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D B Sattelle

Publications and source records attributed to D B Sattelle.

At least 19 recordsLinked to original sources

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.

Animals↗

Alpha7-acetylcholine receptor antibodies in two patients with Rasmussen encephalitis.

Rasmussen encephalitis (RE) sera were screened for antibodies to human alpha7 nicotinic acetylcholine receptors (nAChRs) using electrophysiology, calcium imaging, and ligand binding assays. Sera from two of nine patients with RE blocked ACh-induced currents through alpha7 nAChRs and the ACh-induced rise in intracellular free calcium ([Ca2+]i) and inhibited (125)I-alpha-bungarotoxin binding in cells expressing alpha7 nAChRs. Thus, the alpha7 nAChR is a potential target for pathogenic antibodies in patients with RE.

Adolescent↗

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.

Amino Acid Sequence↗

Edit, cut and paste in the nicotinic acetylcholine receptor gene family of Drosophila melanogaster.

Nicotinic acetylcholine receptors (nAChRs) are important for fast synaptic cholinergic transmission. They are targets of drugs/chemicals for human and animal health as well as for pest control. With the advent of genome sequencing, entire nAChR gene families have now been described for vertebrates and invertebrates. Mostly, these are extensive with a large number of distinct subunits, making possible many nAChR subtypes differing in transmitter affinity, channel conductance, ion selectivity, desensitization, modulation and pharmacology. The smallest nAChR gene family to date is that of the fruit fly, Drosophila melanogaster, with only 10 members. This apparently compact family belies its true diversity as 4 of the 10 subunits show alternative splicing. Also, using Drosophila, A-to-I pre-mRNA editing has been demonstrated for the first time in nAChRs. Such is the extent of this variation, that one subunit alone (Dalpha6) can potentially generate far more isoforms than seen in entire gene families from other species. We present here three-dimensional models constructed for insect nAChRs, which show that many variations introduced by alternative splicing and RNA editing may influence receptor function.

Alternative Splicing↗

Spatiotemporal calcium signaling in a Drosophila melanogaster cell line stably expressing a Drosophila muscarinic acetylcholine receptor.

A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca(2+)](i)) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca(2+)](i) transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.

Animals↗

Cloning and developmental expression analysis of ltd-1, the Caenorhabditis elegans homologue of the mouse kyphoscoliosis (ky) gene.

We have characterized the developmental expression pattern of the Caenorhabditis elegans homologue of the mouse ky gene. The Ky protein has a putative key function in muscle development and has homologues in invertebrates, fungi and a cyanobacterium. The C. elegans Ky homologue gene has been named ltd-1 for LIM and transglutaminase domains gene. The LTD-1::GFP construct is expressed in developing hypodermal cells from the twofold stage embryo through adulthood. These data define the ltd-1 gene as a novel marker for C. elegans epithelial cell development.

Amino Acid Sequence↗

Novel putative nicotinic acetylcholine receptor subunit genes, Dalpha5, Dalpha6 and Dalpha7, in Drosophila melanogaster identify a new and highly conserved target of adenosine deaminase acting on RNA-mediated A-to-I pre-mRNA editing.

Genome analysis of the fruit fly Drosophila melanogaster reveals three new ligand-gated ion channel subunits with the characteristic YXCC motif found only in alpha-type nicotinic acetylcholine receptor subunits. The subunits are designated Dalpha5, Dalpha6, and Dalpha7. Cloning of the Dalpha5 embryonic cDNAs reveals an atypically large N terminus, part of which is without identifiable sequence motifs and is specified by two polymorphic alleles. Embryonic clones from Dalpha6 contain multiple variant transcripts arising from alternative splicing as well as A-to-I pre-mRNA editing. Alternative splicing in Dalpha6 involves exons encoding nAChR functional domains. The Dalpha6 transcript is a target of the Drosophila adenosine deaminase acting on RNA (dADAR). This is the first case for any organism where a nAChR gene is the target of mRNA editing. Seven adenosines could be modified in the extracellular ligand-binding region of Dalpha6, four of which are also edited in the Dalpha6 ortholog in the tobacco budworm Heliothis virescens. The conservation of an editing site between the insect orders Diptera and Lepidoptera makes nAChR editing the most evolutionarily conserved invertebrate RNA editing site so far described. These findings add to our understanding of nAChR subunit diversity, which is increased and regulated by mechanisms acting at the genomic and mRNA levels.

Adenosine Deaminase↗

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.

Animals↗

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.

Alternative Splicing↗

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.

Action Potentials↗

GLC-3: a novel fipronil and BIDN-sensitive, but picrotoxinin-insensitive, L-glutamate-gated chloride channel subunit from Caenorhabditis elegans.

1. We report the cloning and expression of a novel Caenorhabditis elegans polypeptide, GLC-3, with high sequence identity to previously cloned L-glutamate-gated chloride channel subunits from nematodes and insects. 2. Expression of glc-3 cRNA in XENOPUS oocytes resulted in the formation of homo-oligomeric L-glutamate-gated chloride channels with robust and rapidly desensitizing currents, an EC(50) of 1.9+/-0.03 mM and a Hill coefficient of 1.5+/-0.1. GABA, glycine, histamine and NMDA all failed to activate the GLC-3 homo-oligomer at concentrations of 1 mM. The anthelminthic, ivermectin, directly and irreversibly activated the L-glutamate-gated channel with an EC(50) of 0.4+/-0.02 microM. 3. The GLC-3 channels were selective for chloride ions, as shown by the shift in the reversal potential for L-glutamate-gated currents after the reduction of external Cl(-) from 107.6 to 62.5 mM. 4. Picrotoxinin failed to inhibit L-glutamate agonist responses at concentrations up to 1 mM. The polycyclic dinitrile, 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile (BIDN), completely blocked L-glutamate-induced chloride currents recorded from oocytes expressing GLC-3 with an IC(50) of 0.2+/-0.07 microM. The phenylpyrazole insecticide, fipronil, reversibly inhibited L-glutamate-gated currents recorded from the GLC-3 receptor with an IC(50) of 11.5+/-0.11 microM. 5. In this study, we detail the unusual antagonist pharmacology of a new GluCl subunit from C. elegans. Unlike all other native and recombinant nematode GluCl reported to date, the GLC-3 receptor is insensitive to picrotoxinin, but is sensitive to two other channel blockers, BIDN and fipronil. Further study of this receptor may provide insights into the molecular basis of non-competitive antagonism by these compounds.

Amino Acid Sequence↗

Insecticidal and neural activities of candidate photoaffinity probes for neonicotinoid binding sites.

Photoreactive derivatives of imidacloprid and its nitromethylene analogue were synthesized as candidate photoaffinity probes for identifying the amino acid residues of nicotinic acetylcholine receptors (nAChRs) that interact with the neonicotinoid insecticides. When the candidate probes were injected into American cockroaches, the nerve cord neural activity initially increased, then ceased and death of the insect followed. Both the nerve cord and toxicity were enhanced by changing the photoreactive substituent from the para position to the meta position on the spacer benzyl moiety. When tested on a Drosophila SAD/chicken beta2 hybrid, recombinant nAChR expressed in Xenopus oocytes, the nitromethylene candidate probes showed agonist activity similar to that previously observed for imidacloprid.

Anabasine↗

Co-existence in DUM neurones of two GluCl channels that differ in their picrotoxin sensitivity.

Whole cell patch-clamp electrophysiology was used to study the effects of L-glutamate on dissociated cockroach (Periplaneta americana) dorsal unpaired median (DUM) neurones. Application of L-glutamate via pressure-ejection pipette resulted in a two-component hyperpolarization, consisting of an initial transient and a second, prolonged phase. Under voltage-clamp, using isotonic chloride in the saline and intrapipette solutions, two L-glutamate-gated inward currents were characterized. Their reversal potentials were close to the equilibrium potential for chloride ions. One component was selectively activated by ibotenate and was sensitive to picrotoxin (100 microM), BIDN (10 microM) and the phenylpyrazole insecticide fipronil (10 microM), known to be potent blockers of insect GABA-gated chloride channels. The second component was insensitive to picrotoxin (100 microM) and BIDN (10 microM). These findings demonstrated, for the first time, the co-existence of two pharmacologically distinct native insect neuronal L-glutamate-gated chloride channels.

Animals↗

Immunocytochemical localization of putative gamma-aminobutyric acid receptor subunits in the head ganglia of Periplaneta americana using an anti-RDL C-terminal antibody.

A polyclonal antibody raised against a 17 amino acid polypeptide (the predicted C-terminal sequence of the cloned Drosophila melanogaster gamma-aminobutyric acid (GABA) receptor subunit, RDL) was used to investigate the distribution of GABA receptor subunit(s) of this type in the nervous system of the cockroach Periplaneta americana. Intense staining was detected in the calyces of the mushroom bodies, glomeruli of the antennal lobes, lower central body, the corpora cardiaca and several cell layers of the medulla and the lobula regions of the optic lobe. The most intense immunocytochemical staining was in the suboesophageal ganglion. Control sections pre-incubated with the primary antibody and conjugated peptide were not stained. Thus, it appears that a GABA receptor subunit of the RDL type is located in cockroach brain regions involved in the processing of visual, olfactory and mechanosensory inputs to the nervous system. Since the corpora cardiaca reacted to this antiserum, this type of GABA receptor may also be involved in the regulation of neurosecretory activity.

Animals↗

A role for Caenorhabditis elegans in understanding the function and interactions of human disease genes.

A growing number of medical research teams have begun to explore the experimental advantages of using a genetic animal model, the nematode worm Caenorhabditis elegans, with a view to enhancing our understanding of genes underlying human congenital disorders. In this study, we have compared sequences of positionally cloned human disease genes with the C.elegans database of predicted genes. Drawing on examples from spinal muscular atrophy, polycystic kidney disease, muscular dystrophy and Alzheimer's disease, we illustrate how data from C.elegans can yield new insights into the function and interactions of human disease genes.

Amino Acid Sequence↗

Anthelmintic actions on homomer-forming nicotinic acetylcholine receptor subunits: chicken alpha7 and ACR-16 from the nematode Caenorhabditis elegans.

Two homomer-forming nicotinic acetylcholine receptor subunits with 47% identity in their amino acid sequences were employed to compare the actions of cholinergic anthelmintics and ivermectin on expressed vertebrate and nematode nicotinic receptors of known molecular composition. Voltage-clamp electrophysiology was used to study recombinant nicotinic receptors expressed in Xenopus laevis oocytes following nuclear injection of cDNA encoding either chicken alpha7 or Caenorhabditis elegans ACR-16 (Ce21) subunits. Butamisole, morantel and metyridine were without agonist actions on either alpha7 or ACR-16 nicotinic receptors in the range 10nM-1mM. However, butamisole (pIC(50)=4.9 for both alpha7 and ACR-16) and morantel (pIC(50)=5.6 for alpha7 and 5.7 for ACR-16) antagonized responses of both alpha7 and ACR-16 receptors to acetylcholine. Metyridine (1mM) did not affect responses to acetylcholine of either receptor. Oxantel was without agonist actions on ACR-16, but was an acetylcholine antagonist (pIC(50)=5.4). In contrast, it was found to have low efficacy agonist action (pEC(50)=4.4) on alpha7 at concentrations in the range 10-300microM. In agreement with a previous study, ivermectin (30microM), an agonist of L-glutamate-gated chloride channels, enhanced the amplitude of responses to acetylcholine of alpha7 nicotinic receptors. However, this same concentration of ivermectin (30microM) did not potentiate the acetylcholine-induced responses of ACR-16, but rather resulted in a slight attenuation. We conclude that oxantel and ivermectin have identified new pharmacological differences between the chicken alpha7 nicotinic receptor and its C. elegans homologue ACR-16.

Acetylcholine↗

Role of loop D of the alpha7 nicotinic acetylcholine receptor in its interaction with the insecticide imidacloprid and related neonicotinoids.

1. The nitroguanidine insecticide imidacloprid along with a second generation of related compounds including nitenpyram, all nicotinic acetylcholine (ACh) receptor ligands, are used increasingly in many countries. Site-directed mutagenesis and heterologous expression in Xenopus laevis oocytes have been deployed to investigate mutants (G189D and G189E) of the chicken alpha7 homomer-forming nicotinic receptor subunit which are predicted to enhance the negative charge at the negative subsite (loop D) of the ACh binding site. 2. Xenopus oocytes expressing wild-type alpha7 nicotinic receptors respond to imidacloprid with rapid inward currents. Imidacloprid and nitenpyram are partial agonists, whereas ACh, (-)-nicotine and (+)-epibatidine are full agonists. 3. Compared to wild-type alpha7, the mutant G189D and G189E receptors are much less sensitive to the insecticides, whereas their sensitivity to (-)-nicotine, ACh and (+)-epibatidine is only slightly reduced. In contrast, G189N and G189Q mutants are sensitive not only to ACh, (-)-nicotine and (+)-epibatidine, but also to the two insecticides. Thus reduction of the insecticide-sensitivity by the mutations G189D and G189E are attributed to an increase in negativity of loop D. Desnitro-imidacloprid (DN-IMI), an imidacloprid derivative lacking the nitro group is a potent agonist on the G189D and G189E mutants suggesting an important role of loop D in nicotinic receptor interactions with the nitro group of nitroguanidine insecticides.

Animals↗

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.

Animals↗