Determination of residues in the norepinephrine transporter that are critical for tricyclic antidepressant affinity

The norepinephrine (NET) and dopamine (DAT) transporters are highly homolog ous proteins, displaying many pharmacological similarities. Both transport dopamine with higher affinity than norepinephrine and are targets for the p sychostimulants cocaine and amphetamine, However, they strikingly contrast in their affinities for tricyclic antidepressants (TCA). Previous studies, based on chimeric proteins between DAT and NET suggest that domains ranging from putative transmembrane domain (TMD) 5 to 8 are involved in the high a ffinity binding of TCA to NET. We substituted 24 amino acids within this re gion in the human NET with their counterparts in the human DAT, resulting i n 22 different mutants. Mutations of residues located in extra- or intracyt oplasmic loops have no effect on binding affinity of neither TCA nor cocain e. Three point mutations in TMD6 (F316C), -7 (V356S), and -8 (G400L) induce d a loss of TCA binding affinity of 8-, 5-, and 4-fold, respectively, witho ut affecting the affinity of cocaine. The triple mutation F316C/V356S/G400L produced a 40-fold shift in desipramine affinity. These three residues are strongly conserved in all TCA-sensitive transporters cloned in mammalian a nd nonmammalian species. A strong shift in TCA affinity (IC,,) was also obs erved for double mutants F316C/D336T (35-fold) and S399P/G400L (80-fold for nortriptyline and 1000-fold for desipramine), Reverse mutations P401S/L402 G in hDAT did not elicit any gain in TCA affinities, whereas C318F and S358 V resulted in a 3- and 10-fold increase in affinity, respectively. Our resu lts clearly indicate that two residues located in TMD6 and -7 of hNET may p lay an important role in TCA interaction and that a critical region in TMD8 is likely to be involved in the tertiary structure allowing the high affin ity binding of TCA.