Allosteric effects of R- and S-citalopram on the human 5-HT transporter: evidence for distinct high- and low-affinity binding sites
Research output: Contribution to journal › Journal article › Research › peer-review
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.
Original language | English |
---|---|
Journal | European Journal of Pharmacology |
Volume | 567 |
Issue number | 1-2 |
Pages (from-to) | 1-9 |
Number of pages | 8 |
ISSN | 0014-2999 |
DOIs | |
Publication status | Published - 2007 |
Bibliographical note
Keywords: Allosteric Regulation; Animals; Antidepressive Agents; Binding Sites; COS Cells; Cercopithecus aethiops; Citalopram; Humans; Mutagenesis, Site-Directed; Paroxetine; Radioligand Assay; Serotonin Plasma Membrane Transport Proteins; Stereoisomerism
ID: 21702104