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Proceedings of the Estonian Academy of Sciences.
Chemistry
Volume 55 No. 3
September 2006
Interaction
of tritium-labelled dopamine transporter inhibitor PE2I with mice striatal
membrane fragments; 166–172
Vladimir Stepanov and Jaak Järv
Abstract. Interaction of 3H-labelled N-(3-iodoprop-2E-enyl)-2b-carbomethoxy-3b-(4-methylphenyl)nortropane ([3H]PE2I), a novel tritium-labelled ligand for tracing dopamine transporter protein, with mice striatal membrane fragments was studied under equilibrium conditions. Radioligand binding with a homogeneous population of binding sites was observed in these brain membrane fragments and characterized by the Kd value 22 ± 5 nM and Bmax = 0.12 ± 0.02 pmol/mg tissue. The specific binding of [3H]PE2I was effectively displaced by unlabelled PE2I as well as GBR 12935, also known as an inhibitor of the transporter protein. Rather similar pIC50 values, 7.1 ± 0.3 and 6.9 ± 0.3, respectively, were obtained for these ligands in displacement experiments. This is in agreement with similar pharmacological effects of these ligands on dopaminergic neurons. After correction by the Cheng–Prusoff equation the displacement study yielded the Kd value of 40 nM for PE2I. The difference between the Kd values for PE2I obtained from the direct binding study and displacement experiments seems to point to some specific feature of the mechanism of PE2I interaction with the transporter sites and will be clarified through systematic kinetic study of the radioligand binding.
Key words: dopamine transporter, radioligand analysis, [3H]PE2I, DAT inhibitor, GBR 12935.
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