STUDIES OF THE BIOGENIC-AMINE TRANSPORTERS .4. DEMONSTRATION OF A MULTIPLICITY OF BINDING-SITES IN RAT CAUDATE MEMBRANES FOR THE COCAINE ANALOG [I-125] RTI-55

The drug 3 beta-[4'-iodophenyl]tropan-2 beta-carboxylic acid methyl es ter (RTI-55) is a cocaine congener with high affinity for the dopamine transporter (K-d < 1 nM). The present study characterized [I-125]RTI- 55 binding to membranes prepared from rat, monkey and human caudates a nd COS cells transiently expressing the cloned rat dopamine (DA) trans porter. Using the method of binding surface analysis, two binding site s were resolved in rat caudate: a high-capacity binding site (site 1, B-max = 11,900 fmol/mg of protein) and a low-capacity site (site 2, B- max = 846 fmol/mg of protein). The Kd (or Ki) values of selected drugs at the two sites were as follows: (K-i for high-capacity site and K-i for low-capacity site, respectively): RTI-55 (0.76 and 0.21 nM), phen yl-methoxy)ethyl]-4-(3-phenylpropyl)piperazine (0.79 and 358 nM), mazi ndol (37.6 and 631 nM), 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tro pane (45.0 and 540 nM) and cocaine (341 and 129 nM). Nisoxetine, a sel ective noradrenergic uptake blocker, had low affinity for both sites. Serotonergic uptake blockers had a high degree of selectivity and high affinity for the low-capacity binding site (K-i of citalopram = 0.38 nM; K-i of paroxetine = 0.033 nM). The i.c.v. administration of 5,7-di hydroxytryptamine to rats pretreated with nomifensine (to protect dopa minergic and noradrenergic nerve terminals) selectively decreased the B-max of site 2, strongly supporting the idea that site 2 is a binding site on the serotonin (5-HT) transporter. This serotonergic lesion al so increased the affinity of [I-125]RTI-55 for the DA transporter by 1 0-fold. The ligand selectivity of the caudate 5-HT transporter was dif ferent from the [I-125]RTI-55 binding site on the 5-HT transporter pre sent in membranes prepared from whole rat brain minus caudate. The [I- 125]RTI-55 binding to the DA transporter was further resolved into two components, termed sites 1a and 1b, by using human and monkey (Macaca mulatta) caudate membranes but not the membranes prepared from rat ca udate or COS cells that transiently expressed the cloned cocaine-sensi tive DA transporter complementary DNA. Similar experiments also resolv ed two components of the caudate 5-HT transporter. Viewed collectively , these data provide evidence that [I-125]RTI-55 labels multiple bindi ng sites associated with the DA and 5-HT transporters.