Is Cocaine ProtonatedWhen it Binds to the DopamineTransporter?

There has been much controversy about whether the well-known alkaloid and tertiary amine base cocaine (p<i>K</i>_a = 8.5) binds to the human dopamine transporter (DAT) in its protonated form. Most potent DAT inhibitors are also strong aminesyet there are some noteworthy examples where neutral cocaine analogues have high affinity, while the quaternary ammonium analog of cocaine, cocaine methiodide, is a comparatively poor inhibitor. In this paper, we show that a fluorescent cocaine analog, with a lower p<i>K</i>_a than cocaine, becomes protonated in the DAT binding site and conclude that similar behavior must be expected from cocaine. By determining the p<i>K</i>_a of the aspartate residue in DAT believed to interact with the amine of cocaine, we are able to explain the apparently contradictory structure–activity data of cocaine analogues.