Voltage Clamp Method Research Articles

Abstract P427: Novel SNVs of NBCe1, D405E and I803M mutants, reduced cell-surface expression and transport activity.

Introduction: The electrogenic Na + /HCO 3 - cotransporter NBCe1 is mainly expressed in proximal renal tubule, and regulate pH homeostasis and plasma volume. NBCe1 homozygous mutation causes many symptoms, including severe hypotension, proximal renal tubular acidosis (RTA), and extrarenal phenotype (migraine, mental retardation, mineral bone disorder and ocular abnormalities). Hypothesis: Undetermined single nucleotide variants (SNVs) in NBCe1 will be crucial role of sodium and bicarbonate transport. Methods: We identified the missense mutations (D405E and I803M) in NBCe1 variant A (kidney type) from the NCBI database. We conducted a comparative analysis of cellular localization and cell protein expression using confocal microscopy and western blotting. Furthermore, we performed functional analysis in D405E and I803M SNVs using two-electrode voltage clamp method. Results: Immunofluorescence analysis with confocal microscopy revealed that the D405E and I803M variants were present predominantly in the cell-surface in HEK293 cells. Western blotting in HEK293 cells confirmed that the D405E and I803M variants were significantly reduced protein expression level (25% in D405E and 57% in I803M) compared with WT, respectively. Moreover, electrophysiological analysis revealed that D405E and I803M variants were significantly reduced transport activity. These consisting data suggest that cell-expression level and transport activity are comparable. Conclusion: These data illustrate the diverse physiological consequences of distinct SNVs and underscore the importance of functional characterization in membrane transport proteins.

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.

Nanoemulsification of epigallocatechin gallate for debitterization, antibrowning, and inhibition of intracellular lipid accumulation

This study addressed the instability of epigallocatechin gallate (EGCG), a green tea-derived polyphenol, by developing a robust EGCG oil-in-water nanoemulsion. The nanoemulsion comprised lecithin (dispersed phase), pectin (continuous phase), and gallic acid (antibrowning agent). Using a blend of phases via high-energy ultrasonication, we achieved an 88.9% encapsulation efficiency. The nanoemulsion's physical properties improved, yielding a small droplet size (diameter: 169.3 nm) and low polydispersity index (0.16). The nanoemulsion exhibited long-term stability, 82% DPPH radical scavenging activity and 63% browning prevention compared with EGCG. Upon expressing the human taste receptor TAS2R16 + GIRK1/4 in Xenopus oocytes, the nanoemulsion elicited a 50% decrease in bitterness compared with EGCG, as quantified by the oocytes' membrane potential using the two-electrode voltage clamp method. Treating 3T3-L1 cells with the nanoemulsion resulted in a 49% reduction in lipid accumulation and 58% enhancement of triglyceride degradation, as confirmed via Oil Red O staining and lipolysis studies, respectively. This study provides insights into establishing an effective emulsification system for EGCG, enhancing its role as a functional food ingredient.

Protein kinase C epsilon-mediated modulation of T-type calcium channels underlies alcohol withdrawal hyperexcitability in the midline thalamus.

Millions of people struggle with alcohol use disorder (AUD). Abrupt abstinence after a period of chronic alcohol use can precipitate the alcohol withdrawal syndrome (AWS), which includes hyperexcitability and, potentially, seizures. We have shown that T-type Ca2+ channels are novel, sensitive targets of alcohol, an effect that is dependent upon protein kinase C (PKC). The purpose of this study was to (1) understand midline thalamic neuronal hyperexcitability during alcohol withdrawal and its dependence on PKC; (2) characterize T channel functional changes using both current clamp and voltage clamp methods; and (3) determine which PKC isoform may be responsible for alcohol withdrawal (WD) effects. Whole-cell patch clamp recordings were performed in midline thalamic neurons in brain slices prepared from C57bl/6 mice that underwent chronic intermittent alcohol exposure in a standard vapor chamber model. The recordings were compared to those from air-exposed controls. T-channel inactivation curves and burst responses were acquired through voltage-clamp and current-clamp recordings, respectively. Whole-cell voltage clamp recordings of native T-type current exhibited a depolarizing shift in the voltage-dependency of inactivation during alcohol withdrawal compared to air-exposed controls. A PKCε translocation inhibitor peptide mitigated this change. Current clamp recordings demonstrated more spikes per burst during alcohol withdrawal. Consistent with voltage clamp findings, the PKCɛ translocation inhibitor peptide reduced the number of spikes per burst after WD. We found that alcohol WD produces T channel-mediated hyperexcitability in the midline thalamus, produced in part by a shift in the inactivation curve consistent with greater availability of T current. WD effects on T current inactivation were reduced to control levels by blocking PKCε translocation. Our results demonstrate that PKCε translocation plays an important role in the regulation of alcohol withdrawal-induced hyperexcitability in midline thalamic circuitry.

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction.

Although it is particularly valuable in revealing membrane potential changes, intracellular recording has a number of limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of miniature synaptic potentials (minis) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only under low-release conditions when nonlinear summation is not a major issue. As the EJP amplitude increases, it creates a "ceiling effect," because the same amount of transmitter will be less effective in depolarizing the membrane when the potential is approaching the reversal potential of glutamate receptors/channels. To overcome these limitations, the voltage-clamp technique can be used, which uses negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In voltage-clamp mode, the amplitude and kinetics of membrane currents can be determined. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval neuromuscular junction preparation.

A charged existence: A century of transmembrane ion transport in plants.

If the past century marked the birth of membrane transport as a focus for research in plants, the past 50 years has seen the field mature from arcane interest to a central pillar of plant physiology. Ion transport across plant membranes accounts for roughly 30% of the metabolic energy consumed by a plant cell, and it underpins virtually every aspect of plant biology, from mineral nutrition, cell expansion, and development to auxin polarity, fertilization, plant pathogen defense, and senescence. The means to quantify ion flux through individual transporters, even single channel proteins, became widely available as voltage clamp methods expanded from giant algal cells to the fungus Neurospora crassa in the 1970s and the cells of angiosperms in the 1980s. Here, I touch briefly on some key aspects of the development of modern electrophysiology with a focus on the guard cells of stomata, now without dispute the premier plant cell model for ion transport and its regulation. Guard cells have proven to be a crucible for many technical and conceptual developments that have since emerged into the mainstream of plant science. Their study continues to provide fundamental insights and carries much importance for the global challenges that face us today.

New aryl and acylsulfonamides as state-dependent inhibitors of Nav1.3 voltage-gated sodium channel

Voltage-gated sodium channels (Navs) play an essential role in neurotransmission, and their dysfunction is often a cause of various neurological disorders. The Nav1.3 isoform is found in the CNS and upregulated after injury in the periphery, but its role in human physiology has not yet been fully elucidated. Reports suggest that selective Nav1.3 inhibitors could be used as novel therapeutics to treat pain or neurodevelopmental disorders. Few selective inhibitors of this channel are known in the literature. In this work, we report the discovery of a new series of aryl and acylsulfonamides as state-dependent inhibitors of Nav1.3 channels. Using a ligand-based 3D similarity search and subsequent hit optimization, we identified and prepared a series of 47 novel compounds and tested them on Nav1.3, Nav1.5, and a selected subset also on Nav1.7 channels in a QPatch patch-clamp electrophysiology assay. Eight compounds had an IC50 value of less than 1 μM against the Nav1.3 channel inactivated state, with one compound displaying an IC50 value of 20 nM, whereas activity against the inactivated state of the Nav1.5 channel and Nav1.7 channel was approximately 20-fold weaker. None of the compounds showed use-dependent inhibition of the cardiac isoform Nav1.5 at a concentration of 30 μM. Further selectivity testing of the most promising hits was measured using the two-electrode voltage-clamp method against the closed state of the Nav1.1-Nav1.8 channels, and compound 15b displayed small, yet selective, effects against the Nav1.3 channel, with no activity against the other isoforms. Additional selectivity testing of promising hits against the inactivated state of the Nav1.3, Nav1.7, and Nav1.8 channels revealed several compounds with robust and selective activity against the inactivated state of the Nav1.3 channel among the three isoforms tested. Moreover, the compounds were not cytotoxic at a concentration of 50 μM, as demonstrated by the assay in human HepG2 cells (hepatocellular carcinoma cells). The novel state-dependent inhibitors of Nav1.3 discovered in this work provide a valuable tool to better evaluate this channel as a potential drug target.

Dynamic optical clamp: A novel electrophysiology tool and a technique for closed-loop stimulation

Optical methods of neuromodulation show great promise. Recent research has shown that optogenetic and electrical stimulation interact both in vitro and in vivo to facilitate action potentials. With standard electrophysiology approaches, it is not possible to directly compare the cell’s response to a test pulse after an optical or electrical stimulation that lead to the same depolarisation. This paper describes the development of a new “dynamic optical clamp” technique, which aims to build on existing voltage clamp methods, but using light as the clamping input in place of electrical currents. We propose a technique based on combinations of optical and electrical stimulation to clamp membrane potential optically. After illustrating the problem in the context of optogenetically-active spiral ganglion neurons from transgenic mice, the proposed optical clamp was first tested within a NEURON simulation and then demonstrated in vitro with NG108-15 cells transfected with a Channelrhodopsin-2 plasmid. Testing of different control schemes within the NEURON simulation showed that a proportional–integral–derivative controller was appropriate for this application. The dynamic optical clamp technique allowed the membrane voltage of optogenetically-modified NG108-15 cells to be controlled using only light during electrophysiology studies. In order to translate optical approaches into implantable devices, a greater understanding of both the mechanisms of optical modulation and the engineering limitations is desirable. The dynamic optical clamp can be adapted for use in closed-loop neuronal control in future generations of neuroprosthetic devices. The enhanced control afforded by this technique could facilitate future investigations of ion channel dynamics during optical stimulation.

Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology

The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI–MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (− 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies.Graphical abstract

Decreased heat sensitivity of lungfish TRPV1 revealed by the heterologous expression system

In this study, we focused on TRPV1 of African lungfish, Protopterus annectens. During drought at high temperature, African lungfish can survive by undergoing into aestivation in mud cocoons. Therefore, lungfish is considered to have some specialized heat-sensor, TRPV1, for heat tolerance. Further, lungfish which shares similarities with fishes and amphibians, is one of important species for investigating the fish-tetrapod transition. Since fish TRPV1 and tetrapod TRPV1 have some differences, character of lungfish TRPV1 attracts attention. Here, we first cloned TRPV1 paralogue from lungfish, Protopterus annectens (lfTRPV1) and determined the chemical and thermal sensitivities of lfTRPV1 by two-electrode voltage clamp method using frog oocytes. We detected activation of lfTRPV1 by acid and 2-APB, but capsaicin-induced activation was not observed. The sensitivity to acid of lfTRPV1 was similar to that of rat TRPV1 (rTRPV1), but the 2-APB sensitivity of lfTRPV1 was relatively weaker than rTRPV1. Heat stimulation up to 44 °C did not activate lfTRPV1 and the heat-activation was not detected even on acid condition of pH6. This dramatically decreased heat-sensitivity of TRPV1 may contribute the heat tolerance of African lungfish. Moreover, this might be the property of ancient tetrapod-type TRPV1 gene.

Atypical antidepressant mirtazapine inhibits 5-hydroxytryptamine3 receptor currents in NCB-20 cells

Mirtazapine, an atypical antidepressant, is known to enhance serotonergic transmission by inhibiting the 5-hydroxytryptamine (5-HT)1A, 5-HT2C, and 5-HT3 receptors. However, the mechanism of action on the 5-HT3 receptor remains unclear. We investigated the inhibitory mechanisms of mirtazapine on 5-HT3 receptors of NCB20 neuroblastoma cells using the whole-cell voltage-clamp method. Mirtazapine inhibited the 5-HT3 receptor currents in a concentration-dependent manner, and the inhibitory effect was influenced by the concentration of 5-HT. When mirtazapine was co-applied to 5-HT, the maximal response of the 5-HT3 receptor current was reduced and EC50 was increased, suggesting that mirtazapine might act as a non-competitive inhibitor. Inhibition of 5-HT3 current by mirtazapine was stronger in pre-application than in co-application, which suggests that mirtazapine might act as a closed state inhibitor. This finding was further supported by no use-dependency of the mirtazapine for 5-HT3 receptor inhibition. Finally, mirtazapine accelerated the desensitization and deactivation process in a concentration-dependent manner. The difference in recovery time showed that mirtazapine drastically influences the desensitization process than the deactivation process. These mechanistic characteristics of mirtazapine support the understanding of the relationship between the 5-HT3 receptor and atypical antidepressants.

Characterizing Lithium-Mediated Nucleoside Transport by Human Concentrative Nucleoside Transporters 1 and 3 (hCNT1 and hCNT3)


 
 The human concentrative nucleoside transporters (hCNTs) utilize coupling and movement of cations down their electrochemical gradient to mediate inward transport of nucleosides against their concentration gradient. Of the three hCNT family members, hCNT3 exhibits the broadest nucleoside selectivity and cation specificity, utilizing Na+, Li+, and H+ to drive nucleoside transport. Xenopus laevis oocytes were injected with hCNT1 and hCNT3 RNA transcripts and incubated for 48 hours to allow protein expression and localization to the plasma membrane. The two-electrode voltage clamp (TEVC) method was used to perform electrophysiological studies. Oocytes were voltage-clamped, and currents were measured in response to various cation conditions, addition of different nucleosides and changes in membrane potential. Voltage-dependence of hCNT1- and hCNT3-mediated uridine uptake and hCNT3-mediated nucleoside uptake-induced currents was determined using NaCl, ChCl and LiCl transport media. Li+ addition resulted in decreases in uptake of uridine both by hCNT1 and hCNT3. hCNT1 and hCNT3 exhibited similar trends in voltage-dependence of uridine transport; currents generated increased as potentials became more negative. It was shown that whereas Na+-coupled hCNT3 has broad nucleoside selectivity, Li+-coupled transport varied widely and was greatest for uridine, and least for thymidine (uridine > cytidine > guanosine > adenosine > inosine > thymidine). Our results indicate a possible role of the nucleoside nitrogenous base ring structure in the differential uptake of various nucleosides. Despite no significant role of Li+ in vivo, further understanding of hCNT3 interactions with this cation may lead to a better understanding it’s transport mechanism and assist in the future design and delivery of effective anti-cancer drugs and anti-viral nucleoside drugs. 
 
 
 Keywords: electrophysiology, nucleoside transport, Xenopus laevis, two-electrode voltage clamp, secondary active transporters, influx rates

Involvement of spinal G-protein inwardly rectifying potassium (GIRK) channels in the enhanced antinociceptive effects of the activation of both μ-opioid and cannabinoid CB1 receptors

Neuropathic pain is refractory to opioid analgesics. Since there are functional linkages between μ-opioid receptors (MOR) and cannabinoid receptors (CBR), the present study was designed to investigate the interactions between MOR and CB1R based on antinociceptive effects for neuropathic pain mediated through G protein-coupled inwardly-rectifying potassium channels (GIRKs). The antinociceptive effects against pseudonociceptive response or neuropathic pain of MOR and CBR agonists were assessed in mice with or without partial sciatic nerve ligation. To investigate the functional interaction between MOR and CB1R, electrophysiological recording through GIRK was performed using the two-electrode voltage-clamp method in oocytes along with Western blotting in the spinal cord of mice. Co-administration of the MOR agonist DAMGO and the CB1R agonist CP55,940 augmented inwardly rectifying K+ currents in Xenopus oocytes co-expressing MOR, CB1R and GIRK1/2. Further, combination of morphine and the CBR agonist WIN-55,212-2 produced prominent antinociceptive effects in an i.t. GIRK1 inhibitor-reversible manner. Furthermore, CB1R was upregulated under neuropathic pain in the spinal cord, and such upregulation and antinociceptive effects were not altered by repeated treatment with morphine plus WIN-55,212-2. Our findings suggest that co-administration of MOR and CBR agonists could enhance their antinociceptive effects through GIRK1 in the spinal cord of mice.

Dihydropyridines Potentiate ATP-Induced Currents Mediated by the Full-Length Human P2X5 Receptor.

The P2X5 receptor, an ATP-gated cation channel, is believed to be involved in tumor development, inflammatory bone loss and inflammasome activation after bacterial infection. Therefore, it is a worthwhile pharmacological target to treat the corresponding diseases, especially in minority populations that have a gene variant coding for functional homotrimeric P2X5 channels. Here, we investigated the effects of dihydropyridines on the human full-length P2X5 receptor (hP2X5FL) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage clamp method. Agonist dependency, kinetics and permeation behavior, including Cl− permeability, were similar to hP2X5FL expressed in HEK293 or 1321N1 cells. Additionally, 1,4-dihydropyridines have been shown to interact with various other purinergic receptors, and we have examined them as potential hP2X5 modulators. Of seven commercially available and four newly synthesized dihydropyridines tested at hP2X5FL, only amlodipine exerted an inhibitory effect, but only at a high concentration of 300 µM. Isradipine and—even more—nimodipine stimulated ATP-induced currents in the low micromolar range. We conclude that common dihydropyridines or four new derivatives of amlodipine are not suitable as hP2X5 antagonists, but amlodipine might serve as a lead for future synthesis to increase its affinity. Furthermore, a side effect of nimodipine therapy could be a stimulatory effect on inflammatory processes.

Identification and pharmacological characterization of histamine-gated chloride channels in the fall armyworm, Spodoptera frugiperda

Histamine-gated chloride channels (HACls) mediate fast inhibitory neurotransmission in invertebrate nervous systems and have important roles in light reception, color processing, temperature preference and light-dark cycle. The fall armyworm, Spodoptera frugiperda is a main destructive pest of grain and row crops. However, the pharmacological characterization of HACls in S. frugiperda remain unknown. In this study, we identified two cDNAs encoding SfHACl1 and SfHACl2 in S. frugiperda. They had similar expression patterns and were most abundantly expressed in the head of larvae and at the egg stage. Electrophysiological analysis with the two-electrode voltage clamp method showed that histamine (HA) and γ-aminobutyric acid (GABA) activated inward currents when SfHACls were singly or collectively expressed with different ratios in Xenopus laevis oocytes. These channels were ≥2000-fold more sensitive to HA than to GABA. They were anion-selective channels, which were highly dependent on changes in external chloride concentrations, but insensitive to changes in external sodium concentrations. The insecticides abamectin (ABM) and emamectin benzoate (EB) also activated these channels with the EC50 to SfHACl1 lower than that to SfHACl2. And the EC50s of ABM and EB to the co-expressed channels gradually increased with increase in the injection ratio of SfHACl2 cRNA. Homology models and docking simulations revealed that HA bound to the large amino-terminal extracellular domain of SfHACl1 and SfHACl2 by forming 4 and 2 hydrogen bonds, respectively. The docking simulations of ABM and EB had similar binding sites in the transmembrane regions. Overall, these findings indicated that HACls act as targets for macrolide, and this study provides theoretical guidance for further derivatization of abamectin insecticides.

Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds.

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson’s disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.

Use of an electrophysiological technique for stepwise detection of trace agonist constituents of Hochuekkito in Xenopus oocytes injected with serotonin 2C receptor mRNA.

An electrophysiological bioassay was used to isolate the active compound from Hochuekkito (HET), which the current authors previously described as having potent agonist action against serotonin 2C receptors (5-HT2CR). Synthetic 5-HT2CR mRNA was injected into Xenopus oocytes to specifically express these receptors. Crude extracts and purified products were subjected to an electrophysiological bioassay using the voltage clamp method. HET stimulated a 5-HT2CR-induced current response, whereas Juzentaohoto (JTT), which has anti-depressive action similar to that of HET, did not. Current responses were not observed with an extract mixed with five types of herbal medicines common to HET and JTT but were detected with an extract with the five types of herbal medicines found in HET alone (Hoc5). When the responses to each of the five types of Hoc5 were examined, current responses were noted with Cimicifugae rhizoma (CR) and Citrus unshiu Markovich extracts. Since efficacy and the EC50 value were higher for CR, its constituents were separated using three-dimensional high-performance liquid chromatography and the current response at each of the isolated peaks was examined. One constituent displayed a strong response and was identified as a single substance with a molecular weight of 283.1393 based on liquid chromatography/mass spectrometry. These results will contribute to the isolation of 5-HT2CR-stimulating constituents in HET and the identification of trace constituents with agonist action.

5-(Indol-2-yl)pyrazolo[3,4-b]pyridines as a New Family of TASK-3 Channel Blockers: A Pharmacophore-Based Regioselective Synthesis.

TASK channels belong to the two-pore-domain potassium (K2P) channels subfamily. These channels modulate cellular excitability, input resistance, and response to synaptic stimulation. TASK-channel inhibition led to membrane depolarization. TASK-3 is expressed in different cancer cell types and neurons. Thus, the discovery of novel TASK-3 inhibitors makes these bioactive compounds very appealing to explore new cancer and neurological therapies. TASK-3 channel blockers are very limited to date, and only a few heterofused compounds have been reported in the literature. In this article, we combined a pharmacophore hypothesis with molecular docking to address for the first time the rational design, synthesis, and evaluation of 5-(indol-2-yl)pyrazolo[3,4-b]pyridines as a novel family of human TASK-3 channel blockers. Representative compounds of the synthesized library were assessed against TASK-3 using Fluorometric imaging plate reader—Membrane Potential assay (FMP). Inhibitory properties were validated using two-electrode voltage-clamp (TEVC) methods. We identified one active hit compound (MM-3b) with our systematic pipeline, exhibiting an IC50 ≈ 30 μM. Molecular docking models suggest that compound MM-3b binds to TASK-3 at the bottom of the selectivity filter in the central cavity, similar to other described TASK-3 blockers such as A1899 and PK-THPP. Our in silico and experimental studies provide a new tool to predict and design novel TASK-3 channel blockers.

The anticonvulsant zonisamide positively modulates recombinant and native glycine receptors at clinically relevant concentrations

GABAA and glycine receptors mediate fast synaptic inhibitory neurotransmission. Despite studies showing that activation of cerebral glycine receptors could be a potential strategy in the treatment of epilepsy, few studies have assessed the effects of existing anticonvulsant therapies on recombinant or native glycine receptors. We, therefore, evaluated the actions of a series of anticonvulsants at recombinant human homo-oligomeric glycine receptor α1, α2 and α3 subtypes expressed in Xenopus oocytes using two-electrode voltage-clamp methods, and then assessed the most effective drug at native glycine receptors from entorhinal cortex neurons using whole-cell voltage-clamp recordings. Ganaxolone, tiagabine and zonisamide positively modulated glycine induced currents at recombinant homomeric glycine receptors. Of these, zonisamide was the most efficacious and exhibited an EC50 value ranging between 450 and 560 μM at α1, α2 and α3 subtypes. These values were not significantly different indicating a non-selective modulation of glycine receptors. Using a therapeutic concentration of zonisamide (100 μM), the potency of glycine was significantly shifted from 106 to 56 μM at α1, 185 to 112 μM at α2, and 245 to 91 μM at α3 receptors. Furthermore, zonisamide (100 μM) potentiated exogenous homomeric and heteromeric glycine mediated currents from layer II pyramidal cells of the lateral or medial entorhinal cortex. As therapeutic concentrations of zonisamide positively modulate recombinant and native glycine receptors, we propose that the anticonvulsant effects of zonisamide may, at least in part, be mediated via this action.

Nicotinic acetylcholine receptors in the synganglion of the tick Ixodes ricinus: Functional characterization using membrane microtransplantation

Nicotinic acetylcholine receptors are an important class of excitatory receptors in the central nervous system of arthropods. In the ticks Ixodes ricinus, the functional and pharmacological properties of nicotinic receptors located in their neurons are still unknown. The objective of this study was to characterize the pharmacological properties of tick nicotinic receptors using membrane microtransplantation in Xenopus laevis oocytes and two-electrodes voltage clamp method. The membranes microtransplanted were extracted from the tick synganglion. We found that oocytes microtransplanted with tick synganglion membranes expressed nicotinic acetylcholine receptor subtypes which were activated by acetylcholine (1 mM) and nicotine (1 mM). Currents induced by pressure application of acetylcholine and nicotine were diminished by 10 nM α-bungarotoxin and methyllycaconitine, suggesting that they expressed two subtypes of nicotinic receptors, α-bungarotoxin-sensitive and -insensitive, respectively. In addition, we found that nicotine receptors expressed in the synganglion membranes were poorly sensitive to the neonicotinoid insecticides clothianidin (CLT), imidacloprid (IMI), acetamiprid (ACE) and thiamethoxam (TMX), in agreement with their lack of activity as acaricides. Interestingly, current amplitudes were strongly potentialized in the presence of 1 μM PNU-120596. CLT was more active as an agonist than IMI, TMX and ACE. Finally, we demonstrated that microtransplantation of purified membrane from the tick synganglion can be a valuable tool for the development and screening of compounds targeting tick nicotinic acetylcholine receptor subtypes.