Site-specific acylation of GABA-gated Cl- channels: effects on 36Cl- uptake.

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.