Determination of residues in the norepinephrine transporter that are critical for tricyclic antidepressant affinity.
The norepinephrine (NET) and dopamine (DAT) transporters are highly homologous proteins, displaying many pharmacological similarities. Both transport dopamine with higher affinity than norepinephrine and are targets for the psychostimulants 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 affinity binding of TCA to NET. We substituted 24 amino acids within this region in the human NET with their counterparts in the human DAT, resulting in 22 different mutants. Mutations of residues located in extra- or intracytoplasmic loops have no effect on binding affinity of neither TCA nor cocaine. Three point mutations in TMD6 (F316C), -7 (V356S), and -8 (G400L) induced a loss of TCA binding affinity of 8-, 5-, and 4-fold, respectively, without 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 and nonmammalian species. A strong shift in TCA affinity (IC(50)) was also observed for double mutants F316C/D336T (35-fold) and S399P/G400L (80-fold for nortriptyline and 1000-fold for desipramine). Reverse mutations P401S/L402G in hDAT did not elicit any gain in TCA affinities, whereas C318F and S358V resulted in a 3- and 10-fold increase in affinity, respectively. Our results clearly indicate that two residues located in TMD6 and -7 of hNET may play 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 affinity binding of TCA.
Roubert
C
C
INSERM U-513, Neurobiologie et Psychiatrie, Faculté de Médecine de Créteil, 8, rue du Général Sarrail, F-94000 Créteil, France.
Cox
P J
PJ
Bruss
M
M
Hamon
M
M
Bönisch
H
H
Giros
B
B
D000595:Amino Acid Sequence; D000929:Antidepressive Agents, Tricyclic / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003042:Cocaine / Q000494:pharmacology; D003891:Desipramine / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism; D008969:Molecular Sequence Data; D050484:Norepinephrine Plasma Membrane Transport Proteins; D009661:Nortriptyline / Q000494:pharmacology; D027981:Symporters; D014162:Transfection
D000929:Antidepressive Agents, Tricyclic; D002352:Carrier Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D027981:Symporters; D009661:Nortriptyline; D003042:Cocaine; D003891:Desipramine; D004298:Dopamine