Two-electrode Voltage Clamp Recordings Research Articles

Neuropeptide Modulation of Bidirectional Internetwork Synapses.

Oscillatory networks underlying rhythmic motor behaviors, and sensory and complex neural processing, are flexible, even in their neuronal composition. Neuromodulatory inputs enable neurons to switch participation between networks, or participate in multiple networks simultaneously. Neuromodulation of internetwork synapses can both recruit and coordinate a switching neuron in a second network. We previously identified an example in which a neuron is recruited into dual-network activity via peptidergic modulation of intrinsic properties. We now ask whether the same neuropeptide also modulates internetwork synapses for internetwork coordination. The crab (Cancer borealis) stomatogastric nervous system contains two well-defined feeding-related networks (pyloric, food filtering, ~1 Hz; gastric mill, food chewing, ~0.1 Hz). The projection neuron MCN5 uses the neuropeptide Gly1-SIFamide to recruit the pyloric-only LPG neuron into dual pyloric plus gastric mill-timed bursting via modulation of LPG's intrinsic properties. Descending input is not required for a coordinated rhythm, thus intra-network synapses between LPG and its second network must underlie coordination among these neurons. However, synapses between LPG and gastric mill neurons have not been documented. Using two-electrode voltage clamp recordings, we found that graded synaptic currents between LPG and gastric mill neurons (LG, IC, DG) were primarily negligible in saline, but were enhanced by Gly1-SIFamide. Further, LPG and gastric mill neurons entrain each other during Gly1-SIFamide application, indicating bidirectional, functional connectivity. Thus, a neuropeptide mediates neuronal switching through parallel actions, modulating intrinsic properties for recruitment into a second network and as shown here, also modulating bidirectional internetwork synapses for coordination.

A bupropion modulatory site in the Gloeobacter violaceus ligand-gated ion channel

Bupropion is an atypical antidepressant and smoking cessation drug that causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels, such as nicotinic acetylcholine and serotonin type 3A receptors, at clinically relevant concentrations. Here, we demonstrate that bupropion also inhibits a prokaryotic homolog of pentameric ligand-gated ion channels, the Gloeobacter violaceus ligand-gated ion channel (GLIC). Using the GLIC as a model, we used molecular docking to predict binding sites for bupropion. Bupropion was found to bind to several sites within the transmembrane domain, with the predominant site being localized to the interface between transmembrane segments M1 and M3 of two adjacent subunits. Residues W213, T214, and W217 in the first transmembrane segment, M1, and F267 and I271 in the third transmembrane segment, M3, most frequently reside within a 4 Å distance from bupropion. We then used single amino acid substitutions at these positions and two-electrode voltage-clamp recordings to determine their impact on bupropion inhibitory effects. The substitution T214F alters bupropion potency by shifting the half-maximal inhibitory concentration to a 13-fold higher value compared to wild-type GLIC. Residue T214 is found within a previously identified binding pocket for neurosteroids and lipids in the GLIC. This intersubunit binding pocket is structurally conserved and almost identical to a binding pocket described for neurosteroids in γ-aminobutyric acid type A receptors. Our data thus suggest that the T214 that lines a previously identified lipophilic binding pocket in GLIC and γ-aminobutyric acid type A receptors is also a modulatory site for bupropion interaction with the GLIC.

Synthesis, Activity, and Application of Fluorescent Analogs of [D1G, Δ14Q]LvIC Targeting α6β4 Nicotinic Acetylcholine Receptor.

α6β4* nicotinic acetylcholine receptor (nAChR) (* represents the possible presence of additional subunits) is mainly distributed in the central and peripheral nervous system and is associated with neurological diseases, such as neuropathic pain; however, the ability to explore its function and distribution is limited due to the lack of pharmacological tools. As one of the analogs of α-conotoxin (α-CTx) LvIC from Conus lividus, [D1G, Δ14Q]LvIC (Lv) selectively and potently blocks α6/α3β4 nAChR (α6/α3 represents a chimera). Here, we synthesized three fluorescent analogs of Lv by connecting fluorescent molecules 6-carboxytetramethylrhodamine succinimidyl ester (6-TAMRA-SE, R), Cy3 NHS ester (Cy3, C) and BODIPY-FL NHS ester (BDP, B) to the N-terminus of the peptide and obtained Lv-R, Lv-C, and Lv-B, respectively. The potency and selectivity of three fluorescent peptides were evaluated using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes, and the potency and selectivity of Lv-B were almost maintained with the half-maximal inhibition (IC50) of 64 nM. Then, we explored the stability of Lv-B in artificial cerebrospinal fluid and stained rat brain slices with Lv-B. The results indicated that the stability of Lv-B was slightly improved compared to that of native Lv. Additionally, we detected the distribution of the α6β4* nAChR subtype in the cerebral cortex using green fluorescently labeled peptide and fluorescence microscopy. Our findings not only provide a visualized pharmacological tool for exploring the distribution of the α6β4* nAChR subtype in various situ tissues and organs but also extend the application of α-CTx [D1G, Δ14Q]LvIC to demonstrate the involvement of α6β4 nAChR function in pathophysiology and pharmacology.

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.

Loss of Binding Capabilities in an Ecologically Important Odorant Receptor of the Fall Armyworm, Spodoptera frugiperda, by a Single Point Mutation.

Olfaction plays a crucial role in locating food sources, mates, and spawning sites in the fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae). In the current study, SfruOR14, a highly conserved odorant receptor (OR) in lepidopteran species, was newly uncovered in S. frugiperda. In two-electrode voltage clamp recordings, the SfruOR14/Orco complex was narrowly tuned to six volatile compounds including phenylacetaldehyde (PAA), benzaldehyde, heptaldehyde, (E)-2-hexen-1-al, cinnamaldehyde, and 2-phenylethanol, among which PAA showed the strongest binding affinity. Subsequent homology modeling and molecular docking revealed that Phe79, His83, Tyr149, Pro176, Gln177, Leu202, and Thr348 in SfruOR14 were the key binding residues against the six ligands. Finally, as a result of site-directed mutagenesis, the SfruOR14His83Ala mutant completely lost its binding capabilities toward all ligands. Taken together, our findings provide valuable insights into understanding the interaction between SfruOR14 and the chemical ligands including PAA, which can help to design novel olfactory modulators for pest control.

The Potential Antidepressant Compound Org 34167 Modulates HCN Channels Via a Novel Mode of Action.

Org 34167 is a small molecule hyperpolarization-activated cyclic nucleotide-gated (HCN) channel modulator that has been trialed in humans for its potential antidepressant activity. The precise action of Org 34167 is not fully understood. Here we use two-electrode voltage clamp recordings and an allosteric model to explore the interaction of Org 34167 with human HCN1 channels. The impact of Org 34167 on channel function included a hyperpolarizing shift in activation voltage dependence and a slowing of activation kinetics. Furthermore, a reduction in the maximum open probability at extreme hyperpolarization argued for an additional voltage-independent mechanism. Org 34167 had a similar impact on a truncated HCN1 channel lacking the C-terminal nucleotide binding domain, thus ruling out an interaction with this domain. Fitting a gating model, derived from a 10-state allosteric scheme, predicted that Org 34167 strongly reduced the equilibrium constant for the voltage-independent pore domain to favor a closed pore, as well as reducing the voltage sensing domain-pore domain coupling and shifting the zero voltage equilibrium constant of the voltage sensing domain to favor the inactive state. SIGNIFICANCE STATEMENT: The brain penetrant small molecule Org 34167 has been reported to have an antidepressant action by targeting HCN channels; however, its mode of action is unknown. We used heterologously expressed human HCN1 channels to show that Org 34167 inhibits channel activity by modulating kinetic parameters associated with the channel pore domain, voltage sensing domain, and interdomain coupling.

Cation leak: a common functional defect causing HCN1 developmental and epileptic encephalopathy.

Pathogenic variants in HCN1 are an established cause of developmental and epileptic encephalopathy (DEE). To date, the stratification of patients with HCN1-DEE based on the biophysical consequence on channel function of a given variant has not been possible. Here, we analysed data from eleven patients carrying seven different de novo HCN1 pathogenic variants located in the transmembrane domains of the protein. All patients were diagnosed with severe disease including epilepsy and intellectual disability. The functional properties of the seven HCN1 pathogenic variants were assessed using two-electrode voltage-clamp recordings in Xenopus oocytes. All seven variants showed a significantly larger instantaneous current consistent with cation leak. The impact of each variant on other biophysical properties was variable, including changes in the half activation voltage and activation and deactivation kinetics. These data suggest that cation leak is an important pathogenic mechanism in HCN1-DEE. Furthermore, published mouse model and clinical case reports suggest that seizures are exacerbated by sodium channel blockers in patients with HCN1 variants that cause cation leak. Stratification of patients based on their 'cation leak' biophysical phenotype may therefore provide key information to guide clinical management of individuals with HCN1-DEE.

Discovery, Characterization, and Engineering of LvIC, an α4/4-Conotoxin That Selectively Blocks Rat α6/α3β4 Nicotinic Acetylcholine Receptors.

α6β4 nicotinic acetylcholine receptors (nAChRs) are expressed in the central and peripheral nervous systems, but their functions are not fully understood, largely because of a lack of specific ligands. Here, we characterized a novel α-conotoxin, LvIC, and designed a series of analogues to probe structure-activity relationships at the α6β4 nAChR. The potency and selectivity of these conotoxins were tested using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes. One of the analogues, [D1G,ΔQ14]LvIC, potently blocked α6/α3β4 nAChRs (α6/α3 is a chimera) with an IC50 of 19 nM, with minimal activity at other nAChR subtypes, including the structurally similar α6/α3β2β3 and α3β4 subtypes. Using NMR, molecular docking, and receptor mutation, structure-activity relationships of [D1G,ΔQ14]LvIC at the α6/α3β4 nAChR were defined. It is a potent and specific antagonist of α6β4 nAChRs that could potentially serve as a novel molecular probe to explore α6β4 nAChR-related neurophysiological and pharmacological functions.

Evolutionary conservation of Zinc-Activated Channel (ZAC) functionality in mammals: a range of mammalian ZACs assemble into cell surface-expressed functional receptors.

In contrast to the other pentameric ligand-gated ion channels in the Cys-loop receptor superfamily, the ZACN gene encoding for the Zinc-Activated Channel (ZAC) is exclusively found in the mammalian genome. Human ZAC assembles into homomeric cation-selective channels gated by Zn2+, Cu2+ and H+, but the function of the receptor in human physiology is presently poorly understood. In this study, the degree of evolutionary conservation of a functional ZAC in mammals was probed by investigating the abilities of a selection of ZACs from 10 other mammalian species than human to be expressed at the protein level and assemble into cell surface-expressed functional receptors in mammalian cells and in Xenopus oocytes. In an enzyme-linked immunosorbent assay, transient transfections of tsA201 cells with cDNAs of hemagglutinin (HA)-epitope-tagged versions of these 10ZACs resulted in robust total expression and cell surface expression levels of all proteins. Moreover, injection of cRNAs for 6 of these ZACs in oocytes resulted in the formation of functional receptors in two-electrode voltage-clamp recordings. The ZACs exhibited robust current amplitudes in response to Zn2+ (10mM) and H+ (pH 4.0), and the concentration-response relationships displayed by Zn2+ at these channels were largely comparable to that at human ZAC. In conclusion, the findings suggest that the functionality of ZAC at the molecular level may be conserved throughout mammalian species, and that the channel thus may govern physiological functions in mammals, including humans.

Identification of the Acid-Sensitive Site Critical for Chloral Hydrate (CH) Activation of the Proton-Activated Chloride Channel.

The transmembrane protein TMEM206 was recently identified as the molecular basis of the extracellular proton-activated Cl- channel (PAC), which plays an essential role in neuronal death in ischemia-reperfusion. The PAC channel is activated by extracellular acid, but the proton-sensitive mechanism remains unclear, although different acid-sensitive pockets have been suggested based on the cryo-EM structure of the human PAC (hPAC) channel. In the present study, we firstly identified two acidic amino acid residues that removed the pH-dependent activation of the hPAC channel by neutralization all the conservative negative charged residues located in the extracellular domain of the hPAC channel and some positively charged residues at the hotspot combined with two-electrode voltage-clamp (TEVC) recording in the Xenopus oocytes system. Double-mutant cycle analysis and double cysteine mutant of these two residues proved that these two residues cooperatively form a proton-sensitive site. In addition, we found that chloral hydrate activates the hPAC channel depending on the normal pH sensitivity of the hPAC channel. Furthermore, the PAC channel knock-out (KO) male mice (C57BL/6J) resist chloral hydrate-induced sedation and hypnosis. Our study provides a molecular basis for understanding the proton-dependent activation mechanism of the hPAC channel and a novel drug target of chloral hydrate.SIGNIFICANCE STATEMENT Proton-activated Cl- channel (PAC) channels are widely distributed in the nervous system and play a vital pathophysiological role in ischemia and endosomal acidification. The main discovery of this paper is that we identified the proton activation mechanism of the human proton-activated chloride channel (hPAC). Intriguingly, we also found that anesthetic chloral hydrate can activate the hPAC channel in a pH-dependent manner. We found that the chloral hydrate activates the hPAC channel and needs the integrity of the pH-sensitive site. In addition, the PAC channel knock-out (KO) mice are resistant to chloral hydrate-induced anesthesia. The study on PAC channels' pH activation mechanism enables us to better understand PAC's biophysical mechanism and provides a novel target of chloral hydrate.

Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

TransMEMbrane 16A (TMEM16A) is a Ca2+-activated Cl− channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca2+, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P2 head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca2+ to excised inside–out patches, we found that TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P2 analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P2 reduces TMEM16A-conducted currents. Application of PIP2 analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 4′ position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P2 binds to and potentiates TMEM16A channels.

Stoichiometry-Selective Antagonism of α4β2 Nicotinic Acetylcholine Receptors by Fluoroquinolone Antibiotics.

Quinolone antibiotics disrupt bacterial DNA synthesis by interacting with DNA gyrase and topoisomerase IV. However, in addition, they have been shown to act as inhibitors of pentameric ligand-gated ion channels such as GABAA receptors and the α7 nicotinic acetylcholine receptor (nAChR). In the present study, we have examined the effects of quinolone antibiotics on the human α4β2 nAChR, an important subtype that is widely expressed in the central nervous system. A key feature of α4β2 nAChRs is their ability to coassemble into two distinct stoichiometries, (α4)2(β2)3 and (α4)3(β2)2, which results in differing affinities for acetylcholine. The effects of nine quinolone antibiotics were examined on both stoichiometries of the α4β2 receptor by two-electrode voltage-clamp recording. All compounds exhibited significant inhibition of α4β2 nAChRs. However, all of the fluoroquinolone antibiotics examined (ciprofloxacin, enoxacin, enrofloxacin, difloxacin, norfloxacin, pefloxacin, and sparfloxacin) were significantly more potent inhibitors of (α4)2(β2)3 nAChRs than of (α4)3(β2)2 nAChRs. This stoichiometry-selective effect was most pronounced with pefloxacin, which inhibited (α4)2(β2)3 nAChRs with an IC50 of 26.4 ± 3.4 μM but displayed no significant inhibition of (α4)3(β2)2 nAChRs. In contrast, two nonfluorinated quinolone antibiotics (cinoxacin and oxolinic acid) exhibited no selectivity in their inhibition of the two stoichiometries of α4β2. Computational docking studies suggest that pefloxacin interacts selectively with an allosteric transmembrane site at the β2(+)/β2(−) subunit interface, which is consistent with its selective inhibition of (α4)2(β2)3. These findings concerning the antagonist effects of fluoroquinolones provide further evidence that differences in the subunit stoichiometry of heteromeric nAChRs can result in substantial differences in pharmacological properties.

The COVID-19 viral 3a protein forms a potassium channel that can be inhibited by antiarrhythmic drugs

Funding AcknowledgementsType of funding sources: Public Institution(s). Main funding source(s): Deutsche Gesellschaft für Kardiologie /German Cardiac Society Joachim Herz Stiftung /Joachim Herz FoundationBackground/IntroductionSupraventricular and ventricular arrhythmias can often be observed in patients with COVID-19 infection. Both, the clinically observed increase in cardiac biomarkers as well as histological studies indicate virus replication within cardiomyocytes. The 3a open reading frame of the viral genome encodes for a transmembrane protein that is transported to the cell membrane where it can serve as a potassium channel.PurposeThe aim of this study was to investigate whether COVID-19 infected induced pluripotent stem cell (iPSC)-derived cardiomyocytes also express the 3a protein and whether the potassium currents that are conducted through the 3a protein can be inhibited by clinically used antiarrhythmic drugs.Methods and ResultsiPSC-derived cardiomyocytes were infected with COVID-19 and subsequently subjected to immunoblotting, where expression of the 3a protein could be observed. Plasmid DNA, encoding the COVID-19 3a protein, was generated by gene synthesis and used for in vitro transcription of cRNA. 3–5 days after intracytoplasmic injection of the 3a protein cRNA into Xenopus laevis oocytes, potassium currents could be measured by two-electrode voltage clamp recordings. While class I and class IV antiarrhythmic drugs showed only minor effects on the potassium currents of the 3a protein, a robust inhibition by several beta-blockers and by class III antiarrhythmic drugs could be observed. The strongest effects were found with dofetilide (58.1 % inhibition at 100 µM) and amiodarone (50.1 % inhibition at 100 µM, IC50 level 4.7 µM). An in silico docking analysis, based on the recently revealed crystal structure of the 3a protein, identified the amino acid residues K61 and D142 as part of the binding site of amiodarone. After deactivation of these amino acid residues by site-directed mutagenesis, the inhibition by amiodarone was significantly attenuated.ConclusionThe COVID-19 viral 3a protein is expressed in COVID-19-infected iPS-derived cardiomyocytes and forms a potassium channel that can be inhibited by antiarrhythmic drugs.

Xenopus Oocytes: A Tool to Decipher Molecular Specificity of Insecticides towards Mammalian and Insect GABA-A Receptors.

The number of insect GABA receptors (GABAr) available for expression studies has been recently increased by the cloning of the Acyrthosiphon pisum (pea aphid) RDL subunits. This large number of cloned RDL subunits from pest and beneficial insects opens the door to parallel pharmacological studies on the sensitivity of these different insect GABAr to various agonists or antagonists. The resulting analysis of the molecular basis of the species-specific GABAr responses to insecticides is necessary not only to depict and understand species toxicity, but also to help at the early identification of unacceptable toxicity of insecticides toward beneficial insects such as Apis mellifera (honeybees). Using heterologous expression in Xenopus laevis oocytes, and two-electrode voltage-clamp recording to assess the properties of the GABAr, we performed a comparative analysis of the pharmacological sensitivity of RDL subunits from A. pisum, A. mellifera and Varroa destructor GABAr to three pesticides (fipronil, picrotoxin and dieldrin). These data were compared to similar characterizations performed on two Homo sapiens GABA-A receptors (α2β2γ2 and α2β2γ2). Our results underline a global conservation of the pharmacological profiles of these receptors, with some interesting species specificities, nonetheless, and suggest that this approach can be useful for the early identification of poorly specific molecules.

A female-biased odorant receptor tuned to the lepidopteran sex pheromone in parasitoid Microplitis mediator guiding habitat of host insects

IntroductionThe parasitoid wasp Microplitis mediator is an important natural enemy of the turnip moth Agrotis segetum and other Noctuidae pests. In our field observation, it was fortuitously discovered that sex pheromone traps used for A. segetum also attract female wasps, verified by a simulated field condition dual-choice laboratory assay. Therefore, it was hypothesized that olfactory recognition could be crucial in this process. In this regard, a female-biased odorant receptor of the wasp, MmedOR49, attracted our attention. ObjectivesTo unravel the significance of the female-biased MmedOR49 regulating host pheromone recognition. MethodsExpression analysis (fluorescence in situ hybridization; quantitative realtime PCR), in vitro (two-electrode voltage-clamp recordings) and in vivo (RNAi combined with behavioral assessments) functional studies, and bioinformatics (structural modeling and molecular docking) were carried out to investigate the characteristics of MmedOR49. ResultsMmedOR49 expression was detected in the antennae of females by FISH. Quantification indicated that the expression level of MmedOR49 increased significantly after adult emergence. In vitro functional study revealed that MmedOR49 was specifically tuned to cis-5-decenyl acetate (Z5-10:Ac), the major sex pheromone component of A. segetum. Molecular docking showed that Z5-10:Ac strongly bound to the key amino acid residues His 80, Ile 81, and Arg 84 of MmedOR49 through hydrogen bonding. Behavioral assays indicated that female wasps were significantly attracted by Z5-10:Ac in a three-cage olfactometer. RNAi targeting further confirmed that MmedOR49 was necessary to recognize Z5-10:Ac, as female wasps lost their original behavioral responses to Z5-10:Ac after down-regulation of the MmedOR49 transcript. ConclusionAlthough M. mediator is a larval endoparasitoid, female wasps have a behavioral preference for a sex pheromone component of lepidopteran hosts. In this behavior, for female M. mediator, MmedOR49 plays an important role in guiding the habitat of host insects. These data provide a potential target for enhancing natural enemy utilization and pest control.

Antenna-Biased Odorant Receptor HvarOR25 in Hippodamia variegata Tuned to Allelochemicals from Hosts and Habitat Involved in Perceiving Preys.

Odorant receptors (ORs) of ladybird Hippodamia variegata play vital chemosensory roles in searching and locating preys. In the current study, 37 ORs were initially identified from the antennal transcriptome of H. variegata. The quantitative polymerase chain reaction demonstrated that several HvarORs including HvarOR25 were specific or enriched in ladybird antennae. In two-electrode voltage clamp recordings, recombinant HvarOR25 was narrowly tuned to six chemical ligands including aphid-induced, aphid-derived, and plant-derived volatiles. In electroantennogram assays, all six volatiles elicited electrophysiological responses. Among the six volatiles, cis-3-hexenyl acetate, hexyl butyrate, hexyl hexanoate, and 3-methyl-3-buten-1-ol were attractive for both sexes of H. variegata. Additionally, molecular docking indicated that HvarOR25 was bound to all ligands with high binding affinities. Taken together, HvarOR25 facilitates perception of preys by recognizing relevant allelochemicals from hosts and habitat. Our findings provide valuable insights into understanding biological functions of HvarORs and help to develop a novel biocontrol strategy based on olfactory-active compounds.

The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release.

Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far. Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Å resolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST. We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities.

Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors

SummaryLigand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering.

Oxidation sensitizes TRPV2 to chemical and heat stimuli, but not mechanical stimulation

The transient receptor potential vanilloid 2 (TRPV2) ion channel is activated by a chemical ligand (2-aminoethoxydiphenyl borate; 2-APB), noxious heat and mechanical stimulation. In a heterologous mammalian cell expression system, the oxidant chloramine T (ChT) sensitizes TRPV2 activation in response to 2-APB and heat by oxidation of methionine residues at positions 528 and 607 in rat TRPV2. Here, we used a Xenopus oocyte expression system to determine whether ChT-mediated oxidation can also sensitize TRPV2 to mechanical stimulation. In this system, we confirmed that ChT sensitized TRPV2 activation in response to 2-APB and heat, but we detected no sensitization to mechanical stimulation. This result suggests that the activation mechanism of TRPV2 by a chemical ligand and heat differs from that for mechanical stimulation. Further, we demonstrated that two-electrode voltage clamp recording in the Xenopus oocyte expression system is an excellent format for high throughput analysis of oxidization of redox-sensitive TRP channels.

GRIN2A Variants Associated With Idiopathic Generalized Epilepsies.

Objective: The objective of this study is to explore the role of GRIN2A gene in idiopathic generalized epilepsies and the potential underlying mechanism for phenotypic variation.Methods: Whole-exome sequencing was performed in a cohort of 88 patients with idiopathic generalized epilepsies. Electro-physiological alterations of the recombinant N-methyl-D-aspartate receptors (NMDARs) containing GluN2A mutants were examined using two-electrode voltage-clamp recordings. The alterations of protein expression were detected by immunofluorescence staining and biotinylation. Previous studies reported that epilepsy related GRIN2A missense mutations were reviewed. The correlation among phenotypes, functional alterations, and molecular locations was analyzed.Results: Three novel heterozygous missense GRIN2A mutations (c.1770A > C/p.K590N, c.2636A > G/p.K879R, and c.3199C > T/p.R1067W) were identified in three unrelated cases. Electrophysiological analysis demonstrated R1067W significantly increased the current density of GluN1/GluN2A NMDARs. Immunofluorescence staining indicated GluN2A mutants had abundant distribution in the membrane and cytoplasm. Western blotting showed the ratios of surface and total expression of the three GluN2A-mutants were significantly increased comparing to the wild type. Further analysis on the reported missense mutations demonstrated that mutations with severe gain-of-function were associated with epileptic encephalopathy, while mutations with mild gain of function were associated with mild phenotypes, suggesting a quantitative correlation between gain-of-function and phenotypic severity. The mutations located around transmembrane domains were more frequently associated with severe phenotypes and absence seizure-related mutations were mostly located in carboxyl-terminal domain, suggesting molecular sub-regional effects.Significance: This study revealed GRIN2A gene was potentially a candidate pathogenic gene of idiopathic generalized epilepsies. The functional quantitative correlation and the molecular sub-regional implication of mutations helped in explaining the relatively mild clinical phenotypes and incomplete penetrance associated with GRIN2A variants.