Genetically-encoded BRET probes shed light on ligand bias-induced variable ion selectivity in TRPV1 and P2X5/7.

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

Evaluation of mitochondrial stress following ultraviolet radiation and 5G radiofrequency field exposure in human skin cells.

Whether human cells are impacted by environmental electromagnetic fields (EMF) is still a matter of debate. With the deployment of the fifth generation (5G) of mobile communication technologies, the carrier frequency is increasing and the human skin becomes the main biological target. Here, we evaluated the impact of 5G-modulated 3.5 GHz radiofrequency (RF) EMF on mitochondrial stress in human fibroblasts and keratinocytes that were exposed for 24 h at specific absorption rate of 0.25, 1, and 4 W/kg. We assessed cell viability, mitochondrial reactive oxygen species (ROS) production, and membrane polarization. Knowing that human skin is the main target of environmental ultraviolet (UV), using the same read-out, we investigated whether subsequent exposure to 5G signal could alter the capacity of UV-B to damage skin cells. We found a statistically significant reduction in mitochondrial ROS concentration in fibroblasts exposed to 5G signal at 1 W/kg. On the contrary, the RF exposure slightly but statistically significantly enhanced the effects of UV-B radiation specifically in keratinocytes at 0.25 and 1 W/kg. No effect was found on mitochondrial membrane potential or apoptosis in any cell types or exposure conditions suggesting that the type and amplitude of the observed effects are very punctual.

In Vivo Functional Ultrasound (fUS) Real-Time Imaging and Dosimetry of Mice Brain Under Radiofrequency Exposure.

This study aims to analyze in real-time the potential modifications induced by low-level continuous-wave and Global System for Mobile Communications radiofrequency (RF) exposure at 1.8 GHz on brain activation in anesthetized mice. A specific in vivo experimental setup consisting of a dipole antenna for the local exposure of the brain was fully characterized. A unique neuroimaging technique based on a functional ultrasound (fUS) probe was used to observe the areas of mice brain activation simultaneously to the RF exposure with unprecedented spatial and temporal resolution (~100 µm, 1 ms) following manual whisker stimulation using a brush. Numerical and experimental dosimetry was carried out to characterize the exposure and to guarantee the validity of the biological results. Our results show that the fUS probe can be efficiently used during in vivo exposure without interference with the dipole. In addition, we conclude that exposure to brain-averaged specific absorption rate levels of 2 and 6 W/kg does not introduce significant changes in the time course of the evoked fUS response in the left barrel field cortex. The proposed technique represents a valuable instrument for providing new insights into the possible effects induced on brain activation under RF exposure. For the first time, brain activity under mobile phone exposure was evaluated in vivo with fUS imaging, paving the way for more realistic exposure configurations, i.e. awake mice and new signals such as the 5 G networks. © 2022 Bioelectromagnetics Society.

Label-Free Study of the Global Cell Behavior during Exposure to Environmental Radiofrequency Fields in the Presence or Absence of Pro-Apoptotic or Pro-Autophagic Treatments.

It remains controversial whether exposure to environmental radiofrequency signals (RF) impacts cell status or response to cellular stress such as apoptosis or autophagy. We used two label-free techniques, cellular impedancemetry and Digital Holographic Microscopy (DHM), to assess the overall cellular response during RF exposure alone, or during co-exposure to RF and chemical treatments known to induce either apoptosis or autophagy. Two human cell lines (SH-SY5Y and HCT116) and two cultures of primary rat cortex cells (astrocytes and co-culture of neurons and glial cells) were exposed to RF using an 1800 MHz carrier wave modulated with various environmental signals (GSM: Global System for Mobile Communications, 2G signal), UMTS (Universal Mobile Telecommunications System, 3G signal), LTE (Long-Term Evolution, 4G signal, and Wi-Fi) or unmodulated RF (continuous wave, CW). The specific absorption rates (S.A.R.) used were 1.5 and 6 W/kg during DHM experiments and ranged from 5 to 24 W/kg during the recording of cellular impedance. Cells were continuously exposed for three to five consecutive days while the temporal phenotypic signature of cells behavior was recorded at constant temperature. Statistical analysis of the results does not indicate that RF-EMF exposure impacted the global behavior of healthy, apoptotic, or autophagic cells, even at S.A.R. levels higher than the guidelines, provided that the temperature was kept constant.

High-Throughput Screening of Transient Receptor Potential Channel 1 Ligands in the Light of the Bioluminescence Resonance Energy Transfer Technique.

Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening. SIGNIFICANCE STATEMENT: This study screened a chemical library against TRPV1 ion channel using bioluminescence resonance energy transfer (BRET) molecular probes and compared the results with the ones obtained using reference techniques such as automated calcium assay and automated patch-clamp. Multiparametric analysis of our results shed light on the capability of calmodulin antagonists to inhibit chemical activation of TRPV1 and indicates that BRET probes may advantageously be included in ion channel drug screening campaigns.

Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels.

Multiplexed bioluminescence resonance energy transfer (BRET) assays were developed to monitor the activation of several functional transient receptor potential (TRP) channels in live cells and in real time. We probed both TRPV1 intramolecular rearrangements and its interaction with Calmodulin (CaM) under activation by chemical agonists and temperature. Our BRET study also confirmed that: (1) capsaicin and heat promoted distinct transitions, independently coupled to channel gating, and that (2) TRPV1 and Ca2+-bound CaM but not Ca2+-free CaM were preassociated in resting live cells, while capsaicin activation induced both the formation of more TRPV1/CaM complexes and conformational changes. The BRET assay, based on the interaction with Calmodulin, was successfully extended to TRPV3 and TRPV4 channels. We therefore developed a full-spectral three-color BRET assay for analyzing the specific activation of each of the three TRPV channels in a single sample. Such key improvement in BRET measurement paves the way for the simultaneous monitoring of independent biological pathways in live cells.

CNIH4 interacts with newly synthesized GPCR and controls their export from the endoplasmic reticulum.

The molecular mechanisms regulating G protein-coupled receptors (GPCRs) trafficking from their site of synthesis in the endoplasmic reticulum (ER) to their site of function (the cell surface) remain poorly characterized. Using a bioluminescence resonance energy transfer-based proteomic screen, we identified a novel GPCR-interacting protein; the human cornichon homologue 4 (CNIH4). This previously uncharacterized protein is localized in the early secretory pathway where it interacts with members of the 3 family of GPCRs. Both overexpression and knockdown expression of CNIH4 caused the intracellular retention of GPCRs, indicating that this ER-resident protein plays an important role in GPCR export. Overexpression of CNIH4 at low levels rescued the maturation and cell surface expression of an intracellularly retained mutant form of the ß2-adrenergic receptor, further demonstrating a positive role of CNIH4 in GPCR trafficking. Taken with the co-immunoprecipitation of CNIH4 with Sec23 and Sec24, components of the COPII coat complex responsible for ER export, these data suggest that CNIH4 acts as a cargo-sorting receptor, recruiting GPCRs into COPII vesicles.

The chemokine CXC4 and CC2 receptors form homo- and heterooligomers that can engage their signaling G-protein effectors and ßarrestin.

G-protein-coupled receptors have been shown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo- and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo- and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of Gαi1, Gα13, Gαz, and ßarrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents Gi activation, also affects the CCR2-promoted engagement of Gαi1 and ßarrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo- and heteromultimeric CXCR4 and CCR2 can form functional signaling complexes that have unique properties.

Effects of 50 Hz magnetic fields on gap junctional intercellular communication in NIH3T3 cells.

The present study focused on gap junctional intercellular communication (GJIC) as a target for biological effects of extremely low-frequency (ELF) magnetic field (MF) exposure. Fluorescence recovery after photobleaching microscopy (FRAP) was used to visualize diffusion of a fluorescent dye between NIH3T3 fibroblasts through gap junctions. The direct effect of 24 h exposure to 50 Hz MF at 0.4 or 1 mT on GJIC function was assessed in one series of experiments. The potential synergism of MF with an inhibitor of GJIC, phorbol ester (TPA), was studied in another series by observing FRAP when NIH3T3 cells were incubated with TPA for 1 h following 24 h exposure to MF. In contrast to other reports of ELF-MF effects on GJIC, under our experimental conditions we observed neither direct inhibition of GJIC nor synergism with TPA-induced inhibition from 50 Hz MF exposures.

In vitro exposure of neuronal networks to the 5G-3.5 GHz signal.

Introduction: The current deployment of the fifth generation (5G) of wireless communications raises new questions about the potential health effects of exposure to radiofrequency (RF) fields. So far, most of the established biological effects of RF have been known to be caused by heating. We previously reported inhibition of the spontaneous electrical activity of neuronal networks in vitro when exposed to 1.8 GHz signals at specific absorption rates (SAR) well above the guidelines. The present study aimed to assess the effects of RF fields at 3.5 GHz, one of the frequencies related to 5G, on neuronal activity in-vitro. Potential differences in the effects elicited by continuous-wave (CW) and 5G-modulated signals were also investigated. Methods: Spontaneous activity of neuronal cultures from embryonic cortices was recorded using 60-electrode multi-electrode arrays (MEAs) between 17 and 27 days in vitro. The neuronal cultures were subjected to 15 min RF exposures at SAR of 1, 3, and 28 W/kg. Results: At SAR close to the guidelines (1 and 3 W/kg), we found no conclusive evidence that 3.5 GHz RF exposure impacts the activity of neurons in vitro. On the contrary, CW and 5G-modulated signals elicited a clear decrease in bursting and total firing rates during RF exposure at high SAR levels (28 W/kg). Our experimental findings extend our previous results, showing that RF, at 1.8 to 3.5 GHz, inhibits the electrical activity of neurons in vitro at levels above environmental standards.

Effects of 5G-modulated 3.5 GHz radiofrequency field exposures on HSF1, RAS, ERK, and PML activation in live fibroblasts and keratinocytes cells.

The potential health risks of exposure to radiofrequency electromagnetic fields from mobile communications technologies have raised societal concerns. Guidelines have been set to protect the population (e.g. non-specific heating above 1 °C under exposure to radiofrequency fields), but questions remain regarding the potential biological effects of non-thermal exposures. With the advent of the fifth generation (5G) of mobile communication, assessing whether exposure to this new signal induces a cellular stress response is one of the mandatory steps on the roadmap for a safe deployment and health risk evaluation. Using the BRET (Bioluminescence Resonance Energy-Transfer) technique, we assessed whether continuous or intermittent (5 min ON/ 10 min OFF) exposure of live human keratinocytes and fibroblasts cells to 5G 3.5 GHz signals at specific absorption rate (SAR) up to 4 W/kg for 24 h impact basal or chemically-induced activity of Heat Shock Factor (HSF), RAt Sarcoma virus (RAS) and Extracellular signal-Regulated Kinases (ERK) kinases, and Promyelocytic Leukemia Protein (PML), that are all molecular pathways involved in environmental cell-stress responses. The main results are (i), a decrease of the HSF1 basal BRET signal when fibroblasts cells were exposed at the lower SARs tested (0.25 and 1 W/kg), but not at the highest one (4 W/kg), and (ii) a slight decrease of As2O3 maximal efficacy to trigger PML SUMOylation when fibroblasts cells, but not keratinocytes, were continuously exposed to the 5G RF-EMF signal. Nevertheless, given the inconsistency of these effects in terms of impacted cell type, effective SAR, exposure mode, and molecular cell stress response, we concluded that our study show no conclusive evidence that molecular effects can arise when skin cells are exposed to the 5G RF-EMF alone or with a chemical stressor.

Comparative study between radiofrequency-induced and muscimol-induced inhibition of cultured networks of cortical neuron.

Previous studies have shown that spontaneously active cultured networks of cortical neuron grown planar microelectrode arrays are sensitive to radiofrequency (RF) fields and exhibit an inhibitory response more pronounced as the exposure time and power increase. To better understand the mechanism behind the observed effects, we aimed at identifying similarities and differences between the inhibitory effect of RF fields (continuous wave, 1800 MHz) to the γ-aminobutyric acid type A (GABAA) receptor agonist muscimol (MU). Inhibition of the network bursting activity in response to RF exposure became apparent at an SAR level of 28.6 W/kg and co-occurred with an elevation of the culture medium temperature of ~1°C. Exposure to RF fields preferentially inhibits bursting over spiking activity and exerts fewer constraints on neural network bursting synchrony, differentiating it from a pharmacological inhibition with MU. Network rebound excitation, a phenomenon relying on the intrinsic properties of cortical neurons, was observed following the removal of tonic hyperpolarization after washout of MU but not in response to cessation of RF exposure. This implies that hyperpolarization is not the main driving force mediating the inhibitory effects of RF fields. At the level of single neurons, network inhibition induced by MU and RF fields occurred with reduced action potential (AP) half-width. As changes in AP waveform strongly influence efficacy of synaptic transmission, the narrowing effect on AP seen under RF exposure might contribute to reducing network bursting activity. By pointing only to a partial overlap between the inhibitory hallmarks of these two forms of inhibition, our data suggest that the inhibitory mechanisms of the action of RF fields differ from the ones mediated by the activation of GABAA receptors.

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca2+-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca2+ responses, single-channel currents, and Ca2+ influx through the channel were measured using Ca2+ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity.

Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes.

Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor-G protein complexes. Using the data obtained for the alpha(2A)AR-G alpha(i1) beta1gamma2 complex and the available crystal structures of G alpha(i1) beta1gamma2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor-G protein complex, leading the G alpha-G betagamma interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the G alpha subunit, thus reflecting the initial activation event.

Effects of radiofrequency field exposure on proteotoxic-induced and heat-induced HSF1 response in live cells using the bioluminescence resonance energy transfer technique.

As of today, only acute effects of RF fields have been confirmed to represent a potential health hazard and they are attributed to non-specific heating (≥ 1 °C) under high-level exposure. Yet, the possibility that environmental RF impact living matter in the absence of temperature elevation needs further investigation. Since HSF1 is both a thermosensor and the master regulator of heat-shock stress response in eukaryotes, it remains to assess HSF1 activation in live cells under exposure to low-level RF signals. We thus measured basal, temperature-induced, and chemically induced HSF1 trimerization, a mandatory step on the cascade of HSF1 activation, under RF exposure to continuous wave (CW), Global System for Mobile (GSM), and Wi-Fi-modulated 1800 MHz signals, using a bioluminescence resonance energy transfer technique (BRET) probe. Our results show that, as expected, HSF1 is heat-activated by acute exposure of transiently transfected HEK293T cells to a CW RF field at a specific absorption rate of 24 W/kg for 30 min. However, we found no evidence of HSF1 activation under the same RF exposure condition when the cell culture medium temperature was fixed. We also found no experimental evidence that, at a fixed temperature, chronic RF exposure for 24 h at a SAR of 1.5 and 6 W/kg altered the potency or the maximal capability of the proteasome inhibitor MG132 to activate HSF1, whatever signal used. We only found that RF exposure to CW signals (1.5 and 6 W/kg) and GSM signals (1.5 W/kg) for 24 h marginally decreased basal HSF1 activity.

Search for tumor-specific frequencies of amplitude-modulated 27 MHz electromagnetic fields in mice with hepatocarcinoma xenografted tumors.

Aim: The Pasche research group has reported that tumor-specific electromagnetic field frequencies have physiological and potential anti-tumor effects in cells, animals, and humans. Our aim was to investigate whether these fields have similar effects on physiological parameters in murine tumor models.Methods: Human HuH7 or HEPG2 cells were implanted in the right flank of 8-week-old female RAG gamma 2 C immunodeficient mice. An oximeter was used to record systolic blood pressure (pulse) in free-roaming conscious mice. Mice pulses were recorded and analyzed using a in-house software that also controlled the low-frequency generator for modulating the 27.12 MHz carrier wave at selected frequencies.Results: We performed exposures using both systematic scans at low frequencies and at the pre-determined frequencies reported by the Pasche group as altering both pulse and tumor growth in humans. Those exposures produced no detectable change in physiological parameters of tumor-bearing mice.Conclusion: No tumor-related frequencies were found, neither using systematic scans of frequencies nor published specific frequencies. There might obviously be differences between animal and human models, but our approach did not confirm the physiological data of the human Pasche group data.

Effects of radiofrequency fields on RAS and ERK kinases activity in live cells using the bioluminescence resonance energy transfer technique.

Purpose: The present study was conducted to re-evaluate the effect of low-level 1800 MHz RF signals on RAS/MAPK activation in live cells.Material and methods: Using Bioluminescence Resonance Energy Transfer technique (BRET), we assessed the effect of Continuous wave (CW) and Global System for Mobile (GSM)-modulated 1800 MHz signals (up to 2 W/kg) on ERK and RAS kinases' activity in live HuH7 cells.Results: We found that radiofrequency field (RF) exposure for 24 h altered neither basal level of RAS and ERK activation nor the potency of phorbol-12-myristate-13-acetate (PMA) to activate RAS and ERK kinases. However, we found that exposure to GSM-modulated 1800 MHz signals at 2 W/kg decreased the PMA maximal efficacy to activate both RAS and ERK kinases' activity. Exposure with CW 1800 MHz signal at 2 W/kg only decreased maximal efficacy of PMA to activate ERK but not RAS. No effects of RF exposure at 0.5 W/kg was observed on maximal efficacy of PMA to activate either RAS or ERK whatever the signal used.Conclusions: Our results indicate that RF exposure decreases the efficiency of the cascade of events, which, from the binding of PMA to its receptor(s), leads to the activation of RAS and ERK kinases.

Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure.

The existence of effects of radiofrequency field exposure at environmental levels on living tissues and organisms remains controversial, in particular regarding potential "nonthermal" effects produced in the absence of temperature elevation. Therefore, we investigated whether TRPV1, one of the most studied thermosensitive channels, can be activated by the heat produced by radiofrequency fields and by some specific nonthermal interaction with the fields. We have recently shown that TRPV1 activation can be assessed in real-time on live cells using the bioluminescence resonance energy transfer technique. Taking advantage of this innovative assay, we monitored TRPV1 thermal and chemical modes of activation under radiofrequency exposure at 1800 MHz using different signals (CW, GSM, UMTS, LTE, Wi-Fi and WiMAX) at specific absorption rates between 8 and 32 W/kg. We showed that, as expected, TRPV1 channels were activated by the heat produced by radiofrequency field exposure of transiently-transfected HEK293T cells, but found no evidence of TRPV1 activation in the absence of temperature elevation under radiofrequency field exposure. There was no evidence either that, at fixed temperature, radiofrequency exposure altered the maximal efficacy of the agonist Capsaicin to activate TRPV1.

Dosimetric Characteristics of an EMF Delivery System Based on a Real-Time Impedance Measurement Device.

In this paper, the dosimetric characterization of an EMF exposure setup compatible with real-time impedance measurements of adherent biological cells is proposed. The EMF are directly delivered to the 16-well format plate used by the commercial xCELLigence apparatus. Experiments and numerical simulations were carried out for the dosimetric analysis. The reflection coefficient was less than -10 dB up to 180 MHz and this exposure system can be matched at higher frequencies up to 900 and 1800 MHz. The specific absorption rate (SAR) distribution within the wells containing the biological medium was calculated by numerical finite-difference time domain simulations and results were verified by temperature measurements at 13.56 MHz. Numerical SAR values were obtained at the microelectrode level where the biological cells were exposed to EMF including 13.56, 900, and 1800 MHz. At 13.56 MHz, the SAR values, within the cell layer and the 270-µL volume of medium, are 1.9e3 and 3.5 W/kg/incident mW, respectively.