ligand Binding to the Serotonin Transporter: Equilibria, Kinetics, and Ion Dependence (original) (raw)
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Neuroscience Letters, 2005
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-affinitybinding 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.
European Journal of Pharmacology: Molecular Pharmacology, 1991
The dissociations of [3H]imipramine, [3H]paroxetine and [3H]citalopram from the 5-HT (serotonin 5-hydroxytryptamine) transporter were found to be markedly influenced by several drugs, although concentrations in the/~M range were needed. Most of these drugs attenuated the dissociation rate, i.e. increased the affinity between the ligand and the binding site. A few increased the dissociation rate however. The binding of drugs to the affinity-modulating site was specific, although of low affinity and probably changing the conformation of the high-affinity binding site, thereby changing the fit between the ligand and the interacting amino acid side-chains.
European Journal of Pharmacology: Molecular Pharmacology, 1991
The dissociations of [3H]imipramine, [3H]paroxetine and [3H]citalopram from the 5-HT (serotonin 5-hydroxytryptamine) transporter were found to be markedly influenced by several drugs, although concentrations in the/~M range were needed. Most of these drugs attenuated the dissociation rate, i.e. increased the affinity between the ligand and the binding site. A few increased the dissociation rate however. The binding of drugs to the affinity-modulating site was specific, although of low affinity and probably changing the conformation of the high-affinity binding site, thereby changing the fit between the ligand and the interacting amino acid side-chains.
Molecular pharmacology, 2015
The membrane transporters for the monoamines serotonin (SERT) and dopamine (DAT) are prominent targets of various psychoactive substances, including competitive inhibitors such as tricyclic antidepressants, methylphenidate, and cocaine. Upon rapid application of substrate, SERT and DAT display an inwardly directed current comprised of a peak- and a steady-state component. Binding of a competitive inhibitor to the transporter leads to reduction of the peak current amplitude, because occupancy of the transporter by an inhibitor prevents the induction of the peak current by substrate. We show that the inhibitory effect on the peak current can be used to study kon, koff, and KD of chemically distinct SERT and DAT inhibitors with high temporal precision and without the need of high-affinity radioligands as surrogate. We exemplify our approach by measuring the kinetics of cocaine, methylphenidate, and desipramine binding to SERT and DAT. Our analysis revealed that the selectivity of methy...
Molecular Mechanism of Citalopram and Cocaine Interactions with Neurotransmitter Transporters
Journal of Pharmacology and Experimental Therapeutics, 2003
The selective serotonin reuptake inhibitors (SSRIs) and cocaine bind to the neural serotonin (5-HT) transporter (SERT) and thus inhibit presynaptic reuptake of 5-HT and elevate its concentration in the synaptic cleft. Cocaine also binds to the dopamine transporter (DAT) and to the noradrenaline transporter (NET) and inhibits presynaptic reuptake of dopamine and noradrenaline. SERT, DAT and NET belong to the sodium/neurotransmitter symporter family, which is predicted to have a molecular structure with 12 transmembrane α-helices (TMHs) and intracellular amino-and carboxy terminals. We used an electron density projection map of the Escherichia coli Na + /H + antiporter, and site-directed mutagenesis data on DAT and SERT to construct 3-dimensional molecular models of SERT, DAT and NET. These models were used to simulate the molecular interaction mechanisms of the SSRI, Scitalopram, its less potent enantiomer, R-citalopram and of cocaine with the transporters. In the SERT model, a single amino acid (Tyr95) in TMH1 determined the transporter selectivity of S-citalopram for SERT over DAT and NET. A dipole-dipole interaction was formed between the hydroxy group of Tyr95 in SERT and the nitril group of S-citalopram, but could
Life Sciences, 1983
Previous studies have demonstrated a close functional and structural relationship between the "high affinity" binding site for [JH]imipramine and the presynaptic and platelet uptake site(s) for serotonin. Recently we have synthesized several nitro derivatives of imipramine which have a very high affinity for the imipramine binding site and which dissociate very slowly when incubations are performe~ at 0-4°C. In this report, we describe the characteristics of [~H]2-nitroimipramine binding to platelet and brain membranes. Our results support the relative utility of this ligand for studying the impramine binding site (serotonin transporter) since this :nalogue has both^a higher affinity and specific activity than [JH]imipramine.
Characterization of an allosteric citalopram-binding site at the serotonin transporter
Journal of Neurochemistry, 2005
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. In the present study we show that the dissociation rate, of [ 3 H]S-citalopram from human SERT, is retarded by the presence of serotonin, as well as by several antidepressants, when present in the dissociation buffer. Dissociation of [ 3 H]S-citalopram from SERT is most potently inhibited by S-citalopram followed by R-citalopram, sertraline, serotonin and paroxetine. EC 50 values for S-and R-citalopram are 3.6 ± 0.4 lM and 19.4 ± 2.3 lM, respectively. Fluoxetine, venlafaxine and duloxetine have no significant effect on the dissociation of [ 3 H]Scitalopram. Allosteric modulation of dissociation is independ-ent of temperature, or the presence of Na + in the dissociation buffer. Dissociation of [ 3 H]S-citalopram from a complex with the SERT double-mutant, N208Q/N217Q, which has been suggested to be unable to self-assemble into oligomeric complexes, is retarded to an extent similar to that found with the wild-type, raising the possibility that the allosteric mechanism is mediated within a single subunit. A species-scanning mutagenesis study comparing human and bovine SERT revealed that Met180, Tyr495 and Ser513 are important residues in mediating the allosteric effect, as well as contributing to high-affinity binding at the primary site.
European Journal of Pharmacology: Molecular Pharmacology, 1990
Citalopram binds with high affinity to a specific binding site located or, the serotonin (5-HT) transporter in 5-HT neurons. The binding affinity of [3H]citalopram was found to incre~c with increasing concentration of eitalopram. This may be a homotropic positive allosteric effect, thus indicating the presence of an allosteric binding site for citalopram. The molecular weight of the proteins containing the high-affinity binding sites for citalopram and paroxetine, as well as the allosteric binding site for eitalopram were determined by the irradiation method. The molecular weights of the three binding site proteins were found to be the same. suggesting that all three binding sites are located on the same protein molecule in the 5-HT transporter. The results support a hypothesis that the binding area for [3H]citalopram is located deeper in the transport channel than the [3H]paroxetine binding area. Thus the two high-affinity binding sites probably cover different, but overlapping, parts of the protein raolecule. The allostefic binding site may be located elsewhere on the protein where it induces conformational changes of the 5-HT transporter with the result that high-affinity bound figands get trapped in the transport channel, thereby explaining the increase in affinity.
Journal of Neurochemistry, 1987
The nature of interaction between the site labeled by [3H]imipramine (IMI) and the 5-hydroxytryptamine (5-HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N-ethylmaleimide (NEM) differentially affected [3H]5-HT uptake and [3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished [3H]5-HT uptake only minimally reduced [3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet [3H]5-HT uptake revealed significant reductions in maximal velocity (V, , J without altering affinity (K,,,). values for selected uptake blockers on [3H]IMI binding and [3H]5-HT uptake were determined. ICso values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of [3H]IMI binding whereas fluoxetine, CL 2 16,303, pyrilamine, and bicifadine were preferential [3H]5-HT uptake blockers. 5-HT was a weak displacer of [3H]IMI binding = 3.0 p M) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the [3H]IMI binding site and the 5-HT transporter complex in human platelets.
Journal of Pharmacology and Experimental Therapeutics, 2005
Previous studies identified partial inhibitors of serotonin (5-HT) transporter and dopamine transporter binding. We report here on a partial inhibitor of 5-HT transporter (SERT) binding identified among a group of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine analogs (4-[2-[bis(4-fluorophenyl)methoxy]ethyl]-1-(2-trifluoromethyl-benzyl)-piperidine; TB-1-099). Membranes were prepared from rat brains or human embryonic kidney cells expressing the cloned human dopamine (hDAT), serotonin (hSERT), and norepinephrine (hNET) transporters. -(4Ј-125 Iodophenyl)tropan-2-carboxylic acid methyl ester ([ 125 I]RTI-55) binding and other assays followed published procedures. Using rat brain membranes, TB-1-099 weakly inhibited DAT binding (K i ϭ 439 nM), was inactive at NET binding ([ 3 H]nisoxetine), and partially inhibited SERT binding with an extrapolated plateau ("A" value) of 20%. Similarly, TB-1-099 partially inhibited [ 125 I]RTI-55 binding to hSERT with an extrapolated plateau (A value) of 14%. Upon examining the effect of increasing concentrations of TB-1-099 on the apparent K d and B max of [ 125 I]RTI-55 binding to hSERT, we found that TB-1-099 decreased the B max in a dose-dependent manner and affected the apparent K d in a manner well described by a sigmoid dose-response curve. TB-1-099 increased the K d but not to the magnitude expected for a competitive inhibitor. In rat brain synaptosomes, TB-1-099 noncompetitively inhibited [ 3 H]5-HT, but not [ 3 H]dopamine, uptake. Dissociation experiments indicated that TB-1-099 promoted the rapid dissociation of a small component of [ 125 I]RTI-55 binding to hSERT. Association experiments demonstrated that TB-1-099 slowed [ 125 I]RTI-55 binding to hSERT in a manner unlike that of the competitive inhibitor indatraline. Viewed collectively, these results support the hypothesis that TB-1-099 allosterically modulates hSERT binding and function. The serotonin (5-HT) transporter (SERT) is a member of the twelve transmembrane-spanning transporter family that transports 5-HT across cell membranes in a sodium-dependent manner (Amara and Kuhar, 1993; Uhl and Johnson, 1994). SERT, in the central nervous system, is an important target for a wide range of medications used to treat a variety of psychiatric conditions such as anxiety, depression, and obsessive-compulsive disorder (Gorman and Kent, 1999; Zohar and Westenberg, 2000). Drugs that interact with transporters generally interact with this protein in two distinct ways. Reuptake inhibitors bind to transporter proteins but are not transported. These drugs elevate extracellular concentrations of transmitter by blocking transporter-mediated uptake of transmitters from the synapse. Substrate-type releasers bind to transporter proteins and are subsequently transported into the cytoplasm of nerve terminals. Releasers elevate extracellular Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.