Unraveling the ligand–protein interaction between cocaine and the dopamine transporter

Chemical structures of cocaine, CFT, and RTI 82.

What do schizophrenia, attention deficit hyperactivity disorder, Parkinson’s disease, bipolar disorder, autism spectrum disorder and cocaine all have in common? It turns out they are all linked to the role of the dopamine transporter, or DAT, which is an integral membrane protein responsible for the reuptake of dopamine from the synapse. Drugs that bind to DAT to prevent the reuptake of dopamine are used to treat the diseases mentioned above, among others.

However, cocaine, which is also a DAT blocker, leads to profoundly negative effects, such as addiction and psychomotor stimulation. Understanding how different DAT blockers produce distinct behavioral and chemical responses could be the key to developing better drugs to treat dopaminergic disorders and also addiction to DAT blockers like cocaine.

Despite the wide use of DAT blockers, there is still relatively little known about how the drugs interact with DAT. The authors of a paper in the Journal of Biological Chemistry sought to determine precisely how cocaine interacts with DAT, thereby leading to a better understanding of how different responses are generated.

 Image courtesy of The Drug Enforcement Administration

Rejwi Acharya Dahal of the University of North Dakota School of Medicine and Health Sciences and collaborators used computational and biochemical approaches to identify the site of cocaine binding to DAT. The authors performed irreversible labeling with a cocaine analog, RTI 82, which has a 4′-azido-3′-iodophenylethyl, or AIP, moiety that forms a covalent bond with the protein upon ultraviolet irradiation. Using computational modeling and small-molecule docking, they were able to narrow down the possible site of AIP adduction to the Phe319 residue of the transmembrane domain TM6.

To verify their model, the authors used several biochemical approaches: methionine substitution of the residues flanking Phe319 and cyanogen bromide mapping of the mutants, followed by substituted cysteine accessibility method protection, or SCAM, analysis.

They concluded that the binding of the tropane pharmacophore of the analog occurs within the DAT S1 site, which is highly conserved in mammalian transporters. The binding leads to the AIP adduction to the Phe319 residue, which is situated at the interface between the S1 and S2 sites. This likely plays a key role in transitioning of the transporter between conformational states, and the authors predict that cocainelike molecules block transport by inhibiting these transitions.

By better understanding how cocaine binds to DAT, future research can begin to focus on developing improved strategies for treating cocaine addiction.

Jen McGlaughonJen McGlaughon ( jla254@cornell.edu) is a graduate student in the molecular biology and genetics department at Cornell University.