A possible explanation for a neurotoxic effect of the anticancer agent oxaliplatin on neuronal voltage-gated sodium channels.

Oxaliplatin, a new widely used anticancer drug, displays frequent, sometimes severe, acute sensory neurotoxicity accompanied by neuromuscular signs that look like the symptoms observed in tetany and myotonia. The whole cell patch-clamp technique was employed to investigate the oxaliplatin effects on the electrophysiological properties of short-term cultured dorsal unpaired median (DUM) neurons isolated from the CNS of the cockroach Periplaneta americana. Within the clinical concentration range, oxaliplatin (40-500 microM), applied intracellularly, decreased the amplitude of the voltage-gated sodium current resulting in a reduction of half the amplitude of the action potential. For comparison, two other platinum derivatives, cisplatin and carboplatin, were found to be ineffective at reducing the sodium current amplitude. In addition, we compared the oxaliplatin action to those of its metabolites dichloro-diaminocyclohexane platinum (dach-Cl(2)-platin) and oxalate. Oxalate (500 microM) was found to be effective, like oxaliplatin, at reducing the inward sodium current amplitude, whereas dach-Cl(2)-platin (500 microM) failed to change the current amplitude. Interestingly, the effect of oxalate or oxaliplatin could be mimicked by using intracellularly applied 10 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), known as chelator of calcium ions. We concluded that oxaliplatin was capable of altering the voltage-gated sodium channels through a pathway involving calcium ions probably immobilized by its metabolite oxalate. The medical interest of preventing acute neurotoxic side effects of oxaliplatin by infusing Ca(2+) and Mg(2+) is discussed.

Electrophysiological analysis of the neurotoxic action of a funnel-web spider toxin, delta-atracotoxin-HV1a, on insect voltage-gated Na+ channels.

The effects of delta-ACTX-Hv1a, purified from the venom of the funnel-web spider Hadronyche versuta, were studied on the isolated giant axon and dorsal unpaired median (DUM) neurones of the cockroach Periplaneta americana under current- and voltage-clamp conditions using the double oil-gap technique for single axons and the patch-clamp technique for neurones. In parallel, the effects of the toxin were investigated on the excitability of rat dorsal root ganglion (DRG) neurones. In both DRG and DUM neurones, delta-ACTX-Hv1a induced spontaneous repetitive firing accompanied by plateau potentials. However, in the case of DUM neurones, plateau action potentials were facilitated when the membrane was artificially hyperpolarized. In cockroach giant axons, delta-ACTX-Hv1a also produced plateau action potentials, but only when the membrane was pre-treated with 3-4 diaminopyridine. Under voltage-clamp conditions, delta-ACTX-Hv1a specifically affected voltage-gated Na+ channels in both axons and DUM neurones. Both the current/voltage and conductance/voltage curves of the delta-ACTX-Hv1a-modified inward current were shifted 10 mV to the left of control curves. In the presence of delta-ACTX-Hv1a, steady-state Na+ channel inactivation became incomplete, causing the appearance of a non-inactivating component at potentials more positive than -40 mV. The amplitude of this non-inactivating component was dependent on the holding potential. From this study, it is concluded that, in insect neurones, delta-ACTX-Hv1a mainly affects Na+ channel inactivation by a mechanism that differs slightly from that of scorpion alpha-toxins.

Indoxacarb, an oxadiazine insecticide, blocks insect neuronal sodium channels.

1. 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. 2. 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. 3. In voltage-clamp, DCJW resulted in a dose-dependent inhibition (IC(50) 28 nM) of the peak sodium current. Based on IC(50) 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. 4. 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 (IC(50) 30 microM) with various concentrations of DCJW produced a reduction of the apparent potency of DCJW, suggesting that DCJW and lidocaine acted at the same site. 5. DCJW (100 nM) did not affect inward calcium or outward potassium currents. 6. This study describes, for the first time, the action on insect neuronal voltage-dependent sodium channels of Indoxacarb, a new class of insecticides.

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 RDL(ac) Drosophila melanogaster homomer-forming ionotropic GABA receptor subunit, a product of the resistance to dieldrin gene, RDL: GABA (50 microM) 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 microM 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 (E(GABA)=-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 microM fipronil or 1 microM 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 IC(50) 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 P(o) value. Thus both BIDN and fipronil shorten the duration of wild-type RDL(ac) GABA receptor channel openings but appear to act at distinct sites.

Dorsal unpaired median neurones in the insect central nervous system: towards a better understanding of the ionic mechanisms underlying spontaneous electrical activity.

The efferent dorsal unpaired median (DUM) neurones, which include octopaminergic neurones, are among the most intensively studied neurones in the insect central nervous system. They differ from other insect neurones in generating endogenous spontaneous overshooting action potentials. The second half of the 1980s is certain to be considered a turning point in the study of the ion channels underlying the electrical activity of DUM neurones. Recent advances made using the patch-clamp technique have stimulated an increasing interest in the understanding of the biophysical properties of both voltage-dependent and voltage-independent ion channels. Patch-clamp studies of DUM neurones in cell culture demonstrate that these neurones express a wide variety of ion channels. At least five different types of K(+) channel have been identified: inward rectifier, delayed rectifier and A-like channels as well as Ca(2+)- and Na(+)-activated K(+) channels. Moreover, besides voltage-dependent Na(+) and Ca(2+)-sensitive Cl(-) channels, DUM neurones also express four types of Ca(2+) channel distinguished on the basis of their kinetics, voltage range of activation and pharmacological profile. Finally, two distinct resting Ca(2+) and Na(+) channels have been shown to be involved in maintaining the membrane potential and in regulating the firing pattern. In this review, we have also attempted critically to evaluate these existing ion channels with regard to their specific functions in the generation of the different phases of the spontaneous electrical activity of the DUM neurone.

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

A scorpion alpha-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. LqhalphaIT (10-8 m) 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 LqhalphaIT-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). LqhalphaIT 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 x higher than LqhalphaIT. At 10-7 m, LqhalphaIT 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 LqhalphaIT transformed a beating pacemaker activity into a rhythmic bursting.

Nicotine increases [Ca2+]i and regulates electrical activity in insect neurosecretory cells (DUM neurons) via an acetylcholine receptor with 'mixed' nicotinic-muscarinic pharmacology.

An increase in intracellular free calcium concentration ([Ca2+]i) was observed following the application of nicotine to isolated adult dorsal unpaired median (DUM) neurons of the cockroach (Periplaneta americana) terminal abdominal ganglion (TAG) using Fura-2 fluorescence measurements. Bath-applied nicotine (1 mM) induced a transient increase in [Ca2+]i. Calcium responses to bath-applied nicotine were blocked completely by alpha-bungarotoxin (100 nM) and were reduced by 50% in the presence of pirenzepine (1 microM). The sensitivity of the response to both nicotinic and muscarinic antagonists suggested that it was mediated by an acetylcholine receptor with 'mixed' pharmacology. In whole cell current-clamp experiments, nicotine reduced the frequency of evoked action potentials by decreasing the slope of the predepolarization in the last two-thirds of the pacemaker potential. Voltage-clamp studies revealed that nicotine modified the inactivation properties of the maintained low-voltage-activated (LVA) calcium current increasing the rate of relaxation of this current and transforming a U-shaped voltage dependence of inactivation into a monotonic relationship to voltage. These effects were blocked when isolated DUM neurons were pretreated with 0.5 microM alpha-bungarotoxin. Our findings suggested a novel calcium-dependent regulation of firing behavior in TAG DUM neurons following activation of an acetylcholine receptor with 'mixed' pharmacology, resulting in a rise in [Ca2+]i which reduces firing frequency by modulating a maintained LVA calcium current responsible for the action potential predepolarization.

A new scorpion venom toxin paralytic to insects that affects Na+ channel activation. Purification, structure, antigenicity and mode of action.

A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Periplaneta americana and DUM (dorsal unpaired median) neurons, has been purified from the venom of the scorpion Buthus occitanus tunetanus. Its structural, antigenic and pharmacological properties are compared to those of three other groups of neurotoxins found in Buthidae scorpion venoms. Like excitatory, depressant and alpha-type insect-selective neurotoxins, BotIT2 is toxic to insects, but shows the following common and distinctive characteristics. (a) As alpha-type toxins, BotIT2 lack strict selectivity to insects; they have measurable but low toxicity to mice. (b) As depressant toxins and unlike alpha-type toxins, BotIT2 is able to displace iodinated AaHIT from its binding sites in insect neuronal membranes. This indicates that the binding site for BotIT2 is identical, contiguous or in allosteric interaction with that of AaHIT and depressant toxins. (c) The BotIT2 amino acid sequence shows strong similarity to depressant toxins. However, unexpectedly, despite this high sequence similarity, BotIT2 shares moderate cross-antigenic reactivity with depressant toxins. (d) Voltage and current-clamp studies show that BotIT2 induces limited depolarization concomitantly with the development of depolarizing after potential, repetitive activity and later plateau potentials terminated by bursts. Under voltage-clamp conditions, BotIT2 specifically acts on Na+ channels by decreasing the peak Na+ current and by simultaneously inducing a new current with very slow activation/deactivation kinetics. The voltage dependence of this slow current is not significantly different from that of the control current. These observations indicate that BotIT2 chiefly modifies the kinetics of axonal and DUM neuronal membrane Na(+)-channel activation.

Two distinct low-voltage-activated Ca2+ currents contribute to the pacemaker mechanism in cockroach dorsal unpaired median neurons.

1. The contribution of Ca2+ currents to the endogenous firing properties of cockroach isolated adult dorsal unpaired median neurons was investigated using whole cell patch-clamp technique with 5 mM Ca2+ as the charge carrier. At least three types of Ca2+ currents, a high-voltage-activated Ca2+ current and two low-voltage-activated (LVA) Ca2+ currents, have been found in these neurons. This study focused on the LVA Ca2+ currents, which are suitable candidates in the generation of the slow predepolarization because of their low threshold of activation. 2. The global LVA Ca2+ current could be dissociated by means of nickel sensitivity, deactivation time constant and voltage dependence of time to peak, tail current amplitude and inactivation, as transient and maintained LVA Ca2+ currents. 3. The transient LVA Ca2+ current, sensitive to 100 microM Ni2+, was isolated by using a subtraction procedure. It was activated at -70 mV and half-inactivated at -59.5 mV. The inactivation was purely voltage dependent. Current-clamp experiments performed with 150 microM Ni2+ indicated that this current was involved in the initial part of the predepolarization. 4. The maintained LVA Ca2+ current, resistant to 100 microM Ni2+, was activated in a range of potential 10 mV more positive than the transient LVA Ca2+ current, and its voltage dependence of inactivation displayed a U-shaped-curve. 5. Replacing Ca2+ with Ba2+ in equimolar amount or low internal Ca2+ concentration [5 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) in the pipette] induced a monotonic voltage dependence of inactivation and increased the rate of relaxation of this current. These effects were mimicked by high internal Ca2+ concentration [0.1 mM Ca2+ and no ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette]. This demonstrated an unusual Ca2+-sensitive inactivation process that varied over a narrow range of Ca2+ concentrations. 6. Current-clamp experiments performed under 150 microM Ni2+, with 15 mM external Ca2+ concentration (which potentiated the maintained LVA current within 30 s of superfusion) or with 5 mM BAPTA in the pipette demonstrated the participation of this current in the last two-thirds of the slow predepolarizing phase. 7. Our findings demonstrated, for the first time in neurosecretory cells, the coexistence of two distinct LVA Ca2+ currents, which have specialized function in the generation of the pacemaker activity.

Separation and identification of multiple potassium currents regulating the pacemaker activity of insect neurosecretory cells (DUM neurons).

1. Whole cell voltage-clamp studies performed in isolated adult neurosecretory cells identified as dorsal unpaired median (DUM) neurons of the terminal abdominal ganglion of the cockroach Periplaneta americana have allowed us to reveal a complex voltage-dependent outward current regulating the pacemaker activity. 2. The global outward current remaining after tetrodotoxin treatment was activated by depolarization above -50 mV, showing steep voltage dependence and outward rectification. 3. We used tail current analysis to determine the ionic selectivity of this outward current. The reversal potentials for two extracellular potassium concentrations (-92.7 and -65.4 mV for 3.1 and 10 mM, respectively) is consistent with the expected equilibrium potential for potassium ions. 4. Both peak and sustained components of the global outward K+ current were reduced by external application of 20 mM tetraethylammonium chloride, 10 nM iberiotoxin, 1 nM charybdotoxin (CTX) and 1 mM cadmium chloride. Subtraction of current recorded in CTX solution from that in control solution revealed an unusual biphasic Ca(2+)-dependent K+ current. The fast transient current resistant to 5 mM 4-aminopyridine (4-AP) is distinguished by its dependence on holding potential and time course from the late sustained current. 5. In addition, two other components of CTX-resistant outward K+ current could be separated by sensitivity to 4-AP, time course, and voltage dependence. Beside a calcium-independent delayed outwardly rectifying current, a 4-AP-sensitive fast transient current resembling the A-current has been also identified. It activates at negative potential (about -65 mV) and unlike the A-current of other neurons, it inactivates rapidly with complex inactivation kinetics. A-like current is half-inactivated at -63.5 mV and half-activated at -35.6 mV. 6. Our findings demonstrate for the first time in DUM neuron cell bodies the existence of multiple potassium currents underlying the spontaneous electrical activity. Their identification and characterization represent a fundamental step in further understanding the pacemaker properties of these insect neurosecretory cells.

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.