Worm Breeder's Gazette 16(5): 42 (February 1, 2001)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Biology & Biochemistry, University of Bath, Bath BA2 7AY, UK|
|2||MRC Functional Genetics Unit, Department of Human Anatomy & Genetics, University of Oxford, Oxford OX1 3QX, UK|
Glutamate-gated chloride channels (GluCl) are a class of ligand-gated ion channel receptor found only in invertebrates, including nematodes. They are the targets of the avermectin/milbemycin anthelmintics, including ivermectin. Four C. elegans genes (glc-1, glc-2, avr-14 [which has also been called gbr-2] and avr-15) have been demonstrated to encode GluCl subunits. A further predicted C. elegans gene, ZC317.3, has high sequence identity to the GluCl genes. We have cloned and expressed the gene product and shown that it does indeed encode a further GluCl subunit. We have therefore named the gene glc-3. Expression of glc-3 cRNA in Xenopus oocytes resulted in the formation of homo-oligomeric L-glutamate-gated chloride channels with robust and rapidly desensitising currents, an EC50 of 1.9mM and a Hill coefficient of 1.5. GABA, glycine, histamine and NMDA all failed to activate the GLC-3 homo-oligomer at concentrations of 1mM. Ivermectin directly and irreversibly activated the L-glutamate-gated channels with an EC50 of 0.4microM. The channels were selective for chloride ions, as shown by a shift in the reversal potential for L-glutamate-gated currents after the reduction of external Cl- from 107.6mM to 62.5mM. These properties are similar to those of other GluCl alpha subunits encoded by the avr-14 and avr-15 genes. Picrotoxinin, a non-competitive antagonist of most other native and recombinant GluCl, failed to inhibit L-glutamate agonist responses at concentrations up to 1mM. However, 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 IC50 of 0.2microM. The phenylpyrazole insecticide, fipronil, reversibly inhibited L-glutamate-gated currents recorded from the GLC-3 receptor with an IC50 of 11.5microM. BIDN and fipronil have previously been reported to be specific blockers of GABA-gated channels. This antagonist pharmacology differs from that reported for the other GluCl alpha subunits.