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• Skeletal muscle dihydropyridine receptor functions as a voltage-gated Ca2+ channel. • N617D mutation in the DHPRα 1S subunit abolishes Ca2+ permeation through the channel. • Ba2+ and Mn2+ ions are found to permeate the N617D mutant channel. • External Ca2+ strongly blocks Ba2+ currents through the mutant channel. • N617D mutation located outside the selectivity filter influences channel permeation. In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca2+ channel that conducts L-type Ca2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca2+ influx in mammalian muscle, a knock-in (ki) mouse model (nc DHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα 1S subunit abolishing Ca2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca2+, Mn2+ entry and subsequent quenching did occur because Mn2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba2+ and Ba2+ currents were strongly blocked by external Ca2+. Ba2+ permeation was smaller, current kinetics slower and Ca2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca2+ impeding in this way permeation. Because Ba2+ binds with lower affinity to D, Ba2+ currents occur, but with reduced amplitudes as compared to Ba2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment. [ABSTRACT FROM AUTHOR]

The pacemaker channel (HCN) is responsible for electrical activity in a wide range of excitable cells, including those of invertebrates. Using Xenopus oocytes and HEK cell, we show here that HCN-channel from Apis mellifera is activated by hyperpolarization, modulated by cAMP, and blocked by cesium. Its PNa/PK relative permeability is 1:3, and its unitary conductance is 1.5 pS, which is similar to that of the mammalian HCN2 channel. Moreover, bee h-current is blocked by high concentrations of ZD7288, and organochlorine pesticide chlordecone reduces Ih amplitude in a dose-dependent manner (IC50 value was 9.37 µM) and diminishes HCN conductance, while preserving voltage dependence. In contrast, Deltamethrin exhibits no discernible impact. Molecular docking of Chlordecone in a homology model of bee HCN generated by AlphaFold3 suggests a binding site located at the end of the S6 helix that could explain the conductance inhibition caused by Chlordecone., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

The current risk assessment framework for insecticides suffers from certain shortcomings in adequately addressing the effects of low doses on off-target species. To remedy this gap, a combination of behavioural assays and in vitro cellular approaches are required to refine the precision of toxicity assessment. The domestic honey bee has long been standing as an emblematic pollinator in ecotoxicology, and once more, it provides us with a practical testing model for this purpose. First, newly emerged bees (D1) were found more vulnerable than 6 days-old bees (D6) to deltamethrin, a widely used α-cyano-3-phenoxybenzyle pyrethroid. In D1 bees, the range of doses inducing mortality was shifted towards lower values (∼2-fold) with a correspondingly lower LD 50 (11 ng/bee). Moreover, at low doses that do not induce mortality in laboratory conditions, the locomotor behaviour of D1 bees was more impacted than in D6 bees. This was evidenced by an increase in immobility time and a decrease in locomotor performance across all tested doses for D1 bees (0.75, 1.5 and 3 ng/bee) during automated 21 h-long observations. Behavioural disorders are linked to deltamethrin's disruption of voltage-gated sodium channels (Na V s) functions, as quantified in cultured neuronal cells. In the presence of deltamethrin, patch-clamp experiments revealed a concentration- and a use-dependent slowing of Na V kinetics. Channel's deactivation is slowed by three orders of magnitude at 10 μM deltamethrin. Two additional phenoxybenzyle pyrethroids, including the commonly used cypermethrin, elicited quantitatively similar effects on Na V kinetics. The integration of in vitro cellular assays and behavioural assays may facilitate a deeper understanding and prediction of insecticides toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

DSC1, a Drosophila channel with sequence similarity to the voltage-gated sodium channel (NaV), was identified over 20 years ago. This channel was suspected to function as a non-specific cation channel with the ability to facilitate the permeation of calcium ions (Ca2+). A honeybee channel homologous to DSC1 was recently cloned and shown to exhibit strict selectivity for Ca2+, while excluding sodium ions (Na+), thus defining a new family of Ca2+ channels, known as CaV4. In this study, we characterize CaV4, showing that it exhibits an unprecedented type of inactivation, which depends on both an IFM motif and on the permeating divalent cation, like NaV and CaV1 channels, respectively. CaV4 displays a specific pharmacology with an unusual response to the alkaloid veratrine. It also possesses an inactivation mechanism that uses the same structural domains as NaV but permeates Ca2+ ions instead. This distinctive feature may provide valuable insights into how voltage- and calcium-dependent modulation of voltage-gated Ca2+ and Na+ channels occur under conditions involving local changes in intracellular calcium concentrations. Our study underscores the unique profile of CaV4 and defines this channel as a novel class of voltage-gated Ca2+ channels., (© 2024 Bertaud et al.)

Cav2.1 channels are expressed throughout the brain and are the predominant Ca 2+ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations (p.A405T in I-II loop and p.R1359W, p.R1667W and p.S1799L in IIIS4, IVS4, and IVS6 helices, respectively) identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1-e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells.

Heterologous expression systems (e.g., Xenopus laevis oocytes) are useful to study the biophysical properties and pharmacology of ionotropic receptors such as ionotropic glutamate (iGLuRs) and nicotinic acetylcholine (nAChRs) receptors. However, insect receptors often require the co-expression of chaperone proteins to be functional. Only few iGluRs and nAChRs have been successfully expressed in such systems. Here, we compared the efficiency of chaperone proteins to promote the functional expression of one Apis mellifera iGluR and several nAChR subunit combinations (α1α8β1, α7, α2α8β1 and α2α7α8β1) in Xenopus oocytes. To this end, we cloned a new iGluR (GluR-1) and potential chaperone proteins (e.g., SOL-1, Neto, NACHO) and tested more than 40 combinations of human, nematode and honeybee proteins. We obtained robust expression of GluR-1 and α1α8β1 when co-expressed with honeybee chaperone proteins and found that nAChR expression critically depended on the α1 subunit N-terminal sequence. We recorded small ACh-gated currents in few oocytes when the α7 subunit was co-expressed with Caenorhabditis elegans RIC-3, but none of the chaperone proteins allowed efficient expression of α2α8β1 or α2α7α8β1. Our results show that only some protein combinations can reconstitute functional receptors in Xenopus oocytes and that protein combination efficient in one species is not always efficient in another species., (© 2022 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Several mutations on neuronal voltage-gated Ca 2+ channels (VGCC) have been shown to cause neurological disorders and contribute to the initiation of epileptic seizures, migraines, or cerebellar degeneration. Analysis of the functional consequences of these mutations mainly uses heterologously expressed mutated channels or transgenic mice which mimic these pathologies, since direct electrophysiological approaches on brain samples are not easily feasible. We demonstrate that mammalian voltage-gated Ca 2+ channels from membrane preparation can be microtransplanted into Xenopus oocytes and can conserve their activity. This method, originally described to study the alteration of GABA receptors in human brain samples, allows the recording of the activity of membrane receptors and channels with their native post-translational processing, membrane environment, and regulatory subunits. The use of hippocampal, cerebellar, or cardiac membrane preparation displayed different efficacy for transplanted Ca 2+ channel activity. This technique, now extended to the recording of Ca 2+ channel activity, may therefore be useful in order to analyze the calcium signature of membrane preparations from unfixed human brain samples or normal and transgenic mice.

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 (α 2 β 2 γ 2 and α 2 β 2 γ 2 ). 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.

Gamma-L-glutamyl-L-glutamate (γ-Glu-Glu) was synthetized and further characterized for its activity on cultured neurons. We observed that γ-Glu-Glu elicited excitatory effects on neurons likely by activating mainly the N-methyl-D-aspartate (NMDA) receptors. These effects were dependent on the integrity of synaptic transmission as they were blocked by tetrodotoxin (TTX). We next evaluated its activity on NMDA receptors by testing it on cells expressing these receptors. We observed that γ-Glu-Glu partially activated NMDA receptors and exhibited better efficacy for NMDA receptors containing the GluN2B subunit. Moreover, at low concentration, γ-Glu-Glu potentiated the responses of glutamate on NMDA receptors. Finally, the endogenous production of γ-Glu-Glu was measured by LC-MS on the extracellular medium of C6 rat astroglioma cells. We found that extracellular γ-Glu-Glu concentration was, to some extent, directly linked to GSH metabolism as γ-Glu-Glu can be a by-product of glutathione (GSH) breakdown after γ-glutamyl transferase action. Therefore, γ-Glu-Glu could exert excitatory effects by activating neuronal NMDA receptors when GSH production is enhanced., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sebih, Mokrane, Fontanel, Kayatekin, Kaabeche, Guiramand, Cohen-Solal, Cens, Rousset, Charnet, De Jésus Ferreira, Thibaud, Ménard, Cantel, Rolland, Vignes and Roussel.)

The regulation of the redox status involves the activation of intracellular pathways as Nrf2 which provides hormetic adaptations against oxidative stress in response to environmental stimuli. In the brain, Nrf2 activation upregulates the formation of glutathione (GSH) which is the primary antioxidant system mainly produced by astrocytes. Astrocytes have also been shown to be themselves the target of oxidative stress. However, how changes in the redox status itself could impact the intracellular Ca 2+ homeostasis in astrocytes is not known, although this could be of great help to understand the neuronal damage caused by oxidative stress. Indeed, intracellular Ca 2+ changes in astrocytes are crucial for their regulatory actions on neuronal networks. We have manipulated GSH concentration in astroglioma cells with selective inhibitors and activators of the enzymes involved in the GSH cycle and analyzed how this could modify Ca 2+ homeostasis. IP 3 -mediated store-operated calcium entry (SOCE), obtained after store depletion elicited by G q -linked purinergic P 2 Y receptors activation, are either sensitized or desensitized, following GSH depletion or increase, respectively. The desensitization may involve decreased expression of the proteins STIM2, Orai1, and Orai3 which support SOCE mechanism. The sensitization process revealed by exposing cells to oxidative stress likely involves the increase in the activity of Calcium Release-Activated Channels (CRAC) and/or in their membrane expression. In addition, we observe that GSH depletion drastically impacts P 2 Y receptor-mediated changes in membrane currents, as evidenced by large increases in Ca 2+ -dependent K + currents. We conclude that changes in the redox status of astrocytes could dramatically modify Ca 2+ responses to Gq-linked GPCR activation in both directions, by impacting store-dependent Ca 2+ -channels, and thus modify cellular excitability under purinergic stimulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mokrane, Snabi, Cens, Guiramand, Charnet, Bertaud, Menard, Rousset, de Jesus Ferreira, Thibaud, Cohen-Solal, Vignes and Roussel.)

Control of infestation by cosmopolitan lice (Pediculus humanus) is increasingly difficult due to the transmission of parasites resistant to pediculicides. However, since the targets for pediculicides have no been identified in human lice so far, their mechanisms of action remain largely unknown. The macrocyclic lactone ivermectin is active against a broad range of insects including human lice. Isoxazolines are a new chemical class exhibiting a strong insecticidal potential. They preferentially act on the γ-aminobutyric acid (GABA) receptor made of the resistant to dieldrin (RDL) subunit and, to a lesser extent on glutamate-gated chloride channels (GluCls) in some species. Here, we addressed the pediculicidal potential of isoxazolines and deciphered the molecular targets of ivermectin and the ectoparasiticide lotilaner in the human body louse species Pediculus humanus humanus. Using toxicity bioassays, we showed that fipronil, ivermectin and lotilaner are efficient pediculicides on adult lice. The RDL (Phh-RDL) and GluCl (Phh-GluCl) subunits were cloned and characterized by two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Phh-RDL and Phh-GluCl formed functional homomeric receptors respectively gated by GABA and L-glutamate with EC50 values of 16.0 μM and 9.3 μM. Importantly, ivermectin displayed a super agonist action on Phh-GluCl, whereas Phh-RDL receptors were weakly affected. Reversally, lotilaner strongly inhibited the GABA-evoked currents in Phh-RDL with an IC50 value of 40.7 nM, whereas it had no effect on Phh-GluCl. We report here for the first time the insecticidal activity of isoxazolines on human ectoparasites and reveal the mode of action of ivermectin and lotilaner on GluCl and RDL channels from human lice. These results emphasize an expected extension of the use of the isoxazoline drug class as new pediculicidal agents to tackle resistant-louse infestations in humans., Competing Interests: The authors have declared that no competing interests exist.

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca 2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca 2+ channel that conducts L-type Ca 2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca 2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα 1S subunit abolishing Ca 2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn 2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca 2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca 2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca 2+ , Mn 2+ entry and subsequent quenching did occur because Mn 2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba 2+ and Ba 2+ currents were strongly blocked by external Ca 2+ . Ba 2+ permeation was smaller, current kinetics slower and Ca 2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca 2+ impeding in this way permeation. Because Ba 2+ binds with lower affinity to D, Ba 2+ currents occur, but with reduced amplitudes as compared to Ba 2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background and Purpose: Despite a growing awareness, annual losses of honeybee colonies worldwide continue to reach threatening levels for food safety and global biodiversity. Among the biotic and abiotic stresses probably responsible for these losses, pesticides, including those targeting ionotropic GABA receptors, are one of the major drivers. Most insect genomes include the ionotropic GABA receptor subunit gene, Rdl, and two GABA-like receptor subunit genes, Lcch3 and Grd. Most studies have focused on Rdl which forms homomeric GABA-gated chloride channels, and a complete analysis of all possible molecular combinations of GABA receptors is still lacking., Experimental Approach: We cloned the Rdl, Grd, and Lcch3 genes of Apis mellifera and systematically characterized the resulting GABA receptors expressed in Xenopus oocytes, using electrophysiological assays, fluorescence microscopy and co-immunoprecipitation techniques., Key Results: The cloned subunits interacted with each other, forming GABA-gated heteromeric channels with particular properties. Strikingly, these heteromers were always more sensitive than AmRDL homomer to all the pharmacological agents tested. In particular, when expressed together, Grd and Lcch3 form a non-selective cationic channel that opens at low concentrations of GABA and with sensitivity to insecticides similar to that of homomeric Rdl channels., Conclusion and Implications: For off-target species like the honeybee, chronic sublethal exposure to insecticides constitutes a major threat. At these concentration ranges, homomeric RDL receptors may not be the most pertinent target to study and other ionotropic GABA receptor subtypes should be considered in order to understand more fully the molecular mechanisms of sublethal toxicity to insecticides., (© 2020 The British Pharmacological Society.)

Spider venoms represent an original source of novel compounds with therapeutic and agrochemical potential. Whereas most of the research efforts have focused on large mygalomorph spiders, araneomorph spiders are equally promising but require more sensitive and sophisticated approaches given their limited size and reduced venom yield. Belonging to the latter group, the genus Lycosa ("wolf spiders") contains many species widely distributed throughout the world. These spiders are ambush predators that do not build webs but instead rely strongly on their venom for prey capture. Lycosa tarantula is one of the largest species of wolf spider, but its venom composition is unknown. Using a combination of RNA sequencing of the venom glands and venom proteomics, we provide the first overview of the peptides and proteins produced by this iconic Mediterranean spider. Beside the typical small disulfide rich neurotoxins, several families of proteins were also identified, including cysteine-rich secretory proteins (CRISP) and Hyaluronidases. Proteomic analysis of the electrically stimulated venom validated 30 of these transcriptomic sequences, including nine putative neurotoxins and eight venom proteins. Interestingly, LC-MS venom profiles of manual versus electric stimulation, as well as female versus male, showed some marked differences in mass distribution. Finally, we also present some preliminary data on the biological activity of L. tarantula crude venom.

The sequence and genomic organization of the CACNA1A gene that encodes the Cav2.1 subunit of both P and Q-type Ca 2+ channels are well conserved in mammals. In human, rat and mouse CACNA1A, the use of an alternative acceptor site at the exon 46-47 boundary results in the expression of a long Cav2.1 splice variant. In transfected cells, the long isoform of human Cav2.1 produces a C-terminal fragment, but it is not known whether this fragment affects Cav2.1 expression or functional properties. Here, we cloned the long isoform of rat Cav2.1 (Cav2.1(e47)) and identified a novel variant with a shorter C-terminus (Cav2.1(e47s)) that differs from those previously described in the rat and mouse. When expressed in Xenopus laevis oocytes, Cav2.1(e47) and Cav2.1(e47s) displayed similar functional properties as the short isoform (Cav2.1). We show that Cav2.1 isoforms produced short (CT1) and long (CT1(e47)) C-terminal fragments that interacted in vivo with the auxiliary Cavβ4a subunit. Overexpression of the C-terminal fragments did not affect Cav2.1 expression and functional properties. Furthermore, the functional properties of a Cav2.1 mutant without the C-terminal Cavβ4 binding domain (Cav2.1ΔCT2) were similar to those of Cav2.1 and were not influenced by the co-expression of the missing fragments (CT2 or CT2(e47)). Our results exclude a functional role of the C-terminal fragments in Cav2.1 biophysical properties in an expression system widely used to study this channel., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Although immune checkpoint blockers have yielded significant clinical benefits in patients with different malignancies, the efficacy of these therapies is still limited. Here, we show that disruption of transmembrane protein 176B (TMEM176B) contributes to CD8 + T cell-mediated tumor growth inhibition by unleashing inflammasome activation. Lack of Tmem176b enhances the antitumor activity of anti-CTLA-4 antibodies through mechanisms involving caspase-1/IL-1β activation. Accordingly, patients responding to checkpoint blockade therapies display an activated inflammasome signature. Finally, we identify BayK8644 as a potent TMEM176B inhibitor that promotes CD8 + T cell-mediated tumor control and reinforces the antitumor activity of both anti-CTLA-4 and anti-PD-1 antibodies. Thus, pharmacologic de-repression of the inflammasome by targeting TMEM176B may enhance the therapeutic efficacy of immune checkpoint blockers., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

In insects, γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and GABA-gated ion channels are the target of different classes of insecticides, including fipronil. We report here the cloning of six subunits (four RDL, one LCCH3, and one GRD) that constitute the repertoire of the GABA-gated ion channel family of the Varroa mite ( Varroa destructor ), a honey bee ectoparasite. We also isolated a truncated GRD subunit with a premature stop codon. We found that when expressed in Xenopus laevis oocytes, three of the four RDL subunits (VdesRDL1, VdesRDL2, and VdesRDL3) formed functional, homomultimeric anionic receptors, whereas GRD and LCCH3 produced heteromultimeric cationic receptors. These receptors displayed specific sensitivities toward GABA and fipronil, and VdesRDL1 was the most resistant to the insecticide. We identified specific residues in the VdesRDL1 pore-lining region that explain its high resistance to fipronil. VdesRDL4 did not form a functional receptor when expressed alone, but it assembled with VdesRDL1 to form a heteromultimeric receptor with properties distinct from those of the VdesRDL1 homomultimeric receptor. Moreover, VdesRDL1 physically interacted with VdesRDL3, generating a heteromultimeric receptor combining properties of both subunits. On the other hand, we did not detect any functional interaction between VdesLCCH3 and the VdesRDL subunits, an observation that differed from what was previously reported for Drosophila melanogaster In conclusion, this study provides insights relevant to improve our understanding of the precise role of GABAergic signaling in insects and new tools for the development of Varroa mite-specific insecticidal agents that do not harm honey bees., (© 2018 Ménard et al.)

Type 1 hypokalemic periodic paralysis (HypoPP1) is a poorly understood genetic neuromuscular disease characterized by episodic attacks of paralysis associated with low blood K + The vast majority of HypoPP1 mutations involve the replacement of an arginine by a neutral residue in one of the S4 segments of the α1 subunit of the skeletal muscle voltage-gated Ca 2+ channel, which is thought to generate a pathogenic gating pore current. The V876E HypoPP1 mutation has the peculiarity of being located in the S3 segment of domain III, rather than an S4 segment, raising the question of whether such a mutation induces a gating pore current. Here we successfully transfer cDNAs encoding GFP-tagged human wild-type (WT) and V876E HypoPP1 mutant α1 subunits into mouse muscles by electroporation. The expression profile of these WT and V876E channels shows a regular striated pattern, indicative of their localization in the t-tubule membrane. In addition, L-type Ca 2+ current properties are the same in V876E and WT fibers. However, in the presence of an external solution containing low-Cl - and lacking Na + and K + , V876E fibers display an elevated leak current at negative voltages that is increased by external acidification to a higher extent in V876E fibers, suggesting that the leak current is carried by H + ions. However, in the presence of Tyrode's solution, the rate of change in intracellular pH produced by external acidification was not significantly different in V876E and WT fibers. Simultaneous measurement of intracellular Na + and current in response to Na + readmission in the external solution reveals a rate of Na + influx associated with an inward current, which are both significantly larger in V876E fibers. These data suggest that the V876E mutation generates a gating pore current that carries strong resting Na + inward currents in physiological conditions that are likely responsible for the severe HypoPP1 symptoms associated with this mutation., (© 2017 Fuster et al.)

Metaflumizone is the latest addition to the armamentarium of the Na + channel inhibitor insecticide family. We used the Xenopus oocyte expression system and a Markovian model to assess the effect of metaflumizone on Apis mellifera Na + channels (AmNa V 1). Our results reveal that metaflumizone inhibits AmNa V 1 channels by targeting the kinetics of recovery from slow inactivation. Multistate modeling of fast and slow inactivation of the AmNa V 1 channel made it possible to study the effects of metaflumizone on a set of rate constants underlying the transition between the open and inactivated conformations and provided insights into their specificity. We conclude that the methods we used could be extended to assessing the toxicity of other Na + channel inhibitor insecticides., (© 2017 Federation of European Biochemical Societies.)

l-Theanine (or l-γ-N-ethyl-glutamine) is the major amino acid found in Camellia sinensis. It has received much attention because of its pleiotropic physiological and pharmacological activities leading to health benefits in humans, especially. We describe here a new, easy, efficient, and environmentally friendly chemical synthesis of l-theanine and l-γ-N-propyl-Gln and their corresponding d-isomers. l-Theanine, and its derivatives obtained so far, exhibited partial coagonistic action at N-methyl-d-aspartate (NMDA) receptors, with no detectable agonist effect at other glutamate receptors, on cultured hippocampal neurons. This activity was retained on NMDA receptors expressed in Xenopus oocytes. In addition, both GluN2A and GluN2B containing NMDA receptors were equally modulated by l-theanine. The stereochemical change from l-theanine to d-theanine along with the substitution of the ethyl for a propyl moiety in the γ-N position of l- and d-theanine significantly enhanced the biological efficacy, as measured on cultured hippocampal neurons. l-Theanine structure thus represents an interesting backbone to develop novel NMDA receptor modulators.

Voltage-gated Ca 2+ channels are key transducers of cellular excitability and participate in several crucial physiological responses. In vertebrates, 10 Ca 2+ channel genes, grouped in 3 families (Ca V 1, Ca V 2 and Ca V 3), have been described and characterized. Insects possess only one member of each family. These genes have been isolated in a limited number of species and very few have been characterized although, in addition to their crucial role, they may represent a collateral target for neurotoxic insecticides. We have isolated the 3 genes coding for the 3 Ca 2+ channels expressed in Apis mellifera. This work provides the first detailed characterization of the honeybee T-type Ca V 3 Ca 2+ channel and demonstrates the low toxicity of inhibiting this channel. Comparing Ca 2+ currents recorded in bee neurons and myocytes with Ca 2+ currents recorded in Xenopus oocytes expressing the honeybee Ca V 3 gene suggests native expression in bee muscle cells only. High-voltage activated Ca 2+ channels could be recorded in the somata of different cultured bee neurons. These functional data were confirmed by in situ hybridization, immunolocalization and in vivo analysis of the effects of a Ca V 3 inhibitor. The biophysical and pharmacological characterization and the tissue distribution of Ca V 3 suggest a role in honeybee muscle function., Competing Interests: The authors declare no competing financial interests.

Bilaterian voltage-gated Na(+) channels (NaV) evolved from voltage-gated Ca(2+) channels (CaV). The Drosophila melanogaster Na(+) channel 1 (DSC1), which features a D-E-E-A selectivity filter sequence that is intermediate between CaV and NaV channels, is evidence of this evolution. Phylogenetic analysis has classified DSC1 as a Ca(2+)-permeable Na(+) channel belonging to the NaV2 family because of its sequence similarity with NaV channels. This is despite insect NaV2 channels (DSC1 and its orthologue in Blatella germanica, BSC1) being more permeable to Ca(2+) than Na(+) In this study, we report the cloning and molecular characterization of the honeybee (Apis mellifera) DSC1 orthologue. We reveal several sequence variations caused by alternative splicing, RNA editing, and genomic variations. Using the Xenopus oocyte heterologous expression system and the two-microelectrode voltage-clamp technique, we find that the channel exhibits slow activation and inactivation kinetics, insensitivity to tetrodotoxin, and block by Cd(2+) and Zn(2+) These characteristics are reminiscent of CaV channels. We also show a strong selectivity for Ca(2+) and Ba(2+) ions, marginal permeability to Li(+), and impermeability to Mg(2+) and Na(+) ions. Based on current ion channel nomenclature, the D-E-E-A selectivity filter, and the properties we have uncovered, we propose that DSC1 homologues should be classified as CaV4 rather than NaV2. Indeed, channels that contain the D-E-E-A selectivity sequence are likely to feature the same properties as the honeybee's channel, namely slow activation and inactivation kinetics and strong selectivity for Ca(2+) ions., (© 2016 Gosselin-Badaroudine et al.)

Objective: The pathophysiology of idiopathic hypersomnia (IH) remains unclear. Recently, cerebrospinal fluid (CSF)-induced enhancement of γ-aminobutyric acid (GABA)-A receptor activity was found in patients with IH compared to controls., Methods: Fifteen unrelated patients (2 males and 13 females) affected with typical IH, 12 patients (9 males and 3 females) with narcolepsy type 1, and 15 controls (9 males and 6 females) with unspecified hypersomnolence (n = 7) and miscellaneous neurological conditions (n = 8) were included. A lumbar puncture was performed in all participants to measure CSF hypocretin-1 and GABA-A response. We used a voltage-clamp assay on Xenopus oocytes injected with the RNAs that encode the α1 β2 γ2 or the α2 β2 γ2 subunits of the human GABA-A receptor. A sequence of 6 different applications (GABA, GABA/CSF, and CSF alone) with 2 to 4 oocytes per CSF sample was performed in a whole-cell voltage-clamp assay., Results: Representative current traces from oocytes expressing human α1 β2 γ2 or α2 β2 γ2 GABA-A receptors were recorded in response to 6 successive puffs of GABA diluted in the survival medium (SM), showing stable and reliable response. GABA puffs diluted in SM/CSF solution or SM/CSF solution alone showed no significant differences in the CSF of IH, narcolepsy, or control groups. No associations were found between GABA responses, demographic features, disease duration, or disease severity in the whole population or within groups., Interpretation: Using the Xenopus oocyte assay, we found an absence of GABA-A receptor potentiation with CSF from patients with central hypersomnolence disorders, with no significant differences between hypocretin-deficient and non-hypocretin-deficient patients compared to controls. Ann Neurol 2016;80:259-268., (© 2016 American Neurological Association.)

Retinoid-related orphan receptor gamma t (RORγt) is a master transcription factor central to type 17 immunity involving cells such as T helper 17, group 3 innate lymphoid cells or IL-17-producing γδ T cells. Here we show that the intracellular ion channel TMEM176B and its homologue TMEM176A are strongly expressed in these RORγt(+) cells. We demonstrate that TMEM176A and B exhibit a similar cation channel activity and mainly colocalise in close proximity to the trans-Golgi network. Strikingly, in the mouse, the loss of Tmem176b is systematically associated with a strong upregulation of Tmem176a. While Tmem176b single-deficiency has no effect on the course of experimental autoimmune encephalomyelitis, T cell or DSS-induced colitis, it significantly reduces imiquimod-induced psoriasis-like skin inflammation. These findings shed light on a potentially novel specific process linked to post-Golgi trafficking for modulating the function of RORγt(+) cells and indicate that both homologues should be simultaneously targeted to clearly elucidate the role of this intracellular ion flow.

The toxicity of pesticides used in agriculture towards non-targeted organisms and especially pollinators has recently drawn the attention from a broad scientific community. Increased honeybee mortality observed worldwide certainly contributes to this interest. The potential role of several neurotoxic insecticides in triggering or potentiating honeybee mortality was considered, in particular phenylpyrazoles and neonicotinoids, given that they are widely used and highly toxic for insects. Along with their ability to kill insects at lethal doses, they can compromise survival at sublethal doses by producing subtle deleterious effects. In this study, we compared the bee's locomotor ability, which is crucial for many tasks within the hive (e.g. cleaning brood cells, feeding larvae…), before and after an acute sublethal exposure to one insecticide belonging to the two insecticide classes, fipronil and thiamethoxam. Additionally, we examined the locomotor ability after exposure to pyrethroids, an older chemical insecticide class still widely used and known to be highly toxic to bees as well. Our study focused on young bees (day 1 after emergence) since (i) few studies are available on locomotion at this stage and (ii) in recent years, pesticides have been reported to accumulate in different hive matrices, where young bees undergo their early development. At sublethal doses (SLD48h, i.e. causing no mortality at 48 h), three pyrethroids, namely cypermethrin (2.5 ng/bee), tetramethrin (70 ng/bee), tau-fluvalinate (33 ng/bee) and the neonicotinoid thiamethoxam (3.8 ng/bee) caused a locomotor deficit in honeybees. While the SLD48h of fipronil (a phenylpyrazole, 0.5 ng/bee) had no measurable effect on locomotion, we observed high mortality several days after exposure, an effect that was not observed with the other insecticides. Although locomotor deficits observed in the sublethal range of pyrethroids and thiamethoxam would suggest deleterious effects in the field, the case of fipronil demonstrates that toxicity evaluation requires information on multiple endpoints (e.g. long term survival) to fully address pesticides risks for honeybees. Pyrethroid-induced locomotor deficits are discussed in light of recent advances regarding their mode of action on honeybee ion channels and current structure-function studies.

High-Voltage-Activated (HVA) Ca(2+) channels are known regulators of synapse formation and transmission and play fundamental roles in neuronal pathophysiology. Small GTPases of Rho and RGK families, via their action on both cytoskeleton and Ca(2+) channels are key molecules for these processes. While the effects of RGK GTPases on neuronal HVA Ca(2+) channels have been widely studied, the effects of RhoA on the HVA channels remains however elusive. Using heterologous expression in Xenopus laevis oocytes, we show that RhoA activity reduces Ba(2+) currents through CaV2.1, CaV2.2 and CaV2.3 Ca(2+) channels independently of CaVβ subunit. This inhibition occurs independently of RGKs activity and without modification of biophysical properties and global level of expression of the channel subunit. Instead, we observed a marked decrease in the number of active channels at the plasma membrane. Pharmacological and expression studies suggest that channel expression at the plasma membrane is impaired via a ROCK-sensitive pathway. Expression of constitutively active RhoA in primary culture of spinal motoneurons also drastically reduced HVA Ca(2+) current amplitude. Altogether our data revealed that HVA Ca(2+) channels regulation by RhoA might govern synaptic transmission during development and potentially contribute to pathophysiological processes when axon regeneration and growth cone kinetics are impaired., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Pollination is important for both agriculture and biodiversity. For a significant number of plants, this process is highly, and sometimes exclusively, dependent on the pollination activity of honeybees. The large numbers of honeybee colony losses reported in recent years have been attributed to colony collapse disorder. Various hypotheses, including pesticide overuse, have been suggested to explain the disorder. Using the Xenopus oocytes expression system and two microelectrode voltage-clamp, we report the functional expression and the molecular, biophysical, and pharmacological characterization of the western honeybee's sodium channel (Apis Mellifera NaV1). The NaV1 channel is the primary target for pyrethroid insecticides in insect pests. We further report that the honeybee's channel is also sensitive to permethrin and fenvalerate, respectively type I and type II pyrethroid insecticides. Molecular docking of these insecticides revealed a binding site that is similar to sites previously identified in other insects. We describe in vitro and in silico tools that can be used to test chemical compounds. Our findings could be used to assess the risks that current and next generation pesticides pose to honeybee populations.

Voltage-gated Ca(2+) channels allow the influx of Ca(2+) ions from the extracellular space upon membrane depolarization and thus serve as a transducer between membrane potential and cellular events initiated by Ca(2+) transients. Most insects are predicted to possess three genes encoding Cavα, the main subunit of Ca(2+) channels, and several genes encoding the two auxiliary subunits, Cavβ and Cavα2δ; however very few of these genes have been cloned so far. Here, we cloned three full-length cDNAs encoding the three Cavα subunits (AmelCav1a, AmelCav2a and AmelCav3a), a cDNA encoding a novel variant of the Cavβ subunit (AmelCavβc), and three full-length cDNAs encoding three Cavα2δ subunits (AmelCavα2δ1 to 3) of the honeybee Apis mellifera. We identified several alternative or mutually exclusive exons in the sequence of the AmelCav2 and AmelCav3 genes. Moreover, we detected a stretch of glutamine residues in the C-terminus of the AmelCav1 subunit that is reminiscent of the motif found in the human Cav2.1 subunit of patients with Spinocerebellar Ataxia type 6. All these subunits contain structural domains that have been identified as functionally important in their mammalian homologues. For the first time, we could express three insect Cavα subunits in Xenopus oocytes and we show that AmelCav1a, 2a and 3a form Ca(2+) channels with distinctive properties. Notably, the co-expression of AmelCav1a or AmelCav2a with AmelCavβc and AmCavα2δ1 produces High Voltage-Activated Ca(2+) channels. On the other hand, expression of AmelCav3a alone leads to Low Voltage-Activated Ca(2+) channels., (Copyright © 2015 Elsevier Ltd. All rights reserved.)