title: Characterization of [3H]ethynylbicycloorthobenzoate ([3H]EBOB) binding and the action of insecticides on the gamma-aminobutyric acid-gated chloride channel in cultured cerebellar granule neurons.
abstract: [3H]Ethynylbicycloorthobenzoate ([3H]EBOB) is a recently developed specific radioligand for the noncompetitive blocker site of the gamma-aminobutyric acid (GABA) receptor, which is the target of many polychlorocycloalkane and heterocyclic insecticides. This study characterizes [3H]EBOB binding and the action of insecticides on the GABA-gated chloride channel of cerebellar granule neurons in primary culture. Specific [3H]EBOB binding increases rapidly between days 3 and 7 and then remains constant for days 7 to 12 in culture. A single, high-affinity and saturable binding site is involved with Kd = 0.51 nM and Bmax = 239 fmol/mg protein. GABA and muscimol are 6- to 12-fold more potent as inhibitors of [3H]EBOB binding to membranes from cultured cerebellar granule neurons than from cerebellar tissue, possibly because of localization of specific subunits in the granule cells. GABA-stimulated 36Cl- influx is also inhibited by seven insecticidal compounds (including EBOB, 12-ketoendrin and alpha-endosulfan) as well as picrotoxinin and t-butylbicyclophosphorothionate with IC50 levels ranging from 290 to 15300 nM, which correlate well (r = 0.92) with their inhibitory potencies for [3H]EBOB binding (1 to 773 nM). These findings validate the use of [3H]EBOB binding in cultured cerebellar granule cells for pharmacological studies on insecticides and other toxicants acting at the noncompetitive blocker or coupled site of the GABAA receptor/chloride channel complex.
authors.affiliation: Department of Environmental Science, Policy, and Management, University of California, Berkeley, USA.
authors.forename: J
authors.initials: J
authors.lastname: Huang
authors.forename: J E
authors.initials: JE
authors.lastname: Casida
mesh_terms: D000818:Animals; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects* / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects* / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D007306:Insecticides / Q000494:pharmacology*; D015640:Ion Channel Gating / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D014316:Tritium; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D018118:Chloride Channels; D007306:Insecticides; D014316:Tritium; C091518:1-(4-ethynylphenyl)-4-propyl-2,6,7-trioxabicyclo(2.2.2)octane; D005680:gamma-Aminobutyric Acid