Interaction of tyrosine 151 in norepinephrine transporter with the 2β group of cocaine analog RTI-113

Cocaine binds and inhibits dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter. The residues forming cocaine binding sites are unknown. RTI-113, a cocaine analog, is 100× more potent at inhibiting DAT than inhibiting NET. Here we show that removing the hydroxyl group from residue Tyr151 in NET by replacing it with Phe, the corresponding residue in DAT, increased the sensitivity of NET to RTI-113, while the reverse mutation in DAT decreased the sensitivity of DAT to RTI-113. In contrast, RTI-31, another cocaine analog having the same structure as RTI-113 but with the phenyl group at the 2β position replaced by a methyl group, inhibits the transporter mutants equally well whether a hydroxyl group is present at the residue or not. The data suggest that this residue contributes to cocaine binding site and is close to the 2β position of cocaine analogs. These results are consistent with our previously proposed cocaine-DAT binding model where cocaine initially binds to a site that does not overlap with, but is close to, the dopamine-binding site. Computational modeling and molecular docking yielded a binding model that explains the observed changes in RTI-113 inhibition potencies.

► Mutation Y151F in NET greatly increased its sensitivity to cocaine analog RTI-113.
► The reverse mutation, F155Y in DAT decreased its affinity to RTI-113.
► Replacing a phenyl group in RTI-113 at the 2β position with a methyl group abolished such effects.
► Computational modeling indicates the removal of the Tyr hydroxyl group makes a tighter binding.
► The results support a initial cocaine binding site not overlapping with the substrate binding site.