Identification of the attachment site for the cocaine analog [125I]JHC 2‐48 on the dopamine transporter (803.7)

Abstract

Cocaine is a highly addictive drug that targets the presynaptic dopamine transporter (DAT) and blocks the reuptake of dopamine resulting in heightened dopaminergic neurotransmission. The DAT is a 12 transmembrane (TM) spanning protein with residues from TMs 1, 3, 6, and 8 comprising the substrate and putative inhibitor binding sites; although recent studies indicate the presence of an inhibitor binding site in the external vestibule located above the substrate‐binding site. Despite decades of research, the binding site of cocaine has yet to be fully characterized. To address this question, we are mapping the attachment sites of irreversible cocaine analogs, [125I]MFZ 2‐24, [125I] RTI 82, and [125I]JHC 2‐48, using insight gained from in silico docking with DAT comparative models. These compounds have a cocaine pharmacophore with analog specific azido group that adducts to the protein. To determine the adduction site of [125I]JHC 2‐48 we are using methionine substitution mutagenesis to generate custom protease sites for cleavage by cyanogen bromide (CNBr). The fragments generated from the CNBr proteolysis of [125I]JHC 2‐48 labeled DAT were visualized via SDS‐PAGE and autoradiography. The preliminary results obtained thus far have determined the [125I]JHC 2‐48 attachment site to be C‐terminal to TM7. Peptide map analyses of other cocaine analogs, [125I]MFZ 2‐24 and [125I] RTI 82, have determined [125I]MFZ 2‐24 adducts to TM1 and [125I]RTI 82 adducts to TM6. Taken together, our data strongly indicate cocaine binds near the substrate binding site.Grant Funding Source: Supported by: DA027845 and NIDA‐IRP