title: Allosteric effects of R- and S-citalopram on the human 5-HT transporter: evidence for distinct high- and low-affinity binding sites.
abstract: The human 5-HT transporter (hSERT) has two binding sites for 5-HT and 5-HT uptake inhibitors: the orthosteric high-affinity site and a low-affinity allosteric site. Activation of the allosteric site increases the dissociation half-life for some uptake inhibitors. The objectives of this study were 1) to identify hSERT mutations that inactivate the high-affinity site without affecting the allosteric site and 2) to observe allosteric effects in which hSERT binds R-citalopram with higher affinity than S-citalopram. Wild-type and mutant (Y95F, I172M, and Y95F/I172M) hSERTs were expressed in COS-7 cells, and their 5-HT uptake and uptake inhibitor-binding abilities were studied. The hSERT mutations did not alter affinities for 5-HT or paroxetine, but high-affinity binding of S-citalopram was severely affected, particularly by the I172M, and Y95F/I172M mutations - K(i) respectively 4 nM (wild-type), 35 nM, 1000 nM, and 17.100 nM (mutants). The allosteric site however, in wild-type hSERT and the three mutants was unaffected by the mutations as attenuation of the dissociation rate of the [(3)H]-paroxetine:hSERT complex in the presence of S-citalopram or paroxetine was the same for wild-type hSERT and the three mutants. Further, R-citalopram previously thought of as an inactive enantiomer strongly attenuated dissociation of the wild-type [(3)H]-imipramine:hSERT complex, whereas S-citalopram had almost no effect on this complex. These results suggest that 1: The allosteric site on hSERT is distinct from the site to which S-citalopram binds with high affinity. 2: The allosteric effects of R-citalopram on the dissociation of [(3)H]-imipramine from hSERT indicate that R-citalopram introduces a conformational change in hSERT.
authors.affiliation: Laboratory of Neuropsychiatry, Department of Neuroscience and Pharmacology, Rigshospitalet-6102, Blegdamsvej, DK-2100 Copenhagen, Denmark. plenge@rh.dk
authors.forename: Per
authors.initials: P
authors.lastname: Plenge
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
authors.forename: Søren G
authors.initials: SG
authors.lastname: Rasmussen
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D000928:Antidepressive Agents / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D015283:Citalopram / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017374:Paroxetine / Q000494:pharmacology; D011869:Radioligand Assay; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D000928:Antidepressive Agents; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D015283:Citalopram; D017374:Paroxetine