TRPV2: a universal regulator in cellular physiology with a yet poorly defined thermosensitivity.

Transient receptor potential (TRP) ion channels serve as sensors for variations in ambient temperature, modulating both thermoregulation and temperature responsive cellular processes. Among these, the vanilloid TRP subfamily (TRPV) comprises six members and at least four of these members (TRPV1-TRPV4) have been associated with thermal sensation. TRPV2 has been described as a sensor for noxious heat, but subsequent studies have unveiled a more complex role for TRPV2 beyond temperature perception. This comprehensive review aims to elucidate the intricate thermosensitivity of TRPV2 by synthesizing current knowledge on its biophysical properties, expression pattern and known physiological functions associated with thermosensation.

Protein-free media for cardiac differentiation of hPSCs in 2000 mL suspension culture.

BACKGROUND: Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). METHODS AND RESULTS: Both media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 109 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. CONCLUSIONS: This study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration.

A Possible Role of Tetrodotoxin-Sensitive Na+ Channels for Oxidation-Induced Late Na+ Currents in Cardiomyocytes.

An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na+ currents by removing the inactivation of voltage-gated Na+ channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na+ currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na+ currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na+ currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na+ currents significantly stronger as compared to peak Na+ currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na+ channels expressed in cardiomyocytes may be relevant for the generation of late Na+ currents following oxidative stress.

Transient stabilization of human cardiovascular progenitor cells from human pluripotent stem cells in vitro reflects stage-specific heart development in vivo.

AIMS: Understanding the molecular identity of human pluripotent stem cell (hPSC)-derived cardiac progenitors and mechanisms controlling their proliferation and differentiation is valuable for developmental biology and regenerative medicine. METHODS AND RESULTS: Here, we show that chemical modulation of histone acetyl transferases (by IQ-1) and WNT (by CHIR99021) synergistically enables the transient and reversible block of directed cardiac differentiation progression on hPSCs. The resulting stabilized cardiovascular progenitors (SCPs) are characterized by ISL1pos/KI-67pos/NKX2-5neg expression. In the presence of the chemical inhibitors, SCPs maintain a proliferation quiescent state. Upon small molecules, removal SCPs resume proliferation and concomitant NKX2-5 up-regulation triggers cell-autonomous differentiation into cardiomyocytes. Directed differentiation of SCPs into the endothelial and smooth muscle lineages confirms their full developmental potential typical of bona fide cardiovascular progenitors. Single-cell RNA-sequencing-based transcriptional profiling of our in vitro generated human SCPs notably reflects the dynamic cellular composition of E8.25-E9.25 posterior second heart field of mouse hearts, hallmarked by nuclear receptor sub-family 2 group F member 2 expression. Investigating molecular mechanisms of SCP stabilization, we found that the cell-autonomously regulated retinoic acid and BMP signalling is governing SCP transition from quiescence towards proliferation and cell-autonomous differentiation, reminiscent of a niche-like behaviour. CONCLUSION: The chemically defined and reversible nature of our stabilization approach provides an unprecedented opportunity to dissect mechanisms of cardiovascular progenitors' specification and reveal their cellular and molecular properties.

The dynamic TRPV2 ion channel proximity proteome reveals functional links of calcium flux with cellular adhesion factors NCAM and L1CAM in neurite outgrowth.

TRPV2 voltage-insensitive, calcium-permeable ion channels play important roles in cancer progression, immune response, and neuronal development. Despite TRPV2's physiological impact, underlying endogenous proteins mediating TRPV2 responses and affected signaling pathways remain elusive. Using quantitative peroxidase-catalyzed (APEX2) proximity proteomics we uncover dynamic changes in the TRPV2-proximal proteome and identify calcium signaling and cell adhesion factors recruited to the molecular channel neighborhood in response to activation. Quantitative TRPV2 proximity proteomics further revealed activation-induced enrichment of protein clusters with biological functions in neural and cellular projection. We demonstrate a functional connection between TRPV2 and the neural immunoglobulin cell adhesion molecules NCAM and L1CAM. NCAM and L1CAM stimulation robustly induces TRPV2 [Ca2+]I flux in neuronal PC12 cells and this TRPV2-specific [Ca2+]I flux requires activation of the protein kinase PKCα. TRPV2 expression directly impacts neurite lengths that are modulated by NCAM or L1CAM stimulation. Hence, TRPV2's calcium signaling plays a previously undescribed, yet vital role in cell adhesion, and TRPV2 calcium flux and neurite development are intricately linked via NCAM and L1CAM cell adhesion proteins.

Functional and structural insights into activation of TRPV2 by weak acids.

Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.

[Opioid-free anesthesia : Wrong track or meaningful exit from the era of opioid-based analgesia?] / Opioidfreie Anästhesie : Irrweg oder sinnvoller Ausweg aus der Ära der opioidbasierten Analgesie?

The limitations and disadvantages of opioids in anesthesia are very well known but the advantages combined with a lack of effective alternatives even now still prevents refraining from using opioids as part of an adequate pain therapy. For decades, pain research has had the declared goal of replacing opioids with new substances which have no serious side effects; however, currently this goal seems to be a long way off. Due to the media coverage of the "opioid crisis" in North America, the use of opioids for pain management is also increasingly being questioned by the patients. Measures to contain this crisis are only slowly taking effect in view of the increasing number of deaths, which is why the triggers are still being sought. The perioperative administration of opioids is not only a possible gateway to addiction and abuse but it can also cause outcome-relevant complications, such as respiratory depression, postoperative nausea and vomiting and an increase in postoperative pain. Therefore, these considerations gave rise to the idea of an opioid-free anesthesia (OFA), i.e., opioids are not administered as part of anesthesia to carry out surgical procedures. Although this idea may make sense at first glance, a rapid introduction of this concept appears to be risky as it entails significant changes for the entire anesthesiological management. Based on relatively robust data from clinical studies, this concept can now be evaluated and discussed not only emotionally but also objectively. This review article presents arguments for or against the complete avoidance of intraoperative or even perioperative opioids. The current conditions in Germany are primarily taken into account, so that the perioperative pain therapy is transferable to the established standards. The results from current clinical studies on the implementation of an opioid-free anesthesia are summarized and discussed.

Molecular details of ruthenium red pore block in TRPV channels.

Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

Free heme and hemopexin in acute kidney injury after cardiopulmonary bypass and transient renal ischemia.

Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro-inflammatory. Most studies on nephrotoxicity of hemolysis-derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non-protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo-horseradish peroxidase-based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger-protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.

Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region.

BACKGROUND: Bisulfite sequencing has long been considered the gold standard for measuring DNA methylation at single CpG resolution. However, in recent years several new approaches like nanopore sequencing have been developed due to hints for a partial error-proneness of bisulfite sequencing. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter from our previous studies obtained by bisulfite sequencing. METHODS: We compared methylation rates determined by direct bisulfite sequencing and nanopore sequencing following Cas9-mediated PCR-free enrichment. RESULTS: We could show that CpG methylation levels above 20% corroborate well with our previous data. Within the range between 0 and 20% methylation, however, Sanger sequencing data have to be interpreted cautiously, at least in the investigated region of interest (TRPA1 promotor region). CONCLUSION: Based on the investigation of the TRPA1- region as an example, the present work can help in choosing the right method out of the two current main approaches for methylation analysis for different individual settings regarding many factors like cohort size, costs and prerequisites that should be fulfilled for each method. All in all, both methods have their raison d'être. Furthermore, the present paper contains and illustrates some important basic information and explanation of how guide RNAs should be located for an optimal outcome in Cas9 mediated PCR free target enrichment.

[Does medicinal cannabis or cannabis consumption have an influence on the perioperative anesthesiological management?] / Hat medizinisches Cannabis oder Cannabiskonsum einen Einfluss auf das perioperative anästhesiologische Management?

The greatly increasing use of medicinal cannabis products as well as the upcoming legalization of cannabis not only require a general re-evaluation of how these substances might be classified as illegal drugs, but also enforce a critical view on the possible consequences that cannabis consumption might have on the anesthesiological strategies applied for surgical procedures. Although high-quality clinical studies are still lacking, several clinical studies meanwhile indicate that an active preoperative cannabis consumption seems to be associated with relevant pathophysiological aspects. Patients who regularly consume high doses of cannabis show an increased risk of cardiovascular and respiratory complications as well as the postoperative nausea and vomitting (PONV) associated with anesthesia. This also applies to relatively young patients. Moreover, the requirements for general anesthetics and analgesics seem to be increased in the context of cannabis consumption, e.g., these patients may require additional efforts when it comes to monitoring the depth of anesthesia and providing a personalized multimodal postoperative pain therapy. It therefore appears to be meaningful to carefully assess and document the extent and duration of the preoperative cannabis consumption during the preoperative assessment. Furthermore, the possibility to perform a preoperative dose reduction of cannabis products in cases with high doses should at least be considered. As the consumption of cannabis is not only increasing in Germany but also worldwide, important future insights will offer a guide towards a safe handling of cannabis in perioperative medicine in the coming years.

Pain-causing stinging nettle toxins target TMEM233 to modulate NaV1.7 function.

Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.

Molecular mechanisms of MrgprA3-independent activation of the transient receptor potential ion channels TRPA1 and TRPV1 by chloroquine.

BACKGROUND AND PURPOSE: Itch is associated with several pathologies and is a common drug-induced side effect. Chloroquine (CQ) is reported to induce itch by activating the Mas-related G protein-coupled receptor MrgprA3 and subsequently TRPA1. In this study, we demonstrate that CQ employs at least two MrgprA3-independent mechanisms to activate or sensitize TRPA1 and TRPV1. EXPERIMENTAL APPROACH: Patch clamp and calcium imaging were utilized to examine effects of CQ on TRPA1 and TRPV1 expressed in HEK 293T cells. KEY RESULTS: In calcium imaging, CQ induces a concentration-dependent but MrgprA3-independent activation of TRPA1 and TRPV1. Although CQ itself inhibits TRPA1 and TRPV1 in patch clamp recordings, co-application of CQ and ultraviolet A (UVA) light evokes membrane currents through both channels. This effect is inhibited by the reducing agent dithiothreitol (DTT) and is reduced on mutants lacking cysteine residues accounting for reactive oxygen species (ROS) sensitivity. The combination of CQ and UVA light triggers an accumulation of intracellular ROS, removes fast inactivation of voltage-gated sodium currents and activates TRPV2. On the other hand, CQ is a weak base and induces intracellular alkalosis. Intracellular alkalosis can activate TRPA1 and TRPV1, and CQ applied at alkaline pH values indeed activates both channels. CONCLUSION AND IMPLICATIONS: Our data reveal novel pharmacological properties of CQ, allowing activation of TRPA1 and TRPV1 via photosensitization as well as intracellular alkalosis. These findings add more complexity to the commonly accepted dogma that CQ-induced itch is specifically mediated by MrgprA3 coupling to TRPA1.

Cell-Free Hemoglobin in Acute Kidney Injury after Lung Transplantation and Experimental Renal Ischemia/Reperfusion.

Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.

Local Anesthetic Like Inhibition of the Cardiac Na+ Channel Nav1.5 by Chloroquine and Hydroxychloroquine.

Introduction: Chloroquine (CQ) and its derivate hydroxychloroquine (HCQ) are successfully deployed for different diseases beyond the prophylaxis and treatment of malaria. Both substances exhibit antiviral properties and have been proposed for prophylaxis and treatment of COVID-19 caused by SARS-CoV-2. CQ and HCQ cause similar adverse events including life-threatening cardiac arrhythmia generally based on QT-prolongation, which is one of the most reported adverse events for both agents associated with the treatment of COVID-19. Various drugs known to induce QT-prolongation have been proven to exert local anesthetic (LA)-like properties regarding their impact on the cardiac Na+ channel Nav1.5. Inhibition of Nav1.5 is considered as the primary mechanism of cardiotoxicity caused by LAs. However, the mechanism of the arrhythmogenic effects of CQ and HCQ related to Nav1.5 has not yet been fully investigated. Therefore, the exact mechanism of how CQ and HCQ affect the sodium currents generated by Nav1.5 need to be further elucidated. Objective: This in vitro study aims to investigate the effects of CQ and HCQ on Nav1.5-generated sodium currents to identify possible LA-like mechanisms that might contribute to their arrhythmogenic properties. Methods: The effects of CQ and HCQ on Nav1.5-generated sodium currents by HEK-293 cells expressing either wild-type human Nav1.5 or mutant Nav1.5 F1760A are measured using the whole-cell patch-clamp technique. Results: Both agents induce a state-dependent inhibition of Nav1.5. Furthermore, CQ and HCQ produce a use-dependent block of Nav1.5 and a shift of fast and slow inactivation. Results of experiments investigating the effect on the LA-insensitive mutant Nav1.5-F1760A indicate that both agents at least in part employ the proposed LA-binding site of Nav1.5 to induce inhibition. Conclusion: This study demonstrated that CQ and HCQ exert LA-typical effects on Nav1.5 involving the proposed LA binding site, thus contributing to their arrhythmogenic properties.

The coumarin osthole is a non-electrophilic agonist of TRPA1.

The naturally occurring coumarin osthole has antipruritic properties, and recent reports suggest that this effect is due an inhibition or desensitization of the cation channels TRPV1 and TRPV3. Osthole was also suggested to activate TRPA1, an effect that should rather be pruritic than antipruritic. Here we characterized the effects of osthole on TRPA1 by means of ratiometric calcium imaging and patch clamp electrophysiology. In HEK 293 expressing human (h) TRPA1, osthole induced a concentration-dependent increase in intracellular calcium that was inhibited by the TRPA1-inhibitor A967079. In mouse dorsal root ganglion (DRG) cells, osthole induced a strong calcium-influx that was partly mediated by TRPA1. Osthole evoked fully reversible membrane currents in whole-cell as well as cell-free inside-out recordings on hTRPA1. Osthole failed to activate the mutant hTRPA1-S873V/T874L, a previously described binding site for the non-electrophilic TRPA1-agonists menthol and carvacrol. The combined application of osthole and carvacrol diminished channel activation, suggesting a competitive binding. Finally, osthole failed to activate TRPM8 and TRPV4 but induced a modest activation of hTRPV1 expressed in HEK 293 cells. We conclude that osthole is a potent non-electrophilic agonist of TRPA1. The relevance of this property for the antipruritic effects needs to be further explored.

Antidepressants Are Poor Inhibitors of Heat-Evoked Ion Currents Mediated by TRPM2.

INTRODUCTION: The heat and redox-sensitive ion channel TRPM2 was reported to be a causative mechanism for depression in a mouse model and to be upregulated in the hippocampus in patients suffering from depressive disorders. TRPM2 may thus be a novel target for antidepressants, but so far, selective TRPM2-inhibitors have not yet been developed. In this in vitro study, we examined the inhibitory effects of several established antidepressants on heat-evoked inward currents of TRPM2. METHODS: Human (h) TRPM2 expressed in HEK293 cells was examined by means of whole-cell patch clamp recordings. Effects of duloxetine, amitriptyline, sertraline, fluoxetine, paroxetine, citalopram, escitalopram, ketamine, pregabalin, lidocaine, and QX-314 were explored on heat-evoked currents in cells pretreated with ADP-ribose (ADPR). RESULTS: While inward currents induced by 1 mM ADPR in the pipette solution displayed a strong rundown hampering pharmacological experiments, heat-evoked currents in cells loaded with 200 µM APDR remained stable upon repetitive activation. Among all substances examined, only inhibition induced by duloxetine displayed a clear concentration-dependency. Thirty micromolar duloxetine was required for 50% inhibition, the same degree of inhibition was also induced by 30 µM amitriptyline, fluoxetine, and paroxetine. While citalopram, escitalopram, ketamine, and pregabalin failed to robustly modify TRPM2, sertraline and low concentrations of lidocaine even potentiated heat-evoked currents. CONCLUSION: Our data indicate that some, but not all established antidepressants inhibit hTRPM2 when it is activated by heat and ADPR in vitro, e.g., presumably relevant endogenous agonists. However, none of the examined substances exhibited a potent inhibition which is likely to translate into a clinically relevant effect at effective plasma concentrations. Whether or not TRPM2 may be a relevant target for antidepressants cannot be conclusively assessed by a single in vitro study, thus further studies are required along these lines. Nevertheless, future studies may get simplified by the novel approach we developed for in vitro pharmacological analysis of TRPM2.

Low potency inhibition of NaV1.7 by externally applied QX-314 via a depolarizing shift in the voltage-dependence of activation.

QX-314 is a quaternary permanently charged lidocaine derivative that inhibits voltage-gated sodium channels (NaV). As it is membrane impermeable, it is generally considered that QX-314 applied externally is inactive, unless it can gain access to the internal local anesthetic binding site via another entry pathway. Here, we characterized the electrophysiological effects of QX-314 on NaV1.7 heterologously expressed in HEK293 cells, and found that at high concentrations, external QX-314 inhibited NaV1.7 current (IC50 2.0 ± 0.3 mM) and shifted the voltage-dependence to more depolarized potentials (ΔV50 +10.6 mV). Unlike lidocaine, the activity of external QX-314 was not state- or use-dependent. The effect of externally applied QX-314 on NaV1.7 channel biophysics differed to that of internally applied QX-314, suggesting QX-314 has an additional externally accessible site of action. In line with this hypothesis, disruption of the local anesthetic binding site in a [F1748A]NaV1.7 mutant reduced the potency of lidocaine by 40-fold, but had no effect on the potency or activity of externally applied QX-314. Therefore, we conclude, using an expression system where QX-314 was unable to cross the membrane, that externally applied QX-314 is able to inhibit NaV1.7 peak current at low millimolar concentrations.

Structural insights into TRPV2 activation by small molecules.

Transient receptor potential vanilloid 2 (TRPV2) is involved in many critical physiological and pathophysiological processes, making it a promising drug target. Here we present cryo-electron microscopy (cryo-EM) structures of rat TRPV2 in lipid nanodiscs activated by 2-aminoethoxydiphenyl borate (2-APB) and propose a TRPV2-specific 2-ABP binding site at the interface of S5 of one monomer and the S4-S5 linker of the adjacent monomer. In silico docking and electrophysiological studies confirm the key role of His521 and Arg539 in 2-APB activation of TRPV2. Additionally, electrophysiological experiments show that the combination of 2-APB and cannabidiol has a synergetic effect on TRPV2 activation, and cryo-EM structures demonstrate that both drugs were able to bind simultaneously. Together, our cryo-EM structures represent multiple functional states of the channel, providing a native picture of TRPV2 activation by small molecules and a structural framework for the development of TRPV2-specific activators.

The antipyretic effectiveness of dipyrone in the intensive care unit: A retrospective cohort study.

INTRODUCTION: Dipyrone (metamizol) is regularly used in critical care for pain and fever treatment, especially in Germany and Spain. However, indication for antipyretic therapy in critically ill patients is currently unclear and data for both the risk and benefit of dipyrone treatment in the intensive care environment are scarce. We hypothesized that antipyretic efficiency of dipyrone would not exceed antipyretic efficiency of acetaminophen. We therefore aimed to compare temperature courses in critically ill patients receiving either intravenous dipyrone, acetaminophen or no antipyretic medication. MATERIAL AND METHODS: We included 937 intensive care unit (ICU) patients with body temperature recordings of at least 37.5°C. We investigated temperature decrease associated with dipyrone or acetaminophen and additionally compared it to an untreated control group. RESULTS: Within the eight-hour study interval, maximum body temperature decrease in patients without antipyretic medication was -0.6°C (IQR: -1.0 to -0.4°C; n = 315). Maximal decrease in body temperature was higher both with dipyrone (-0.8°C (IQR: -1.2 to -0.4°C); p = 0.016; n = 341) and acetaminophen (-0.9°C (IQR: -1.6 to -0.6°C); p<0.001; n = 71), but did not differ between dipyrone and acetaminophen (p = 0.066). As compared to untreated patients, dipyrone only led to a marginal additional decrease in body temperature of only -0.1°C. Maximum of antipyretic effectiveness was reached four hours after administration. CONCLUSION: Antipyretic effectiveness of dipyrone in ICU patients may be overestimated. Given the lack of prospective data, clinical evidence for antipyretic dipyrone therapy in the ICU is insufficient and warrants further critical evaluation.