Orthosteric muscarinic receptor activation by the insect repellent IR3535 opens new prospects in insecticide-based vector control

Abstract

The insect repellent IR3535 is one of the important alternative in the fight against mosquito-borne disease such as malaria, dengue, chikungunya, yellow fever and Zika. Using a multidisciplinary approach, we propose the development of an innovative insecticide-based vector control strategy using an unexplored property of IR3535. We have demonstrated that in insect neurosecretory cells, very low concentration of IR3535 induces intracellular calcium rise through cellular mechanisms involving orthosteric/allosteric sites of the M1-muscarinic receptor subtype, G protein βγ subunits, background potassium channel inhibition generating depolarization, which induces voltage-gated calcium channel activation. The resulting internal calcium concentration elevation increases nicotinic receptor sensitivity to the neonicotinoid insecticide thiacloprid. The synergistic interaction between IR3535 and thiacloprid contributes to significantly increase the efficacy of the treatment while reducing concentrations. In this context, IR3535, used as a synergistic agent, seems to promise a new approach in the optimization of the integrated vector management for vector control.

Figure 1

The insect repellent IR3535 induces a non-monotonic concentration-dependent intracellular calcium rise in insect DUM neurosecretory cell body. (a) The chemical structure of acetylcholine is shown together with the structure of the repellent IR3535, the selective M1- mAChR subtype agonist oxotremorine-M, the selective M1-mAChR subtype antagonist pirenzepine and the selective positive allosteric modulator of the M1-mAChR subtype (BQCA). (b) Bath application of IR3535 produces a multiphasic semi-logarithmic concentration-response curve with the first component reaching a maximum effect obtained at very low concentration (i.e., 10 nM IR3535; square and arrow) as illustrated in comparative histogram in (c). (d) Images of Fura-2 fluorescence of a single DUM neuron cell body after application of 10 nM IR3535. The elevation in intracellular calcium concentration is measured in parallel at the membrane cell body level (1) and at the intracellular medium level (2). Note that low concentration of IR3535 (10 nM) only induces calcium response in the membrane cell body region. (e) Comparative histograms illustrating that higher concentrations of IR3535 (i.e., 10 µM) produce intracellular calcium rise at both membrane cell body and intracellular medium levels. Data are means ± S.E.M. (n = 5); statistical test is Student unpaired t-test **p < 0.01; ns, non significant. Scale bar 55 µm.

Figure 2

IR3535 increases intracellular calcium concentration through interaction with M1- mAChR subtype and voltage-gated calcium channel activation. (a) Histogram summarizing the blocking effects of the pretreatment with EGTA-buffered calcium-free solution and ω-CgTX GVIA, the potent N-type high voltage-activated calcium channel inhibitor, on IR3535-induced elevation of intracellular calcium concentration. (b,c) illustrate the inhibitory action of pirenzepine (PZP, (b) the selective M1-mAChR subtype antagonist, on intracellular calcium rise produced by IR3535 whereas TMB-8, the IP3 receptor inhibitor (b) and caffeine (c) do not produce any effect on the intracellular calcium elevation. (d) is a representative example of the DUM neuron cell body beating pacemaker activity recorded using the whole-cell patch-clamp technique in control and in the presence of bath applied low concentration of IR3535 (10 nM). (e,f,g,h) Comparative histograms showing the effects of IR3535 (10 nM) on different beating pacemaker electrophysiological parameters including membrane potential (e), action potential discharge frequency in control (f), with pirenzepine (PZP) (g) and with U73122, an inhibitor of PLC (h). (i) Hypothetic model illustrating the participation of the membrane molecular events identified as M1 muscarinic acetylcholine receptor (mAChR) subtype and high voltage-activated calcium channels (HVACC) involved in the intracellular calcium elevation induced by the insect repellent IR3535 used at low concentration (10 nM). Average data is shown as mean ± S.E.M. (n = 6); statistical test is Student unpaired t-test **p < 0.01; *p < 0.05; ns, non significant.

Figure 3

Involvement of the background potassium channel inhibition through a Gβγ-dependent mechanism in the IR3535-induced calcium rise. (a) IR3535 (10 nM) induces an increase of the input membrane resistance studied under current-clamp in response to a 100-ms hyperpolarizing current pulse. Reduced equivalent circuit of the DUM neuron cell body plasma membrane obtained by combining a fixed capacitance (Cm) in parallel with ion-specific pathway (Rm). The reciprocal of electrical membrane resistance (Rm) is conductance (G). If the resistance to movement of ion across the membrane is high then the conductance of the membrane to this particular ion is low. (b) Histogram summarizing the membrane potential recorded in response to a hyperpolarizing current pulse (100 ms in duration) in control and in the presence of IR3535 (10 nM) and pirenzepine (PZP). (c,d) Involvement of background potassium channels (BgKC) in the effect of IR3535. IR3535-induced increase of the input membrane resistance is completely inhibited by the potassium channel blocker TEA-Cl (c). Because BgKC, open at the resting state (i.e., −50mV), mediates a permanent spontaneous outward potassium conductance, the resulting steady-state inwardly directed current recorded, under voltage-clamp, in the presence of IR3535 (10 nM) correlate well with the switch from the open to closed state of potassium channels (d). (d,e) This inward current amplitude is strongly reduced when the holding membrane potential is hyperpolarized to −100mV, which corresponds to the calculated Nernstian equilibrium potential for potassium ions (−100.8 mV, in our experimental conditions) (d) and in the presence of SP-related peptide (0.1 mg/mL), known to inhibit binding of G proteins to mAChR (e). (f) Histograms illustrating the involvement of Gβγ protein in IR3535-induced increase of the input membrane resistance recorded following intracellularly applied GDP-β-S and gallein, a specific Gβγ dimer signaling inhibitor. (g) Hypothetic scheme illustrating the participation of the molecular events involved in the effect of IR3535 via M1 muscarinic acetylcholine receptor subtype (M1-mAChR) activation. IR3535 induces inhibition of BgKC through Gβγ-dependent mechanism. This produces membrane depolarization (Vm > 0) and activation of high voltage-activated calcium channel (HVACC), resulting in the intracellular calcium rise. Individual controls were normalized as percentage of control. Data are means ± S.E.M. (n = 5); statistical test is Student unpaired t-test *p < 0.05; ns, non significant.

Figure 4

Computational docking results of ligands interactions at the orthosteric site of M1 mACh receptor. The highest affinity poses of: (a) IR3535 (black circle), (b) pirenzepine (red sticks) and (c) oxotremorine-M (black sticks) located at the orthosteric site of M1-mAChR. The mean value and standard deviation of binding energy are calculated for each ligand based on the five runs. The energy of binding is respectively: (−6.2 ± 0.4) kcal/mol for IR3535, (−7.9 ± 0.4) kcal/mol for pirenzepine and (−5.9 ± 0.6) kcal/mol for oxotremorine-M. (d) Close-up view of the highest affinity pose of IR3535 from the top view of M1-mAChR. Residues observed in the close vicinity of IR3535 are displayed in stick representation and coded by the color of transmembrane helices: TM1 - orange, TM2 - green, TM3 - dark blue, TM4 - yellow, TM5 - red, TM6 - magenta, TM7 - light blue. (e) Residues with the highest contribution to the energy of binding of IR3535 at the orthosteric site of M1-mAChR.

Figure 5

High concentration of IR3535 generates outward potassium current following the early inward current. (a) Under current-clamp, in control condition (1) and in the presence of pirenzepine (PZP,100 nM), IR3535 (100 nM) produces a dual effect on DUM neuron action potential discharge frequency. The first effect corresponds to an increase of the action potential frequency (2), followed by an important reduction (3). (b) Histogram representing the dual effect of IR3535 (100 nM) on spontaneous electrical activity, recorded at different times of exposure, as indicated. (c) Under voltage-clamp, at a holding potential of −50mV and in the presence of pirenzepine (PZP, 100 nM), IR3535 (100 nM) induces an early steady-state inward current (2) followed by an outwardly directed current (3). (d) The outward current (3) is reduced in the presence of TEA-Cl (10 mM) and when the resting membrane potential is hyperpolarized to −100mV, corresponding to the calculated Nernstian equilibrium potential for potassium ions (−100.8 mV). (e,f) IR3535 (100 nM) induces an increase (2) and a decrease (3) in the input membrane resistance studied under current-clamp in response to a 100-ms hyperpolarizing current pulse. Both inward (2) and outward (3) currents correlate well with the increase (2) and decrease (3) in the input membrane resistance, which thereby influence the action potential discharge frequency. (g) Spontaneous action potentials recorded in the presence of pirenzepine (PZP; 500 nM) and after bath application of IR3535 (100 nM). (h) Histogram illustrating the effects of bath application of IR3535 (100 nM) on DUM neuron cell body action potential discharge frequency, pretreated with PZP (500 nM; (1) control). After 15 s of exposure, the pacemaker activity decreases (2) and then finally stoppes (3). The increase in the action potential discharge frequency is never observed with PZP used at 500 nM. Data are shown as mean ± S.E.M. (n = 6–8), statistical test is Student unpaired t-test *p < 0.05; ns, non significant. (i,j) Schemes suggesting that high concentration of IR3535 could interact with two distinct sites (i.e., orthosteric (OS) and putative allosteric (AS) sites) on M1-mAChR. In addition, high concentration of IR3535 (100 nM) can displace PZP from orthosteric site (j) indicating that more concentration of PZP is required to get full effect on orthosteric site (i,j).

Figure 6

High concentration of IR3535 interacts with the allosteric site on M1-mAChR. (a) Comparative histogram showing the inhibition of the outward potassium current, recorded under voltage-clamp at a holding potential of −50mV, induced by IR3535 (100 nM) by pretreatment with the well known M1-mAChR allosteric modulator BQCA (100 nM). In the absence of orthosteric site agonist, BQCA (100 nM) applied alone has no significant effect. It is interesting to note that in the presence of BQCA, IR3535 (100 nM) never generates inward current, reflecting the activation of orthosteric site. Average data is shown as mean ± S.E.M. (n = 5–7), statistical test is Student unpaired t-test *p < 0.05; ns, non significant. (b–d) Hypothetic schemes illustrating that i) the high selective allosteric modulator of M1-mAChR, BQCA completely inhibits the outward current induced by IR3535 (100 nM) and ii) high concentration of IR3535 (100 nM) could also interacts with a different site than the M1-mAChR orthosteric site on M1-mAChR, reinforcing the involvement of allosteric binding site in the complex effect of IR3535.

Figure 7

Computational docking results of ligands interactions at the allosteric site of M1 mACh receptor. The highest affinity poses of: IR3535 (red circle) (a) and BQCA (blue sticks in red circle) (b) located at the allosteric site of M1-mAChR. In panel (a,b) the pose of IR3535 at the orthosteric site is also displayed for clarity (below red circle). The mean value and standard deviation of binding energy has been calculated for each ligand based on the five runs. The energy of binding is: (−5.1 ± 0.1) kcal/mol for IR3535 and (−8.5 ± 0.1) kcal/mol for BQCA, respectively. (c) Close-up view of the highest affinity pose of IR3535 from the top view of M1-mAChR. Residues observed in the close vicinity of IR3535 are displayed in stick representation and coded by the color of transmembrane helices: TM1 - orange, TM2 - green, TM3 - dark blue, TM4 - yellow, TM5 - red, TM6 - magenta, TM7 - light blue. (d) Residues with the highest contribution to the energy of binding of IR3535 at the allosteric site of M1-mAChR.

Figure 8

Allosteric site activation limits ligand effect with orthosteric site of M1-mAChR in DUM neurons. (a) Comparative histogram illustrating the inward current amplitude generated by the well known M1-mAChR agonist oxotremorine-M (OXO) and IR3535 recorded under voltage-clamp at a holding potential of −50mV. Number in brackets indicated above each bar represent the different experimental conditions. (b–f) Hypothetic models based on the results obtained in (a). Number in brackets mentioned below each schemes correlates well with those indicated in (a). Average data is shown as mean ± S.E.M. (n = 3–11); statistical test is Student unpaired t-test, **p < 0.01; *p < 0.05; ns, non significant.).

Figure 9

Low concentration oncentration of IR3535, used as synergistic agent, increases the effect of thiacloprid, a neonicotinoid insecticide. (a,b,c) Histograms illustrating the concentration-dependent amplitudes of the thiacloprid-induced inward currents recorded under voltage-clamp conditions at a steady-state holding potential of −50 mV, applied alone at 100 nM (a), 1 µM (b) and 10 µM (c) and after pretreatment with 10 nM IR3535. Note that IR3535 significantly potentiates the amplitude of the current produced by thiacloprid (a,b) whereas it is strongly decreased for high concentration of thiacloprid (c). Data are means ± S.E.M. (n = 4–7), statistical test is Student unpaired t-test *p < 0.05; **p < 0.01. (d) Superimposed semi-logarithmic concentration-response curves for the inward current induced by thiacloprid applied alone and in the presence of 10 nM IR3535. Inset represents the chemical structure of the neonicotinoid insecticide, thiacloprid. (e) Bath application of 10 µM thiacloprid increases intracellular calcium concentration in Fura-2 loaded DUM neuron cell body. (f) Histogram illustrating that CdCl₂ (1 mM), an inorganic calcium channel blocker counteracts the effect of IR3535 (10 nM) applied in combination with high concentration of thiacloprid (10 µM). Data are means ± S.E.M. (n = 3–9), statistical test is Student unpaired t-test, *p < 0.05; **p < 0.01.

Figure 10

Diagram representing the molecular events involved in the activation of M1 muscarinic acetylcholine receptor by IR3535. (a) IR3535, depending on the concentration tested, binds to distinct sites (M1-mAChR orthosteric and allosteric sites) that couple to the heterotrimeric G protein underlying the signal transduction that leads to IR3535-induced modulation of BgKC. (b) At low concentration, IR3535 interacts with M1-mAChR orthosteric site, which stimulation leads to trigger Gαβγ dissociation. The release of Gβγ subunits inhibits background potassium current, resulting in membrane depolarization, which thereby activates high-voltage-activated calcium channels governing calcium influx. (c) At higher concentration, IR3535 induces dual effect on both M1-mAChR orthosteric and allosteric sites, depending on the time of exposure. Within the first seconds, IR3535 stimulates the orthosteric site (1) before interacting with the allosteric site (2). This last effect renders the G protein inactivated (3), which maintains the BgKC functional resulting in the hyperpolarized potential where high-voltage-activated calcium channels remain closed. Vm > 0 and Vm < 0 mean depolarization and hyperpolarization, respectively. M1-mAChR, M1 muscarinic acetylcholine receptor subtype; HVACC, high-voltage-activated calcium channels; BgKC, background potassium channels; OS and AS orthosteric and allosteric sites, respectively.