Molecular biology and expression of some ligand-gated ion channels from the nematodes Ascaris suum and Caenorhabditis elegans
Molecular biology and expression of some ligand-gated ion channels from the nematodes Ascaris suum and Caenorhabditis elegans
The nematodes Ascaris suum and Caenorhabditis elegans, though very different in size and life-style, bear strong morphological and pharmacological similarities to each other. Here, we have utilised the similarities between the two species to study aspects of their nervous systems, and hence the actions of some anthelmintic drugs.
We have used a variety of molecular probes to screen the A.suum genome for acetylcholine receptor subunits, and glutamate-gated chloride channel subunits. We have screened two different A.suum cDNA libraries and used a variety of PCR (Polymerase Chain Reaction) techniques to search for ligand-gated ion channel subunit fragments.
The Xenopus laevis oocyte expression system has been used to characterize the C.elegans glutamate-gated chloride channel α1 and β subunits. The EC50 for glutamate for the heteromeric receptor in this system is 1.8±0.3mM, which is significantly different to that of he GluClβ homomer (0.8±0.8mM; P<0.001). The EC50 for the ivermectin (22,23, dihydroavermectin B1A) response of the heteromer is 1.02±0.06μM, similar to that of the α1 homomer. The reversal potential is -21±0.9mV, which is the same as EC1 in these conditions. The non-competitive GABAA antagonist picrotoxin blocks the glutamate response of this receptor (IC50 of 18.5±1.0μM). The non-specific chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; IC50 = 6.03±0.81μM) and 4,4-dinitrostilbene-2,2-disulphonic acid (DNDS; IC50 = 1.58±0.34μM) also block the glutamate and ivermectin responses of this receptor. DNDS and NPPB are more potent than picrotoxin. The barbiturate compound amobarbital (IC50 = 2.04±0.5μM) is more potent on the GluClα1β heteromer than pentobarbital (IC50 = 17.56±2.16μM). Pentobarbital is more potent on the GluC1β homomer (IC50 = 0.59±0.09μM) than on the heteromer. The benzodiazepine flurazepan does not block the glutamate response of GluClαlβ. We have demonstrated the presence of binding sites on the C.elegans glutamate-gated chloride channel for classical mammalian GABAA receptor modulators.
University of Southampton
1998
Bush, Elizabeth Rosina
(1998)
Molecular biology and expression of some ligand-gated ion channels from the nematodes Ascaris suum and Caenorhabditis elegans.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The nematodes Ascaris suum and Caenorhabditis elegans, though very different in size and life-style, bear strong morphological and pharmacological similarities to each other. Here, we have utilised the similarities between the two species to study aspects of their nervous systems, and hence the actions of some anthelmintic drugs.
We have used a variety of molecular probes to screen the A.suum genome for acetylcholine receptor subunits, and glutamate-gated chloride channel subunits. We have screened two different A.suum cDNA libraries and used a variety of PCR (Polymerase Chain Reaction) techniques to search for ligand-gated ion channel subunit fragments.
The Xenopus laevis oocyte expression system has been used to characterize the C.elegans glutamate-gated chloride channel α1 and β subunits. The EC50 for glutamate for the heteromeric receptor in this system is 1.8±0.3mM, which is significantly different to that of he GluClβ homomer (0.8±0.8mM; P<0.001). The EC50 for the ivermectin (22,23, dihydroavermectin B1A) response of the heteromer is 1.02±0.06μM, similar to that of the α1 homomer. The reversal potential is -21±0.9mV, which is the same as EC1 in these conditions. The non-competitive GABAA antagonist picrotoxin blocks the glutamate response of this receptor (IC50 of 18.5±1.0μM). The non-specific chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; IC50 = 6.03±0.81μM) and 4,4-dinitrostilbene-2,2-disulphonic acid (DNDS; IC50 = 1.58±0.34μM) also block the glutamate and ivermectin responses of this receptor. DNDS and NPPB are more potent than picrotoxin. The barbiturate compound amobarbital (IC50 = 2.04±0.5μM) is more potent on the GluClα1β heteromer than pentobarbital (IC50 = 17.56±2.16μM). Pentobarbital is more potent on the GluC1β homomer (IC50 = 0.59±0.09μM) than on the heteromer. The benzodiazepine flurazepan does not block the glutamate response of GluClαlβ. We have demonstrated the presence of binding sites on the C.elegans glutamate-gated chloride channel for classical mammalian GABAA receptor modulators.
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Published date: 1998
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Local EPrints ID: 463332
URI: http://eprints.soton.ac.uk/id/eprint/463332
PURE UUID: d7dc4a23-25b5-4aed-a87c-fadf41581f36
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Date deposited: 04 Jul 2022 20:50
Last modified: 04 Jul 2022 20:50
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Author:
Elizabeth Rosina Bush
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