Dual incorporation of photoaffinity ligands on dopamine transporters implicates proximity of labeled domains

We have recently developed novel high-affinity blockers for the dopamine tr ansporter (DAT) by carrying out structure-activity studies of GBR 12909 mol ecule piperidine analogs. To investigate the molecular basis of binding of these compounds in comparison to known sites of action of GBR 12909, cocain e, and benztropine analogs, we developed a piperidine-based photoaffinity l abel [I-125]4-[2-(diphenylmethoxy)ethyl]-1-[(4-azido-3-iodophenyl)methyl]-p iperidine[I-125]AD-96-129), and used proteolysis and epitope-specific immun oprecipitation to identify the protein domains that interact with the ligan d. [I-125]AD-96-129 became incorporated into two different regions of the D AT primary sequence, an N-terminal site containing transmembrane domains (T Ms) 1 to 2, and a second site containing TMs 4 to 6. Both of these regions have been identified previously as sites involved in the binding of other D AT photoaffinity labels. However, in contrast to the previously characteriz ed ligands that showed nearly complete specificity in their binding site in corporation, [I-125]AD-96-129 became incorporated into both sites at compar able levels. These results suggest that the two domains may be in close thr ee-dimensional proximity and contribute to binding of multiple uptake block ers. We also found that DATs labeled with [I-125]AD-96-129 or other photoaf finity labels displayed distinctive sensitivities to proteolysis of a site in the second extracellular loop, with protease resistance related to the e xtent of ligand incorporation in the TM4 to 6 region. These differences in protease sensitivity may indicate the relative proximity of the ligands to the protease site or reflect antagonist-induced conformational changes in t he loop related to transport inhibition.