Potential misconceptions in dopamine transporter assays arising from the binding of [125I]RTI-121 to filters: effect of ions and cocaine.
Binding of the cocaine analog 3 beta-(4-[125I]iodophenyl)tropane-2 beta-carboxylic acid isopropyl ester ([125I]RTI-121) to filters was studied in order to assess its contribution to labeling dopamine transporters on rat striatal synaptosomal membranes in filtration assays. Filter binding (FB) decreased with increasing Na+. Cocaine (30 and 100 microM) substantially reduced the FB at low Na+ with much less of an effect at higher Na+. Similar results were observed with K+. At 10 mM Na+, RTI-121 (1 microM) displaced the FB to the same degree as cocaine (100 microM); mazindol (10 microM), BTCP (1 microM), and dopamine (1 mM) did so to a lesser degree; and GBR12935 (1 microM) did not. If the specific binding was calculated without deducting the FB displaced with cocaine (DFB), the DFB accounted for 15-19% of the 'specific binding' at 10 mM Na+ in the assay. This additional binding population resulted in an upward curvilinear Scatchard plot and incorrect estimation of equilibrium binding parameters and ion potencies. At 10 mM Na+, without deduction of DFB, the high-affinity component had a Kd of 3.4 nM and Bmax of 2.4 pmol/mg protein, and the respective values for the low-affinity component were 84 nM and 16 pmol/mg protein; when DFB was deducted, one component was observed with a Kd of 4.4 nM and Bmax of 3.3 pmol/mg protein. The presence of higher Na+ in the assay diminished these artifacts. Thus, at 150 mM Na+, without deduction of DFB, there was one binding component with a Kd of 3.9 nM and Bmax of 4.6 pmol/mg protein; these values became 3.3 nM and 3.8 pmol/mg protein when DFB was deducted.
Chen
N H
NH
Department of Pharmacology, School of Basic Medical Sciences, Nanjing Medical University, People's Republic of China.
Ding
J H
JH
Wang
Y L
YL
Reith
M E
ME
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D003042:Cocaine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D050483:Dopamine Plasma Membrane Transport Proteins; D006651:Histocytochemistry / Q000379:methods*; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism; D014462:Ultrafiltration
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C079213:RTI 121; D012964:Sodium; D003042:Cocaine