TY - JOUR

T1 - Species-scanning mutagenesis of the serotonin transporter reveals residues essential in selective, high-affinity recognition of antidepressants

AU - Mortensen, O V

AU - Kristensen, A S

AU - Wiborg, O

PY - 2001/10

Y1 - 2001/10

N2 - The serotonin transporter (SERT) is a high-affinity sodium/chloride-dependent neurotransmitter transporter responsible for reuptake of serotonin from the extracellular space. SERT is a selective target of several clinically important antidepressants. In a cross-species analysis comparing human and bovine SERTs, the kinetic parameters for serotonin uptake were found to be similar, however, the pharmacological profiles of the two transporters differ. Following transient expression in COS-1 cells, IC(50) values were determined for several antidepressants and psychostimulants. The potencies of the antidepressants citalopram, fluoxetine, paroxetine and imipramine were several-fold higher at hSERT compared with bSERT. No species selectivity was observed for the antidepressants fluvoxamine, and sertraline or for the psychostimulants cocaine, the cocaine analogue beta-carbomethoxy-3beta-(4-iodophenyl)tropane, or for 3,4-methylenedioxymethamphetamine (MDMA). Analysis of six hSERT/bSERT chimeras and subsequent species-scanning mutagenesis of each isoform revealed methionine-180, tyrosine-495, and phenylalanine-513 to be responsible for the increase in citalopram and paroxetine potencies at hSERT and methionine-180 and phenylalanine-513 to confer species selectivity at hSERT for fluoxetine and imipramine. Results were obtained by doing the forward, bovine to human, mutations and confirmed by doing the reverse mutations. Citalopram analogues were used to define the roles of methionine-180, tyrosine-495, and phenylalanine-513 and to reveal molecular interactions with individual functional groups of citalopram. We suggest that methionine-180 interacts with the heterocyclic nucleus of citalopram or stabilizes the binding pocket and phenylalanine-513 to be a steric blocker of antidepressant recognition.

AB - The serotonin transporter (SERT) is a high-affinity sodium/chloride-dependent neurotransmitter transporter responsible for reuptake of serotonin from the extracellular space. SERT is a selective target of several clinically important antidepressants. In a cross-species analysis comparing human and bovine SERTs, the kinetic parameters for serotonin uptake were found to be similar, however, the pharmacological profiles of the two transporters differ. Following transient expression in COS-1 cells, IC(50) values were determined for several antidepressants and psychostimulants. The potencies of the antidepressants citalopram, fluoxetine, paroxetine and imipramine were several-fold higher at hSERT compared with bSERT. No species selectivity was observed for the antidepressants fluvoxamine, and sertraline or for the psychostimulants cocaine, the cocaine analogue beta-carbomethoxy-3beta-(4-iodophenyl)tropane, or for 3,4-methylenedioxymethamphetamine (MDMA). Analysis of six hSERT/bSERT chimeras and subsequent species-scanning mutagenesis of each isoform revealed methionine-180, tyrosine-495, and phenylalanine-513 to be responsible for the increase in citalopram and paroxetine potencies at hSERT and methionine-180 and phenylalanine-513 to confer species selectivity at hSERT for fluoxetine and imipramine. Results were obtained by doing the forward, bovine to human, mutations and confirmed by doing the reverse mutations. Citalopram analogues were used to define the roles of methionine-180, tyrosine-495, and phenylalanine-513 and to reveal molecular interactions with individual functional groups of citalopram. We suggest that methionine-180 interacts with the heterocyclic nucleus of citalopram or stabilizes the binding pocket and phenylalanine-513 to be a steric blocker of antidepressant recognition.

KW - Animals

KW - Antidepressive Agents

KW - Antidepressive Agents, Second-Generation

KW - COS Cells

KW - Carrier Proteins

KW - Cattle

KW - Chimera

KW - Citalopram

KW - Humans

KW - Membrane Glycoproteins

KW - Membrane Transport Proteins

KW - Mutagenesis, Site-Directed

KW - Nerve Tissue Proteins

KW - Serotonin

KW - Serotonin Plasma Membrane Transport Proteins

KW - Serotonin Uptake Inhibitors

KW - Species Specificity

M3 - Journal article

C2 - 11677251

VL - 79

SP - 237

EP - 247

JO - Journal of Neurochemistry

JF - Journal of Neurochemistry

SN - 0022-3042

IS - 2

ER -