Radiolabeling of dopamine uptake sites in mouse striatum: comparison of binding sites for cocaine,mazindol, and GBR 12935

Summary This study addressed the possibility of a unique binding interaction between cocaine and the dopamine transporter as compared with other blockers of dopamine uptake. Cocaine binding sites in a fresh P₂ fraction of mouse striatum were labeled with ³H]CFT, a phenyltropane analog of cocaine also known as WIN 35,428, and compared with sites labeled with ³H]mazindol or ³H]GBR 12935. Under the conditions used, homogeneous binding was observed that was inhibited monophasically by cocaine, CFT, and mazindol; the same potencies were observed with the three radioligands. Saturation analysis in the presence and in the absence of unlabeled inhibitor (CFT, mazindol, cocaine) indicated a change in the K_d but not the B_max, consonant with a competitive mechanism. Tris-HCl reduced the affinity of each radioligand and unlabeled inhibitor without changing the B_max. N-Ethylmaleimide reduced the binding of all radioligands equally and cocaine offered protection. The dissociation rate of ³H]CFT and ³H]mazindol binding was not affected by the presence of mazindol and CFT, respectively. The B_max of ³H]CFT and ³H]mazindol binding was the same; the relatively higher value for ³H]GBR 12935 binding in analyses involving varying tritiated GBR 12935 only, was due primarily to an underestimation of the specific activity of ³H]GBR 12935. All results are in agreement with a one-site model in which cocaine, CFT, mazindol, and GBR 12935 share a common binding site in mouse striatum.