High- and low-affinity binding of S-citalopram to the human serotonin transporter mutated at 20 putatively important amino acid positions.

The serotonin transporter (SERT) is responsible for terminating or modulating the action of serotonin released from the presynaptic neuron and is the major target for most antidepressants including the tricyclic antidepressants and the selective serotonin uptake inhibitors. Two binding sites for uptake inhibitors and serotonin (5-HT) have been found on SERT. At one site, uptake inhibitors bind with high-affinity to SERT, thereby blocking the uptake of 5-HT. The other site is a low-affinity allosteric site, which influences the dissociation of uptake inhibitors, such as imipramine, paroxetine, and citalopram from the first site, when occupied by 5-HT and a few uptake inhibitors like paroxetine and citalopram. In this study, the connection between the high-affinity binding site and the allosteric affinity-modulating site was investigated by introducing 20 single amino acid substitutions into positions of presumed importance. Binding of S-citalopram, both to the high-affinity-binding site and to the allosteric binding site, was measured in these mutants with the purpose of investigating the connection between the two binding sites. The amino acid substitutions did not introduce large changes in the two binding sites, but the results indicate that the two binding sites are independent as mutants were found in which the two binding sites were affected differently. Mutations were found which destabilised the high-affinity binding without changing the allosteric effect (e.g., G128A); mutations which destabilised the high-affinity binding but increased the allosteric effect (e.g., G100A), and mutations which were without effect on the high-affinity binding, but which increased the allosteric effect (e.g., Q562A). It is concluded that the allosteric binding site is independent of the high-affinity-binding site; it may therefore represent a new drug target.