Characterization of [125I]RTI-121 binding to dopamine transporter in vitro
Abstract
AIM: To characterize the binding of [125I]3 beta-(4-iodophenyl) tropane-2
beta-carboxylic acid isopropyl ester (RTI-121) to the dopamine transporter (DAT)
under physiologically relevant conditions.
METHODS: [125I]RTI-121 was used to label the DAT on fresh rat striatum
synaptosomal membranes in artificial cerebrospinal fluid (ACSF) at 37 degrees C.
RESULTS: [125I]RTI-121 binding reached equilibrium within 3 min and remained at
its plateau value for at least 9 min. The data from kinetic, saturation, and
competition studies supported a one-site model for the binding of [125I]RTI-121
to the DAT. Various DAT blockers (oocaine, GBR12935, and BTCP) and substrates
(dopamine and d-amphetamine) competitively inhibited the binding of
[125I]RTI-121. Compared with NaPhos-KCl-NaCl assay buffer, ACSF containing Ca2+
and Mg2+ markedly increased the IC50 of DAT blockers for inhibiting [125I]RTI-121
binding with less effect on that of substrates. Various D2 receptor ligands
(pergolide, quinirole, sulpiride, and l-stepholidine) had no direct effect on the
binding of [125I]RTI-121.
CONCLUSION: [125I]RTI-121 binding under physiologically relevant conditions
fulfills the basic criteria for DAT binding assay.
Keywords:
beta-carboxylic acid isopropyl ester (RTI-121) to the dopamine transporter (DAT)
under physiologically relevant conditions.
METHODS: [125I]RTI-121 was used to label the DAT on fresh rat striatum
synaptosomal membranes in artificial cerebrospinal fluid (ACSF) at 37 degrees C.
RESULTS: [125I]RTI-121 binding reached equilibrium within 3 min and remained at
its plateau value for at least 9 min. The data from kinetic, saturation, and
competition studies supported a one-site model for the binding of [125I]RTI-121
to the DAT. Various DAT blockers (oocaine, GBR12935, and BTCP) and substrates
(dopamine and d-amphetamine) competitively inhibited the binding of
[125I]RTI-121. Compared with NaPhos-KCl-NaCl assay buffer, ACSF containing Ca2+
and Mg2+ markedly increased the IC50 of DAT blockers for inhibiting [125I]RTI-121
binding with less effect on that of substrates. Various D2 receptor ligands
(pergolide, quinirole, sulpiride, and l-stepholidine) had no direct effect on the
binding of [125I]RTI-121.
CONCLUSION: [125I]RTI-121 binding under physiologically relevant conditions
fulfills the basic criteria for DAT binding assay.