t-[3H]butylbicycloorthobenzoate: new radioligand probe for the gamma-aminobutyric acid-regulated chloride ionophore.
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 degrees 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 degrees C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37 degrees 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 gamma-aminobutyric acid receptor-ionophore complex.
Lawrence
L J
LJ
Palmer
C J
CJ
Gee
K W
KW
Wang
X
X
Yamamura
H I
HI
Casida
J E
JE
D000731:Androstanes / Q000494:pharmacology; D000818:Animals; D001378:Azasteroids / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000378:metabolism; D001643:Bridged Bicyclo Compounds / Q000138:chemical synthesis / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002712:Chlorides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
D000731:Androstanes; D001378:Azasteroids; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001952:Bridged-Ring Compounds; D002712:Chlorides; D007476:Ionophores; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; C037476:tert-butylbicyclophosphorothionate; C046308:tert-butylbicyclo-2-benzoate; C032061:RU 5135