Biology subjects

Voets, T.

Publications and source records attributed to Voets, T..

8 recordsLinked to original sources

bioRxiv2025

Stereoselectivity and functional plasticity of a common ligand-binding pocket in TRPM3

The transient receptor potential melastatin 3 (TRPM3) channel is a key mediator of peripheral pain signaling, and pathogenic mutations in TRPM3 are linked to neurodevelopmental delay and epilepsy. Despite the therapeutic promise of TRPM3 modulators, the molecular mechanisms by which ligands modulate channel gating remain poorly understood. Here, we combine cryo-electron microscopy (cryo-EM) with functional analyses to characterize a promiscuous ligand-binding pocket formed by transmembrane helices S1-S4. This pocket accommodates several chemically diverse plant-derived and synthetic agonists and antagonists. We uncover unanticipated stereoselectivity of TRPM3 for the non-natural enantiomer of the flavonoid antagonist isosakuranetin and the (R)-enantiomer of the synthetic agonist CIM0216. Mutations within this pocket--including newly identified patient variants--alter ligand affinity and, in some cases, invert the functional outcome of ligand binding. These findings reveal the stereoselectivity and functional plasticity of the TRPM3 ligand-binding pocket, highlighting how subtle changes in the molecular interactions can produce divergent effects on channel gating, with important ramifications for TRPM3-targeted drug development and therapy.

biochemistry↗

bioRxiv2025

A high-throughput assay for the measurement of Ca2+-oscillations and insulin release from uniformly sized β-cell spheroids.

Diabetes mellitus is a rapidly growing global health challenge, necessitating the development of more effective anti-diabetic therapies, including drugs that improve insulin release from pancreatic {beta}-cells. Traditional high-throughput screening methods typically rely on 2D {beta}-cell cultures, but such cultures do not mimic the 3D organisation and cell-to-cell communication of {beta}-cells in pancreatic islets of Langerhans. Existing 3D {beta}-cell culture models are hindered by high costs, technical complexity, and limited compatibility with high-throughput screening platforms. In this work, we developed an approach for generating 19 homogeneously shaped pancreatic {beta}-cell spheroids in each well of a 96-well plate, using micropatterned polyethylene glycol (PEG)-based hydrogels and MIN6 insulinoma cells. The uniform shape and positioning of the individual spheroids enabled the simultaneous, real-time imaging of Ca2+ signals in up to 1824 independent spheroids in response to glucose and various test compounds. Using this approach, we show that increasing glucose causes concentration-dependent Ca2+ oscillations in individual spheroids, that these Ca2+ oscillations are sensitive to modulators of ATP-sensitive K+ channels, and that the frequency of Ca2+ oscillations correlates with insulin secretion. Finally, we demonstrate that the neurosteroid pregnenolone sulphate, an agonist of the cation channel TRPM3, increases the frequency of glucose-induced Ca2+ oscillations and enhances insulin release; the TRPM3 antagonist isosakuranetin inhibited these responses. In conclusion, we established a cost-effective and scalable 3D {beta}-cell platform for high-throughput screening of insulin release-modifying compounds, with potential applications in drug development and personalized medicine for the management of diabetes mellitus.

bioengineering↗

bioRxiv2024

Cell-surface receptor-mediated regulation of synaptic organelle distribution controls dendritic spine maturation

The spine apparatus (SA), an endoplasmic reticulum-related organelle present in a subset of mature dendritic spines, plays a key role in postsynaptic development and has been implicated in various neurological disorders. However, the molecular mechanisms that dictate SA localization at selected synapses remain elusive. Here, we identify a postsynaptic signaling complex comprising the GPCR-like receptor GPR158 and a largely uncharacterized phospholipase C (PLC), PLCXD2, that controls SA abundance. Sparse genetic manipulations in vivo demonstrate that in the absence of GPR158, unrestrained PLCXD2 activity impedes postsynaptic SA incorporation and hampers dendritic spine maturation. Finally, we show that extracellular heparan sulfate proteoglycan (HSPG) binding modulates the GPR158-PLCXD2 interaction. Together, our findings reveal how a postsynaptic receptor signaling complex regulates the local lipid microenvironment to control SA abundance required for the proper maturation of dendritic spines.

neuroscience↗

bioRxiv2024

Direct modulation of TRPM8 ion channels by rapamycin and analog macrolide immunosuppressants

Rapamycin (sirolimus), a macrolide compound isolated from the bacterium Streptomyces hygroscopicus, is widely used as oral medication for the prevention of transplant rejection and the treatment of lymphangioleiomyomatosis. It is also incorporated in coronary stent coatings to prevent restenosis and in topical preparations for the treatment of skin disorders. Rapamycins in vivo activities are generally ascribed to its binding to the protein FKBP12, leading to potent inhibition of the mechanistic target of rapamycin kinase (mTOR) by the FKBP12-rapamycin complex. The specific rapamycin-induced interaction between domains from mTOR and FKBP12 is also frequently employed in cell biological research, for rapid chemically-induced protein dimerization strategies. Here we show that rapamycin activates TRPM8, a cation channel expressed in sensory nerve endings that serves as the primary cold sensor in mammals. Using a combination of electrophysiology, Saturation Transfer Triple-Difference (STTD) NMR spectroscopy and molecular docking-based targeted mutagenesis, we demonstrate that rapamycin directly binds to TRPM8. We identify a rapamycin-binding site in the groove between voltage sensor-like domain and the pore domain, distinct from the interaction sites of cooling agents and known TRPM8 agonists menthol and icilin. Related macrolide immunosuppressants act as partial TRPM8 agonists, competing with rapamycin for the same binding site. These findings identify a novel molecular target for rapamycin and provide new insights into the mechanisms of TRPM8 activation, which may assist in the development of therapies targeting this ion channel. Moreover, our findings also indicate that caution is needed when using molecular approaches based on rapamycin-induced dimerization to study ion channel regulation.

neuroscience↗

bioRxiv2022

TRPM3 as novel target to alleviate acute oxaliplatin-induced peripheral neuropathic pain

Chemotherapy-induced peripheral neuropathic pain (CIPNP) is an adverse effect observed in up to 80% of cancer patients upon treatment with cytostatic drugs including paclitaxel and oxaliplatin. CIPNP can be so severe that it limits dose and choice of chemotherapy, and has a significant negative consequences on the quality of life of survivors. Current treatments for CIPNP are limited and unsatisfactory. TRPM3 is a Ca2+-permeable ion channel functionally expressed in peripheral sensory neurons involved in the detection of thermal stimuli. Here, we focus on the possible involvement of TRPM3 in acute oxaliplatin-induced mechanical allodynia and cold hypersensitivity. In vitro calcium microfluorimetry and whole-cell patch-clamp experiments showed that TRPM3 is functionally upregulated in both heterologous and homologous expression systems after acute (24h) oxaliplatin treatment, while direct application of oxaliplatin was without effect. In vivo behavioral studies using an acute oxaliplatin model for CIPNP showed the development of cold and mechano-hypersensitivity in control mice, which was lacking in TRPM3 deficient mice. In addition, the levels of pERK, a marker for neuronal activity, were significantly reduced in DRG neurons derived from TRPM3 deficient mice compared to control after oxaliplatin administration. Moreover, intraperitoneal injection of a TRPM3 antagonist, isosakuranetin, effectively reduced the oxaliplatin-induced pain behavior in response to cold and mechanical stimulation in mice with an acute form of oxaliplatin-induced peripheral neuropathy. In summary, TRPM3 thus represents a potential new target for the treatment of neuropathic pain in patients undergoing chemotherapy.

neuroscience↗

bioRxiv2022

Machine learning-assisted fluoroscopy of bladder function in awake mice

Understanding the lower urinary tract (LUT) and development of highly needed novel therapies to treat LUT disorders depends on accurate techniques to monitor LUT (dys)function in preclinical models. We recently developed videocystometry in rodents, which combines intravesical pressure measurements with X-ray-based fluoroscopy of the LUT, allowing the in vivo analysis of the process of urine storage and voiding with unprecedented detail. Videocystometry relies on the precise contrast-based determination of the bladder volume at high temporal resolution, which can readily be achieved in anesthetized or otherwise motion-restricted mice but not in awake and freely moving animals. To overcome this limitation, we developed a machine-learning method, in which we trained a neural network to automatically detect the bladder in fluoroscopic images, allowing the automatic analysis of bladder filling and voiding cycles based on large sets of time-lapse fluoroscopic images (>3 hours at 30 images/second) from behaving mice and in a non-invasive manner. With this approach, we found that urethane, an injectable anesthetic that is commonly used in preclinical urological research, has a profound, dose-dependent effect on urethral relaxation and voiding duration and that implantation of a suprapubic catheter, as is standardly performed for cystometric analyses, leads to a [~]4-fold reduction in bladder capacity. Our findings provide a paradigm for the non-invasive, in vivo monitoring of a hollow organ in behaving animals and pinpoint important limitations of the current gold standard techniques to study the LUT in mice.

physiology↗

bioRxiv2020

Upregulation of TRPM3 drives hyperexcitability in nociceptors innervating inflamed tissue.

Genetic ablation or pharmacological inhibition of the heat-activated cation channel TRPM3 alleviates heat hyperhyperalgesia in animal models of inflammation, but the mechanisms whereby the channel contributes to inflammatory pain are unknown. Here, we induced unilateral inflammation of the hind paw in mice, and directly compared expression and function of TRPM3 and two other heat-activated TRP channels (TRPV1 and TRPA1) in sensory neurons innervating the ipsilateral and contralateral paw. We detected increased Trpm3 mRNA levels in dorsal root ganglion neurons innervating the inflamed paw, as well as augmented TRP channel-mediated calcium responses, both in the cell bodies and the intact peripheral endings of nociceptors. Notably, inflammation provoked a pronounced increase in nociceptors co-expressing functional TRPM3 with TRPV1 and TRPA1, and pharmacological inhibition of TRPM3 caused normalization of TRPV1- and TRPA1-mediated responses. These new insights into the mechanisms underlying inflammatory heat hypersensitivity provide a rationale for developing TRPM3 antagonists to treat pathological pain.

neuroscience↗

bioRxiv2020

Gain of channel function and modified gating properties in TRPM3 mutants causing intellectual disability and epilepsy

Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of disorders characterized by epilepsy with comorbid intellectual disability. Recently, two de novo heterozygous mutations in the gene encoding TRPM3, a calcium permeable ion channel, were identified as the cause of DEE in eight probands, but the functional consequences of the mutations remained elusive. Here we demonstrate that both mutations (V990M and P1090Q) have distinct effects on TRPM3 gating, including increased basal activity, higher sensitivity to stimulation by the endogenous neurosteroid pregnenolone sulphate (PS) and heat, and altered response to ligand modulation. Most strikingly, the V990M mutation affected the gating of the non-canonical pore of TRPM3, resulting in large inward cation currents via the voltage sensor domain in response to PS stimulation. Taken together, these data indicate that the two DEE mutations in TRPM3 result in a profound gain of channel function, which may lie at the basis of epileptic activity and neurodevelopmental symptoms in the patients.

neuroscience↗