Neurodevelopmental disorders caused by variants in TRPM3.

Developmental and epileptic encephalopathies (DEE) are a broad and varied group of disorders that affect the brain and are characterized by epilepsy and comorbid intellectual disability (ID). These conditions have a broad spectrum of symptoms and can be caused by various underlying factors, including genetic mutations, infections, and other medical conditions. The exact cause of DEE remains largely unknown in the majority of cases. However, in around 25 % of patients, rare nonsynonymous coding variants in genes encoding ion channels, cell-surface receptors, and other neuronally expressed proteins are identified. This review focuses on a subgroup of DEE patients carrying variations in the gene encoding the Transient Receptor Potential Melastatin 3 (TRPM3) ion channel, where recent data indicate that gain-of-function of TRPM3 channel activity underlies a spectrum of dominant neurodevelopmental disorders.

Targeting TRP channels for pain relief: A review of current evidence from bench to bedside.

Several decades of research support the involvement of transient receptor potential (TRP) channels in nociception. Despite the disappointments of early TRPV1 antagonist programs, the TRP family remains a promising therapeutic target in pain disorders. High-dose capsaicin patches are already in clinical use to relieve neuropathic pain. At present, localized injections of the side-directed TRPV1 agonist capsaicin and resiniferatoxin are undergoing clinical trials in patients with osteoarthritis and bone cancer pain. TRPA1, TRPM3, and TRPC5 channels are also of significant interest. This review discusses the role of TRP channels in human pain conditions.

ß-Lactam TRPM8 Antagonists Derived from Phe-Phenylalaninol Conjugates: Structure-Activity Relationships and Antiallodynic Activity.

The protein transient receptor potential melastatin type 8 (TRPM8), a non-selective, calcium (Ca2+)-permeable ion channel is implicated in several pathological conditions, including neuropathic pain states. In our previous research endeavors, we have identified ß-lactam derivatives with high hydrophobic character that exhibit potent and selective TRPM8 antagonist activity. This work describes the synthesis of novel derivatives featuring C-terminal amides and diversely substituted N'-terminal monobenzyl groups in an attempt to increase the total polar surface area (TPSA) in this family of compounds. The primary goal was to assess the influence of these substituents on the inhibition of menthol-induced cellular Ca2+ entry, thereby establishing critical structure-activity relationships. While the substitution of the tert-butyl ester by isobutyl amide moieties improved the antagonist activity, none of the N'-monobencyl derivatives, regardless of the substituent on the phenyl ring, achieved the activity of the model dibenzyl compound. The antagonist potency of the most effective compounds was subsequently verified using Patch-Clamp electrophysiology experiments. Furthermore, we evaluated the selectivity of one of these compounds against other members of the transient receptor potential (TRP) ion channel family and some receptors connected to peripheral pain pathways. This compound demonstrated specificity for TRPM8 channels. To better comprehend the potential mode of interaction, we conducted docking experiments to uncover plausible binding sites on the functionally active tetrameric protein. While the four main populated poses are located by the pore zone, a similar location to that described for the N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist cannot be discarded. Finally, in vivo experiments, involving a couple of selected compounds, revealed significant antinociceptive activity within a mice model of cold allodynia induced by oxaliplatin (OXA).

pH-dependent modulation of TRPV1 by modality-selective antagonists.

BACKGROUND AND PURPOSE: Antagonists of TRPV1 that inhibit all activation modes cause hyperthermia, hampering their medical use as novel analgesics. TRPV1 antagonists that do not (fully) inhibit responses to low pH do not cause hyperthermia, but it remains incompletely understood how such antagonists affect channel gating. We tested the hypothesis that pH-sparing antagonists act in a modality-selective manner on TRPV1, differentially affecting channel activation by protons and capsaicin. EXPERIMENTAL APPROACH: Using whole-cell patch-clamp and calcium imaging to measure channel activity in cells expressing wild type human TRPV1 or the pH-insensitive mutant F660A. Responses to protons and capsaicin were measured at different pH values in the presence of antagonists that reportedly partially spare (A-1165442) or potentiate (AMG7905) acid-evoked channel activation. KEY RESULTS: At pH 5.5, A-1165442 was equipotent at blocking acid- and capsaicin-evoked responses of wild type TRPV1. Its potency to inhibit acid-evoked responses was attenuated at pH ≤ 5.0. AMG7905, at a concentration (1 µM) that fully inhibits capsaicin-evoked responses, potentiated proton-evoked (pH 5.5) responses of wild type TRPV1. In the F660A mutant, the inhibitory efficacy of A-1165442 and AMG7905 towards capsaicin-evoked responses was reduced at lower pH values and AMG7905 acted as a partial agonist. CONCLUSION AND IMPLICATIONS: Our findings show that A-1165442 and AMG7905 interact in a pH-dependent manner with TRPV1, but this pH dependence is not strictly modality-selective. Reduced TRPV1 antagonism at acidic pH may limit analgesic efficacy in injured tissue and needs to be considered in models explaining the effects of antagonists on core body temperature.

Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2.

BACKGROUND: Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive. METHODS: To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression. RESULTS: We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the 'Window of implantation' markers in human endometrial epithelial cells. CONCLUSIONS: These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.

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

ABSTRACT: Chemotherapy-induced peripheral neuropathic pain (CIPNP) is an adverse effect observed in up to 80% of patients of cancer on treatment with cytostatic drugs including paclitaxel and oxaliplatin. Chemotherapy-induced peripheral neuropathic pain can be so severe that it limits dose and choice of chemotherapy and has significant negative consequences on the quality of life of survivors. Current treatment options for CIPNP are limited and unsatisfactory. TRPM3 is a calcium-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 (24 hours) oxaliplatin treatment, whereas the 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 protein ERK, a marker for neuronal activity, were significantly reduced in dorsal root ganglion neurons derived from TRPM3 deficient mice compared with 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 represents a potential new target for the treatment of neuropathic pain in patients undergoing chemotherapy.

Primidone improves symptoms in TRPM3-linked developmental and epileptic encephalopathy with spike-and-wave activation in sleep.

Developmental and epileptic encephalopathy with continuous spike-and-wave activation in sleep (CSWS) or DEE-SWAS is an age-dependent disease, often accompanied by a decline in cognitive abilities. Early successful treatment of CSWS is associated with a better cognitive outcome. We retrospectively analyzed the clinical, electrophysiological, radiological, and genetic data of children with DEE-SWAS associated with melastatin-related transient receptor type 3 gene (TRPM3) missense variants. We report two unrelated children with pharmacoresistant DEE-SWAS and developmental delay/regression and different heterozygous de novo missense variants in the TRPM3 gene (NM_001366145.2; c.3397 T > C/p.Ser1133Pro, c.2004G > A/p.Val1002Met). The variant p.Val1002Met (previously known as p.Val990Met or p.Val837Met) and p.Ser1133Pro were recently shown to result in a gain-of-function effect. Based on this finding, previous drug resistance, and the experimentally demonstrated inhibitory effect of primidone on TRPM3, we initiated an individualized therapy with this drug. In both children, developmental regression was stopped, psychomotor development improved, and CSWS was no longer detectable. To our knowledge, this is the first report of a treatment with primidone in TRPM3-associated CSWS. Our results highlight the importance of early genetic diagnosis in patients with epilepsy and the possibility of precision medicine, which should be considered in the future in individuals with a TRPM3-linked DEE-SWAS.

Clotrimazole-Based Modulators of the TRPM3 Ion Channel Reveal Narrow Structure-Activity Relationship.

TRPM3 is an ion channel that is highly expressed in nociceptive neurons and plays a key role in pain perception. In the presence of the endogenous TRPM3 ligand, pregnenolone sulfate (PS), the antifungal compound clotrimazole (Clt) augments Ca2+ signaling and opens a non-canonical pore, permeable to Na+, which aggravates TRPM3-induced pain. To date, little is known about structural features that govern the Clt modulatory effect of TRPM3. Here, we synthesized and evaluated several Clt analogues in order to gain insights into their structure-activity relationship. Our results reveal a tight SAR with the three phenyl rings on the trityl moiety being essential for the activity, as well as the presence of fluorine or chlorine substituents on the trityl group. Imidazole as a heterocycle is also necessary for activity. Interestingly, we identified a pentafluoro-trityl analogue (29a) that is able to act as a TRPM3 agonist in the absence of PS. The compounds we report in this work will be useful tools for the further study of TRPM3 modulation and its effect on pain perception.

Gain-of-function variants in the ion channel gene TRPM3 underlie a spectrum of neurodevelopmental disorders.

TRPM3 is a temperature- and neurosteroid-sensitive plasma membrane cation channel expressed in a variety of neuronal and non-neuronal cells. Recently, rare de novo variants in TRPM3 were identified in individuals with developmental and epileptic encephalopathy, but the link between TRPM3 activity and neuronal disease remains poorly understood. We previously reported that two disease-associated variants in TRPM3 lead to a gain of channel function . Here, we report a further 10 patients carrying one of seven additional heterozygous TRPM3 missense variants. These patients present with a broad spectrum of neurodevelopmental symptoms, including global developmental delay, intellectual disability, epilepsy, musculo-skeletal anomalies, and altered pain perception. We describe a cerebellar phenotype with ataxia or severe hypotonia, nystagmus, and cerebellar atrophy in more than half of the patients. All disease-associated variants exhibited a robust gain-of-function phenotype, characterized by increased basal activity leading to cellular calcium overload and by enhanced responses to the neurosteroid ligand pregnenolone sulfate when co-expressed with wild-type TRPM3 in mammalian cells. The antiseizure medication primidone, a known TRPM3 antagonist, reduced the increased basal activity of all mutant channels. These findings establish gain-of-function of TRPM3 as the cause of a spectrum of autosomal dominant neurodevelopmental disorders with frequent cerebellar involvement in humans and provide support for the evaluation of TRPM3 antagonists as a potential therapy.

Inhibition of TRPM8 by the urinary tract analgesic drug phenazopyridine.

BACKGROUND: and purpose: Phenazopyridine (PAP) is an over-the-counter drug widely used to provide symptomatic relief of bladder pain in conditions such as cystitis or bladder pain syndrome (BPS). Whereas the analgesic effect of PAP has been attributed to a local effect on the mucosa of the lower urinary tract (LUT), the molecular targets of PAP remain unknown. We investigated the effect of PAP on pain-related Transient Receptor Potential (TRP) channels expressed in sensory neurons that innervate the bladder wall. EXPERIMENTAL APPROACH: The effects of PAP on the relevant TRP channels (TRPV1, TRPA1, TRPM8, TRPM3) expressed in HEK293 or CHO cells was investigated using Fura-2-based calcium measurements and whole-cell patch-clamp recordings. Activity of PAP on TRPM8 was further analysed using Fura-2-based calcium imaging on sensory neurons isolated from lumbosacral dorsal root ganglia (DRG) of mice. KEY RESULTS: PAP rapidly and reversibly inhibits responses of TRPM8 expressed in HEK293 cells to cold and menthol, with IC50 values between 2 and 10 µM. It acts by shifting the voltage dependence of channel activation towards positive potentials, opposite to the effect of menthol. PAP also inhibits TRPM8-mediated, menthol-evoked calcium responses in lumbosacral DRG neurons. At a concentration of 10 µM, PAP did not significantly affect TRPA1, TRPV1, or TRPM3. CONCLUSION AND IMPLICATIONS: PAP inhibits TRPM8 in a concentration range consistent with PAP levels in the urine of treated patients. Since TRPM8 is expressed in bladder afferent neurons and upregulated in patients with painful bladder disorders, TRPM8 inhibition may underlie the analgesic activity of PAP.

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 hr at 30 images/s) from behaving mice and in a noninvasive 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. Moreover, both in awake and in anesthetized mice, the bladder capacity was decreased ~fourfold when cystometry was performed acutely after surgical implantation of a suprapubic catheter. Our findings provide a paradigm for the noninvasive, 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.

Healthy adults empty their bladder many times a day with little thought. This seemingly simple process requires communication between the lower urinary tract and the central nervous system. About one in five adults experience conditions like urinary incontinence, urgency, or bladder pain caused by impairments in their lower urinary tract. Despite the harmful effects these conditions have on people's health and well-being, few good treatments are available. Mice are often used to study lower urinary tract conditions and treatments. One common technique is to fill a mouse's bladder using a catheter and measure changes in pressure as the bladder empties and refills. But these procedures and the anesthesia used during them may affect bladder function and skew results. Here, De Bruyn et al. have developed a new technique that allows scientists to measure bladder function in awake, freely moving mice. The mice's bladders were photographed using a specialized X-ray based fluoroscope that captured 30 images per second over the course of three hours. A machine learning algorithm was then applied which can automatically detect the circumference of the bladder in each captured image (over 30,000 in total) and quantify its volume. This makes it is possible to measure the bladder as it empties and fills even if the mice move between time frames. The new approach showed that 'gold standard' commonly used methods have a profound effect on the bladder. Surgical implantation of a catheter reduced the bladder to a quarter of its capacity. In addition, one of the most widely used anesthetic drugs in urinary tract research was found to affect the bladder's ability to drain. The technique created by De Bruyn et al. provides a new way to study lower urinary tract function and disease in awake, moving animals. This tool would be easy for other academic and pharmaceutical laboratories to implement, and may help scientists discover new therapies for lower urinary tract conditions.

TRPM4 inhibition by meclofenamate suppresses Ca2+-dependent triggered arrhythmias.

AIMS: Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca2+ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca2+ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca2+-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear. METHODS AND RESULTS: In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca2+-dependent proarrhythmic models, freely moving Trpm4-/- mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca2+ overload-induced background current, thereby regulating cell excitability in Ca2+ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4-/- mice, 10 µM meclofenamate inhibited the Ca2+ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner. CONCLUSION: The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca2+-dependent triggered arrhythmias.

Loratadine, an antihistaminic drug, suppresses the proliferation of endometrial stromal cells by inhibition of TRPV2.

The transient receptor potential (TRP) channel TRPV2 is widely expressed in a variety of different cell types and tissues. However, elucidating the exact biological functions of TRPV2 is significantly hampered by the lack of selective pharmacological tools to modulate channel activity in vitro and in vivo. This study aimed to identify new compounds that modify TRPV2 activity via the use of a plate-based calcium imaging approach to screen a drug repurposing library. Three antihistaminic drugs, loratadine, astemizole and clemizole were identified to reduce calcium-influx evoked by the TRPV2 agonist tetrahydrocannabivarin in HEK293 cells expressing murine TRPV2. Using single-cell calcium-microfluorimetry and whole-cell patch clamp recordings, we further confirmed that all three compounds induced a concentration-dependent block of TRPV2-mediated Ca2+ influx and whole-cell currents, with loratadine being the most potent antagonist of TRPV2. Moreover, this study demonstrated that loratadine was able to block both the human and mouse TRPV2 orthologs, without inhibiting the activity of other closely related members of the TRPV superfamily. Finally, loratadine inhibited TRPV2-dependent responses in a primary culture of mouse endometrial stromal cells and attenuated cell proliferation and migration in in vitro cell proliferation and wound healing assays. Taken together, our study revealed that the antihistaminic drugs loratadine, astemizole and clemizole target TRPV2 in a concentration-dependent manner. The identification of these antihistaminic drugs as blockers of TRPV2 may form a new starting point for the synthesis of more potent and selective TRPV2 antagonists, which could further lead to the unravelling of the physiological role of the channel.

Phenotypic spectrum of the recurrent TRPM3 p.(Val837Met) substitution in seven individuals with global developmental delay and hypotonia.

TRPM3 encodes a transient receptor potential cation channel of the melastatin family, expressed in the central nervous system and in peripheral sensory neurons of the dorsal root ganglia. The recurrent substitution in TRPM3: c.2509G>A, p.(Val837Met) has been associated with syndromic intellectual disability and seizures. In this report, we present the clinical and molecular features of seven previously unreported individuals, identified by exome sequencing, with the recurrent p.(Val837Met) variant and global developmental delay. Other shared clinical features included congenital hypotonia, dysmorphic facial features (broad forehead, deep-set eyes, and down turned mouth), exotropia, and musculoskeletal issues (hip dysplasia, hip dislocation, scoliosis). Seizures were observed in two of seven individuals (febrile seizure in one and generalized tonic-clonic seizures with atonic drops in another), and epileptiform activity was observed in an additional two individuals. This report extends the number of affected individuals to 16 who are heterozygous for the de novo recurrent substitution p.(Val837Met). In contrast with the initial report, epilepsy was not a mandatory feature observed in this series. TRPM3 pathogenic variation should be considered in individuals with global developmental delays, moderate-severe intellectual disability with, or without, childhood-onset epilepsy.

Pharmacological properties of TRPM3 isoforms are determined by the length of the pore loop.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin 3 (TRPM3) is a non-selective cation channel that plays a pivotal role in the peripheral nervous system as a transducer of painful heat signals. Alternative splicing gives rise to several TRPM3 variants. The functional consequences of these splice isoforms are poorly understood. Here, the pharmacological properties of TRPM3 variants arising from alternative splicing in the pore-forming region were compared. EXPERIMENTAL APPROACH: Calcium microfluorimetry and patch clamp recordings were used to compare the properties of heterologously expressed TRPM3α1 (long pore variant) and TRPM3α2-α6 (short pore variants). Furthermore, site-directed mutagenesis was done to investigate the influence of the length of the pore loop on the channel function. KEY RESULTS: All short pore loop TRPM3α variants (TRPM3α2-α6) were activated by the neurosteroid pregnenolone sulphate (PS) and by nifedipine, whereas the long pore loop variant TRPM3α1 was insensitive to either compound. In contrast, TRPM3α1 was robustly activated by clotrimazole, a compound that does not directly activate the short pore variants but potentiates their responses to PS. Clotrimazole-activated TRPM3α1 currents were largely insensitive to established TRPM3α2 antagonists and were only partially inhibited upon activation of the µ opioid receptor. Finally, by creating a set of mutant channels with pore loops of intermediate length, we showed that the length of the pore loop dictates differential channel activation by PS and clotrimazole. CONCLUSION AND IMPLICATIONS: Alternative splicing in the pore-forming region of TRPM3 defines the channel's pharmacological properties, which depend critically on the length of the pore-forming loop. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

TRP channel expression correlates with the epithelial-mesenchymal transition and high-risk endometrial carcinoma.

Transient receptor potential (TRP) channels excel in cellular sensing as they allow rapid ion influx across the plasma membrane in response to a variety of extracellular cues. Recently, a distinct TRP mRNA expression signature was observed in stromal cells (ESC) and epithelial cells (EEC) of the endometrium, a tissue in which cell phenotypic plasticity is essential for normal functioning. However, it is unknown whether TRP channel mRNA expression is subject to the phenotypic switching that occurs during epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET), and whether TRP channel mRNA expression is associated with aggressive phenotypes in endometrial cancer (EC). Here, we induced EMT and MET in vitro using in primary EEC and ESC, respectively, and analyzed expression and functionality of TRP channels using RT-qPCR and intracellular Ca2+ imaging. The outcome of these experiments showed a strong association between TRPV2 and TRPC1 mRNA expression and the mesenchymal phenotype, whereas TRPM4 mRNA expression correlated with the epithelial phenotype. In line herewith, increased TRPV2 and TRPC1 mRNA expression levels were observed in both primary and metastatic EC biopsies and in primary EC cells with a high EMT status, indicating an association with an aggressive tumor phenotype. Remarkably, TRPV2 mRNA expression in primary EC biopsies was associated with tumor invasiveness and cancer stage. In contrast, increased TRPM4 mRNA expression was observed in EC biopsies with a low EMT status and less aggressive tumor phenotypes. Taken together, this dataset proved for the first time that TRP channel mRNA expression is strongly linked to cellular phenotypes of the endometrium, and that phenotypic transitions caused by either experimental manipulation or malignancy could alter this expression in a predictable manner. These results implicate that TRP channels are viable biomarkers to identify high-risk EC, and potential targets for EC treatment.

Partial Agonistic Actions of Sex Hormone Steroids on TRPM3 Function.

Sex hormone steroidal drugs were reported to have modulating actions on the ion channel TRPM3. Pregnenolone sulphate (PS) presents the most potent known endogenous chemical agonist of TRPM3 and affects several gating modes of the channel. These includes a synergistic action of PS and high temperatures on channel opening and the PS-induced opening of a noncanonical pore in the presence of other TRPM3 modulators. Moreover, human TRPM3 variants associated with neurodevelopmental disease exhibit an increased sensitivity for PS. However, other steroidal sex hormones were reported to influence TRPM3 functions with activating or inhibiting capacity. Here, we aimed to answer how DHEAS, estradiol, progesterone and testosterone act on the various modes of TRPM3 function in the wild-type channel and two-channel variants associated with human disease. By means of calcium imaging and whole-cell patch clamp experiments, we revealed that all four drugs are weak TRPM3 agonists that share a common steroidal interaction site. Furthermore, they exhibit increased activity on TRPM3 at physiological temperatures and in channels that carry disease-associated mutations. Finally, all steroids are able to open the noncanonical pore in wild-type and DHEAS also in mutant TRPM3. Collectively, our data provide new valuable insights in TRPM3 gating, structure-function relationships and ligand sensitivity.

X-ray videocystometry for high-speed monitoring of urinary tract function in mice.

Lower urinary tract dysfunction (LUTd) represents a major health care problem with a high, unmet medical need. Design of additional therapies for LUTd requires precise tools to study bladder storage and voiding (dys)function in animal models. We developed videocystometry in mice, combining intravesical pressure measurements with high-speed fluoroscopy of the urinary tract. Videocystometry substantially outperforms current state-of-the-art methods to monitor the urine storage and voiding process, by enabling quantitative analysis of voiding efficiency, urethral flow, vesicoureteral reflux, and the relation between intravesical pressure and flow, in both anesthetized and awake, nonrestrained mice. Using videocystometry, we identified localized bladder wall micromotions correlated with different states of the filling/voiding cycle, revealed an acute effect of TRPV1 channel activation on voiding efficiency, and pinpointed the effects of urethane anesthesia on urine storage and urethral flow. Videocystometry has broad applications, ranging from the elucidation of molecular mechanisms of bladder control to drug development for LUTd.

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging.

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable. For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited. This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.