Xenopus Oocyte Expression System Research Articles

Contributions of multiple transport mechanisms to intestinal uptake of serotonin

This study aimed to analyze the contributions of multiple transport mechanisms to the intestinal uptake of serotonin (5-HT) by employing a variety of in vitro experimental techniques, focusing on organic cation transporters expressed in the gastrointestinal (GI) tract, such as SERT, PMAT, THTR2, OCT3, and OCTN2. Analysis of the concentration dependence of 5-HT uptake by Caco-2 cells revealed multi-affinity kinetics with high-affinity and low-affinity components, suggesting that multiple transporters are involved in the intestinal 5-HT uptake. Comparative analysis of transporters using Km values obtained in Xenopus oocyte expression systems suggested that SERT is responsible for the high-affinity transport, while PMAT, THTR2, and OCT3 contribute to the low-affinity transport. Further analysis indicated that the relative contributions of SERT and PMAT to the intestinal 5-HT uptake (0.01 µM) are approximately 94.9% and 1.1%, respectively. Interestingly, at the concentration of 10 µM, the reported steady-state concentration of 5-HT in the human colon, the contributions of SERT, PMAT, THTR2, and OCT3 were estimated to be approximately 37.0%, 1.0%, 18.2%, and 20.5%, respectively. In conclusion, the present study indicated that the contributions of multiple transporters to 5-HT uptake in the GI tract are dependent upon the colon luminal concentration of 5-HT.

The role of OR5, which is highly expressed in the winged grain aphid Sitobion miscanthi, in specific recognition of EBF

Winged parthenogenetic aphids are mainly responsible for migration and dispersal. Aphid alarm pheromone (E)-β-Farnesene (EBF) has dual effects on repelling and stimulating wing differentiation in aphids. Previous studies have shown that the odorant coreceptor SmisOrco is involved in the perception of EBF by S. miscanthi; however, its EBF-specific odorant receptor (OR) and the difference between winged and wingless aphids remain unclear. In this study, the Xenopus oocyte expression system and RNAi technology were used to detect the transmission of EBF signals, and it was found that the olfactory receptor SmisOR5 is an EBF-specific OR in S. miscanthi and is specifically highly expressed in the antennae of winged aphids. Furthermore, when OR5 was silenced with dsRNA, the repellent effect of EBF was weakened, and aphids showed more active aimless movements. Therefore, as a specific OR for EBF, the high expression level of SmisOR5 in winged aphids suggests a molecular basis for its high sensitivity to EBF. This study advances our understanding of the molecular mechanisms of aphid EBF perception and provides novel ideas for effective management and prevention of the migration of winged aphids.

The Ubiquitin Ligase Adaptor NDFIP1 Interacts with TRESK and Negatively Regulates the Background K+ Current.

The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the Xenopus oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K+ channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K+ current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K+ current.

Functional Characterization of an Odorant Receptor Expressed in Newly Hatched Larvae of Fall Armyworm Spodoptera frugiperda.

In the past decade, Spodoptera frugiperda has emerged as a significant invasive pest globally, posing a serious threat to agriculture due to its broad diet, migratory behavior, and ability to cause extensive plant damage. While extensive research has focused on the olfactory capabilities of adult S. frugiperda, understanding of the olfactory process in larvae remains limited, despite larvae playing a crucial role in crop damage. To address this gap, we identified an odorant receptor (OR), SfruOR40, expressed in the first-instar larvae through phylogenetic analysis. Using quantitative real-time PCR, we compared SfruOR40 expression levels in larvae and adults. We then characterized the function of SfruOR40 against 67 compounds using the Xenopus oocyte expression system and found that SfruOR40 responded to three plant volatiles. Further, behavioral experiments revealed a larval attraction to (-)-trans-Caryophyllene oxide. This study elucidates SfruOR40's role in the olfactory recognition of newly hatched S. frugiperda larvae, expanding our knowledge of such mechanisms in Noctuid moths. Furthermore, it highlights the potential of plant-derived natural products for biological pest control from a behavioral ecology perspective.

3,3',4,4'-tetrachlorobiphenyl (PCB77) enhances human Kv1.3 channel currents and alters cytokine production.

Polychlorinated biphenyls (PCBs) were once used throughout various industries; however, because of their persistence in the environment, exposure remains a global threat to the environment and human health. The Kv1.3 and Kv1.5 channels have been implicated in the immunotoxicity and cardiotoxicity of PCBs, respectively. We determined whether 3,3',4,4'-tetrachlorobiphenyl (PCB77), a dioxin-like PCB, alters human Kv1.3 and Kv1.5 currents using the Xenopus oocyte expression system. Exposure to 10 nM PCB77 for 15 min enhanced the Kv1.3 current by approximately 30.6%, whereas PCB77 did not affect the Kv1.5 current at concentrations up to 10 nM. This increase in the Kv1.3 current was associated with slower activation and inactivation kinetics as well as right-shifting of the steady-state activation curve. Pretreatment with PCB77 significantly suppressed tumor necrosis factor-α and interleukin-10 production in lipopolysaccharide-stimulated Raw264.7 macrophages. Overall, these data suggest that acute exposure to trace concentrations of PCB77 impairs immune function, possibly by enhancing Kv1.3 currents.

Molecular Basis of CO2 Sensing in Hyphantria cunea.

Carbon dioxide (CO2) released by plants can serve as a cue for regulating insect behaviors. Hyphantria cunea is a widely distributed forestry pest that may use CO2 as a cue for foraging and oviposition. However, the molecular mechanism underlying its ability to sense CO2 has not been elucidated. Our initial study showed that CO2 is significantly attractive to H. cunea adults. Subsequently, 44 H. cunea gustatory receptors (GRs) were identified using transcriptome data, and 3 candidate CO2 receptors that are specifically expressed in the labial palps were identified. In vivo electrophysiological assays revealed that the labial palp is the primary organ for CO2 perception in H. cunea, which is similar to findings in other lepidopteran species. By using the Xenopus oocyte expression system, we showed that the HcunGR1 and HcunGR3 co-expressions produced a robust response to CO2, but HcunGR2 had an inhibitory effect on CO2 perception. Finally, immunohistochemical staining revealed sexual dimorphism in the CO2-sensitive labial pit organ glomerulus (LPOG). Taken together, our results clarified the mechanism by which H. cunea sense CO2, laying the foundation for further investigations into the role of CO2 in the rapid spread of H. cunea.

Cab39 is the major scaffolding protein for SPAK-mediated NCC activation in the mouse kidney

STE20/SPS1-related proline/alanine-rich kinase (SPAK) phosphorylates the sodium-chloride cotransporter (NCC) to stimulate sodium reabsorption in the Distal Convoluted Tubule (DCT) for K+ homeostasis and blood pressure. The scaffold proteins, Calcium-binding protein 39 (Cab39) and Cab39-like (Cab39l) activate SPAK in a Xenopus oocyte expression system. Still, it has not been determined whether Cab39 or Cab39-like are required for NCC phosphorylation in the DCT. Here, we developed DCT-specific Cab39 knockout (KO), global Cab39l-KO, and Cab39/Cab39l double knockout (DKO) mice to assess the role of the two adaptor proteins in the Kidney. The SPAK-NCC signaling axis was evaluated by Western Blot and immunofluorescence microscopy. Physiological consequences of altered signaling were evaluated with blood and urine electrolyte measurements. Cab39-KO mice had significantly lower total NCC (tNCC) and phosphorylated NCC (pNCC) abundance than wild-type mice. Phospho-activation of NCC with low K+ diet was also blunted in the Cab39 KO mice. By contrast, the Cab39l-KO mice did not show differences in total or phosphorylated NCC signals. However, when Cab39 was eliminated on the Cab39l KO background (DKO), pNCC abundance further decreased to almost undetectable levels. Remarkably, the DKO did not affect the phospho-activation of SPAK. Instead, SPAK failed to move to the apical membrane and accumulated into cytoplasmic puncta in the DKO mice. In summary, these data indicate that Cab39 and Cab39l act as major and minor regulators of SPAK-NCC signaling, providing an adaptor for translocating SPAK to the apical membrane for NCC interaction and phosphorylation. NIH grant R01DK093501 to Professor Eric Delpire and Professor Paul Welling, The Leducq Foundation grant 17CVD05 to Professor Eric Delpire and Professor Paul Welling, and a Crawford Long Innovation grant to Professor Eric Delpire. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Use of Carboxyfluorescein Reveals the Transport Function of MCT6/SLC16A5 Associated with CD147 as a Chloride-Sensitive Organic Anion Transporter in Mammalian Cells

Oral drug absorption involves drug permeation across the apical and basolateral membranes of enterocytes. Although transporters mediating the influx of anionic drugs in the apical membranes have been identified, transporters responsible for efflux in the basolateral membranes remain unclear. Monocarboxylate transporter 6 (MCT6/SLC16A5) has been reported to localize to the apical and basolateral membranes of human enterocytes and to transport organic anions such as bumetanide and nateglinide in the Xenopus oocyte expression system; however, its transport functions have not been elucidated in detail. In this study, we characterized the function of MCT6 expressed in HEK293T cells and explored fluorescent probes to more easily evaluate MCT6 function. The results illustrated that MCT6 interacts with CD147 to localize at the plasma membrane. When the uptake of various fluorescein derivatives was examined in NaCl-free uptake buffer (pH 5.5), the uptake of 5-carboxyfluorescein (5-CF) was significantly greater in MCT6 and CD147-expressing cells. MCT6-mediated 5-CF uptake was saturable with a Km of 1.07 mM and inhibited by several substrates/inhibitors of organic anion transporters and extracellular Cl ion with an IC50 of 53.7 mM. These results suggest that MCT6 is a chloride-sensitive organic anion transporter that can be characterized using 5-CF as a fluorescent probe.

Ionotropic Receptor IR75q.2 Mediates Avoidance Reaction to Nonanoic Acid in the Fall Armyworm Spodoptera frugiperda (Lepidoptera, Noctuidae).

Ionotropic receptors (IRs) play an important role in olfaction, but little is known in nondrosophila insects. Here, we report in vitro and in vivo functional characterization of IR75q.2 in the invasive moth pest Spodoptera frugiperda. First, 13 IRs (including four coreceptor IRs) were found specifically or highly expressed in adult antennae. Second, these IRs were tested for responding profiles to 59 odorants using the Xenopus oocyte expression system, showing that only SfruIR75q.2 responded to 8-10C fatty acids and their corresponding aldehydes, with SfruIR8a as the only coreceptor. Third, the three acids (especially nonanoic acid) showed repellent effects on moth's behavior and oviposition, but the repellence significantly reduced to the insects with IR75q.2 knockout by CRISPR/Cas9. Taken together, our study reveals the function of SfruIR75q.2 in perception of acid and aldehyde odorants and provides the first in vivo evidence for olfactory function of an odor-specific IR in Lepidoptera.

An outward-rectifying plant K+ channel SPORK2 exhibits temperature-sensitive ion-transport activity

Temperature sensing is critical for the survival of living organisms.1,2 Thermosensitive transient receptor-potential (TRP) cation channels function as thermosensors in mammals.2,3,4,5,6 In contrast to animals, landplants lack TRP genes.7,8,9 Previous patch-clamp studies in plant cells suggested the presence of ion channels whose activities are related to temperature, implying the presence of TRP-like channels.10,11,12,13,14 However, the molecular entities of such temperature-sensitive ion channels were still unknown in land plants. In this study, we observed that the unique rainfall-induced leaf-folding movement of the legumetree Samanea saman15 was temperature-sensitive by using a rainfall-mimicking assay. Chilling-induced leaf folding in S.saman was shown to be related to the swelling of the motor cells16,17 at the base of the leaflet. This swelling suggested involvement of temperature-sensitive inactivation of K+ currents, independent of fluctuations in ion channel gene expression in motor cells. These findings led us to examine the temperature sensitivity of an outward-rectifying K+ channel, SPORK2, which was reported as an ion channel responsible for the nyctinastic (circadian-rhythmic) leaf movement of S.saman.18 We alsodiscovered that SPORK2 exhibits temperature-sensitive K+ transport activity in the Xenopus oocyte expression system. Using chimeric channels, we showed that two domains of SPORK2 regulated the temperaturesensitivity. Furthermore, heterologously expressed SPORK2 in Arabidopsis guard cells induced temperature-dependent stomatal closure. Therefore, SPORK2 is an ion channel in land plantswith temperature-sensitive ion-transport activity that functions similarly to mammalian TRP channels. Our current findings advance the molecular understanding of temperature-sensing mechanisms in plants.

Acute Inhibition of the Human Kv1.5 Channel by H1 Receptor Antagonist Dimenhydrinate: Mode of Action.

Dimenhydrinate, an H1 receptor antagonist, is generally used for the prevention and treatment of nausea and vomiting. However, cardiac arrhythmias have been reported to be associated with the overdose of histamine H1 receptor antagonists, indicating the probable effect of antihistamines on ion channels. By using a two-microelectrode voltage clamp, we have herein studied the electrophysiological effects of dimenhydrinate on the human Kv1.5 channel in the Xenopus oocyte expression system. Dimenhydrinate acutely and reversibly suppressed the amplitudes of the peak and the steady-state current, within 6 min. The inhibitory effect of dimenhydrinate on the peak and the steady-state Kv1.5 currents increased progressively from -10 to +50 mV. At each test voltage, the drug suppressed both the peak and the steady-state currents to a similar extent. When the oocytes were stimulated at the rates of 5- and 30-s intervals, dimenhydrinate-induced a use-dependent blockade of the human Kv1.5 channel. Dimenhydrinate expedited the timecourse of the Kv1.5 channel activation more effectively than the timecourse of its inactivation. However, the activation and inactivation curves of the channel were not altered by the H1 receptor antagonist. In conclusion, we found that dimenhydrinate inhibits the human Kv1.5 channel by changing the channel's activation mode, thereby possibly increasing the possibility of triggering cardiac arrhythmias and affecting atrial fibrillation.

An odorant receptor tuned to an attractive plant volatile vanillin in Spodoptera litura

The insect olfaction plays crucial roles in many important behaviors, in which ORs are key determinants for signal transduction and the olfactory specificity. Spodoptera litura is a typical polyphagous pest, possessing a large repertoire of ORs tuning to broad range of plant odorants. However, the specific functions of those ORs remain mostly unknown. In this study, we functionally characterized one S. litura OR (OR51) that was highly expressed in the adult antennae. First, by using Xenopus oocyte expression and two-electrode voltage clamp recording system (XOE-TEVC), OR51 was found to be strongly and specifically responsive to vanillin (a volatile of S. litura host plants) among 77 tested odorants. Second, electroantennogram (EAG) and Y-tube behavioral experiment showed that vanillin elicited significant EAG response and attraction behavior especially of female adults. This female attraction was further confirmed by the oviposition experiment, in which the soybean plants treated with vanillin were significantly preferred by females for egg-laying. Third, 3D structural modelling and molecular docking were conducted to explore the interaction between OR51 and vanillin, which showed a high affinity (−4.46 kcal/mol) and three residues (Gln163, Phe164 and Ala305) forming hydrogen bonds with vanillin, supporting the specific binding of OR51 to vanillin. In addition, OR51 and its homologs from other seven noctuid species shared high amino acid identities (78–97%) and the same three hydrogen bond forming residues, suggesting a conserved function of the OR in these insects. Taken together, our study provides some new insights into the olfactory mechanisms of host plant finding and suggests potential applications of vanillin in S. litura control.

Identification of transient receptor potential channel genes and functional characterization of TRPA1 in Spodoptera frugiperda

Spodoptera frugiperda is a highly destructive pest that has become a global problem due to its robust reproductive and migratory capabilities. Transient receptor potential (TRP) channels, which constitute a vast ion channel family, play pivotal roles in sensing the external environment and maintaining internal homeostasis in insects. TRP channels have been widely investigated for their critical roles in regulating various insect behaviors in recent years. In this study, we identified 15 TRP gene loci encoding 26 transcripts in the genome of S. frugiperda and analyzed their expression profiles at different developmental stages. The results revealed that S. frugiperda possesses four TRPC genes, six TRPA genes, one TRPM gene, two TRPV genes, one TRPN gene, and one TRPML gene, while a canonical TRPP is absent. Moreover, the SfruTRPA1 was functionally characterized using the Xenopus oocyte expression system. The results showed that SfruTRPA1 is activated by temperature increases from 20 to 45°C, and there is no significant desensitization after repeated stimuli within the same temperature range. Additionally, SfruTRPA1 is activated by certain natural chemicals, including allyl isothiocyanate (AITC) and cinnamaldehyde (CA). These findings provide valuable insights to the TRP genes in S. frugiperda.

Functional Characterization of Pheromone Receptors in the Beet Webworm, Loxostege sticticalis (Lepidoptera: Pyralidae).

Lepidopteran insects mainly rely on sex pheromones to complete sexual communications. Pheromone receptors (PRs) are expressed on the olfactory receptor neurons (ORNs) of the sensilla trichodea and play an essential role in sexual communication. Despite extensive investigations into the mechanisms of peripheral recognition of sex pheromones in Lepidoptera, knowledge about these mechanisms in L. sticticalis remains limited. In this study, five candidate LstiPRs were analyzed in a phylogenetic tree with those of other Lepidopteran insects. Electroantennography (EAG) assays showed that the major sex pheromone component E11-14:OAc elicited a stronger antennal response than other compounds in male moths. Moreover, two types of neurons in sensilla trichodea were classified by single sensillum recordings, of which the "a" neuron specifically responded to E11-14:OAc. Five candidate PRs were functionally assayed by the heterologous expression system of Xenopus oocytes, and LstiPR2 responded to the major sex pheromone E11-14:OAc. Our findings suggest that LstiPR2 is a PR sensitive to L. sticticalis's major sex pheromone compound, E11-14:OAc. Furthermore, this study offers valuable insights into the sexual communication behavior of L. sticticalis, forming a foundation for further analysis of the species' central nervous system.

Atorvastatin Exerts More Selective Inhibitory Effects on hMCT2 than on hMCT1 and hMCT4.

Human monocarboxylate transporter 1 (hMCT1), hMCT2, and hMCT4 transport monocarboxylates, such as L-lactate and pyruvate, with pH dependency. They are often over-expressed in various cancer cells and mediate the energy balance and pH homeostasis. Therefore, hMCT inhibitors can potentially be used as anticancer drugs. However, isoform-selective inhibitors have not yet been well-characterized. In addition, several statins and 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors have been reported to inhibit hMCTs, but their selectivity has not yet been evaluated. In this study, we aimed to determine whether statins could inhibit hMCT1, hMCT2, and hMCT4. We expressed hMCT1, hMCT2, and hMCT4 in a heterologous expression system of Xenopus oocytes and performed inhibitory experiments with various statins (fluvastatin, atorvastatin, simvastatin, rosuvastatin, pravastatin, and pitavastatin). As the three-dimensional structure of hMCT2 has been recently reported, docking simulations of statins and their structures were also performed to estimate the inhibition site. All statins inhibited the transport activities of hMCT1, hMCT2, and hMCT4. In addition, atorvastatin was found to be a potent isoform-selective inhibitor of hMCT2. Docking simulation indicated that atorvastatin could interact with a site surrounded by transmembrane (TM)-2, TM11, and intracellular helix in the TM6/7loop. Therefore, targeting this site may lead to the discovery of more potent hMCT2-selective inhibitors. Atorvastatin exerts selective inhibitory effects on hMCT2. These findings provide insights into the inhibitory mechanism of statins against hMCT1, hMCT2, and hMCT4 and may aid in the development of novel anticancer agents.

Inhibitory effects of N-methyl-D-aspartate (NMDA) and α1-adrenergic receptor antagonist ifenprodil on human Kv1.5 channel.

Ifenprodil has been known to reduce cardiac contractility and cerebral vasodilation by antagonizing α1-adrenergic and N-methyl D-aspartate receptor-mediated intracellular signals. This study aimed to investigate the direct effect of ifenprodil on the human voltage-gated Kv1.5 channel (hKv1.5) by using a Xenopus oocyte expression system and a two-microelectrode voltage clamp technique. The amplitudes of hKv1.5 currents, including peak and steady state, were suppressed in a concentration-dependent manner (IC50; 43.1 and 35.5μM, respectively) after 6min of ifenprodil treatment. However, these effects were ~ 80% reversed by washout, suggesting that ifenprodil directly inhibited the hKv1.5 independent of membrane receptors or intracellular signals. The inhibition rate of steady state showed voltage dependence, wherein the rates increased according to test voltage depolarization. Ifenprodil reduced the time constants of hKv1.5 inactivation but has higher effects on activation. hKv1.5 inhibition by ifenprodil showed use dependency because the drug more rapidly reduced the current at the higher activation frequencies, and subsequent reduction in frequency after high activation frequency caused a partial channel block relief. Therefore, ifenprodil directly blocked the hKv1.5 in an open state and accelerated the time course of the channel inactivation, which provided a biophysical mechanism for the hKv1.5 blocking effects of ifenprodil.

Functional analysis of plasma membrane Ca2+ATPase 3 and its primary aldosteronism-associated mutations.

Primary aldosteronism (PA) is the most common form of secondary hypertension and can lead to higher rates of cardiovascular complications and death than essential hypertension. Aldosterone-producing adenomas (APAs) are the most frequent cause of PA. Most APAs have mutations in plasma membrane ion-transport proteins with 0.6%-9% carrying a mutation in the Plasma Membrane Ca2+ ATPase 3 (PMCA3). We have developed a Xenopus oocyte expression system to measure the function of PMCA3 wildtype (PMCA3WT) and an APA associated deletion mutant (PMCA3L425_V426del.) with minimal endogenous contamination. We measured ATPase activity at 37 °C in plasma membrane preparations from oocytes expressing PMCA3. Ca2+-dependent ATPase was observed in preparations from oocytes expressing PMCA3WT, with a [Ca2+] dependence well described with a rectangular hyperbola (K0.5 = 25.6 ± 5.1 μM) (triplicates from three independent membrane preparations). In contrast, membrane preparations from PMCA3L425_V426del. expressing oocytes lacked Ca2+-dependent ATPase activity, indicating loss of enzymatic activity in the mutant. We used two-electrode voltage clamp electrophysiology to evaluate the presence of PMCA3-variant mediated currents. At resting intracellular [Ca2+], PMCA3WT-injected oocytes had currents indistinguishable from those in uninjected oocytes, at all voltages. When bathed by extracellular 125 mM Na+, PMCA3L425_V426del.-injected oocytes presented inward currents at negative membrane potentials and outward currents at positive ones, consistent with induction of an aberrant channel-like current. The currents were outwardly directed upon substitution of external Na+ with N-methyl D-glucamine+ and were insensitive to changes in extracellular [Ca2+]. These results indicate that PMCA3L425_V426del. causes hyperaldosteronism due to the concomitant loss of active Ca2+ transport and induction of a depolarizing Na+-mediated current. Current experiments are evaluating the ATPase and physiological characteristics of other PA-associated PMCA3 mutations. Funded by NSF-MCB 2003251.

Fluorescent Biosensor Imaging of Nitrate in Arabidopsis thaliana.

Nitrate (NO3-) is an essential element and nutrient for plants and animals. Despite extensive studies on the regulation of nitrate uptake and downstream responses in various cells, our knowledge of the distribution of nitrogen forms in different root cell types and their cellular compartments is still limited. Previous physiological models have relied on in vitro biochemistry and metabolite level analysis, which limits the ability to differentiate between cell types and compartments. Here, to address this, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named nlsNitraMeter3.0, for the quantitative visualization of nitrate concentration and distribution at the cellular level in Arabidopsis thaliana. This biosensor was specifically designed for nitrate measurements, not nitrite. Through genetic engineering to create and select sensors using yeast, Xenopus oocyte, and Arabidopsis expression systems, we developed a reversible and highly specific nitrate sensor. This method, combined with fluorescence imaging systems such as confocal microscopy, allows for the understanding and monitoring of nitrate transporter activity in plant root cells in a minimally invasive manner. Furthermore, this approach enables the functional analysis of nitrate transporters and the measurement of nitrate distribution in plants, providing a valuable tool for plant biology research. In summary, we provide a protocol for sensor development and a biosensor that can be used to monitor nitrate levels in plants. Key features This protocol builds upon the concept of FRET biosensors for in vivo visualization of spatiotemporal nitrate levels at a cellular resolution. Nitrate levels can be quantified utilizing the biosensor in conjunction with either a plate reader or a fluorescence microscope.

Verapamil inhibits Kir2.3 channels by binding to the pore and interfering with PIP2 binding

The inwardly rectifying potassium current of the cardiomyocyte (IK1) is the main determinant of the resting potential. Ion channels Kir2.1, Kir2.2, and Kir2.3 form tetramers and are the molecular correlate of macroscopic IK1 current. Verapamil is an antiarrhythmic drug used to suppress atrial and ventricular arrhythmias. Its primary mechanism of action is via blocking calcium channels. In addition, it has been demonstrated to block IK1 current and the Kir2.1 subunit. Its effect on other subunits that contribute to IK1 current has not been studied to date. We therefore analyzed the effect of verapamil on the Kir channels 2.1, 2.2, and 2.3 in the Xenopus oocyte expression system. Kir2.1, Kir2.2, and Kir2.3 channels were heterologously expressed in Xenopus oocytes. Respective currents were measured with the voltage clamp technique and the effect of verapamil on the current was measured. At a concentration of 300 µM, verapamil inhibited Kir2.1 channels by 41.36% ± 2.7 of the initial current, Kir2.2 channels by 16.51 ± 3.6%, and Kir2.3 by 69.98 ± 4.2%. As a verapamil effect on kir2.3 was a previously unknown finding, we analyzed this effect further. At wash in with 300 µM verapamil, the maximal effect was seen within 20 min of the infusion. After washing out with control solution, there was only a partial current recovery. The current reduction from verapamil was the same at − 120 mV (73.2 ± 3.7%), − 40 mV (85.5 ± 6.5%), and 0 mV (61.5 ± 10.6%) implying no voltage dependency of the block. Using site directed mutations in putative binding sites, we demonstrated a decrease of effect with pore mutant E291A and absence of verapamil effect for D251A. With mutant I214L, which shows a stronger affinity for PIP2 binding, we observed a normalized current reduction to 61.9 ± 0.06% of the control current, which was significantly less pronounced compared to wild type channels. Verapamil blocks Kir2.1, Kir2.2, and Kir2.3 subunits. In Kir2.3, blockade is dependent on sites E291 and D251 and interferes with activation of the channel via PIP2. Interference with these sites and with PIP2 binding has also been described for other Kir channels blocking drugs. As Kir2.3 is preferentially expressed in atrium, a selective Kir2.3 blocking agent would constitute an interesting antiarrhythmic concept.

Silencing the gustatory receptor BtGR11 affects the sensing of sucrose in the whitefly Bemisia tabaci.

RNA interference (RNAi) is powerful biotechnology for studying the in vivo functions of key genes. Based on this property, RNAi can also be used for pest control as an effective alternative to chemical pesticides. The management of phloem-sucking pests is a tricky issue in current agricultural and forestry pest control. RNAi can silence key chemoreceptor genes of phloem-sucking pests; thereby regulating the behavior of these pests can be manipulated. So, it is considered to be a promising new type of ecological pest management strategy. In this study, we identified a candidate taste receptor gene, BtGR11, that controls the taste sensitivity to sucrose in the whitefly Bemisia tabaci, which is a serious invasive phloem-sucking pest worldwide. Functional analyses using the Xenopus oocyte expression system and the two-electrode voltage-clamp system revealed that the oocytes expressing BtGR11 responded to sucrose. Furthermore, we found that silencing BtGR11 by RNAi inhibited the function of sensing sucrose in the whitefly. This study reports a key chemoreceptor gene that can be used for the understanding of the gustatory sensing mechanisms of whitefly to deterrent.