Characterization of [³H]CFT binding to the norepinephrine transporter suggests that binding of CFT and nisoxetine is not mutually exclusive.

The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [(3)H]nisoxetine. However, traditional [(3)H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [(3)H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [(3)H]CFT binding to whole cells (at physiological [Na(+)] and 25°C) were similar to potencies observed in the [(3)H]NE uptake assay. Inhibition constants (K(i)) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R(2)=0.99, p<0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [(3)H]nisoxetine binding to membrane preparations (4°C for 3h, [Na(+)] at 295 mM), the potency of nisoxetine and desipramine in inhibiting [(3)H]CFT binding became greater than that measured in a functional assay of [(3)H]NE uptake at physiological [Na(+)]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [(3)H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [(3)H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter.