Recent Advances in the Functional Characterization of Honeybee

Chapter 5

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Recent Advances in the Functional Characterization of Honeybee Voltage-Gated Ca2+ Channels T. Cens, M. Rousset,1 J-B. Thibaud,1 C. Menard,1 C. Collet,2 M. Chahine,3 and P. Charnet*,1 1Biomolecules Institute Max Mousseron (IBMM), UMR 5247, CNRS ENSCM, Montpellier University, bât. CRBM, 1919 Route de Mende, 34293 Montpellier cedex 5, France 2INRA, UR 406 Honeybees and Environment, 228 Route de l’aérodrome, Domaine Saint Paul, Site Agroparc, CS40509, 84914 Avignon cedex 9, France 3Research Center, in Mental health University LAVAL Québec, 2601 Chemin de la Canardière, Québec (Québec) G1J 2G3, Canada *E-mail: [email protected].

Voltage-gated Ca2+ (CaV) channels allow Ca2+ to enter the cell in response to membrane depolarization. This Ca2+ influx is not only necessary for cell excitability, but also triggers, via Ca2+-binding proteins, important biological functions such as contraction, synaptic transmission, or gene expression. Insect CaV channels are encoded by only three genes (against 10 in mammals), and their invalidation or pharmacological blockade is expected to have deleterious effects. They may thus constitute interesting targets for specific insecticides. However, the precise identification of the genes underlying the different Ca2+ currents recorded in different tissues, as well as the heterologous expression of these genes to screen selective molecules, have been proven to be difficult. This chapter reports on the recent successful expression of honeybee Ca2+ channels genes in Xenopus oocytes and reviews pharmacological properties of Ca2+ currents recorded in isolated honeybee neurons and muscle cells.

© 2017 American Chemical Society Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Introduction Membrane ion channels are the targets of numerous alkaloid or peptidic toxins produced by plants or venomous animals to repel predators or paralyze their preys. Voltage-gated Na+ (NaV) and Ca2+ channels, directly involved in neuronal and/or neuromuscular synaptic transmission, are particularly targeted by these molecules. Channel over-activation or blockade by toxins result in an impairment of synaptic transmission and/or of nervous influx propagation. These perturbations result in various peripheral or motor deficits leading for instance to pain, paralysis or even death. Most of the neurotoxic insecticides used by humans as anti-vectorial or agrochemical protection tools have similar modes of action, i.e. they target ion channels and alter the normal function of these membrane-inserted molecules. The molecular structure of neurotoxic insecticides has often been derived from natural products such as plant alkaloids (nicotin, pyrethrins). Among the most used neurotoxic insecticides, pyrethroids, for instance, bind within the channel pore and produce a gating dysfunction of the Na+ channels. This prolongs the pore opening and results in intracellular neuronal Na+ overload, excess membrane depolarization and cellular hyperexcitability (or hypoexcitability if sustained membrane depolarization reaches a sufficient level). Depending on the insecticide dose, the nature/number of vital organs affected (brain, heart, sensory organs), the number of channels modified and on the intensity of channel modification, a gradation of effects can arise, from subtle effects up to highly deleterious and lethal effects, with a slow or quick death of the exposed insect. Noteworthy are some apparently sublethal effects of insecticides that may arise in conjunction with natural environmental stressors (heat, cold, predators) and turn out to become lethal. Some pyrethroids also block the Ca2+ influx in the presynaptic terminals and in muscle cells by inhibiting CaV channels. In insects, these secondary pyrethroid targets have been functionally characterized in a number of cellular preparations, but the genes encoding these channels are still poorly characterized, at variance to mammals, where ten genes have been reported to encode CaV channel α-subunits of three types (CaV1.x, CaV2.y, CaV3.z), with specific biophysical and pharmacological properties. The recent cloning of the honeybee genome and other insects allowed the identification of three genes only (CaV1, CaV2 and CaV3), encoding a single CaV channel α-subunit of each of the types found in mammals (1). Since these sequences emerged to be less conserved among insects than that of the NaV channels, they have been proposed as potential targets for species-specific insecticides able to preserve pollinators. Heterologous expression of these genes has just been reported (1–3). This should allow a more precise characterization of their biophysical and pharmacological properties, and therefore should also open new fields of research dedicated to the design of more species-specific insecticides. Honeybee CaV channels and their recently described functional/pharmacological properties are the subject of the present review.

76 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Physiological Recordings of Ca2+ Currents in Honeybee Neurons and in Muscle Cells The characterization of CaV currents in honeybee tissues has been performed in antennal lobe neurons (ALN) (4), in neurons from thoracic ganglia (Gt2N) (5), in Kenyon cells of the mushroom bodies (MBN) (4, 6), and in skeletal muscle cells (MC) (7). It is only recently that a direct comparison of the functional properties of these currents has been performed under similar experimental conditions (2) the reported biophysical parameters are relatively homogeneous between the different neurons. The current activates around -40 mV, reaches a peak amplitude close to 0 mV, and reverses at voltages greater than +40 mV (Figure 1A). The kinetics of activation are fast, with a peak amplitude at 0 mV reached in around 2-5 ms. The current density is close to 40 pA/pF. All these properties are shared by ALN and MBN Ca2+ currents (Figure 1A). Due to the smaller size of MBN, the current amplitudes recorded in these cells (~150 pA) are therefore smaller than in ALN or Gt2N (~700 pA). The Ca2+ currents recorded in these preparations are often not stable, a pronounced run-down occurring in all cell types, decreasing the current amplitude by about 50% in a few minutes. Interestingly, while the inactivation kinetics of CaV currents was systematically slower than that of NaV currents (~50% decrease of NaV current in ~2-3 ms), a strong variability of this kinetics was systematically observed between ALN, MBN and Gt2N, as well as between different neurons of the same tissue. This variability has been attributed to potential variations in the expression level of different Ca2+ channel-encoding genes (5), but also to differences in the developmental stages of honeybees used to prepare the recorded cells. However, neither the functional/pharmacological properties of the cloned CaV gene products, nor the relative expression levels of these three genes have been analyzed yet. Consequently, there is still no practical means to ascribe a given CaV current recorded in the native context to one of the three types of CaV channels. For example, replacing Ca2+ with Ba2+ has been reported to systematically increase the amplitude of the current ascribed to honeybee CaV channels, but the exact value of the Ba2+/Ca2+ current ratio has never been determined, although this parameter could be used to distinguish the different channel types (8). In contrast to mammalian Ca2+ channels, this change in current amplitude, however, develops in honeybee cells without any marked change in the inactivation kinetics. This suggests either (i) a lack of Ca2+-dependent inactivation for all the Ca2+ channel types expressed in honeybee cells or (ii) a high efficacy of 5-10µM of ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid –EGTA- in buffering intracellular Ca2+ in these cells, with a block of inactivation. In both cases, Ca2+-dependent inactivation would not be a pertinent criterion for discrimination between CaV channels in the native context. It should be noted, however, that LVA Ca2+ channels (able to activate at potential lower than -40 mV) were never reported in honeybee central neurons, in apparent correlation with the difficulty to observe CaV3 staining in in situ hybridation experiments (2).



Figure 1. In vivo and in vitro characterization of voltage-gated Ca2+ channels in honeybees. A. Ba2+ currents recorded in isolated mushroom body neurons (MBN), antennal lobes neurons (ALN), thoracic ganglia neurons (Gt2N) or skeletal muscle cells (MC) voltage-clamped and submitted to a 1 s-long voltage-ramp from -80 mV to +80 mV (MBN, ALN, Gt2N) or to +50 mV (MC). The extracellular divalent cation was 10 mM Ba2+ (2). Note the lower threshold voltage for activation in mucle cells as well as the presence of a hump on the current trace (IV curve) at hyperpolarized voltages (-50/-30 mV). B. TOP. Ba2+ currents recorded in Xenopus oocytes expressing any of the four honeybee voltage-gated Ca2+ channels (CaV1-4) 2-5 days after injection of the corresponding cRNA. CaV1 and CaV2 cRNAs were co-injected with the CaVa2d1 and CaVb cRNAs. Current were recorded during voltage-steps from -100mV to -30 mV (CaV3) or to +10 mV (CaV1, CaV2 or CaV4) in 10 mM Ba2+ external solution. BOTTOM. Corresponding current-voltage curves obtained by plotting the peak current recorded during depolarizing steps from –110 mV to a range of voltage between –80 to +50 mV. For the CaV3 channel, note, please, the fast kinetics of inactivation as well as the activation for hyperpolarized voltages.

The Ca2+ currents recorded in the different tissues also appear to be quite similar from a pharmacological point of view. They are all completely blocked by 50 µM Cd2+, and they are sensitive only to very high doses (~50% block at 100 µM) of classical Ca2+ antagonists such as verapamil or nifedipine (6). The pharmacological profile of these Ca2+ currents remains, however, essentially unknown, while such information has clearly been proven to be useful for channel type distinction. In this situation, further isolation of the different Ca2+ channel types found in invertebrate seems difficult to achieve (9–12). 78 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


The Ca2+ currents recorded in muscle cells, however, display differences with those recorded in neurons. Although the overall current-voltage curves (activation threshold, peak current and reversal potential) are similar between neurons and muscle cells (Figure 1A), the activation and inactivation kinetics are significantly slower (30% decrease in 100ms) (13, 14). More surprisingly, the careful inspection of the current-voltage curve at negative potentials shows a pronounced hump in the curve close to -30 mV (Figure 1A), indicative of the expression of some LVA Ca2+ channels. It should be noted that LVA Ca2+ channels have rarely been seen in insect cells, and this is the first evidence of LVA Ca2+ channel expression in insect muscle cells. Since these cells lack any fast voltage-gated Na+ channels (14), their expression of LVA channels would have an interesting physiological meaning. The conclusion of these biophysical characterizations of CaV channels in honeybees is that, except in muscle cells, where LVA channel expression can be revealed, no clear markers have been found to unambiguously distinguish that of different types of Ca2+ channels. A similar situation is also found in other insect species. The pharmacological properties of the insect channels are markedly different from that of their mammalian counterparts. However, up to now, no specific molecules have been found to be useful for the precise identification of a given channel-type. Finally, the level of expression of LVA channels in the brain, and in muscle, where current amplitudes are small and observed in ~50% of the cells only, is surprising. This suggests a minor role for CaV3 in neuronal physiology, a fact which is correlated with the low toxicity of CaV3 inhibitors in vivo and the light phenotype of Drosophila knocked-out for CaV3 channel expression.

Cloning and Sequence Analysis From a molecular point of view, CaV channels are heteromeric transmembrane proteins composed of a main pore-forming subunit (CaVα) and two regulatory subunits with extracellular (α2-δ subunits) or intracellular (CaVβ subunit) localization. In mammals, ten genes encode CaVα subunits forming Ca2+ channels with distinctive biophysical and pharmacological properties that can be grouped in three families. The CaV1 family with 4 members (CaV1.1-1.4) form L-type Ca2+ channels that are activated by high voltage, have slow inactivation kinetics and are sensitive to dihydropyridines such as nifedipine, nicardipine or Bay-K8644 (15). These channels are ubiquitously found in neurons, muscle or secretory cells and produce an entry of Ca2+ that triggers contraction, secretion, or gene activation (15). The second CaV2 family, contains 3 members (CaV2.1-2.3), that are also activated by high voltage, but display faster kinetics of inactivation and are blocked specifically by spider or conus toxins (ω-AgaIV-A, ω-GVI-A for example). CaV2 channels produce an influx of Ca2+ in presynaptic terminals, responsible for synaptic transmission. The third Ca2+ channel family, CaV3, also called T-type or transient Ca2+ channels contains three members (CaV3.1-3.3) that are activated by low voltage and have relatively fast kinetics of activation and inactivation. These channels are specifically inhibited by mibefradil, or TTA-2, and play a role in rhythmic activity in heart or neurons. The trafficking 79 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


and biophysical properties of CaV1 and CaV2 channels are modulated by the auxiliary CaVα2δ (encoded by four genes CaVα2δ1-4) and CaVβ subunits (four genes: CaVβ1-4). These subunits increase the membrane expression of the pore-forming CaVα subunit but also increase the opening probability of CaV channels and modulate their activation and inactivation properties. There is no known regulatory subunit for CaV3 channels. Worth noting is that proteomic analysis of CaV2 channels suggests that a large number of cellular proteins are able to interact, directly or indirectly, with the Ca2+ channels, some of them displaying strong regulatory properties (16) (see for example the small GTPAse Rem (17–19), or RhoA (20)). In human, mutations affecting the different CaV channel types at various location within the pore-forming subunits (in the voltage-sensor domain, the pore domain or the intracellular loops) result in severe diseases of the nervous or muscular systems (epilepsy, migraine, ataxia, Timothy syndrome, periodic paralysis, etc.). The sequencing of the Apis mellifera genome in 2006 unraveled the repertoire of the different Ca2+ channel subunits in this insect. There are only three poresubunit genes encoding a single member of in each of the CaV1, CaV2 and CaV3 families. Two HVA and 1 LVA Ca2+ channels are thus expected to be expressed in insect cells. In addition, three genes have been identified as encoding CaVα2δ regulatory subunits while only one gene was found to encode a CaVβ regulatory subunit (1, 3). All these genes have been cloned (1). The sequences of the 3 Apis mellifera CaVα subunits display all the typical features of pore-forming subunits of voltage-gated ion channels. They are organized in four homologous domains each containing six transmembrane α helices (S1-S6, see Figure 2A, B and C) (21). The first four α helices (S1-S4) of each domain form the so-called voltage-sensing domain (VSD) with S4 serving as the voltage-sensor and displaying 4-7 regularly spaced positively charged amino-acids (Arg or Lys). The last two α helices (S5-S6) of each domain make the channel pore and the activation gate. The selectivity filter of the channel is formed by a short segment between S5 and S6 called the pore sequence, with an acidic glutamate residue positioned at a conserved location in each domain (in red stick in Figure 2D). Therefore, in the folded configuration of the CaVα protein, these acidic residues from each domain form the EEEE ring or locus (or EEDD in CaV3), which is the pore hallmark of Ca2+ -selective channels (21, 22) (Figure 2D). Two NaV encoding genes can be pinpointed in the sequenced Apis mellifera genome: para and DSC1. The para gene (named AmNaV1) is highly homologous to the para gene of Drosophila melanogaster and encodes a voltage-gated Na+ channel with a typical DEKA sequence for the channel selectivity filter and the expected functional properties (23). Conversely, despite its homology with the para sequences, the AmDSC1 sequence encodes an atypical selectivity filter motif (DEEA, Figure 3), and when heterologously expressed in Xenopus oocytes, features an exclusive permeability to Ca2+ ions (see below) (24), and gating properties similar to HVA CaV channels. The AmDSC1 channel was therefore proposed to be renamed AmCaV4 (24).



Figure 2. Molecular structure of voltage-gated Ca2+ channels. A. Schematic cartoon representing the secondary structure of a typical amino acid sequence of a voltage–gated Ca2+ channel. Within the black square, the voltage-sensing domain (S1, S2, S3 transmembrane a helices in magenta, and S4 helice in red) and the pore domain (S5 and S6 in yellow and the selectivity filter in blue) of the first domain (Domain I) are underlined. B. Side-view of the cristal structure of this first domain with the same color code using the mammalian skeletal CaV1.1 Ca2+ channel structure (PDB Acc numb. 5GJV (21)). C. Side-view of the complete crystal structure of the mammalian skeletal CaV1.1 Ca2+ channel (PDB Acc numb. 5GJV, (21)), with the first domain underlined using the same color code than in A, and the other 3 domains colored in green. D. Top-view of the lower-part of the selectivity filter showing the position of the 4 glutamates (as red sticks) at the narrowest part of the pore. The pore helices of the selectivity filter region of domain I, II, III and IV, as indicated. (see color insert)



Figure 3. Pore sequence and expression of Apis mellifera DSC1/CaV4. A. Agarose gel showing the DNA fragments amplified by RT-PCR using DSC1/CaV4-specific primers and mRNAs from Apis mellifera brain, gut, leg muscle, antenna, thoracic ganglia and whole larva. The PCR-products amplified using actinin1-specific primers and RNA from the same tissues are also shown as control. B. Amino-acid alignement of the pore sequences of Apis mellifera CaV1, CaV2, CaV3, NaV1, and DSC1/CaV4 voltage-gated channels. The EEEE locus (EEEE) and the second outer site (OS) are shaded and charged (negatively, D,E and positively, K, L) or conserved amino acids at these loci and between them are in bold. Note the strictly conserved tryptophan (W) between the EEEE and OS loci (bold, in grey).

The cellular expression of these four CaV channel genes has not been extensively studied yet. RT-PCR analysis, however, revealed an ubiquitous expression in all tested tissues: antennae, skeletal muscles, antennal lobes and mushroom bodies. In situ hybridization of CaV3 however, confirms that the expression level is very low in the brain, with a faint labeling only in the Kenyon cells, suggesting tissue and channel specific regional expression (2).



Functional Properties in Heterologous Systems Functional expression of insect Ca2+ channels has not been achieved until recently. In 2011, we first cloned two isoforms of the honeybee Ca2+channel CaVβ subunit, and used mammalian channel subunits to characterize their properties after expression in Xenopus oocytes (3). This subunit is more homologous to the mammalian CaVβ2a subunit, than to CaVβ1, CaVβ3 or CaVβ4 subunits, although it lacks the typical cysteines at position 3 and 4 responsible for the palmitoylation of CaVβ2a. Nevertheless, this AmCaVβ subunit is able to increase Ca2+ current amplitude, to shift the activation curve toward hyperpolarized potentials and to slow-down channel inactivation, a property typical of the mammalian CaVβ2 subunits either palmitoylated (CaVβ2a) (25, 26) or not (CaVβ2e) (27). The slowing of inactivation was demonstrated to be related to subunit interaction with the inner leaflet of the plasma membrane, thanks to the presence of palmitate in the N-terminal sequence of the mammalian CaVβ2a. For the honeybee subunit, however, this interaction was due to electrostatic interactions with membrane PIP2 as shown also for the CaVβ2e subunit (27). We subsequently cloned the three CaVα and three CaVα2δ subunits expressed in honeybee (1). The heterologous expression of the CaV1 and CaV2 channels appeared to be problematic. When co-injected with the regulatory CaVα2δ and CaVβ subunits, CaVα1 or CaVα2 produced only very small Ca2+ or Ba2+ currents (30-200 nA), while expression of CaVα3 or CaVα4 subunits, under similar experimental conditions, produced robust Ca2+ currents of several hundred nA. When the current-voltage curves of these different Ca2+ channels are plotted together on the same graph (Figure 1B), it is obvious that three of them (CaV1, CaV2 and CaV4) are HVA Ca2+ channels, with variable kinetics of inactivation, slow for CaV1 and CaV2 and rather fast for CaV4. The reversal potential of the CaV4 current was also quite different from that of CaV1 and CaV2 currents, suggesting a slightly different cation selectivity. These 3 types of Ca2+ channels probably contribute to the HVA influx of Ca2+ recorded in the different neuronal preparation, but their relative expression in these tissues remains to be determined. As shown in Figure 1B, the CaVα3 gene encodes a pure LVA Ca2+ channel with hyperpolarized activation and reversal potentials. As expected for a LVA channel, expression of the CaVα3 subunit produced a rapidly inactivating current, with complete inactivation in less than 100 ms. Altogether, these data demonstrate successful expression of honeybee CaV channels in Xenopus oocytes, and suggest the existence of one LVA and three HVA Ca2+ channels with slightly different permeation properties for CaV3 and CaV4, as suggested by their particular pore sequences and deduced from their reversal potentials. Nevertheless, the changes in reversal potential of Cav3 and Cav4 currents in the presence of different Ba2+ concentrations clearly demonstrated that these two channels are genuine Ca2+ channels (Figure 4 A and B)



Figure 4. Biophysical properties of CaV3 and CaV4. A. Typical current trace showing the response of a voltage-clamped oocyte expressing CaV3 channels and submitted to a voltage-ramp from 100mV to +40 mV. The measure of the reversal potential is shown by an arrow. The extracellular solution contained 10 mM Ba2+ (2). B.Plot of the reversal potentials measured using different extracellular solutions with increasing amount of Ba2+ (from 1mM to 40 mM) and recorded on oocytes expressing either the CaV3 or the CaV4 channels. The linear relationship between the reversal potentials and the log([Ba]), with slopes of 29.5 and 31.5 mV respectively, indicates that both channels conducts almost exclusively divalent cations in these conditions. C. Dose-response curves of the effects of Cd2+ on the Ba2+ currents recorded on Xenopus oocytes expressing the Apis mellifera CaV3 or CaV4 Ca2+ channels. Note the higher EC50 for CaV3 (0.22mM versus 0.03mM for CaV4). D. Pharmacological profile of CaV3 Ca2+ channel obtained on voltage-clamped Xenopus oocytes expressing the CaV3 Ca2+ channel, by a 5 min perfusion of various insecticides [Pe: permethrin (50µM), al: allethrin (50µM), Iv: ivermectin (10µM), Pi:picrotoxin (10µM), Fi: fipronil (10µM), Cl: clothianidin (10µM), Ch: chlorantraniliprole (10µM)], Ca2+ antagonists [Mi: mibefradil (10µM), NCC: NCC55-0396 (10µM), TT: TTA-A2 (10µM), Ni: nifedipine (10µM, BK: Bay-K8644 (10µM), Ve: verapamil (10µM), Di: diltiazem (10µM), Am: amiloride (1mM)] or toxins [SNX:SNX482, (10µM), ATx: atrachotoxin (1µM), Aga: w-Aga-IVA (1µM)].



When pharmacological agents were tested against these four CaV channels, none of them displayed any sensitivity to nifedipine, Bay-K8644, ω Agat IV-A or ωGVI-A (at 1-10µM concentrations, not shown). However, due to the poor expression of the CaV1 and CaV2 channels the analysis of their pharmacological profile could not be carried out thoroughly, and only CaV3 and CaV4 were tested. Figure 4C demonstrates that these two channels can be distinguished by their sensitivity to Cd2+, with an IC50 for CaV3 (0.22 mM) being one order of magnitude larger that that for CaV4 (0.03mM). We have built a set of different molecules to be tested against these four Ca2+ channels in order to establish their pharmacological profiles and hopefully find, in each case, one or two specific inhibitors to be used in further physiological characterization in the native context. Commonly used insecticides (permethrin, allethrin, ivermectin, picrotoxin, fipronil, clothianidin, chloranniliprole), Ca2+ antagonists (mibefradil, NCC05, TTA-2, nifedipin, PN200-110, BayK8644, verapamil, diltiazem, amiloride) or toxins (SNX483, ω-atrachotoxin, or ωAga-IV-A) were chosen. The pharmacological profile obtained for CaV3 (Figure 4D) demonstrates that this channel was only blocked by the classical LVA antagonist mibefradil and its degradation product NCC05 (2), but not by the other LVA antagonist TTA2. When tested, all the other molecules were without effects. This profile has now to be compared to the profiles of the other Ca2+ channels types studied in similar experimental conditions. It is clear that the characterization of the different honeybee CaV channels is in its early stages. Now that the clones are available and conditions to record channel activity well established, upcoming studies should provide a better knowledge of the biophysical and pharmacological properties of these channels, of their role(s) in the neuronal and muscle physiology in honeybee, and also of their likely involvement in intoxication by insecticides.

CaV Channels as Alternative Insecticides Target The effect of the different insecticides on insect CaV channels have been poorly studied. Sequence alignment of the CaVα subunits of different insect species shows, however, a relatively low level of conservation (28) (see Table). This latter point suggests that specific inhibitors or activators could be found with species-specific effects, and therefore one can propose Ca2+ channels as an alternative targets for specific pest-control strategies (1–3, 24, 28). Isolation of inhibitors specific for the three types of insect CaV channels will also be of invaluable help to decipher the precise identity of the genes underlying the Ca2+ currents recorded in different tissues, as well as to understand the role of these genes in neuronal and muscle physiology. Expressing Ca2+ channel-encoding genes from different species is therefore mandatory to isolate and characterize new and specific regulators, and the work presented here constitutes a first step toward this goal. Noteworthy is the identification of several toxins from spider venoms able to inhibit specifically insect ions channels (29), and that may therefore serve as bio-insecticides. Limited penetration through the insect cuticle and diffusion throughout the nervous system tissues (29, 30) precludes using these toxins by topic application. The recent construction of fusion proteins, in 85 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


which the toxin is fused with a carrier protein (like the snowdrop lectin (31)) and displays oral toxicity, opens the way to a potential alternative to conventional pesticides with enhanced selectivity. Hundreds of toxins from the venoms of spiders, the most efficient insect predators, can therefore be screened for activity against pest ion channels with this goal in mind. Among the toxins that have been shown to be active on insect ion channels, PLTX-II (from Plectreurys tristis), ω-hexatoxin-Hv1a (from Hadronych versuta), or ωTbo-IT1 (from Tibellus oblongus) are known to inhibit different insect Ca2+ channels, as demonstrated both in vivo and in vitro using isolated neurons (32, 33). Few of these toxins, however, have been directly tested on heterologously-expressed insect Ca2+ channel genes. With the recent report of heterologous expression of several of these channels in Xenopus oocytes (2, 34, 35), and thus the setting of an effective screening tool, the situation is likely to change rapidly.

Acknowledgments Financial support for this work was obtained from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), University of Monpellier and the Agence Nationale de la Recherche (ANR Bee-Channels N° ANR-13-BSV7-0010-0), the Fondation Lune et Miel, the Institut des Biomolécules Max Mousseron (UMR 5247) and NSERC Discovery Grant from the Natural Sciences and Engineering Research Council of Canada. 86 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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