title: An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: kinetic binding studies with the ALI/VFL-SI/TT mutant.
abstract: The human serotonin transporter (hSERT) has primary and allosteric binding sites for escitalopram and R-citalopram. Previous studies have established that the interaction of these two compounds at a low affinity allosteric binding site of hSERT can affect the dissociation of [(3)H]escitalopram from hSERT. The allosteric binding site involves a series of residues in the 10th, 11th, and 12th trans-membrane domains of hSERT. The low affinity allosteric activities of escitalopram and R-citalopram are essentially eliminated in a mutant hSERT with changes in some of these residues, namely A505V, L506F, I507L, S574T, I575T, as measured in dissociation binding studies. We confirm that in association binding experiments, R-citalopram at clinically relevant concentrations reduces the association rate of [(3)H]escitalopram as a ligand to wild type hSERT. We demonstrate that the ability of R-citalopram to reduce the association rate of escitalopram is also abolished in the mutant hSERT (A505V, L506F, I507L, S574T, I575T), along with the expected disruption the low affinity allosteric function on dissociation binding. This suggests that the allosteric binding site mediates both the low affinity and higher affinity interactions between R-citalopram, escitalopram, and hSERT. Our data add an additional structural basis for the different efficacies of escitalopram compared to racemic citalopram reported in animal studies and clinical trials, and substantiate the hypothesis that hSERT has complex allosteric mechanisms underlying the unexplained in vivo activities of its inhibitors.
authors.affiliation: Department of Biological Research, Lundbeck Research USA, Paramus, NJ 07652, USA. HGZ@Lundbeck.com
authors.forename: Huailing
authors.initials: H
authors.lastname: Zhong
authors.forename: Kasper B
authors.initials: KB
authors.lastname: Hansen
authors.forename: Noel J
authors.initials: NJ
authors.lastname: Boyle
authors.forename: Kiho
authors.initials: K
authors.lastname: Han
authors.forename: Galina
authors.initials: G
authors.lastname: Muske
authors.forename: Xinyan
authors.initials: X
authors.lastname: Huang
authors.forename: Jan
authors.initials: J
authors.lastname: Egebjerg
authors.forename: Connie
authors.initials: C
authors.lastname: Sánchez
mesh_terms: D000495:Allosteric Site; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D015283:Citalopram / Q000737:chemistry / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D004336:Drug Antagonism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011869:Radioligand Assay; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D017367:Selective Serotonin Reuptake Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D013237:Stereoisomerism
chemical_list: D015283:Citalopram; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; C493230:SLC6A4 protein, human