Dissection of an Allosteric Mechanism on the Serotonin Transporter: A Cross-Species Study

Laboratory of Molecular Neurobiology, Centre for Basic Psychiatric Research, Aarhus Psychiatric University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark.
Molecular Pharmacology (Impact Factor: 4.13). 05/2006; 69(4):1242-50. DOI: 10.1124/mol.105.018507
Source: PubMed


The serotonin transporter (SERT), which belongs to a family of sodium/chloride-dependent transporters, is the major pharmacological target in the treatment of several clinical disorders, including depression and anxiety. Interaction with a low-affinity allosteric site on SERT modulates the ligand affinity at the high-affinity binding site. Serotonin (5-hydroxytryptamine) and certain SERT inhibitors possess affinity for both sites. In the present study, we report the characterization of a severely attenuated allosteric mechanism at the recently cloned chicken serotonin transporter (gSERT). A cross-species chimera study was performed, followed by species scanning mutagenesis. Residues important for the allosteric mechanism were mapped to the C-terminal part of SERT containing the transmembrane domains 10 to 12. We identified nine residues located in four distinct amino acid segments. The contribution of each segment and individual residues was investigated. Consequently, a gSERT mutant with a restored allosteric mechanism, as well as a human SERT (hSERT) mutant with a severely attenuated allosteric mechanism, was generated. The nine residues confer a functional allosteric mechanism for different combinations of ligands, suggesting that they contribute to a general allosteric mechanism at SERT. The finding of an allosteric mechanism at SERT is likely to be of physiological importance, in that serotonin was also found to act as an allosteric effector at duloxetine, RTI-55 and (S)-citalopram. Furthermore, the allosteric potency of 5-HT was found to be conserved for both hSERT and gSERT.

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    • "It was also confirmed that R-citalopram slowed the association rate of escitalopram binding to WT hSERT at clinically relevant concentrations , given that the effective escitalopram plasma levels during treatment are in the 100 nM region [13]. These data further support the hypothesis that the interaction between R-citalopram, escitalopram , and hSERT is mediated through the allosteric site, and that TM10 ALI and TM12 SI residues, identified previously [16], are required for this allosteric effect. It is interesting that the allosteric site mediates both the low affinity (seen at 24.9 ␮M R-citalopram on inhibition of escitalopram dissociation, Fig. 1) and high affinity (effect seen at 40 nM on association binding, Fig. 3a and d) interac- Fig. 3. "
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    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.
    Full-text · Article · Nov 2009 · Neuroscience Letters
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    ABSTRACT: El transportador de serotonina (SERT) realiza la recaptación de 5-HT al interior celular, regulando así su disponibilidad y unión a receptores. La 5-HT es producida en un 99% en el tracto gastrointestinal donde actúa como regulador. El objetivo de este trabajo ha sido caracterizar la expresión de SERT en la línea celular humana de tipo enterocitario Caco-2 y estudiar su regulación fisiológica y farmacológica y los mecanismos implicados. Los resultados obtenidos muestran que las células Caco-2 expresan endógenamente SERT, que es idéntico a la proteína cerebral humana y que se encuentra regulado por las vías intracelulares mediadas por AMPc y PKC. Asimismo, tanto el tratamiento de las células con fluoxetina como con 5-HT redujo la función de SERT, debido en el caso de la fluoxetina a una internalización de SERT, independiente de PKC y PKA, y en el caso de la 5-HT a una inhibición de tipo alostérico.
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    ABSTRACT: Inhibition of serotonin (5-HT) reuptake has been a central theme in the therapy of depression for half a century. Through the years these therapies have improved, particularly with regard to side effects, and today's selective serotonin reuptake inhibitors (SSRIs) constitute a reasonably effective offer for the patients. However, there is still room for major improvement and considering that almost 20% of the population in the western world will experience a depressive period in their lifetime, there is a large need for improved therapies. A large spectrum of targets and strategies are currently being pursued, but so far none of these new approaches have been successful, mainly due to lack of a deeper understanding of the disease biology. Since inhibition of 5-HT reuptake ensures a certain degree of antidepressant efficacy, there has been a large interest in various combinations with serotonin reuptake inhibitors (SRIs) in order to improve on the shortcomings of treatment with SSRIs. Some of these approaches have resulted in marketed antidepressants, eg combinations of SRI with norepinephrine (NE) reuptake inhibition, whereas other approaches are still at an experimental stage. This review attempts to present the current status of these add-on/combination approaches with particular focus on the medicinal chemistry aspects.
    No preview · Article · Feb 2006 · Current topics in medicinal chemistry
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