Inhibition of transport function and desipramine binding at the human noradrenaline transporter by N-ethylmaleimide and protection by substrate analogs

Abstract.

N-ethylmaleimide (NEM) inhibits [³H]desipramine binding and [³H]noradrenaline uptake at the rat noradrenaline transporter (rNET) by covalently modifying cysteine residues. We report here that NEM also inhibits [³H]desipramine binding and [³H]noradrenaline uptake at the cloned human noradrenaline transporter (hNET) stably expressed in C6 glial cells. The IC₅₀ for NEM inhibition of [³H]noradrenaline uptake was 43.6±5.5 µM. We tested several compounds for their abilities to inhibit [³H]noradrenaline uptake via the hNET and for their abilities to protect against NEM inactivation of [³H]desipramine binding. We found that the substrate analogs bupropion, 3-bromomethcathinone, and 4-bromomethcathinone all inhibit uptake at the hNET with IC₅₀ values of 1370±140, 158±20, and 453±30 nM, respectively. These compounds as well as methamphetamine, methcathinone, and desipramine also protected the hNET from NEM inactivation of [³H]desipramine binding. The ability of substrate analogs and desipramine to protect the [³H]desipramine binding site is consistent with the hypothesis that the desipramine binding site and the substrate binding site are mutually exclusive. It also supports the use of structure-activity relationships derived from substrate analogs in the rational design of hNET uptake inhibitors. The hNET contains 10 cysteine residues whereas the rNET contains 12 cysteine residues. Since the hNET and the rNET are both inhibited by NEM, and because the NEM inhibition is protectable by desipramine and substrate analogs, we conclude that the two additional cysteine residues (C28 and C447) present in the rNET are not likely to be involved in desipramine binding or uptake function.