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Sci Rep
2017 Feb 01;7:41782. doi: 10.1038/srep41782.
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Honeybee locomotion is impaired by Am-CaV3 low voltage-activated Ca2+ channel antagonist.
Rousset M
,
Collet C
,
Cens T
,
Bastin F
,
Raymond V
,
Massou I
,
Menard C
,
Thibaud JB
,
Charreton M
,
Vignes M
,
Chahine M
,
Sandoz JC
,
Charnet P
.
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Voltage-gated Ca2+ channels are key transducers of cellular excitability and participate in several crucial physiological responses. In vertebrates, 10 Ca2+ channel genes, grouped in 3 families (CaV1, CaV2 and CaV3), 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 Ca2+ channels expressed in Apis mellifera. This work provides the first detailed characterization of the honeybee T-type CaV3 Ca2+ channel and demonstrates the low toxicity of inhibiting this channel. Comparing Ca2+ currents recorded in bee neurons and myocytes with Ca2+ currents recorded in Xenopus oocytes expressing the honeybee CaV3 gene suggests native expression in bee muscle cells only. High-voltage activated Ca2+ 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 CaV3 inhibitor. The biophysical and pharmacological characterization and the tissue distribution of CaV3 suggest a role in honeybee muscle function.
Figure 1. Functional characterization of Am-CaV3 in Xenopus oocytes.(a) Ba2+ Current traces recorded from oocytes expressing Am-CaV3. Holding potential: −100 mV, Step potential −30 mV, duration: 150 ms. (b) Averaged activation and inactivation curves recorded from Am-CaV3-injected oocytes. The inactivation curves were obtained using 2.5 s conditioning potentials from −100 to various potentials (10 mV increment) followed by a subsequent test pulse to −30 mV. Voltages for half-activation (Vact): −47 ± 1 mV, activation slope: 6.7 ± 0.3 mV, voltage for half-inactivation (Vinact): −66 ± 1 mV, inactivation slope: 3.5 ± 0.2 mV, percentage of non-inactivating current: 5 ± 1% (n = 12). The window current, is underlined as a dotted surface. (c) Current inactivation time-course was fitted at each potential using a mono-exponential decay with a time constant Tau. The voltage-dependence of Tau displayed an exponential decrease with an e-fold decrease (Vdep.) every 7.9 ± 0.4 mV (n = 10). (d) Increase in Ba2+ current amplitudes recorded on oocytes expressing CaV3 Ca2+ channel produced by the coexpression of the CaVβ subunit20. Currents were normalized to the current amplitude of Am-CaV3 without CaVβ (n = 4). (e) Pharmacological profile of Am- CaV3 Ca2+ channels defined by the response to several calcium antagonists, insecticides and toxins. The residual currents in response to these antagonists are shown relative to the current recorded in control condition. Pe: permethrin 50 μM; Al: allethrin 50 μM: Iv: ivermectine 10 μM; Pi: picrotoxin 10 μM; Fi: fipronil 10 μM; Cl: clothianidine 10 μM; Ch: Chlorantraniliprole 10 μM; Mi: mibefradil 10 μM; NN: NNC 55-0396 10 μM; TT: TTA-A2 10 μM; Ni: nifedipine: 10 μM; BK: bay-k 8644 10 μM; Ve: verapamil 10 μM; Di: diltiazem 10 μM; Am: amiloride 1 mM; At: atrachotoxin 10 μM; Ag: w-aga-IV-A 10 μM; SN: SNX 482 10 μM; Ni: nickel chloride 0.12 mM, CdCl2: cadmium chloride 0.2 mM. (f) Dose-response curve of mibefradil onAm-CaV3 current amplitude. Continuous line is the best fit using the Hill equation (n = 10). The effect of mibefradil (10 μM, holding potential of −100 mV and depolarization to −30 mV, trace marked “Mâ€) is shown on the inset. Rel cur: currents normalized to the current amplitude recorded in the absence of mibefradil.
Figure 2. Honeybee toxicity of mibefradil.(a,b) Dose-mortality curves produced by 3 modes of exposure: oral, topic (a) or injection (b). Toxicity was evaluated at 120âh for oral or contact exposure or at 48âh post-injection. (c) Time course of bee mortality after mibefradil injection at two different concentrations. Mibefradil was injected into the median ocellus at 0.11âμg (circle) and 1.1âμg (hexagon) per bee and the effects were compared to solvent-injected bees (control, square).
Figure 3. Neuronal CaV3 Ca2+ channel and odor learning.(a) In situ hybridization of frontal section of honeybee brain using an Am-CaV3 specific RNA probe. Left, Am-CaV3 probe; right, control with the sens probe. The MBN stained by the probe is marked by an arrowhead. Scale bar 100 μm. (b) Standard PER conditioning assays performed on bees injected with 1.1 μg/bee mibefradil (black squares) or solvent (blue hexagons) depicted mild deficits in acquisition. At 0.11 μg/bee, mibefradil (red circles) had no effect. See Fig. S4a for tests on 1 h memory and olfactory generalization. (c) Staining using Am-CaV3 3CT6 antibody (at 1/100) of Hek-293 cells transfected with the Am-CaV3 or of various honeybee neurons in culture. Staining was performed 2 days after transfection, or at Div 2–6 for cultured neurons. LEFT. Staining of Am-CaV3-expressing HEK 293 cells display a cytoplasmatic and membrane localisation of the channel. RIGHT. Phase contrast images (top) and fluorescent images (bottom) of (from left to right) antennal lobe neurons (ALN), suboesophageal neurons (SBON), mushroom body neurons (MBN) and brain cells other than SBON, ALN and MBN. A few cells amongst the latter brain cells are stained by 3CT6 antibody. Scale bar: 10 μm. (d) Ba2+ currents recorded on MBN, ALN or Gt2N, during voltage ramps from −80 to +80 mV (middle) or during a two voltage-steps protocol from −100 mV to −30 mV and 0 mV (right). DIC (differential interference contrast) images of neurons where Ba2+ currents displayed on the left have been recorded. Scale bar, 10 μm.
Figure 4. Muscle CaV3 Ca2+ channel and locomotion.(a) Confocal images of isolated muscle cells stained with phalloïdin (left, green) and with Am-CaV3 specific antibody 3CT6, (middle, red). Merged staining is shown on the right. CaV3 staining is observed in bands within and between phalloïdin staining, in lines along the plasma membrane (arrow) and in isolated spots (arrowheads). The averaged distances ratio of phalloïdin staining over CaV3 staining was 1.6 ± 0.2 (n = 14). Scale bar, 30 μm. (b) Averaged walking distances covered by bees during 3 minutes in a vertical circular arena after intra-ocellar injection of 0, 0.11 or 1.1 μg/bee of mibefradil. Experiments were performed 6 h after injection (+6 h post-inj.). (c) Ba2+ currents recorded on isolated leg muscle (MTib), during voltage ramps from −80 to +80 mV (middle) or during a two-step voltage-clamp protocol with depolarizations from −100 mV to −30 mV and to 0 mV (right). Left: typical DIC (differential interference contrast) images of the muscle cell where Ba2+ currents have been recorded. The arrow indicates the presence of a hump (see text) in the IV curve at hyperpolarized potentials. (d) Proportion of cells displaying traces with LVA currents evaluated by the presence of a hump on the current-voltage curve for MBN (n = 30), ALN (n = 19), Gt2N (n = 15) and muscle cells (MTib, n = 58) (e) Left. Current density of the Ba2+ currents recorded in muscle cells at −30 mV or 0 mV ([Ba2+] = 2 mM, n = 32). Middle-right. Voltage for peak amplitude of the current-voltage curves for LVA and HVA muscle cell Ba2+ currents (n = 16 and 32). Kinetics of inactivation (Middle-left), quantified as the remaining current at 90 ms after the onset of the depolarization (R90), for Ba2+ currents recorded in muscle at −30 mV or 0 mV (n = 17). Right. Effect of mibefradil (10 μM) on Ba2+ currents recorded onmuscle cells (depolarization to −30 mV or 0 mV). Rel cur: currents normalized to the current in absence of mibefradil.
Cens,
Characterization of the first honeybee Ca²⁺ channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation.
2013, Pubmed,
Xenbase
Cens,
Characterization of the first honeybee Ca²⁺ channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation.
2013,
Pubmed
,
Xenbase
Cens,
Molecular determinant for specific Ca/Ba selectivity profiles of low and high threshold Ca2+ channels.
2007,
Pubmed
Charreton,
A Locomotor Deficit Induced by Sublethal Doses of Pyrethroid and Neonicotinoid Insecticides in the Honeybee Apis mellifera.
2015,
Pubmed
Chong,
The omega-atracotoxins: selective blockers of insect M-LVA and HVA calcium channels.
2007,
Pubmed
Collet,
Excitation-contraction coupling in skeletal muscle fibers from adult domestic honeybee.
2009,
Pubmed
Collet,
Excitable properties of adult skeletal muscle fibres from the honeybee Apis mellifera.
2007,
Pubmed
Demmer,
Intrinsic membrane properties and inhibitory synaptic input of kenyon cells as mechanisms for sparse coding?
2009,
Pubmed
Gielow,
Resolution and pharmacological analysis of the voltage-dependent calcium channels of Drosophila larval muscles.
1995,
Pubmed
Gosselin-Badaroudine,
Biophysical characterization of the honeybee DSC1 orthologue reveals a novel voltage-dependent Ca2+ channel subfamily: CaV4.
2016,
Pubmed
,
Xenbase
Grolleau,
Two distinct low-voltage-activated Ca2+ currents contribute to the pacemaker mechanism in cockroach dorsal unpaired median neurons.
1996,
Pubmed
Grünewald,
Differential expression of voltage-sensitive K+ and Ca2+ currents in neurons of the honeybee olfactory pathway.
2003,
Pubmed
Honeybee Genome Sequencing Consortium,
Insights into social insects from the genome of the honeybee Apis mellifera.
2006,
Pubmed
Husch,
Calcium current diversity in physiologically different local interneuron types of the antennal lobe.
2009,
Pubmed
Iniguez,
Cav3-type α1T calcium channels mediate transient calcium currents that regulate repetitive firing in Drosophila antennal lobe PNs.
2013,
Pubmed
Inoue,
Ionic channel mechanisms mediating the intrinsic excitability of Kenyon cells in the mushroom body of the cricket brain.
2014,
Pubmed
Jacus,
Presynaptic Cav3.2 channels regulate excitatory neurotransmission in nociceptive dorsal horn neurons.
2012,
Pubmed
Jeong,
Ca-α1T, a fly T-type Ca2+ channel, negatively modulates sleep.
2015,
Pubmed
,
Xenbase
Kloppenburg,
Voltage-activated currents from adult honeybee (Apis mellifera) antennal motor neurons recorded in vitro and in situ.
1999,
Pubmed
Laurent,
Whole-cell recording from honeybee olfactory receptor neurons: ionic currents, membrane excitability and odourant response in developing workerbee and drone.
2002,
Pubmed
Menzel,
The honeybee as a model for understanding the basis of cognition.
2012,
Pubmed
Nakasu,
Novel biopesticide based on a spider venom peptide shows no adverse effects on honeybees.
2014,
Pubmed
Perisse,
Early calcium increase triggers the formation of olfactory long-term memory in honeybees.
2009,
Pubmed
Ryglewski,
Ca(v)2 channels mediate low and high voltage-activated calcium currents in Drosophila motoneurons.
2012,
Pubmed
Schäfer,
Ionic currents of Kenyon cells from the mushroom body of the honeybee.
1994,
Pubmed
Spafford,
In vitro characterization of L-type calcium channels and their contribution to firing behavior in invertebrate respiratory neurons.
2006,
Pubmed
Wicher,
Non-synaptic ion channels in insects--basic properties of currents and their modulation in neurons and skeletal muscles.
2001,
Pubmed
Worrell,
Characterization of voltage-dependent Ca2+ currents in identified Drosophila motoneurons in situ.
2008,
Pubmed
Wüstenberg,
Current- and voltage-clamp recordings and computer simulations of Kenyon cells in the honeybee.
2004,
Pubmed
Yan,
Permethrin modulates cholinergic mini-synaptic currents by partially blocking the calcium channel.
2011,
Pubmed
