POTENTIAL MISCONCEPTIONS IN DOPAMINE TRANSPORTER ASSAYS ARISING FROM THE BINDING OF [I-125] RTI-121 TO FILTERS - EFFECT OF IONS AND COCAINE

Binding of the cocaine analog 3 beta-(4-[I-125]iodophenyl)tropane-2 be ta-carboxylic acid isopropyl ester ([I-125]RTI-121) to filters was stu died in order to assess its contribution to labeling dopamine transpor ters on rat striatal synaptosomal membranes in filtration assays. Filt er binding (FB) decreased with increasing Na+. Cocaine (30 and 100 mu M) 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+, R TI-121 (1 mu M) displaced the FB to the same degree as cocaine (100 mu M); mazindol (10 mu M), BTCP (1 mu M), and dopamine (1 mM) did so to a lesser degree; and GBR12935 (1 mu M) did not. If the specific bindin g was calculated without deducting the FB displaced with cocaine (DFB) , the DFB accounted for 15-19% of the 'specific binding' at 10 mM Nain the assay. This additional binding population resulted in an upward curvilinear Scatchard plot and incorrect estimation of equilibrium bi nding 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 compo nent were 84 nM and 16 pmol!mg protein; when DFB was deducted, one com ponent was observed with a K-d of 4.4 nM and B-max of 3.3 pmol/mg prot ein. The presence of higher Na+ in the assay diminished these artifact s. Thus, at 150 mM Na+, without deduction of DFB, there was one bindin g component with a K-d of 3.9 nM and B-max of 4.6 pmol/mg protein; the se values became 3.3 nM and 3.8 pmol/mg protein when DFB was deducted. (C) 1997 Elsevier Science B.V.