Françoise Grolleau

Indoxacarb, an oxadiazine insecticide, blocks insect neuronal sodium channels

Decarbomethoxyllated JW062 (DCJW), the active component of a new oxadiazine insecticide DPX‐JW062 (Indoxacarb), was tested on action potentials and the inward sodium current recorded from short‐term cultured dorsal unpaired median neurones of the cockroach Periplaneta americana. Under whole‐cell current‐clamp conditions, 100 nM DCJW reduced the amplitude of action potentials and induced a large hyperpolarization of the resting membrane potential associated with a 41% increase in input resistance. In voltage‐clamp, DCJW resulted in a dose‐dependent inhibition (IC50 28 nM) of the peak sodium current. Based on IC50 values, the effect of DCJW was about 10 fold less potent than tetrodotoxin (TTX) but 1000 fold more potent than the local anaesthetic lidocaine. DCJW (100 nM) was without effect on activation properties of the sodium current, reversal potential, voltage dependence of sodium conductance and on both fast and slow steady‐state inactivations. TTX (2 nM) resulted in 48% inhibition of the peak inward sodium current. Co‐application of TTX (2 nM) with various concentrations of DCJW produced an additional inhibition of the peak inward current, indicating that the blocking actions of DCJW and TTX were distinct. Co‐application of lidocaine (IC50 30 μM) with various concentrations of DCJW produced a reduction of the apparent potency of DCJW, suggesting that DCJW and lidocaine acted at the same site. DCJW (100 nM) did not affect inward calcium or outward potassium currents. This study describes, for the first time, the action on insect neuronal voltage‐dependent sodium channels of Indoxacarb, a new class of insecticides.

Contryphan-Vn: a modulator of Ca2+-dependent K+ channels ☆

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.

Two distinct calcium-sensitive and -insensitive PKC up- and down-regulate an α-bungarotoxin-resistant nAChR1 in insect neurosecretory cells (DUM neurons)

While there is mounting knowledge about the structure and diversity of insect neuronal nicotinic acetylcholine receptors, less attention has been directed towards their intracellular regulation by calcium‐mediated activation or inhibition of protein phosphorylation. The main goal of this work was to delineate the chain of molecular events that lead to the up‐ and down‐regulation by two protein kinase Cs of an insect neuronal α‐bungarotoxin‐resistant nicotinic acetylcholine receptor (called nAChR1). The native nicotinic acetylcholine receptor intracellular regulation was studied on dissociated adult dorsal unpaired median neurons isolated from the terminal abdominal ganglion of the cockroach Periplaneta americana using whole‐cell patch‐clamp technique and calcium imaging. We report that under 0.5 µmα‐bungarotoxin treatment, the inward current produced by pressure ejection application of nicotine onto the cell body was differentially sensitive to specific protein kinase C activators and inhibitors. The phorbol ester PMA produced a calcium‐dependent increase in current amplitude blocked by chelerythrine. By contrast, the diacylglycerol analogue 1,2‐dioctanoyl‐sn‐glycerol produced a calcium‐independent reduction of the nicotinic response, reversed by rottlerin and chelerythrine. This indicated that two protein kinase C isozymes (‘classical’ and ‘novel’ protein kinase C, named PKC1 and PKC2, respectively) up‐ and down‐regulated nicotinic acetylcholine receptor function. PMA and 1,2‐dioctanoyl‐sn‐glycerol effects were mimicked by pirenzepine‐sensitive M1 muscarinic receptor subtype coupled to phospholipase C second messenger pathway. Low concentration of muscarine elevated internal calcium levels, which thereby activated PKC1. By contrast, a high concentration of muscarine strongly increased [Ca 2+]i, which induced inhibition of PKC1. This effect was reversed by FK506, suggesting the implication of PP2B which unmasked PKC2 activity mediating down‐regulation of nicotinic acetylcholine receptor.

Single channel analysis of the blocking actions of BIDN and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a Drosophila cell line

Single channel recordings were obtained from a Drosophila S2 cell line stably expressing the wild‐type RDLac Drosophila melanogaster homomer‐forming ionotropic GABA receptor subunit, a product of the resistance to dieldrin gene, Rdl. GABA (50 μM) was applied by pressure ejection to outside‐out patches from S2‐RDL cells at a holding potential of −60 mV. The resulting inward current was completely blocked by 100 μM picrotoxin (PTX). The unitary current‐voltage relationship was linear at negative potentials but showed slight inward rectification at potentials more positive than 0 mV. The reversal potential of the current (EGABA=−1.4 mV) was close to the calculated chloride equilibrium potential. The single channel conductance elicited by GABA was 36 pS. A 71 pS conductance channel was also observed when the duration of the pulse, used to eject GABA, was longer than 80 ms. The mean open time distribution of the unitary events was fitted best by two exponential functions suggesting two open channel states. When either 1 μM fipronil or 1 μM BIDN was present in the external saline, the GABA‐gated channels were completely blocked. When BIDN or fipronil was applied at a concentration close to the IC50 value for suppression of open probability (281 nM, BIDN; 240 nM, fipronil), the duration of channel openings was shortened. In addition, the blocking action of BIDN resulted in the appearance of a novel channel conductance (17 pS). The effects of co‐application of BIDN and fipronil were examined. Co‐application of BIDN (300 nM) with various concentrations (100–1000 nM) of fipronil resulted in an additional BIDN‐induced dose‐dependent reduction of the maximum Po value. Thus both BIDN and fipronil shorten the duration of wild‐type RDLac GABA receptor channel openings but appear to act at distinct sites.

Biophysical properties of scorpion α‐toxin‐sensitive background sodium channel contributing to the pacemaker activity in insect neurosecretory cells (DUM neurons)

A scorpion α‐toxin‐sensitive background sodium channel was characterized in short‐term cultured adult cockroach dorsal unpaired median (DUM) neurons using the cell‐attached patch‐clamp configuration. Under control conditions, spontaneous sodium currents were recorded at different steady‐state holding potentials, including the range of normal resting membrane potential. At −50 mV, the sodium current was observed as unclustered, single openings. For potentials more negative than −70 mV, investigated patches contained large unitary current steps appearing generally in bursts. These background channels were blocked by tetrodotoxin (TTX, 100 nm), and replacing sodium with TMA‐Cl led to a complete loss of channel activity. The current–voltage relationship has a slope conductance of 36 pS. At −50 mV, the mean open time constant was 0.22 ± 0.05 ms (n = 5). The curve of the open probability versus holding potentials was bell‐shaped, with its maximum (0.008 ± 0.004; n = 5) at –50 mV. LqhαIT (10–8m) altered the background channel activity in a time‐dependent manner. At −50 mV, the channel activity appeared in bursts. The linear current–voltage relationship of the LqhαIT‐modified sodium current determined for the first three well‐resolved open states gave three conductance levels: 34, 69 and 104 pS, and reversed at the same extrapolated reversal potential (+52 mV). LqhαIT increased the open probability but did not affect either the bell‐shaped voltage dependence or the open time constant. Mammal toxin AaHII induced very similar effects on background sodium channels but at a concentration 100 × higher than LqhαIT. At 10–7m, LqhαIT produced longer silence periods interrupted by bursts of increased channel activity. Whole‐cell experiments suggested that background sodium channels can provide the depolarizing drive for DUM neurons essential to maintain beating pacemaker activity, and revealed that 10–7 m LqhαIT transformed a beating pacemaker activity into a rhythmic bursting.

Transient Na(+)-activated K+ current in beating pacemaker-isolated adult insect neurosecretory cells (dum neurones).

A whole-cell sodium-activated outward current has been identified in adult cockroach dorsal unpaired median (DUM) neurones maintained in short-term culture. Superfusion of 100 nM TTX completely blocked the inward current but also reduced the transient outward component without affecting the sustained outward current. Different experimental procedures indicate that TTX effects could not be due to a voltage clamp artefact. Similar effects were obtained when extracellular sodium was replaced with Tris-HCl. The outward current was unaffected by TTX when the membrane was stepped to potential more positive (+60 mV) than sodium reversal potential and the TTX-sensitive outward current amplitude increased in parallel with the sodium inward current at each potential tested. The tail current analysis was used to determine the ionic selectivity of the TTX-sensitive outward current. Tail currents reversed polarity at -95.5 mV (potassium equilibrium potential: -100.5 mV), indicating that sodium-activated outward current was carried mostly by potassium ions.

Bot IT2, a toxin paralytic to insects from the Buthus occitanus tunetanus venom modifying the activity of insect sodium channels

The effects of insect toxin Bot IT2, purified from the venom of the scorpion Buthus occitanus tunetanus, were investigated on the isolated giant axon and on isolated dorsal unpaired median (DUM) neurone of the cockroach Periplaneta americana under current- and voltage-clamp conditions, using the double-oil-gap technique and the patch-clamp technique, respectively. In both preparations, Bot IT2, induces a limited depolarization together with the development of a repetitive activity in axon and an increase of spontaneous discharge frequency in DUM neurone. After artificial hyperpolarization to the normal resting level, plateau potentials can be evoked in both preparations. Under voltage-clamp conditions, Bot IT2 induces a similar effect in axon and DUM neurone by acting specifically on sodium channels. The peak sodium current is decreased and simultaneously a new current with very slow activation-deactivation kinetics is developed. Voltage dependence of this slow current is not very different from that of the control. The inactivation of the fast component is incomplete because it is masked by the development of the slow component. These results suggest that Bot IT2 modifies the kinetics of insect sodium channel activation, and the transformation of normal fast channels into slow ones is discussed.

Indirect activation of neuronal noncapacitative Ca2+ entry is the final step involved in the neurotoxic effect of Tityus serrulatus scorpion β‐toxin

Interweaving strategies of electrophysiology, calcium imaging and immunocytochemistry bring new insights into the mode of action of the Brazilian scorpion Tityus serrulatusβ‐toxin VII. Pacemaker dorsal unpaired median neurons isolated from the cockroach central nervous system were used to study the effects of toxin VII. In current‐clamp, 50 nm toxin VII produced a membrane depolarization and reduced spiking. At 200 nm, depolarization associated with multiphasic effects was seen. After artificial hyperpolarization, plateau potentials on which spontaneous electrical activity appeared were observed. In voltage clamp, toxin VII induced a negative shift of the voltage dependence of sodium current activation without significant effect on steady‐state inactivation. In addition, toxin VII produced a permanent TTX‐sensitive holding inward current, indicating that background sodium channels were targeted by β‐toxin. Cell‐attached patch recordings indicated that these channels were switched from unclustered single openings to current fluctuating between distinct subconductance levels exhibiting increased open probability and open‐time distribution. Toxin VII also produced a TTX‐sensitive [Ca2+]i rise. Immunostaining with Cav2.2(α1b) antibodies and calcium imaging data obtained with ω‐CgTx GVIA indicated that N‐type high‐voltage‐activated calcium channels initiated calcium influx and were an essential intermediate in the pathway linking toxin VII‐modified sodium channels to the activation of an additional route for calcium entry. By using inhibitors of (i) noncapacitative calcium entry (inhibitor LOE‐908), (ii) NO‐sensitive guanylyl cyclase (ODQ) and (iii) phosphodiesterase 2 (EHNA), together with cGMP antibodies, we demonstrated that noncapacitative calcium entry was the final step in a complex combination of events that was initiated by toxin VII‐alteration of sodium channels and then involved successive activation of other membrane ion channels.

Insect background sodium channel as a new target for scorpion alpha toxin

The effects of an anti-insect scorpion alpha toxin, LqhαIT, isolated from the venom of the Israeli scorpion Leiurus quinquestriatus hebraeus was studied on isolated adult dorsal unpaired median (DUM) neurones isolated from the cockroach Periplaneta americana terminal abdominal ganglion. Using the cell-attached patch-clamp configuration, a new type of sodium channel, called background sodium channel (bNa), was recently characterized. At -50mV, the channel activity was observed as unclustered brief single openings. For hyperpolarized steady-state holding potential (-100mV) the patches contained large unitary current steps, appearing generally in bursts. The open probability (Po) calculated at -50mV was low (0.008 (±0.004), n =5) and displayed a typical bell-shaped voltage dependence. LqhαIT (10 -8 M) altered the bNa activity in a time-dependent manner. At -50mV the channel activity appeared in bursts. Po calculated at -50mV was about 20 times greater than Po calculated in controls and also showed bell-shaped voltage dependence. At 10 -7 M, LqhαIT induced longer silent periods interrupted by bursts of increased channel activity. Whole-cell recordings revealed that 10 -7 M LqhαIT transformed regular beating DUM neurone pacemaker activity into a rhythmic bursting. In this paper we demonstrate, for the first time, that bNa is a new target for anti-insect scorpion toxin.