[3H]WIN 35,428 ([3H]CFT) binds to multiple charge-states of the solubilized dopamine transporter in primate striatum

Caudate-putamen membranes of rhesus monkey were solubilized (1% digitonin; w/v) and [3H]WIN 35,428 ([3H]CFT: 2 beta-carbomethoxy-3- beta-(4-fluorophenyl)-N-[3H]methyltropane) binding assayed. Saturation analysis revealed high- and low-affinity binding components (KHIGH: 7.48 +/- 2.77 nM; KLOW: 292 +/- 788 nM; mean +/- SEM). Monoamine transport inhibitors and neurotransmitters had similar affinities for soluble and membrane [3H]CFT binding sites (r, 0.998; P < .001). The rank order of potency of these compounds for inhibiting [3H]CFT binding (Lu 19-005 > mazindol > CFT > GBR 12909 > (-)-cocaine > talsupram > dopamine > norepinephrine > citalopram) was consistent with [3H]CFT labeling cocaine binding sites on the dopamine transporter. [3H]CFT binding sites were separated into three protein fractions by anion-exchange chromatography. Monoamine transport inhibitors and neurotransmitters inhibited [3H]CFT binding in each fraction with a rank order of potency consistent with binding to the dopamine transporter. Detection of multiple binding components for [3H]CFT labeled sites by these drugs varied in each fraction. Size-exclusion chromatography indicated [3H]CFT bound to a single protein in each fraction (apparent molecular weight, 170 kDa). Therefore, multiple binding components for cocaine reside solely on the dopamine transporter and exhibit different affinities for drugs depending on the charge-state of the transporter.