Steven D. Buckingham

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.

Nicotinic Acetylcholine Receptor Signalling: Roles in Alzheimer's Disease and Amyloid Neuroprotection

Alzheimers disease (AD), the major contributor to dementia in the elderly, involves accumulation in the brain of extracellular plaques containing the β-amyloid protein (Aβ) and intracellular neurofibrillary tangles of hyperphosphorylated tau protein. AD is also characterized by a loss of neurons, particularly those expressing nicotinic acetylcholine receptors (nAChRs), thereby leading to a reduction in nAChR numbers. The Aβ1–42 protein, which is toxic to neurons, is critical to the onset and progression of AD. The discovery of new drug therapies for AD is likely to be accelerated by an improved understanding of the mechanisms whereby Aβ causes neuronal death. We examine the evidence for a role in Aβ1–42 toxicity of nAChRs; paradoxically, nAChRs can also protect neurons when activated by nicotinic ligands. Aβ peptides and nicotine differentially activate several intracellular signaling pathways, including the phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene homolog pathway, the extracellular signal-regulated kinase/mitogen-activated protein kinase, and JAK-2/STAT-3 pathways. These pathways control cell death or survival and the secretion of Aβ peptides. We propose that understanding the differential activation of these pathways by nicotine and/or Aβ1–42 may offer the prospect of new routes to therapy for AD.

Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha‐bungarotoxin (alpha‐Bgt) and kappa‐bungarotoxin (kappa‐Bgt). Reversible block of the expressed insect AChR by mecamylamine, d‐tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha‐Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.

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 Drosphila 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→SS) 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, homo‐oligomeric RDL receptors should facilitate detailed studies of the molecular basis of convulsant/ insecticide antagonist actions on GABA receptors.

Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co‐expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 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. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)‐epibatidine, (−)‐nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)‐nicotine, whereas (+)‐epibatidine and acetylcholine were full agonists. The EC50 of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC50 for (+)‐epibatidine, (−)‐nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. Imidacloprid acted as a weak antagonist of ACh‐mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit.

CHEMISTRY-TO-GENE SCREENS IN CAENORHABDITIS ELEGANS

The nematode worm Caenorhabditis elegans is a genetic model organism linked to an impressive portfolio of fundamental discoveries in biology. This free-living nematode, which can be easily and inexpensively grown in the laboratory, is also a natural vehicle for screening for drugs that are active against nematode parasites. Here, we show that chemistry-to-gene screens using this animal model can define targets of antiparasitic drugs, identify novel candidate drug targets and contribute to the discovery of new drugs for treating human diseases.

Neonicotinoid insecticides display partial and super agonist actions on native insect nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole‐cell patch‐clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P‐CH‐clothianidin on native nAChRs of these neurons. Dose–response data yield an EC50 value for ACh of 19 μm. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10–14% of those observed for ACh. Conversely, clothianidin and P‐CH‐clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 μm ACh in the same neurons. This is the first demonstration of ‘super’ agonist actions of an insecticide on native insect nAChRs. Cell‐attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.

Stable expression of a functional homo-oligomeric Drosophila GABA receptor in a Drosophila cell line

A cloned Drosophila γ-aminobutyric acid (GABA) receptor subunit (Rdl) has been stably expressed as a functional homo-oligomeric ion channel in a Drosophila cell line. Stably-transfected clonal cell lines which expressed high levels of GABA receptor were identified by specific [3H]-muscimol binding. Expression of functional GABA-gated ion channels in these cell lines was demonstrated by electrophysiological recording. Rapid and pronounced rundown of responses to GABA during whole-cell patch clamp recordings was overcome by the inclusion of EGTA in the pipette solution, indicating a possible role for calcium-dependent processes in the functional regulation of this GABA receptor. Relative agonist potencies of the expressed receptor were found to be in the order GABA = TACA > CACA. We have observed a reversible block of the receptor by the convulsant antagonists, picrotoxinin and EBOB, and by the insecticide fipronil. Potentiation of GABA responses was seen with the anaesthetic steroid 5α-pregnan-3α-ol-20-one. No significant effects (either agonist, antagonist or modulatory) were observed with bicuculline (a vertebrate GABAAR antagonist), benzodiazepines or barbiturates (vertebrate GABAAR modulators), or with glycine (agonist of the closely related vertebrate glycine receptors). The suitability of this Drosophila stable expression system for the characterization of receptors and ion channels is discussed.

Actions of the insecticide 2(nitromethylene)tetrahydro-1,3-thiazine on insect and vertebrate nicotinic acetylcholine receptors.

The nitromethylene heterocyclic compound 2(nitromethylene)tetrahydro) 1, 3-thiazine (NMTHT) inhibits the binding of [125I)α-bungarotoxin to membranes prepared from cockroach (Periplaneta americana) nerve cord and fish (Torpedo californica) electric organ. Electrophysiological studies on the cockroach fast coxal depressor motorneuron (Df) reveal a dose-dependent depolarization in response to bath-applied NMTHT. Responses to ionophoretic application of NMTHT on to the cell-body membrane of motorneuron Df are suppressed by bath-applied mecamylamine (1.0 x 10-4 M) and α-bungarotoxin (1.0 x 10-7 M). These findings, together with the detection of a reversal potential close to that estimated for acetylcholine, provide evidence for an agonist action of this nitromethylene on an insect neuronal nicotinic acetylcholine receptor. The binding of [3H]H12-histrionicotoxin to Torpedo membranes was enhanced in the presence of NMTHT indicating an agonist action at this vertebrate peripheral nicotinic acetylcholine receptor. NMTHT is ineffective in radioligand binding assays for rat brain GABAAreceptors, rat brain L-glutamate receptors and insect (Musca domestica) L-glutamate receptors. Partial block of rat brain muscarinic acetylcholine receptors is detected at millimolar concentrations of NMTHT. Thus nitromethylenes appear to exhibit selectivity for acetylcholine receptors and exhibitan agonist action at nicotinic acetylcholine receptors.

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.