title: Site-specific acylation of GABA-gated Cl- channels: effects on 36Cl- uptake.
abstract: Radioligand binding studies indicate that p-isothiocyanato-t-butylbicycloorthobenzoate (p-NCS-TBOB) specifically acylates GABA-gated chloride channels. Preincubation of synaptoneurosomes with p-NCS-TBOB followed by washing resulted in a concentration dependent (63-500 nM) inhibition of both muscimol-stimulated chloride uptake and [355]t-butylbicyclophosphorothionate (TBPS) binding. The extent of acylation (assessed by inhibition of [35S]TBPS binding) was highly correlated (r = 0.89; p less than 0.001) with the inhibition of muscimol-stimulated Cl- uptake. Neither basal Cl- uptake nor [3H]muscimol binding to GABAA receptors were affected by p-NCS-TBOB. Preincubation with the nonacylating 'cage' convulsant t-butylbicycloorthobenzoate (500 nM) followed by washing had no effect on either muscimol-stimulated Cl- uptake or [35S]TBPS binding. These findings indicate that p-NCS-TBOB interferes with the efficacy of muscimol promoted channel openings, but does not affect the recognition qualities of GABAA receptors. p-NCS-TBOB should prove useful in electrophysiological and biochemical studies examining the properties of GABA-gated Cl- channels.
authors.affiliation: Laboratory of Neuroscience, NIDDK, National Institutes of Health, Bethesda, MD 20892.
authors.forename: E J
authors.initials: EJ
authors.lastname: Moody
authors.forename: A H
authors.initials: AH
authors.lastname: Lewin
authors.forename: B R
authors.initials: BR
authors.lastname: de Costa
authors.forename: K C
authors.initials: KC
authors.lastname: Rice
authors.forename: P
authors.initials: P
authors.lastname: Skolnick
mesh_terms: D000215:Acylation; D000818:Animals; D001693:Biological Transport, Active; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009118:Muscimol / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008024:Ligands; D008565:Membrane Proteins; D011963:Receptors, GABA-A; C069730:4-(t-butyl)-1-(4-isothiocyanatophenyl)-2,6,7-trioxabicyclo(2.2.2)octane; D009118:Muscimol; D005680:gamma-Aminobutyric Acid