Edited GluR2, a gatekeeper for motor neurone survival?

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disorder of motor neurones. Although the genetic basis of familial forms of ALS has been well explored, the molecular basis of sporadic ALS is less well understood. Recent evidence has linked sporadic ALS with the failure to edit key residues in ionotropic glutamate receptors, resulting in excessive influx of calcium ions into motor neurones which in turn triggers cell death. Here we suggest that edited AMPA glutamate (GluR2) receptor subunits serve as gatekeepers for motor neurone survival.

Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study.

Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation.

Sgbeta1, a novel locust (Schistocerca gregaria) non-alpha nicotinic acetylcholine receptor-like subunit with homology to the Drosophila melanogaster Dbeta1 subunit.

The cloning, sequencing and functional expression of Sgbeta1, a novel locust (Schistocerca gregaria) non-alpha nicotinic acetylcholine receptor (nAChR) subunit is described. This subunit shows 80% identity with the Drosophila melanogaster Dbeta1 and 92% identity with the Locusta migratoria beta1, non-alpha subunits but only 38% identity to Sgalpha1 (also referred to as alphaL1), a previously cloned S. gregaria nAChR alpha-subunit. When expressed in Xenopus laevis oocytes, Sgbeta1 does not respond to nicotine. Responses to nicotine are observed, however, in oocytes co-expressing Sgalpha1 and Sgbeta1, but the pharmacology is indistinguishable from that of currents produced by expressing Sgalpha1 alone. We conclude that either Sgbeta1 does not co-assemble with Sgalpha1, or that it is unable to contribute to the functional properties of the receptor, in the Xenopus oocyte expression system.

Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors.

Imidacloprid is increasingly used worldwide as an insecticide. It is an agonist at nicotinic acetylcholine receptors (nAChRs) and shows selective toxicity for insects over vertebrates. Recent studies using binding assays, molecular biology and electrophysiology suggest that both alpha- and non-alpha-subunits of nAChRs contribute to interactions of these receptors with imidacloprid. Electrostatic interactions of the nitroimine group and bridgehead nitrogen in imidacloprid with particular nAChR amino acid residues are likely to have key roles in determining the selective toxicity of imidacloprid. Chemical calculation of atomic charges of the insecticide molecule and a site-directed mutagenesis study support this hypothesis.

Alternative splicing of a Drosophila GABA receptor subunit gene identifies determinants of agonist potency.

Alternative splicing of the Drosophila melanogaster Rdl gene yields four ionotropic GABA receptor subunits. The two Rdl splice variants cloned to date, RDL(ac) and RDL(bd) (DRC17-1-2), differ in their apparent agonist affinity. Here, we report the cloning of a third splice variant of Rdl, RDL(ad). Two-electrode voltage clamp electrophysiology was used to investigate agonist pharmacology of this expressed subunit following cRNA injection into Xenopus laevis oocytes. The EC(so) values for GABA and its analogues isoguvacine, muscimol, isonipecotic acid and 3-amino sulphonic acid on the RDL(ad) homomeric receptor differed from those previously described for RDL(ac) and DRC17-1-2 receptors. In addition to providing a possible physiological role for the alternative splicing of Rdl, these data delineate a hitherto functionally unassigned region of the N-terminal domain of GABA receptor subunits, which affects agonist potency and aligns closely with known determinants of potency in nicotinic acetylcholine receptors. Thus, using expression in Xenopus oocytes, we have demonstrated differences in agonist potency for the neurotransmitter GABA (and four analogues) between splice variant products of the Drosophila melanogaster Rdl gene encoding homomer-forming GABA receptor subunits.

K(+) currents in cultured neurones from a polyclad flatworm.

Cells from the brain of the polyclad flatworm Notoplana atomata were dispersed and maintained in primary culture for up to 3 weeks. Whole-cell patch-clamp of presumed neurones revealed outwardly directed K(+) currents that comprised, in varying proportions, a rapidly activating (time constant tau =0.94+/-0.79 ms; N=15) and inactivating ( tau =26.1+/-1.9 ms; N=22) current and a second current that also activated rapidly ( tau =1.1+/-0.2 ms; N=9) (means +/- s.e.m.) but did not inactivate within 100 ms. Both current types activated over similar voltage ranges. Activation and steady-state inactivation overlap and are markedly rightward-shifted compared with most Shaker-like currents (half-activation of 16.9+/-1. 9 mV, N=7, half-inactivation of -35.4+/-3.0 mV, N=5). Recovery from inactivation was rapid (50+/-2.5 ms at -90 mV). Both currents were unaffected by tetraethylammonium (25 mmol l(-1)), whereas 4-aminopyridine (10 mmol l(-1)) selectively blocked the inactivating current. The rapidly inactivating current, like cloned K(+) channels from cnidarians and certain cloned K(+) channels from molluscs and the Kv3 family of vertebrate channels, differed from most A-type K(+) currents reported to date. These findings suggest that K(+) currents in Notoplana atomata play novel roles in shaping excitability properties.

Cultured insect mushroom body neurons express functional receptors for acetylcholine, GABA, glutamate, octopamine, and dopamine.

Fluorescence calcium imaging with fura-2 and whole cell, patch-clamp electrophysiology was applied to cultured Kenyon cells (interneurons) isolated from the mushroom bodies of adult crickets (Acheta domesticus) to demonstrate the presence of functional neurotransmitter receptors. In all cells investigated, 5 microM acetylcholine (ACh, n = 52) evoked an increase in intracellular free calcium ([Ca2+]i). Similar effects were observed in response to 10 microM nicotine. The ACh response was insensitive to atropine (50 microM) but was reduced by mecamylamine (50 microM) and alpha-bungarotoxin (alpha-bgt, 10 microM). ACh-induced inward ion currents (n = 28, EACh approximately 0 mV) were also blocked by 1 microM mecamylamine and by 1 microM alpha-bgt. Nicotine-induced inward currents desensitized more rapidly than ACh responses. Thus functional alpha-bgt-sensitive nicotinic ACh receptors are abundant on all Kenyon cells tested, and their activation leads to an increase in [Ca2+]i. gamma-Aminobutyric acid (GABA, 100 microM) triggered a sustained decrease in [Ca2+]i. Similar responses were seen with a GABAA agonist, muscimol (100 microM), and a GABAB agonist, 3-APPA (1 mM), suggesting that more than one type of GABA receptor can affect [Ca2+]i. This action of GABA was not observed when the extracellular KCl concentration was lowered. All cells tested (n = 26) with patch-clamp electrophysiology showed picrotoxinin (PTX)-sensitive, GABA-induced (30-100 microM) currents with a chloride-sensitive reversal potential. Thus, an ionotropic PTX-sensitive GABA receptor was found on all Kenyon cells tested. Most (61%) of the 54 cells studied responded to -glutamate (100 microM) application either with a biphasic increase in [Ca2+]i or with a single, delayed, sustained [Ca2+]i increase. Nearly all cells tested (95%, n = 19) responded to (100 microM) -glutamate with rapidly desensitizing, inward currents that reversed at approximately -30 mV. Dopamine (100 microM) elicited either a rapid or a delayed increase in [Ca2+]i in 63% of the 26 cells tested. The time course of these responses varied greatly among cells. Dopamine failed to elicit currents in patch-clamped cells (n = 4). A brief decrease in [Ca2+]i was induced by octopamine (100 microM) in approximately 54% of the cells tested (n = 35). However, when extracellular CaCl2 was lowered, octopamine triggered a substantial increase in [Ca2+]i in 35% of the cells tested (n = 26). No octopamine-elicited currents were detected in patched-clamped cells (n = 10).

Antagonist profile and molecular dynamic simulation of a Drosophila melanogaster muscarinic acetylcholine receptor.

A stably-transfected, Drosophila cell line (S2-DMl-1) expressing the Drosophila DMl muscarinic acetylcholine receptor (mAChR) exhibits high-affinity, saturable, specific binding of the radiolabelled muscarinic antagonist [3H]-N-methyl scopolamine ([3H]-NMS) with an equilibrium dissociation constant (Kd) of 0.67 +/- 0.02 and a Bmax of 1.53 +/- 0.3 pmol/mg protein. Displacement of [3H]-NMS by mAChR antagonists results in the pharmacological profile: 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > hexahydrosiladifenidol > p-fluorohexahydrosiladifenidol > nitrocaramiphen > pirenzepine > methoctramine > AFDX-116. This antagonist profile most closely resembles that of the vertebrate M3 mAChR subtype. In this study, however, we have demonstrated that the antagonist profile of DM1 is distinct from those of vertebrate mAChR subtypes. Molecular dynamic simulations of the Drosophila muscarinic receptor are presented in the free, carbamylcholine-bound and NMS-bound forms. Theoretical, quantitative structure-activity relationship models have been developed; a good correlation is observed between the interaction energies of the minimized ligand-receptor complexes and the pharmacological affinities of the antagonists tested.

Functional characterization of a mutated chicken alpha7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2.

1. Site-directed mutagenesis was used to create an altered form of the chicken alpha7 nicotinic acetylcholine (ACh) receptor subunit (alpha7x61) in which a leucine residue was inserted between residues Leu9' and Ser10' in transmembrane domain 2. The properties of alpha7x61 receptors are distinct from those of the wild-type receptor. 2. Oocytes expressing wild-type alpha7 receptors responded to 10 microM nicotine with rapid inward currents that desensitized with a time-constant of 710+/-409 ms (mean+/-s.e.mean, n=5). However in alpha7x61 receptors 10 microM nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684+/-3403 ms (mean+/-s.e.mean, n = 4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild-type receptors was observed. Dihydro-beta-erythroidine (DHbetaE) acted as an antagonist on both receptors. 3. Molecular modelling of the alpha7x61 receptor channel pore formed by a bundle of M2 alpha-helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10 would be replaced by the leucine insertion, Val13' and Phe14' would be replaced, by Thr and Val, respectively. 4 When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild-type and alpha7x61 receptors. However, block was more dependent on membrane potential for the alpha7x61 receptors. 5. We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.

Adult insect mushroom body neurons in primary culture: cell morphology and characterization of potassium channels.

The dissociation and maintenance in culture of cells derived from the mushroom bodies of adult crickets (Acheta domesticus) are described. This primary culture was developed in order to investigate maturation and differentiation of mushroom-body cells including Kenyon cells, the major intrinsic interneurons of mushroom bodies, which have been shown to be involved in learning and memory in insects. Three distinct cell types were observed, all identified as neural cells on the basis of their size, morphology and immunocytochemical staining with horseradish peroxidase. These cells appear to correspond to the three cell types observed in vivo: Kenyon cells, ganglion mother cells and neuroblasts. Some cells showed neurite growth, usually with long unipolar processes, occasionally with either bipolar or, more rarely, multipolar processes. Neuronal cell bodies readily formed seals with patch pipettes, allowing stable, whole-cell, patch-clamp electrophysiological recordings. Depolarization of the cell under voltage-clamp resulted in at least two types of outwardly directed potassium currents: a delayed rectifier-type of current that was sensitive to tetraethylammonium, and a cadmium-sensitive current with rapid inactivation. Neither type of current was affected by quinidine, a blocker of potassium currents recorded from pupal honeybee Kenyon cells. Other ionic currents, which have yet to be characterized, were also observed.

Effects of the alpha subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors.

1. Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (alpha4beta2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal alpha subunit (SAD) with the chicken beta2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (-)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology. 2. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the alpha4beta2 receptor; (+)-epibatidine, (-)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken beta2 receptor, as was (-)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists. 3. The EC50 of imidacloprid was decreased by replacing the chicken alpha4 subunit with the Drosophila SAD alpha subunit. This alpha subunit substitution also resulted in an increase in the EC50 for (+)-epibatidine, (-)-nicotine and acetylcholine. Thus, the Drosophila (SAD) alpha subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. 4. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADbeta2 hybrid receptors and as a weak potentiator of ACh responses mediated by alpha4beta2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the alpha subunit.

Effects of [3H]-BIDN, a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates.

1. The radiolabelled bicyclic dinitrile, [3H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of gamma-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35 +/- 3 nM) and dieldrin (IC50, 93 +/- 7 nM), displaced [3H]-BIDN from rootworm membranes. When tested at 100 microM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]oct ane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicy-clo[2.2.2]octane-1-thio ne (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. 2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and alpha and beta endosulphan displace bound [3H]-BIDN from rootworm membranes by a competitive mechanism. 3. Rat brain membranes were also shown to possess a population of saturable, specific [3H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. 4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA(B) receptors were unaffected. 5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl- channels to which a number of insecticidally-active molecules bind.

Pharmacology of skeletal muscle GABA-gated chloride channels in the cockroach Periplaneta americana.

The pharmacology of -aminobutyric acid (GABA)-gated chloride channels of the coxal levator (182c,d) muscle of the cockroach Periplaneta americana has been investigated and the data compared with similar findings for the cell body of the cockroach fast coxal depressor motor neurone (Df). Muscle GABA receptors resembled those of the motor neurone cell body in their sensitivity to picrotoxinin and insensitivity to bicuculline. However, muscle GABA receptors were insensitive to the neuronal GABA receptor agonists isoguvacine (10(-4) mol l-1) and 3-aminopropane sulphonic acid (10(-3 )mol l-1). The benzodiazepine flunitrazepam, which at 10(-6 )mol l-1 greatly enhances the amplitude of the motor neurone GABA-induced responses, failed to affect muscle responses to GABA when tested at the same and at a higher (10(-4 )mol l-1) concentration. The convulsant t-butylbicyclophosphorothionate was a weak antagonist of cockroach muscle GABA receptors, whereas several cyclodienes were much more effective antagonists. Thus, studies using a benzodiazepine and several convulsant antagonists reveal differences in the pharmacology of muscle and neuronal GABA receptors of the cockroach Periplaneta americana.

Polycyclic dinitriles: a novel class of potent GABAergic insecticides provides a new radioligand, [3H]BIDN.

The polycyclic dinitriles are a potent class of insecticides which are non-competitive GABA (gamma-aminobutyric acid) antagonists acting at the convulsant site. Comparison with other classes of GABA convulsant site ligands using molecular modelling has shown significant structural similarities. We have developed a pharmacophore model which unifies this class and some previous classes of GABA convulsants. Key pharmacophore elements are a polarizable functionality separated by a fixed distance from two H-bond accepting elements. This model is based on information from X-ray crystal structures and Sybyl using the Tripos force field. Using this pharmacophore model, numerous structural modifications were explored to enhance understanding of structure-activity relationships at the GABA receptor convulsant site of insects and mammals. A radiolabelled bicyclic dinitrile, [3H]BIDN [3H]3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile+ ++), was prepared from this area of chemistry and was used as a probe for the interaction of polycyclic dinitriles at the target site.

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.

pH-dependent actions of THIP and ZAPA on an ionotropic Drosophila melanogaster GABA receptor.

The actions of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and ZAPA (Z-3-[(aminoiminomethyl)thio]prop-2-enoic acid) were tested on an ionotropic homo-oligomeric GABA receptor of Drosophila melanogaster. The amplitude of currents activated by THIP and ZAPA declined rapidly during agonist application and a rebound response was observed on washout. By correcting the pH shift induced by these acid salts, responses more typical of GABA agonists were seen. Less striking pH-dependence was observed in the case of GABA responses.

Wild-type and insecticide-resistant homo-oligomeric GABA receptors of Drosophila melanogaster stably expressed in a Drosophila cell line.

RDL is an ionotropic GABA receptor subunit, a product of the Rdl gene, originally identified in the Maryland strain of Drosophila melanogaster. Here, we report the generation of a Drosophila melanogaster cell line (S2-RDLA302S) stably expressing a mutated, dieldrin-resistant (A302S) form of RDL. The properties of this dieldrin-resistant, homo-oligomeric receptor have been compared with those of the stably expressed, wild-type form (S2-RDL). Using these stable lines, a striking reduction in sensitivity to both picrotoxinin and dieldrin was observed for responses to GABA of S2-RDLA302S compared to S2-RDL. To determine if these stable insect cell lines generate results similar to those obtained by transient expression in Xenopus laevis oocytes, we have examined the actions of two widely used convulsants, EBOB and TBPS, and a recently developed convulsant BIDN, on RDL-mediated GABA responses in the two expression systems. In both oocytes and S2 cells, the three convulsants suppressed the amplitude of responses to GABA. Thus, in accord with earlier work on agonist and allosteric sites, the S2-RDL cell line is found to yield similar pharmacological results to those obtained in transient expression studies. Stable cell lines are now available expressing susceptible and resistant forms of an ionotropic receptor by GABAergic insecticides.

Blocking actions of BIDN, a bicyclic dinitrile convulsant compound, on wild-type and dieldrin-resistant GABA receptor homo-oligomers of Drosophila melanogaster expressed in Xenopus oocytes.

The receptor antagonist actions are described for a novel bicyclic dinitrile compound (BIDN, 3,3-bis-(trifluoromethyl)-bicyclo [2.2.1] heptane-2,2-dicarbonitrile) on a Drosophila melanogaster homo-oligomeric GABA receptor expressed in Xenopus oocytes. BIDN blocked the wild-type form of the receptor in a neither purely competitive, nor purely non-competitive manner, being dependent on the GABA concentration yet insurmountable, and block was independent of the membrane potential. BIDN was found to be less effective against a mutant (A(302) --> S) form of the receptor resistant to dieldrin and picrotoxinin. This cross resistance of dieldrin-resistant receptors to BIDN is of interest in the light of recent findings that BIDN binding to insect membranes is displaced competitively by dieldrin, but not by picrotoxinin.

Actions of the insecticide fipronil, on dieldrin-sensitive and- resistant GABA receptors of Drosophila melanogaster.

1. Blocking actions of the novel insecticide, fipronil, were examined on GABA responses recorded from Xenopus oocytes expressing either wild type (dieldrin-sensitive) or mutant (dieldrin-resistant) forms of the Drosophila melanogaster GABA-gated chloride channel homo-oligomer, RDL (the product of the resistance to dieldrin locus: Rdl). 2. In the case of the wild type receptor, fipronil blocked GABA-induced currents inducing both a shift to the right in the GABA dose-response curve and depressing the maximum amplitude of responses to GABA. The potency of fipronil was dependent on the GABA concentration but was unaffected by membrane potential. 3. Mutant RDL GABA-receptors, which have a naturally occurring amino acid substitution (A302-->S) in the putative ion-channel lining region, conferring resistance to dieldrin and picrotoxinin, were markedly less sensitive to fipronil than the wild-type receptors. 4. Fipronil antagonism is qualitatively similar to that produced by the structurally distinct compound, picrotoxinin. As the mutation A302-->S reduces the potency of both fipronil and picrotoxinin, homooligomeric RDL receptors should facilitate detailed studies of the molecular basis of convulsant/insecticide antagonist actions on GABA receptors.

Actions of nitromethylenes on an alpha-bungarotoxin-sensitive neuronal nicotinic acetylcholine receptor.

Nine nitromethylene analogues were tested for their actions on insect neuronal nicotinic acetylcholine receptors (nAChRs). Microelectrode recordings were used to study the actions of nitromethylenes on the cell body of an identified cockroach (Periplaneta americana) motor neurone, the fast coxal depressor (Df) in the metathoracic ganglion. Six nitromethylenes showed potent nAChR agonist actions; others were without nAChR agonist actions. Five nitromethylenes competitively displaced bound [125I]-alpha-bungarotoxin from cockroach nervous system membranes. The rank orders of potency for the compounds determined by their depolarizing actions and their ability to displace [125I]-alpha-bungarotoxin binding were similar. These findings, together with toxicity data obtained on the insects, Nephotettix cinciteps and Nilaparvata lugens, support the hypothesis that insect nAChRs are molecular targets of nitromethylene insecticides. Structure-activity relationships of the nitromethylenes suggest that optimal activity at neuronal nAChRs requires the presence of an electron-withdrawing component in the region of the aryl substituent and an electron-donating component at the 3' position of the imidazolidine ring.