Honeybee locomotion is impaired by Am-CaV3 low voltage-activated Ca2+ channel antagonist

Abstract

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 (Ca_V1, Ca_V2 and Ca_V3), 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 Ca_V3 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 Ca_V3 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 Ca_V3 inhibitor. The biophysical and pharmacological characterization and the tissue distribution of Ca_V3 suggest a role in honeybee muscle function.

Figure 1. Functional characterization of Am-Ca_V3 in Xenopus oocytes.

(a) Ba²⁺ Current traces recorded from oocytes expressing Am-Ca_V3. Holding potential: −100 mV, Step potential −30 mV, duration: 150 ms. (b) Averaged activation and inactivation curves recorded from Am-Ca_V3-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 (V_act): −47 ± 1 mV, activation slope: 6.7 ± 0.3 mV, voltage for half-inactivation (V_inact): −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 Ca_V3 Ca²⁺ channel produced by the coexpression of the Ca_Vβ subunit. Currents were normalized to the current amplitude of Am-Ca_V3 without Ca_Vβ (n = 4). (e) Pharmacological profile of Am- Ca_V3 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 on Am-Ca_V3 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 “M”) 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 120 h for oral or contact exposure or at 48 h post-injection. (c) Time course of bee mortality after mibefradil injection at two different concentrations. Mibefradil was injected into the median ocellus at 0.11 μg (circle) and 1.1 μg (hexagon) per bee and the effects were compared to solvent-injected bees (control, square).

Figure 3. Neuronal Ca_V3 Ca2⁺ channel and odor learning.

(a) In situ hybridization of frontal section of honeybee brain using an Am-Ca_V3 specific RNA probe. Left, Am-Ca_V3 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-Ca_V3 3CT6 antibody (at 1/100) of Hek-293 cells transfected with the Am-Ca_V3 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-Ca_V3-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 Ca_V3 Ca2⁺ channel and locomotion.

(a) Confocal images of isolated muscle cells stained with phalloïdin (left, green) and with Am-Ca_V3 specific antibody 3CT6, (middle, red). Merged staining is shown on the right. Ca_V3 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 Ca_V3 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 on muscle cells (depolarization to −30 mV or 0 mV). Rel cur: currents normalized to the current in absence of mibefradil.