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.
Horoszok
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L
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY.
Raymond
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V
Sattelle
D B
DB
Wolstenholme
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D000969:Antinematodal Agents / Q000494:pharmacology; D000977:Antiparasitic Agents / Q000494:pharmacology; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology*; D017173:Caenorhabditis elegans / Q000187:drug effects* / Q000235:genetics; D018118:Chloride Channels / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D003292:Convulsants / Q000037:antagonists & inhibitors; D018076:DNA, Complementary / Q000032:analysis; D007306:Insecticides / Q000494:pharmacology; D007559:Ivermectin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009570:Nitriles / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010852:Picrotoxin / Q000031:analogs & derivatives / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011720:Pyrazoles / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D054830:Sesterterpenes; D014162:Transfection; D014982:Xenopus laevis; D005466:Fluorocarbons
D000969:Antinematodal Agents; D000977:Antiparasitic Agents; D001643:Bridged Bicyclo Compounds; D018118:Chloride Channels; D003292:Convulsants; D018076:DNA, Complementary; D007306:Insecticides; D007559:Ivermectin; D009570:Nitriles; D010852:Picrotoxin; D011720:Pyrazoles; D054830:Sesterterpenes; C109777:glutamate-gated chloride channels; C098123:3,3-bis(trifluoromethyl)bicyclo(2.2.1)heptane-2,2-dicarbonitrile; D005466:Fluorocarbons; C022961:picrotoxinin; C082360:fipronil