t‐[3H]Butylbicycloorthobenzoate: New Radioligand Probe for the γ‐Aminobutyric Acid–Regulated Chloride Ionophore

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Abstract
t[3H]Butylbicycloorthobenzoate ([3H]TBOB; 22 Ci/mmol) was prepared by reductive dechlorination of its 4-chlorophenyl analog with tritium gas. This new radioligand binds reversibly to fresh washed rat brain P2 membranes in 500 mM NaCl plus 50 mM sodium-potassium phosphate buffer (pH 7.4) at 25°C, with 80–90% specific relative to total binding, a KD of 61 ± 15 nM, and a Bmax of 1.6 ± 0.5 pmol/mg of protein. [3H]TBOB association with its binding site(s) is monophasic, but its dissociation is biphasic. The binding characteristics of [3H]TBOB are essentially identical to those of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) with respect to pH dependence, stimulation by anions, regional distribution in the brain, and pharmacological profile. Saturation analyses and dissociation studies further indicate that TBOB and TBPS have a common binding site. However, binding of the two radioligands differs in respect to temperature effects. In contrast to [35S]TBPS, which exhibits negligible binding at 0°C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37°C with similar KD values. [3H]TBOB with its long radioactive half-life and temperature-independent KD is a valuable supplement to [35S]TBPS in further biochemical and pharmacological characterization of the γ-aminobutyric acid receptor-ionophore complex.

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