Labeling of Dopamine Transporter Transmembrane Domain 1 with the Tropane Ligand N-[4-(4-Azido-3-[¹²⁵I]iodophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane Implicates Proximity of Cocaine and Substrate Active Sites

Abstract

The novel photoaffinity ligand N- [4-(4-azido-3-125 125 I]RTI 82 possess identical tropane pharmacophores and differ only in the placement of the reactive azido moieties, their distinct incorporation profiles identify the regions of the protein adjacent to different aspects of the cocaine molecule. These findings thus strongly support the direct interaction of cocaine on DAT with TM1 and TM6, both of which have been implicated by mutagenesis and homology to a bacterial leucine transporter as active sites for substrates. These results directly establish the proximity of TMs 1 and 6 in DAT and suggest that the mechanism of transport inhibition by cocaine involves close interactions with multiple regions of the substrate permeation pathway.

Sex-Specific Brain Deficits in Auditory Processing in an Animal Model of Cocaine-Related Schizophrenic Disorders

“…Cocaine-induced euphoria is due at least in part to the inhibition of dopamine reuptake in mesocorticolimbic neuronal circuitry of brain and the dopamine transporter is believed to perform the mechanism by which dopamine remains in the synapse. Molecular pharmacology studies show that the tropane ring influences the reinforcing properties of cocaine [5,6]. …”

Evolution of a Compact Photoprobe for the Dopamine Transporter Based on (±)-threo-Methylphenidate

“…For example, [ 125 I]-MFZ-2–24 ( 2 ) has been prepared and found to covalently attach to TM1 of the DAT, 11 while similar results have also been achieved with benztropine probe [ 125 I]-GA-2–34 ( 3 ). 12 However, phenyltropane-based probe [ 125 I]-RTI-82 ( 4 ) covalently ligates to TM6 of the DAT.…”

“…In particular, molecular modeling studies of photoprobe 2 indicate distances of 10.5 and 15.5 Angstroms between the azide, and the pharmacophore tropane nitrogen and 3β-phenyl ring. 11 Given the azide is not directly appended to the tropane pharmacophore for probes 2 – 4 , adduction may occur at a residue near, but not at, a direct inhibitor contact point, thus representing a significant limitation when trying to delineate the discrete molecular interactions between the probe and DAT protein. Towards potentially addressing this point, 3-(4′-azido-3′-iodo-phenyl)-8-methyl-8-aza-bicyclo-[3.2.1]octane-2-carboxylic acid methyl ester ( 5 ) has been synthesized and displays high affinity, wash-resistant binding to the DAT, 15 but fails to label the DAT in subsequent immunoprecipitation and proteolysis experiments (Vaughan et al, unpublished observations).…”

Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling

“…To determine if the DAT was able to be irreversibly labeled with [ 125 I]-(±)- 3a , we used procedures previously developed for the analysis of other DAT photoaffinity labels 13, 14, 17, 20, 28, 30, 40–43…”