To enhance accuracy and reliability, structural and functional predictions derived from the computational models were validated against existing experimental data.

Results

Computational analyses revealed distinct functional disparities in KV10.1 channels between cancerous and healthy cells, particularly regarding altered gating kinetics and conductance properties that may enhance proliferative and metastatic capacities. Specific KV10.1 inhibitors were identified, exhibiting high binding affinities and selective inhibitory effects. Elevated KV10.1 expression correlated with a decrease in membrane potential in breast cancer cells. Ion channel conductance and current measurements further substantiated these findings. Notably, the application of 10 nM tricyclic antidepressants, recognized as KV10.1 blockers, effectively stabilized resting membrane potential, suggesting their potential therapeutic utility.

Conclusions

The findings underscore the pivotal role of KV10.1 channels in the electrophysiological regulation of breast cancer cells, positioning them as promising targets for therapeutic intervention. In silico data suggest that KV10.1 inhibition may disrupt oncogenic signaling pathways, enhance immune system recognition, and impede tumor progression and metastasis. These results advocate for further experimental validation, as selective KV10.1 inhibitors could significantly advance breast cancer therapeutics by mitigating metastatic spread and bolstering anti-tumor immune responses.

_______________________________________________

Mechanism of GLP-1 receptor signaling in human POMC neurons

Simone Mazzaferro, Institute of Metabolic Science, Department of Pharmacology, University of Cambridge, United Kingdom; Hsiao-Jou Cortina Chen, Institute of Metabolic Science, Department of Pharmacology, University of Cambridge, United Kingdom; Olivier Cahn, Imperial College, London, United Kingdom; Andrian Yang European Bioinformatics Institute, European Molecular Biology Laboratory, Cambridge, United Kingdom; Dmytro Shepilov, Institute of metabolic Science, Department of Pharmacology, University of Cambridge, United Kingdom; Jiahui Chen, Institute of Metabolic Science, University of Cambridge, United Kingdom; Constanza Alcaino, Institute of Metabolic Science, University of Cambridge, United Kingdom; Viviana Macarelli, Institute of Metabolic Science, University of Cambridge, United Kingdom; Iman Mali, Institute of Metabolic Science, University of Cambridge, United Kingdom; Fiona Gribble, Institute of Metabolic Science, University of Cambridge, United Kingdom; Frank Reimann, Institute of metabolic Science, University of Cambridge, United Kingdom; John C. Marioni European Bioinformatics Institute, European Molecular Biology Laboratory, Cambridge, United Kingdom, Wellcome Sanger Institute, Cambridge, United Kingdom, Genentech, San Francisco, USA; Florian T. Merkle, Institute of Metabolic Science, Department of Pharmacology, Cambridge, United Kingdom, Cambridge Stem Cell Institute, Cambridge, United Kingdom.

Obesity shortens the lifespan of millions of people worldwide by increasing the risk of severe chronic diseases. Pharmacotherapies in combination with lifestyle interventions are a promising strategy tohelp obese patients reduce their body weight. Specifically, glucagon-like peptide receptor (GLP-1R)
agonists have emerged as an effective and approved therapy to promote weight loss by reducing appetite and food intake. Studies in mice demonstrated that GLP-1R agonists Liraglutide and Semaglutide reach the hypothalamus and activate pro-opiomelanocortin (POMC)neurons, which reduce food intake when stimulated.

How GLP-1R signal is integrated by POMC neurons in humans remains poorly understood, limiting the development of pharmacotherapies that selectively enhance the anorectic function of these cells. To address this need, we generated human POMC neurons from Pluripotent Stem Cell (iPSC) lines and used live imaging, high-content microscopy, electrophysiology, and RNA sequencing to determine the mechanisms and the long-term consequences of activating the GLP-1R signaling in human POMC neurons.

We found that GLP-1R agonists persistently increase the excitability of POMC neurons, and identified candidate mechanisms that sustain this excitability. Additionally, we determined that long-term exposure to GLP-1R agonists regulates the expression of genes that might contribute to appetite regulation.

The study provides insight into the mechanism of action of GLP1R agonists and suggests new targets that could further enhance the appetite-suppressing effects of GLP1R agonists via neurons to reduce feeding and body weight.

_______________________________________________

Cloxiquine is a gating modulator of a voltage and calcium-gated channel in U2OS cells

The human Osteosarcoma cell-line U2OS is a popular model in imaging and other assay technologies due to its large cytosplasm area and ease of transfection. Here, we demonstrate using an automated patch-clamp electrophysiology assay that application of the antimalarial drug Cloxiquine is followed by a large increase in outward currents in U2OS cells.

This effect was dependent on the intracellular concentration of Calcium and could be inhibited by the tool compoundTRAM34, a known blocker of SK4 calcium activated K+ channels.

The large dormant population of calcium activated K+ channels in U2OS makes this cell line liable to false positives when used as a cellular background for K+ flux assays in drug discovery.

Linus Johann Conrad, Mechanistic Biology & Profiling UK/US, BioPharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom; John Linley, Neuroscience, BioPharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom.

_______________________________________________

A robust platform for recombinant production of animal venom toxinmodulators of ion channels

Jose Enrique Gonzalez-Prada, University of Cambridge, Cambridge, United Kingdom; Alex Haworth, Metrion Biosciences, Cambridge, United Kingdom; Jacob Browne, University of Cambridge, Cambridge, United Kingdom; Samantha Salvage, University of Cambridge, Cambridge, United Kingdom; Anne Ritoux, University of Cambridge, Cambridge, United Kingdom; Maya Dannawi, University of Cambridge, Cambridge, United Kingdom; Leonard Lee, University of Oxford, Oxford, United Kingdom; Vasiliki Mavridou, University of Cambridge, Cambridge, United Kingdom; Yin Dong, University of Oxford, Oxford, United Kingdom; Ewan Smith, University of Cambridge, Cambridge, United Kingdom; Anthony P Jackson, University of Cambridge, Cambridge, United Kingdom; Edward Stevens, Metrion Biosciences, Cambridge, United Kingdom; Paul S Miller, University of Cambridge, Cambridge, United Kingdom.

Peptide toxins are an essential component of ion channel drug discovery. Their structural complexity contributes to high potency and selectivity compared to small molecules¹. Since both extraction from the host organism and chemical synthesis can be costly and inaccessible to the standard lab, recombinant expression in heterologous systems is an ideal alternative for producing toxins for SAR and HTS campaigns. However, the disulfide bridges responsible for high stability of many types of snake, spider and scorpion peptide toxins can be scrambled during expression from heterologous systems, and so production fails or yields are low and activity is poor. Fusing toxins to carrier proteins such as antibodies can greatly improve stability and yield, and also imbue favourable in vivo biodistribution2. In this work, we established a strategy for the robust, high-yield production of Fc-Toxin fusions.

A panel of 13 snake, spider and scorpion toxins were genetically fused to a human immunoglobulin γ1 heavy chain in two alternative formats, in a bivalent (Biv) configuration with two toxin payloads, or in a monovalent (Mov) configuration with one toxin payload. After expression and secretion and affinity purification via octahis tags, the bivalent analogues typically had yields at or below 12 μg from each ml of expression media, whereas monovalent yields were robustly above 15 μgfrom each ml of media (n = 3). Furthermore, the expression for both formats was much greater than that of the non-Fcfused toxins. A Fura-2 assay was used to measure the effect of Fc-snake toxins relative to synthetic toxins on the acetylcholine-induced Ca2+ response of TE671 cells endogenously expressing nAChR receptors3. Both monovalent and bivalent Fc domains retained strong inhibition, but with a modest a 4-5-fold loss in potency with respect to the synthetic toxins (n=4). HEK293T cells transiently transfected with nACHRα7, GABARα1β3, or a negative control, preincubated with Fc-αCBTx cell media and subsequently stained with anti-Fc antibody (AF568), revealed clear staining by the Fc-αCBTx against nACHRα7 but not GABARα1β3, for which the toxin has lower affinity for (n=5).

In this work, we have established a robust platform for the production of antibody toxins and demonstrated their utility as functional probes and cell-labelling agents.

1. Montnach J, De Waard S, Nicolas S, et al. Fluorescent- and tagged-protoxin II peptides: potent markers of the Nav1.7channel pain target. Br J Pharmacol. 2021;178(13):2632-2650.
2. Wulff H, Christophersen P, Colussi P, Chandy KG, Yarov-Yarovoy V. Antibodies and venom peptides: new modalities forion channels. Nat Rev Drug Discov. 2019;18(5):339-357.
3. Bencherif M, Lukas RJ. Differential regulation of nicotinic acetylcholine receptor expression byhuman TE671/RD cells following second messenger modulation and sodium butyrate treatments. Mol Cell Neurosci. 1991;2(1):52-65.

_______________________________________________

Integral Membrane Proteins for Biophysics and Cryo-EMapplications – Case Study of the Ion Channel TrpML3

Philip Rawlins, Domainex Limited, Cambridge, United Kingdom; Nathan Zaccai, Domainex Limited, Cambridge, United Kingdom; Alec O’Keeffe, Domainex Limited, Cambridge, United Kingdom; Christopher Morton, Domainex Limited, Cambridge, United Kingdom; Alexandra Parker, Domainex Limited, Cambridge, United Kingdom; Holly Madden, Domainex Limited, Cambridge, United Kingdom; Graeme Sloan, Domainex Limited, Cambridge, United Kingdom; Philip Leonard, Domainex Limited, Cambridge, United Kingdom; Steven Hardwick, University of Cambridge, Cambridge, United Kingdom; Dimitri Chirgadze, University of Cambridge, Cambridge, United Kingdom; Stefanie Reich, Domainex Limited, Cambridge, United Kingdom; Jim Reid, Domainex Limited, Cambridge, United Kingdom; Natalie Winfield, Domainex Limited, Cambridge, United Kingdom.

Ion channels are important drug discovery targets, yet their biophysical and structural analysis remains challenging. The ion channel TrpML3 is a therapeutic target implicated in autophagy.

Following successful protein purification of TrpML3, we confirmed small molecule ligand binding by Grating Coupled Interferometry (GCI).Our structural biologists generated both apo and ligand (ML-SA1) bound Cryo-EM maps for TrpML3, at 2.7 Å and 3.0 Å resolution, respectively. Comparison of the two derived atomic models demonstrated a clear transition from the closed channel apo state to the open channel ligand bound state (Figure 1). Detailed map features in the region of the ligand and its binding pocket were used as a guide to model ligand location and conformation, thereby opening the way for future computer-aided drug design (CADD) and virtual screening efforts.

Figure 1: Comparison of post-processed electron-potential maps of apo (orange, 2.7 Å)and ML-SA1 bound TrpML3 (blue, 3.0 Å). A transition from the closed TrpML3 channel apostate to the open channel ML-SA1 bound state is observed.

_______________________________________________

Characterisation of cancer cell lines with hERG mRNA expression

Malcolm Charangwa, University of Warwick, Coventry, United Kingdom; Linus Conrad, AstraZeneca, Cambridge, United Kingdom.

Drug-induced long QT syndrome is one of the most common safety risks in drug development. Functional effects on the hERG ion channel are ruled out with electrophysiological safety assays that act to capture any acute inhibition of hERG by prototype compounds. However, genetic defects in hERG that also cause long QT-syndrome are most commonly related to the channels trafficking tothe plasma membrane (90% of pathogenic hERG variants). This suggests that drug induced changes to plasma membrane trafficking of hERG may translate into long-QT phenotypes in the clinic that cannot be de-risked with the electrophysiological tests for acute inhibition.

Here, we are characterising a panel of unmodified cell lines with known expression of hERG mRNA using automated patch-clamp to find a suitable model to study the trafficking and functional expression of native hERG channels.

_______________________________________________

Mechanisms Underlying Mesenchymal Stem Cell-Derived Extracellular Vesicle Modulation of Sensory Neuron Excitability

Lanhui Qiu, Universty of Cambridge, Cambridge, United Kingdom; Alexander Cloake, Universty of Cambridge, Cambridge, United Kingdom; Luke Pattison, Universty of Cambridge, Cambridge, United Kingdom; Tim Williams, Universty of Cambridge, Cambridge, United Kingdom; Paula Milan-Rois, Universty of Cambridge, Cambridge, United Kingdom; Ewan Smith, Universty of Cambridge, Cambridge, United Kingdom.

Introduction

Mesenchymal stem cells (MSCs) are promising therapeutic agents in treating different conditions, including osteoarthritis(OA). Recent investigations have underscored the pivotal role played by MSC-derived extracellular vesicles (EVs) that enable biomolecular cargo exchange between cells. We previously demonstrated the capacity of MSC-EVs to alleviate pain in a mouse OA model, which was due to MSC-EV amelioration of OA-induced sensory neuron hyperexcitability, rather than modifying joint pathology, thereby illuminating their potential in pain management. Additionally, MSC-EVs directly reverse nerve growth factor (NGF)-induced nociceptor sensitization in vitro, suggesting a potential mechanism for their analgesic effects. This study investigates how MSC-EVs regulate sensory neuron excitability [LQ2].

Methods

MSC-EVs were isolated via ultracentrifugation of conditioned media. Whole-cell patch-clamp electrophysiology was usedto assess excitability in IB4-negative dorsal root ganglion (DRG) neurons following MSC-EV treatment. EV surfaceproteins were removed using trypsin or proteinase K ("shaving"), and effects on EV internalization and neuronalmodulation were assessed using confocal microscopy and electrophysiology. MSC-EVs and DRG neurons were co-cultured for both short-term (10 min) and long-term (48 hours) durations. RNA sequencing was performed on the MSC-EVs.

Results

MSC-EVs exhibited the expected lipid bilayer structure. Proteinase treatment did not alter EV morphology but inhibited internalization and reduced MSC-EV modulation of neuronal excitability. Short-term MSC-EV treatment (10 min) failed to reverse NGF-induced hyperexcitability, and actinomycin D (a transcription inhibitor) prevented EV-mediated recovery, suggesting a transcriptional mechanism. RNA sequencing of EVs identified several microRNAs implicated in pain relief and transcriptional regulation. To further explore this, we are developing gold nanoparticle-based microRNA delivery systems for co-culture experiments with DRG neurons.

Conclusions

MSC-EV internalization appears necessary for modulating DRG neuron excitability, likely through transcriptional regulation. Further research will clarify the mechanisms underlying MSC-EV effects and their potential for pain management.

_______________________________________________

Analyzing the developmental profile and physiological function of neuronal Kv7 channels

Elif Karabatak, Thomas Budde, University Hospital Münster, Institute of Physiology. Collaboration partners: Guiscard Seebohm (University of Münster), Frank Glorius (University of Münster), Iain Greenwood (City St. George’s, University of London).

Introduction/Background & Aims

Kv7 (KCNQ or M-) channels generate voltage-dependent K+ currents and play a fundamental role in regulating neuronal excitability. These channels comprise five known subtypes (Kv7.1-7.5), with Kv7.2 and Kv7.3 being the primary subunits in most neurons[1,2]. Channel modulators are critical for physiological and pathophysiological conditions, emphasizing the need for a deeper understanding of their developmental expression and functional modulation [3]. Previous studies from our group have highlighted the significance of Kv7 channels within the thalamocortical system [4]. This project aims to investigate (1) the developmental expression profile of Kv7.2 and Kv7.3 channels in thalamic neurons and(2) the modulation of these channels by various compounds.

Methods/Summary of Work

• RT-qPCR was employed to investigate ion channel expression at the mRNA level.
• Western blot analysis was used to assess protein-level expression of Kv7.2 and Kv7.3.
• Cell transfection was performed to generate a HEK293FT cell line expressingfunctional Kv7.2 and Kv7.3 channels.
• Mouse models (C57BL/6 wild-type mice) were used to examine in vivo expression patterns.
• Patch-clamp electrophysiology measured ion channel activity and response to various modulators.

Results/Discussion

• Kv7.2 and Kv7.3 expression was detected as early as postnatal day 10 and remained present throughout development.
• Overexpressed Kv7.2 and Kv7.3 channels in HEK293FT cells were functional.
• Patch-clamp recordings demonstrated significant modulation of M-current bycholesterol and steroid compounds:
  • Cholesterol application significantly reduced current (n=10).
  • Progesterone (50 μM) decreased current amplitude (n=11).
  • β-Estradiol (10 μM) induced a reduction in current (n=7).
  • CHIM-L, an imidazolium-based cholesterol analog, mimicked cholesterol’sinhibitory effect (n=8).

Conclusion(s)

Our findings highlight the developmental expression profile and functional modulation of Kv7.2 and Kv7.3 channels in thalamic neurons. The results underscore the importance of maintaining optimal cholesterol levels to ensure proper Kv7channel function. These insights contribute to understanding Kv7 channels as potential therapeutic targets for neurological disorders.

[1] Dirkx et al., Front. Physiol., 2020, S. 1240.
[2] Brown et al., Br. J. Pharmacol., 2009, 156(8), 1185-1195.
[3] Borgini et al., RSC Med. Chem., 2021, 12.4, 483-537.
[4] Cerina et al., Br. J. Pharmacol., 2015, 172.12, 3126-3140.

_______________________________________________

Triheteormeric NMDA Receptors as drug targets in human neurons?

Oreoluwa Fakeye, South Coast Biosciences DTP, University of Southampton, Southampton, United Kingdom; Midhun Krishnan Vasanthakrishnan, University of Southampton, Southampton, United Kingdom; Kif Liakath-Ali, University of Southampton, Southampton, United Kingdom; Mariana Vargas-Caballero, University of Southampton, Southampton, United Kingdom.

Introduction

NMDA receptors (NMDARs) are tetramers formed of two GluN1 subunits and either identical (diheteromers) or different (triheteromers) GluN2/GluN3 subunits. Current NMDAR targeting drugs are either general antagonists (e.g. D-AP5) or subunit-specific antagonists like TCN201(GluN2A) and Ifenprodil(GluN2B), but none selectively target triheteromers with two different GluN2 subunits.

Here we highlight the role of GluN2A/GluN2B triheteromers in contributing to the slow-decaying NMDAR currents that sustain the recurrent activation of layer V pyramidal neurons in the mouse medial prefrontal cortex(mPFC), facilitating working memory ¹. Additionally, we show that expression in triheteromers may be crucial for the synaptic localisation of a short primate-specific GluN2A isoform (GluN2A-S) ².

Methods

Synaptic currents were obtained by performing whole-cell patch clamp recordings of primary brain slices (mPFC) orcultured hippocampal and cortical neurons transduced with Lentiviral constructs(GluN2A-S), from wild-type C57BL/6 miceor a conditional GluN2A/GluN2B knockout mouse model. Summary data and sample traces were generated from an average of at least 3 NMDAR currents per cell using MATLAB. Statistical significance was assessed using one-tailed T-tests, and graphs were generated using GraphPad prism.

Results

We show a developmental acceleration of the slow decaying NMDAR currents in the mouse mPFC indicating a GluN2Acontribution to recurrent activation, possibly through its incorporation into triheteromers. GluN2A loss-of-function mutations are associated with Schizophrenia [3]. However, research on their effect on working memory focuses on interneurons dueto the perceived dominance of GluN2B in pyramidal neurons. The GluN2A contribution to the slow decaying currents suggests that recurrent activity in pyramidal neurons may also be disrupted. We also show that replacing endogenousGluN2(A/B) subunits in cultured hippocampal neurons with GluN2A-S abolishes the synaptic NMDAR current, suggesting that GluN2A-S cannot be synaptically expressed in diheteromers. Further experiments will investigate how coexpression of GluN2A-S with other GluN2 subunits affects its synaptic expression.

Conclusion

Together, these highlight the importance of developing triheteromer-specific modulators by developing direct molecule interactors or strategies to control their formation.

[1] Wang H, Stradtman GG, Wang XJ, Gao WJ. A specialized NMDA receptor function in layer 5 recurrentmicrocircuitry of the adult rat prefrontal cortex. Proc Natl Acad Sci U S A . 2008;105(43):16791-16796.doi:10.1073/PNAS.0804318105.
[2] Warming H, Pegasiou CM, Pitera AP, et al. A primate-specific short GluN2A-NMDA receptor isoform is expressedin the human brain.
Mol Brain . 2019;12(1):1-8. doi:10.1186/s13041-019-0485-9.
[3] Lu Y, Mu L, Elstrott J, et al. Differential depletion of GluN2A induces heterogeneous schizophrenia-relatedphenotypes in mice. Published online 2024. doi:10.1016/j.ebiom.2024.105045.

_______________________________________________

Lipid invasion as a mechanistic hypothesis for Alzheimer’s disease

Sofia Ioanna Nouka, School of Pharmacy, University of Reading, Reading, United Kingdom; Dr Jonathan Rudge, School of Pharmacy
University of Reading, Reading, United Kingdom; Dr Nandini Vasudevan, School of Biological Sciences, University of Reading, Reading, United Kingdom; Dr Francesco Tamagnini, School of Pharmacy, University of Reading, Reading, United Kingdom.

Introduction

Late-onset AD (LOAD) is influenced by age, genetics and cholesterol metabolism [1]. The lipid invasion model proposes that BBB damage and cholesterol dysregulation drive LOAD. A compromised BBB allows influx of peripheral lipids, including LDL-cholesterol, disrupting brain lipid homeostasis [2]. To explore the mechanistic role of lipids in LOAD we evaluated the effect of amyloid-beta (Aβ42) on neuronal physiology. We further assessed the effect of total cholesterol(TC) on Aβ production and neuro steroid levels in the hippocampus (HP) and prefrontal cortex (PFC).

Methods

HP brain slices, 300μm, were obtained from 7–11-week-old male and female C57BL/6J mice following cervical dislocation and decapitation. After a 1-hour recovery period, the slices were treated with 500nM Aβ42 or vehicle (0.05% NH4OH) for2-5 hours. At the 2-hour timepoint voltage and current clamp recording were conducted to measure electrotonic and electrogenic properties of CA3 interneurons. HP brain slices were obtained from 65–74-week-old female mice that have been carrying the humanized APPNL-G-F gene. Following a 1-hour recovery period, voltage and current clamp recordings of CA1 pyramidal neurons were conducted. Recordings were analysed using MATLAB-R2024b, with statistical significance determined via independent sample T-tests in Origins. 8-11-week-old male and female C57BL/6J mice were sacrificed, and 200μm coronal brain slices from the PFC and HP were obtained. After a 1-hour rest period, the slices were treated with 1ug/1.5mL of TC for 24-hours. Tissue and aCSF were collected for molecular and biochemical analysis. Analysis was conducted in PRISM-10.1.1, with significance determined using 2-way ANOVA.

Results

500nM of Aβ42 does not impair neuronal function of CA3 interneurons. qPCR showed that TC treatment did not alter expression of amyloidogenic genes. ELISA showed no significant changes in 17b-oestradiol following treatment in the PFC or hippocampus. However, testosterone levels increased in the PFC of females.

Discussion

Acute exposure of 500nM of Aβ42 does not impair the physiology of CA3 neurons, though prior findings suggest that the treatment induces hyperexcitation in CA1 pyramidal neurons [3]. This could indicate region-specific or neuron-type selectivity. TC treatment did not alter amyloidogenic gene expression, while is associated with increased testosterone in females. Ongoing research employs patch-clamp electrophysiology to assess chronic exposure to Aβ, using the APPNL-G-F mouse model, and investigate the effect of LDL-cholesterol on neuronal physiology. Molecular and biochemical analysis will evaluate the effect of treatment on lipid markers, and neuro steroid alterations within and between sexes.

[1] Feringa FM, van der Kant R. Cholesterol and Alzheimer’s Disease; From Risk Genes to Pathological Effects. FrontAging Neurosci. 2021;13. doi:10.3389/fnagi.2021.690372.
[2] Rudge JD. A New Hypothesis for Alzheimer’s Disease: The Lipid Invasion Model. J Alzheimers Dis Rep. 2022;6(1):129-161. doi:10.3233/ADR-210299.
[3] Tamagnini F, Scullion S, Brown JT, Randall AD. Intrinsic excitability changes induced by acute treatment ofhippocampal CA1 pyramidal neurons with exogenous amyloid β peptide. Hippocampus. 2015;25(7):786-797.doi:10.1002/hipo.22403.

_______________________________________________

Production and structural/functional characterization of Mucolipin-1 andthe P2X purinoceptor 3 as significant drug targets

David Speck Crelux, Crelux, Gräfelfing, Germany.

Introduction/Background & aims

Mucolipin-1 (also known as TRPML1), encoded by the MCOLN1 gene, is a lysosomal/endosomal cation channel that mediates ion transport (e.g., Fe²⁺ and Ca²⁺) crucial for proper lysosomal function, including vesicular trafficking, exocytosis, and autophagy. Mutations in MCOLN1 cause mucolipidosis type IV (MLIV), a lysosomal storage disorder characterized by psychomotor delay, vision loss, and achlorhydria, primarily due to defective lysosomal ion transport and the accumulation of lipids and proteins.

The P2X purinoceptor 3 (P2X3) is a ligand-gated ion channel activated by extracellular ATP, playing a key role in pain perception by mediating ATP-evoked activation of sensory neurons. It is implicated in chronic pain conditions such as neuropathic, inflammatory, and cancer pain.

Given the therapeutic relevance of TRPML1 and P2X3, CRELUX (a WuXi AppTec company) undertook their production and subsequent structural/functional characterization on behalf of clients.

Method/Summary of work

For initial construct evaluations, both ion channels were expressed in small-scale systems with either an N-terminal His 6 or FLAG tag. The most promising constructs were then expressed on a liter scale and purified through affinity and size-exclusion chromatography. TRPML1 (with and without ligand) was vitrified on cryo-EM grids, and the resulting samples were resolved at resolutions enabling model building. P2X3 was subjected primarily to biophysical characterization via nano differential scanning fluorimetry (nanoDSF) to assess how client compounds affect its thermostability.

Results/Discussion

Cryo-EM analysis of TRPML1 enabled visualization of the binding mode for client-derived ligand, as well as the channel inthe open- and closed conformation. P2X3 exhibited a marked increase in thermostability in nanoDSF assays, depending on both the compound and its concentration.

Conclusions

Through protein engineering approaches and literature-guided optimization, CRELUX successfully expressed and purified TRPML1 and P2X3 from mammalian systems using a multi-step purification strategy. High-resolution cryo-EM structures of TRPML1 were obtained in both open and closed states in complex with a client compound. Meanwhile, P2X3 proved suitable for biophysical screening, demonstrating enhanced thermal stability upon ligand binding. These findings highlight the potential of TRPML1 and P2X3 as valuable platforms for drug discovery targeting lysosomal function as well as pain modulation.

_______________________________________________

Targeting T-type calcium channels to combat pathophysiology of chemotherapy-induced peripheral neuropathy (CIPN)

Anjana Pattila, SCFP, University Of Reading, Reading, United Kingdom; Sofia Fontana-Giusti SCFP, University Of Reading, Reading, United Kingdom; Daniil Luzyanin SCFP, University Of Reading, Reading, United Kingdom; Daniel Allen-Ross SCFP, University Of Reading, Reading, United Kingdom; Francesco Tamagnini SCFP, University Of Reading, Reading, United Kingdom; Maria Maiarú SCFP, University Of Reading, Reading, United Kingdom; Gary J Stephens SCFP, University Of Reading, Reading, United Kingdom.

Introduction

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and dose-limiting side effect of cancertreatment, causing chronic pain and sensory dysfunction that severely impacts patients' quality of life. Despite the highprevalence of CIPN, no FDA-approved therapies currently exist, highlighting the urgent need for novel treatments [1].Recent evidence points to CaV3.2 calcium channels as key players in CIPN pathophysiology, where their upregulationcontributes to neuronal hyperexcitability and aberrant sensory signalling [2]. This study evaluates the effects of the CaV3blocker, TTA-P2, in a mouse model of CIPN.

Methods

Effects of TTA-P2 (1 mg/kg) on in vivo mechanical allodynia pain-like behaviours were measured in the paclitaxel (PTX) model of CIPN in
14-month-old male and female C57Bl/6 mice. Pilot current-clamp recordings were made to investigate effects ofTTA-P2 (1mM) on hippocampal CA3 pyramidal neuron electrophysiological activity in exvivo brain slices from naïve male (6-8 weeks) C57Bl/6 mice.

Results and discussion

In a pilot behavioural study, TTA-P2 restored mechanical sensitivity thresholds to pre-PTX levels(3-way ANOVA for factor ‘treatment’, p=0.00035 and ‘sex’ p=0.099; n=3-4), with a stronger antinociceptive effect whengiven at 14d post-PTX. TTA-P2 improved cold hypersensitivity in females after a single dose at 14d, but in males only aftera second dose at 28d (3-way ANOVA for factor ‘treatment’ p= 0.033 and ‘sex’ p= 0.047; n=3-4). In electrophysiologicalstudies, TTA-P2 increased input resistance from 85 ± 3.8 to 95 ± 3.8 M W (n=5, P<0.05, two-tailed t-test) and increased latency of firing from 0.11 ± 0.16 to 0.68 ± 0.20 s (n=5, P<0.05 two-tailed t-test) at +150 pA current injection, suggesting altered neuronal firing dynamics.
Conclusion: Our preliminary findings suggest that pharmacological inhibition of T-typecalcium currents has anti-nociceptive effects in the CIPN model. TTA-P2 can also affect neuronal excitability. These data will be advanced to investigate the therapeutic potential oftargeting CaV3 channels in CIPN.

[1] Fonseca, M. de C., Marazzi-Diniz, P. H. S., Leite, M. F. & Ehrlich, B. E. (2023) Calciumsignalling in chemotherapy-induced neuropathy. Cell Calcium 113, 102762.
[2] Cai, S., Gomez, K., Moutal, A. &; Khanna, R. (2021) Targeting T-type/CaV3.2 channels for chronic pain. Transl Res234, 20–30.

_______________________________________________

Lysosomal ion channels and transporters as drug targets: a methodological study

Alison Obergrussberger, Nanion Technologies GmbH, Munich, Germany; Markus Rapedius, Nanion Technologies GmbH, Munich, Germany; Nioletta Murciano, Nanion Technologies GmbH, Munich, Germany; Andre Bazzone, Nanion Technologies GmbH, Munich, Germany; Maria Barthmes, Nanion Technologies GmbH, Munich, Germany; Rocco Zerlotti, Nanion Technologies GmbH, Munich, Germany; Niels Fertig, Nanion Technologies GmbH, Munich, Germany.

Introduction

Intracellular ion channels are known to play an essential role in various signaling pathways in health and disease. Over80% of transport processes take place across intracellular membranes. Among the variety of organelles, lysosomal channels and transporters, such as the proton leak channel transmembrane protein 175 (TMEM175), the transient receptor potential cation channel, mucolipin subfamily TRPML1, and the lysosomal two-pore channel (TPC) have received increasing attention in the field. This interest was sparked by genetic studies of the corresponding transporter genes (suchas variants in TMEM175) suggesting lysosomal ion channel (dys-)function plays a crucial role for the susceptibility for Parkinson’s disease and cancer. Consequently, there is great interest in exploring intracellular ion channels and their pharmacology using high-throughput electrophysiology.

Methods

Automated patch clamp (APC) and solid supported membrane electrophysiology (SSME) instruments were employed in this study to record ion channels and transporters in isolated intact lysosomes. The SyncroPatch 384, a high throughput APC device, was used to record ion channels in isolated lysosomes, allowing recordings from hundreds of lysosomes to take place simultaneously while manipulating internal and external recording solutions. In addition, SSME using the SURFE2 R N1 and SURFE2 R 96SE was used to record lysosomal ion channels and transporters in intact lysosomes.

Results/Discussion

We present and compare data on several lysosomal ion channels including TMEM175, TRPML1 and TPC2 recorded using high throughput APC and SSME. We activated TMEM175, TRPML1 and TPC2 using the activators DCPIB, ML-SA1or TPC2-A1-P, respectively, and could also enhance current amplitude by changing pH of the internal solution. We could also block currents using specific or non-specific inhibitors and compared EC50 and IC
50 values obtained using different techniques.

Conclusion

Our findings demonstrate the suitability of high throughput electrophysiological techniques, including APC and SSME, for advancing research into lysosomal ion channel physiology and pharmacology, thus accelerating the discovery of potential therapeutics.

_______________________________________________

Human Induced Pluripotent Stem Cell Derived Dorsal Horn Neurons for the Study and Drug Discovery of Central Pain Targets

Marc Rogers, Albion Drug Discovery Services Ltd, Cambridge, United Kingdom; Vincent Truong, Anatomic Inc, Minneapolis, USA; Asta Arendt-Tranholm, University of Texas at Dallas, Center for Advanced Pain Studies, Dallas, USA; Christian Petroski, Neuroservices Alliance, San Diego, USA; Dong Liu Neuroservices Alliance, San Diego, USA; Sergey Grigoryev, Neuroservices Alliance, San Diego, USA; Miaomiao He, Neuroservices Alliance, San Diego, USA; Bob Petroski, Neuroservices Alliance, San Diego, USA; Theodore Price University of Texas (Dallas), Center for Advanced Pain Studies, Dallas, USA; Patrick Walsh Anatomic Inc, Minneapolis, USA.

Central sensitization, characterized by hypersensitivity within the spinal dorsal horn, plays a pivotal role in the development and persistence of chronic pain [1]. The recent FDA approval of the NaV1.8 inhibitor Suzetrigine exemplifies the current focus of analgesia drug discovery on peripheral nociceptors, but opportunities to explore central mechanisms of pain remain largely unaddressed. The validation of new pain targets within the central nervous system has been limited by the lack of physiologically relevant human models. Here we present a comprehensive molecular and functional characterization of spinal cord dorsal horn neurons (DHNs) derived from human induced pluripotent stem cells (hiPSCs), establishing a novel platform for the investigation of central nociceptive mechanisms and therapeutic target discovery.

Using a novel small molecule and growth factor–based differentiation protocol, multiple batches of DHNs were characterized by immunocytochemistry and qPCR to assess lot-to-lot consistency. Immunocytochemical analysis confirmed a highly enriched neuronal population comprising approximately 80% glutamatergic neurons and 20%GABAergic neurons. qPCR analysis of dorsal horn–specific genes (BRN3A, LMX1B, PAX2, GBX1, DMRT3
and LHX5) demonstrated consistent expression profiles across several production runs. Bulk RNA sequencing revealed a profile similar to primary human dorsal horn tissues, supporting the regional fidelity of the model. To explore the utility of this platform for pain target discovery, we analyzed the expression of nociception-relevant ion channels (Table 1),neurotransmitter receptors, neuropeptides, as well as genes implicated in central sensitization and opioid response, including GRIN1, SCN9A, TAC1, and OPRM1.

To correlate transcriptomic signatures with functional maturation, we performed time course electrophysiology assessments using microelectrode array (MEA) recordings and manual whole-cell patch clamp analysis. MEA recordings from DHNs cultured on Axion Cy to View MEA 24 well plates revealed the emergence of synchronous burst firing activity asearly as two weeks in vitro, indicative of functional network formation and dynamic interplay between excitatory glutamatergic and inhibitory GABAergic neuronal subtypes. Whole-cell patch clamp recordings demonstrated that 72% of neurons exhibited hyperpolarized resting membrane potentials below −50 mV, 86% fired spontaneous action potentials, and 57% generated repetitive spike trains in response to 1-second depolarizing current injections. All recorded neurons displayed large voltage-gated sodium currents (~3,000 pA), and 81% exhibited miniature excitatory postsynaptic currents (mEPSCs), consistent with synaptic activity and advanced electrophysiological maturation.

Together, these findings demonstrate hiPSC-derived DHNs as a scalable and translationally relevant screening tool forinvestigating human-specific mechanisms underlying chronic pain.

[1] Latremoliere A, Woolf CJ. Central sensitization: a generator of pain hypersensitivity by central neural plasticity. J Pain.2009;10(9:895-926. doi:10.1016/j.jpain.2009.06.012.

Table 1. Nociception-relevant ion channels expressed in hiPSC-derived dorsal horn neurons after1 and 3 weeks in culture post-thaw, quantified as transcripts per million (TPM) from bulk RNA-seqdatasets. DNP = Did not populate.

_______________________________________________

Investigating state-dependent effects of diclofenac using a NaV1.x cellline panel

Ellen Braksator, B'SYS GmbH, Witterswil, Switzerland; Simon Hebeisen, B'SYS GmbH, Witterswil, Switzerland; Daniel Konrad, B'SYS GmbH, Witterswil, Switzerland.

Introduction / Background and aims

The aim of this work was to investigate the state-dependent effects of diclofenac on sodium channels expressed in heterologous expression systems.

Method / Summary of work

Automated whole-cell patch-clamp recordings (QPatch HTX, Sophion, Denmark) were performed from CHO or HEK293cells stably expressing human NaV1.1, NaV1.2, NaV1.5, NaV1.6, NaV1.7, NaV18 or NaV1.9. Except for NaV1.9, cells were clamped at -100 mV and depolarized to 0 mV for 10 ms. (resting state). For the evaluation of the fast inactivated state, cells were clamped for 2 s to a potential 10 mV more negative V0.5 (inactivation), followed by a depolarization to 0 mV for10 ms. Cells were clamped to 0 mV for at least 5 s to enter slow inactivated states. To recover all fast inactivated states, ashort pulse to -100 or -110 mV followed, before cells were again depolarized to 0 mV for 10 ms. (slow inactivated state).This test pulse was applied every 30 s. During wash in of diclofenac, cells were clamped to the holding potential for at least 60 s. NaV1.9: cells were clamped at -140 mV and sodium inward currents were elicited by 40 ms voltage steps from-140 mV to -40 mV. and, once stable baseline currents were achieved, cells were continuously perfused with a bathsolution containing a concentration of diclofenac. The amount of current inhibition was calculated as percentage of controland IC
50 values were determined.

Results / Discussion & Conclusion

Differences in NaVsubtype specificity and state-dependence were observed with diclofenac, the results can be summarised as follows:

Table 1: Diclofenac summary, IC₅₀ values (μM) and Hill coefficient. ND, not determined.

_______________________________________________

Potassium Channel Modulation of Murine Gastrointestinal Motility

Abishana Vishuvanathan, University of Hertfordshire, Hatfield, United Kingdom; Christopher Benham, University of Hertfordshire, Hatfield, United Kingdom; Christopher Keating, University of Hertfordshire, Hatfield, United Kingdom.

Introduction

Understanding the mechanisms underlying gut motility and contractility is crucial for elucidating the pathophysiology underpinning gastrointestinal disorders such as irritable bowel syndrome and devising effective therapeutic interventions. Potassium channels are known to regulate motor activity in the gastrointestinal tract. In this study, we investigate the role of Kv7 and TREK channels in modulating murine gastrointestinal motility.

Methods

Colonic, jejunal and ileal segments from C57BL/6J mice were utilized. Electric field stimulation (EFS) was applied as 5-sec pulses (0.1ms, 20V, 0.3Hz colon or 10Hz small intestine) every 5 min to assess neurally invoked smooth muscle contractility and peak contractile responses to EFS were measured. An in-vitro colonic motility model was used to assess gastrointestinal motor function [1]. The motility model produced self-generating reproducible contractile activity in the colon which were termed colonic peristaltic motor complexes (CPMCs). CPMC frequency was measured using time in quiescence (TIQ) while contractile activity was measured using CMPC amplitude. TIQ quantified the neurogenic property of the tissue whereas CPMC amplitude measured myogenic activity. Potassium channel agonists ML213 (Kv7.2/7.4) and ML335 (TREK1/2) were used to assess their effects on the EFS and motility responses, whilst antagonist studies were performed using the Kv7 antagonist XE991. Drug responses were compared to no-drug responses. Data presented as mean ± SEM (n ≥ 4). Statistical significance was assessed using repeat measures of one-way ANOVA, followed by Tukey’s post hoc test.

Results

Table 1. The effect of potassium channel compounds on EFS responses. Kv7 channel activation suppressed EFS-induced responses in both the colon and jejunum, while inhibition of Kv7channels significantly enhanced contractility in the ileum only. TREK channel activation reducedEFS responses in the colon alone.

Table 2. The effect of ML213 and XE991 on colonic motility. Motility studies showed that Kv7activation with ML213 inhibited colonic motility, but the CPMC amplitude was not affected. Inhibition of Kv7 with XE991 showed an increase in TIQ but the CPMC amplitude was not affected. ML213 also significantly blocked the effect of XE991 on TIQ (P<0.05).

Conclusions

These findings indicate that TREK channels appear to have region-specific effects in the gut but that Kv7 channels act in both the jejunum and colon to regulate contractility. The motility studies show that Kv7 appears to be acting primarily through a neuronal role to regulate motility-like behaviour, as activation or inhibition of Kv7 channels had no significant effect on CPMC amplitude but decreased or increased CPMC frequency respectively.
Modulating these channels may offer a potential treatment for motility disorders linked toirritable bowel syndrome and further studies are being undertaken to determine differentialroles of these channels in regulating gastrointestinal smooth muscle function.

[1] Keating C, Martinez V, Ewart L, Gibbons S, Grundy L, Valentin JP, Grundy D. The validation ofan in vitro colonic motility assay as a biomarker for gastrointestinal adverse drug reactions.Toxicol Appl Pharmacol. 2010;245(3):299-309.

_______________________________________________

Extracellular ATP activates P2-receptor mediated Ca2+-signalling and inhibits migration in urothelial cancer cells

Niamh McKerr, Queen's University Belfast, Belfast, United Kingdom; Daniel Crummey, Queen's University Belfast, Belfast, United Kingdom; Conor Breen, Queen's University Belfast, Belfast, United Kingdom; James Boncan, Queen's University Belfast, Belfast, United Kingdom; Karen McCloskey, Queen's University Belfast, Belfast, United Kingdom.

Introduction

Normal urothelial cells express many P2 purinergic receptor subtypes which are activated by ATP and contribute to bladder physiology. The tumour microenvironment has an elevated extracellular ATP concentration(approaching mM), which potentially activates P2 purinergic receptors expressed by cancer cells [1] [2]. P2X7 are sensitive to mM ATP and can activate pore formation and cell death. We aimed to characterise the Ca2+- signalling response of urothelial cancer cells to ATP (mM) and determine whether this initiated pore formation, cell death or affected cell migration.

Methods

SVHUC (normal urothelial cells), HT1376 and T24 (urothelial cancer cells) were used. Fluorescence Ca2+- imaging, flow cytometry, scratch-wound migration assays and pore-forming assays were used, along with P2 receptor pharmacological modulators. Data were analysed and compared with one way analysis of variance, or unpaired t-tests.

Results

SVHUC (N=3), HT1376 (N=4) and T24 (N=5) all responded to ATP (1mM) with Ca2+-transients, exhibiting distinctive shapes and duration profiles. The pan P2-receptor inhibitor, suramin (100μM) reduced the percentage of responding HT1376 (P<0.05, N=3) and decreased Ca2+-transient amplitudes in SVHUC and HT1376 (N=3, P<0.05).

In extracellular Ca2+- free conditions, the duration of ATP-evoked Ca2+-transients was shorter in all cell lines. Furthermore, the percentage of responding HT1376 (P<0.01, N=4) and the amplitude of their Ca2+-transients was reduced (P<0.05,N=4) - these were unchanged in SVHUC (N=3) and T24 (N=3).

ATP (1mM, 5mM, 24 hours) did not evoke cell death in SVHUC or T24 (N=3). Neither ATP (1mM), nor the P2X7 agonist BzATP (500μM) induced pore formation in SVHUC or T24 cells, in contrast to positive control macrophages (BMDM)which formed pores in response to ATP or BzATP (N=3). In scratch-wound migration assays, ATP reduced cell migration in SVHUC (P<0.05, N=8) and T24 (P<0.05, N=8); BzATP had a similar effect on SVHUC (P<0.01, N=8).

Conclusion

Urothelial cancer cells express functional P2-receptors which evoke ATP-Ca2+-transients comprising Ca2+-influx (P2X) and intracellular Ca2+
-release (P2Y). This signalling did not evoke cell death or pore-formation but inhibitedcell migration. This work provides insights into the response of urothelial cancer cells to the typical ATP concentration ofthe tumour microenvironment.

[1] Di Virgilio F, Adinolfi E. Extracellular purines, purinergic receptors and tumor growth. Oncogene. 2017 Jan 19:36:293-303.

[2] Lara R, Adinolfi E, Harwood CA, et al. P2X7 in Cancer: From Molecular Mechanisms to Therapeutics. FrontPharmacol. 2020:11:793.

_______________________________________________

Investigation of store-operated calcium entry in glioblastoma cancer stem cells

Daniel Crummey, Queen's University Belfast, Belfast, United Kingdom; Niamh McKerr, Queen's University Belfast, Belfast, United Kingdom; Kevin Prise, Queen's University Belfast, Belfast, United Kingdom; Karen McCloskey, Queen's University Belfast, Belfast, United Kingdom.

Introduction

Glioblastoma represents the most common high-grade primary brain tumour in adults. It has a poor prognosis with a mean survival time of 12-18 months. Standard of care treatment includes surgery, temozolomide and radiotherapy [1, 2]; however, recurrence is common and is partly attributed to treatment-resistant cancer stem cells (CSC). In this preliminary study, we investigated Ca2+-signalling in radioresistant glioblastoma CSC and focussed on store-operated calcium entry (SOCE) mechanisms.

Methods

E2-CSC (patient-derived glioblastoma cancer stem cells), E2-tumour cells and the U251 cell line were used. Fluorescence Ca2+
-imaging, clonogenic assays, 3D spheroid growth assays and radiation protocols were utilised along with interrogation of publicly available datasets.

Results

E2-CSC were radioresistant compared with E2-tumour cells in clonogenic assays (N=3, P<0.05). Publicly available data (Gene Expression Omnibus, GSE119834) showed that expression of the SOCE genes, ORAI1, ORAI2 andSTIM2 were upregulated in glioblastoma CSC (N=44) compared with glioblastoma tumour cells (N=45, P<0.001). In Ca2+-imaging experiments, E2-CSC (N=2) and U251 (N=5) exhibited SOCE mechanisms as activated by the classic ‘addback’protocol. The SOCE inhibitor, GSK7975a (25 μM) reduced the amplitude of the SOCE Ca2+-events in U251 (N=5,P<0.001). In normal Ca2+conditions, GSK7975a reduced baseline intracellular Ca2+in U251 and E2-CSC (N=2),potentially revealing constitutively active SOCE in glioblastoma models. Radiation (2Gy) reduced the growth of E2-CSCspheroids over a 14-day time course (N=5, P<0.05); furthermore, initial experiments showed that GSK7975a reduced thegrowth of E2-CSC spheroids (N=2).

Conclusions

Glioblastoma CSC have increased expression of SOCE genes and exhibit functional SOCE Ca2+-signalling. Pharmacological inhibition of SOCE reduced the growth of CSC spheroids; indicating that SOCE-dependentpathways contribute to the growth of treatment-resistant glioblastoma.

[1] McBain C, Lawrie TA, Rogozinska E, Kernohan A, Robinson T, Jefferies S. Treatment options for progression orrecurrence of glioblastoma: a network meta-analysis. Cochrane Database Syst Rev. 2021 May 4:5:CD013579.
[2] McKinnon C, Nandhabalan M, Murray SA, Plaha P. Glioblastoma: clinical presentation, diagnosis, andmanagement. BMJ. 2021 Jul 14:
374:n1560.

_______________________________________________

Iterative in silico and in vitro screens identify compounds simultaneously improving CFTR channel function and biogenesis

Maria-Cristina Ardelean, Neuroscience Physiology and Pharmacology, UCL, London, United Kingdom; Sacha Javor, Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland;  Jean-Louis Reymond, Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland; Paola Vergani, Neuroscience Physiology and Pharmacology, UCL, London, United Kingdom.

Introduction/ Background and aims

Cystic fibrosis (CF) is caused by mutations in the CFTR gene, encoding a Type IV ATP-binding cassette (ABC) transporter uniquely functioning as an anion channel. The current CF standard-of-care combination treatment, ETI (Elexacaftor-Tezacaftor-Ivacaftor, Vertex Pharmaceuticals), directly targets CFTR. Different components of ETI are needed to correct both CFTR gating (I) and biogenesis (E and T). ETI has significantly improved patient outcomes but simplifying treatment could be more cost-effective and reduce off-target effects. We conducted an extensive compound screen simultaneously testing effects on CFTR activity and biogenesis, aiming at identifying molecules with the potential of repairing both defects in CF-causing CFTR variants.

Methods/ Summary of work

A computational workflow combining both molecular docking and ligand-based in silico screening identified 109 candidate molecules extracted from a database of 4.9 million compounds (Molport). The compounds were selected based on the structural attributes of G-compounds [1], designed to target the bacterial ABC transporter MsbA (also Type IV) but also active on CFTR [2]. In a first screen, testing acute effects on CFTR gating, one compound (Hit1) emerged as particularly promising. A second round of ligand-based virtual screening of the original 4.9 million compound database selected 30structural neighbours to Hit1. All 139 compounds were subsequently tested under chronic conditions to characterize simultaneously CFTR biogenesis and channel function. An image-based assay [3] was employed, monitoring CFTR folding/trafficking/stability (biogenesis, quantified as membrane proximity, ρ) and anion flux (Area Above the quenching Curve, AAC7), exploiting the co-expression of iodide-sensitive YFP (H148Q/I152L) fused to CFTR, and cytosolic mCherry for precise image segmentation.

Results/ Discussion

Top hit compounds improving CFTR membrane proximity and/or channel function are shown in Table 1. Future research will focus on evaluating the effects of these molecules on F508del-CFTR.

Table 1. Effects of selected compounds on WT-CFTR channel function and membrane proximity.

Conclusion

These results underscore the potential for discovery of more effective CFTR modulators, simultaneously repairing both biogenesis and gating defects.

[1] Ho, H., et al., Structural basis for dual-mode inhibition of the ABC transporter MsbA. Nature, 2018. 557(7704): p. 196-201.
[2] de Jonge, H.R., et al., Strategies for cystic fibrosis transmembrane conductance regulatorinhibition: from molecular mechanisms to treatment for secretory diarrhoeas. FEBS Lett, 2020.594(23): p. 4085-4108.
[3] Prins, S., et al., Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasmamembrane proximity. J Biol Chem, 2020. 295(49): p. 16529-16544.

_______________________________________________

Development of Automated Electrophysiology Assays for the Characterisation of Inhibitors Against Human HCN Ion Channels

Bethany J. Sharp, Metrion, Cambridge, UK; Catherine M. Hodgson, Metrion, Cambridge, UK; Graham D. Smith, Metrion, Cambridge, UK; Gary S. Clark, Metrion, Cambridge, UK.

Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels are a family of four non-selective cation channels that help regulate cardiac and neuronal rhythmicity. HCN channels open upon membrane hyperpolarisation and are differentially modulated by cyclic nucleotides, phosphorylation and neuromodulators. Highly expressed in nociceptors, HCN2 has been implicated as a regulator of inflammatory and neuropathic pain. Inflammatory mediators increase neuronal cyclic adenosine monophosphate (cAMP) production, shifting HCN2 activation to more depolarised potentials, increasing the frequency of nociceptor firing. This physiological role highlights HCN2 as a potential non-opioid drug target in the treatment of chronic inflammatory and neuropathic pain. 

Using manual and automated patch clamp techniques, this poster demonstrates the generation and validation of a functional CHO-hHCN2 cell line with channel biophysics consistent with current knowledge of HCN channels. QPatch 48 and Qube 384 (Sophion Bioscience) hHCN2 assays were successfully developed for the screening of compounds. HCN blockers Zatebradine, Ivabradine and ZD 7288 gave consistent IC₅₀ values across platforms and values similar to those reported previously. These data demonstrate the suitability of these electrophysiological assays for the screening of novel compounds to develop selective HCN2 blockers for use in the treatment of pain.

_______________________________________________