Potential misconceptions in dopamine transporter assays arising from the binding of [ 125I]RTI-121 to filters: effect of ions and cocaine

• Published in: Journal of neuroscience methods 1997-08, Vol.75 (2), p.179-186
• Main Authors: Chen, Nian-Hang, Ding, Jian-Hua, Wang, You-Lin, Reith, Maarten E.A
• Format: Article
• Language: English
• Subjects: [ 125I]RTI-121; Animals; Biological and medical sciences; Carrier Proteins - metabolism; Cocaine - analogs & derivatives; Cocaine - pharmacokinetics; Dopamine Plasma Membrane Transport Proteins; Dopamine transporter; Filter binding; Fundamental and applied biological sciences. Psychology; General aspects. Models. Methods; Histocytochemistry - methods; Iodine Radioisotopes; Ion dependency; Kinetics; Male; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Protein Binding; Rat striatum; Rats; Rats, Sprague-Dawley; Sodium - metabolism; Synaptosomes - metabolism; Ultrafiltration; Vertebrates: nervous system and sense organs
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/S0165-0270(97)00070-8

Binding of the cocaine analog 3 β-(4-[ 125I]iodophenyl)tropane-2 β-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 μM) 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 μM) displaced the FB to the same degree as cocaine (100 μM); mazindol (10 μM), BTCP (1 μM), and dopamine (1 mM) did so to a lesser degree; and GBR12935 (1 μM) 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 K d of 3.4 nM and B max 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 K d of 4.4 nM and B max 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 K d of 3.9 nM and B max of 4.6 pmol/mg protein; these values became 3.3 nM and 3.8 pmol/mg protein when DFB was deducted.