Dopamine Transporter Inhibition by Organic Ions & their Effects on GBR12909 Inhibition

Abstract

Dopamine transporters (DATs) in the presynaptic neuron uptake the remaining dopamine in the synaptic cleft, thereby terminating signal transmission and maintaining presynaptic dopamine storage. DATs co-transport two Na+ and one Cl- with one dopamine; the driving force for dopamine uptake is the concentration gradients of Na+ and Cl-. Consequently, dopamine uptake does not occur when either Na+ or Cl- is not present. Interestingly, however, other inorganic ions act like DAT inhibitors, suggesting they would compete with Na+ or Cl- binding, thus reducing the driving force for dopamine transport. Some organic ions are also known to inhibit dopamine uptake while the underlying mechanism is not yet clear. Here, human DAT (hDAT) inhibition by organic cations (NMDG and choline) and anions (gluconate and isethionate) was studied using fluorescence-based dopamine uptake assays. All tested organic ions inhibited hDAT in a concentration-dependent manner, exhibiting complex inhibitory effects associated with their size and charge (IC50 values ranging from 1 to 37 mM). In particular, NMDG showed a stiff sigmoidal curve with an apparent Hill coefficient (h) of 11 while other ions have h values of 1, suggesting hDAT may have multiple binding sites for NMDG. To investigate further the inhibition mechanism by organic ions, their effects on hDAT inhibition by GBR12909, a DAT specific inhibitor were tested. Interestingly, all tested ions reduced GBR12909 potency by ∼50% in a similar fashion. Among them, isethionate seemed to alter GBR12909 inhibition the most, increasing the IC50 value 3-fold and the h value 2-fold. Since isethionate contains a chemical moiety observed in some atypical DAT inhibitors, it might directly compete with GBR12909 binding. Based on the experimental data and the crystal structure of leucine transporter with ligands, putative organic ion binding sites on hDAT were speculated.