Discovery of G Protein-Biased Ligands against 5-HT7R.

There has been significant attention concerning the biased agonism of G protein-coupled receptors (GPCRs), and it has resulted in various pharmacological benefits. 5-HT7R belongs to a GPCR, and it is a promising pharmaceutical target for the treatment of neurodevelopmental and neuropsychiatric disorders. Based on our previous research, we synthesized a series of 6-chloro-2'-methoxy biphenyl derivatives 1, 2, and 3 with a variety of amine scaffolds. These compounds were evaluated for their binding affinities to 5-HTR subtypes and their functional selectivity toward the Gs protein and the ß-arrestin signaling pathways of 5-HT7R. Among them, 2-(6-chloro-2'-methoxy-[1,1'-biphenyl]-3-yl)-N-ethylethan-1-amine, 2b, was found to be a G-protein-biased ligand of 5-HT7R. In an in vivo study with Shank3 transgenic mice, the self-grooming behavior test was performed with 2b, which increased the duration of self-grooming. The experiments further suggested that 5-HT7R is associated with autism spectrum disorders (ASDs) and could be a therapeutic target for the treatment of stereotypy in ASDs.

Investigation of Potential Antioxidant, Thrombolytic and Neuropharmacological Activities of Homalomena aromatica Leaves Using Experimental and In Silico Approaches.

The leaves of Homalomena aromatica are traditionally used in Bangladesh for the treatment of different chronic ailments. The purpose of this study was to explore in vitro antioxidant, thrombolytic activities, and in vivo neuropharmacological effects of methanolic extract of Homalomena aromatica (MEHA) leaves. Antioxidant activity of MEHA was assessed by a DPPH free radical scavenging assay and total phenolics content, total flavonoids content were also measured. The thrombolytic activity was determined by percentage of clot lysis and neuropharmacological activities by hole board, tail suspension, forced swimming and elevated plus maze tests. The results showed that the IC50 value of the extract against DPPH was 199.51 µg/mL. Quantitative analysis displayed higher contents of phenolics and flavonoids (147.71 mg gallic acid equivalent/g & 66.65 mg quercetin equivalent/g dried extract, respectively). The extract also showed a significant clot lysis (33.31%) activity. In case of anxiolytic activity, the elevate plus maze (EPM) test demonstrated an increase in time spent in open arms, and in case of hole board test, the number of head dipping was also significantly increased (p < 0.05). All the test compared with control (1% Tween in water) and standard (diazepam 1 mg/kg), significant dose (200 & 400 mg/kg) dependent anxiolytic activity was found. In antidepressant activity, there was a significant decrease in period of immobility in both test models (tail suspension and forced swimming) (p < 0.05). Moreover, 13 compounds were identified as bioactive, showed good binding affinities to xanthine oxidoreductase, tissue plasminogen activator receptor, potassium channel receptor, human serotonin receptor targets in molecular docking experiments. Furthermore, ADME/T analysis revealed their drug-likeness, likely pharmacological actions and non-toxic upon consumption. Taken together, our finding support the traditional medicinal use of this plant, which may provide a potential source for future drug discovery.

Ursolic acid enhances stress resistance, reduces ROS accumulation and prolongs life span in C. elegans serotonin-deficient mutants.

INTRODUCTION: Depression and anxiety disorders contribute to the global disease burden. Ursolic acid (UA), a natural compound present in many vegetables, fruits and medicinal plants, was tested in vivo for its effect on (1) enhancing resistance to stress and (2) its effect on life span. METHODS: The compound was tested for its antioxidant activity in C. elegans. Stress resistance was tested in the heat and osmotic stress assay. Additionally, the influence on normal life span was examined. RT-PCR was used to assess possible serotonin targets. RESULTS: UA prolonged the life span of C. elegans. Additionally, UA significantly lowered reactive oxygen species (ROS). Molecular docking studies, PCR analysis and microscale thermophoresis (MST) supported the results that UA acts through serotonin receptors to enhance stress resistance. DISCUSSION: Considering the urgent need for new and safe medications in the treatment of depression and anxiety disorders, our results indicate that UA may be a promising new drug candidate.

Serotonin Receptor Binding Characteristics of Geissoschizine Methyl Ether, an Indole Alkaloid in Uncaria Hook.

BACKGROUND: Geissoschizine methyl ether (GM) is one of the indole alkaloids in Uncaria hook, and an active ingredient of yokukansan (YKS) that improves behavioral and psychological symptoms of dementia (BPSD) in patients with several types of dementia. The pharmacological action of GM has been related to various serotonin (5-HT) receptor subtypes. OBJECTIVE: The aim of this article is to review the binding characteristics of GM to the 5-HT receptor subtypes in the brains using our own data and previous findings. METHOD: Competitive receptor-binding and agonist/antagonist activity assays for several 5-HT receptor subtypes were performed. Moreover, the articles describing pharmacokinetics and brain distribution of GM were searched in PubMed. RESULTS: GM bound the following 5-HT receptor subtypes: 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, 5- HT4, 5-HT5A, 5-HT6, and 5-HT7. Among these receptors, GM had partial agonistic activity for 5-HT1A receptors and antagonistic activity for 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT7 receptors. Also, GM was metabolized by various CYP isoforms, mainly CYP3A4. Parent/unchanged GM was detected in both the blood and brain of rats after oral administration of YKS. In the brains, GM was presumed to bind to 5- HT1A, 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT7 receptors on neuron-like large cells mainly in the frontal cortex. CONCLUSION: These results suggest that GM is a pharmacologically important alkaloid that regulates various serotonergic activities or functions by binding to multiple 5-HT receptor subtypes. Thus, this review provides recent 5-HT receptor-related evidence that GM is partly responsible for pharmacological effects of YKS.

Dreamlike effects of LSD on waking imagery in humans depend on serotonin 2A receptor activation.

RATIONALE: Accumulating evidence indicates that the mixed serotonin and dopamine receptor agonist lysergic acid diethylamide (LSD) induces an altered state of consciousness that resembles dreaming. OBJECTIVES: This study aimed to test the hypotheses that LSD produces dreamlike waking imagery and that this imagery depends on 5-HT2A receptor activation and is related to subjective drug effects. METHODS: Twenty-five healthy subjects performed an audiorecorded guided mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally) and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally). Cognitive bizarreness of guided mental imagery reports was quantified as a standardised formal measure of dream mentation. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC) questionnaire. RESULTS: LSD, compared with placebo, significantly increased cognitive bizarreness (p < 0.001). The LSD-induced increase in cognitive bizarreness was positively correlated with the LSD-induced loss of self-boundaries and cognitive control (p < 0.05). Both LSD-induced increases in cognitive bizarreness and changes in state of consciousness were fully blocked by ketanserin. CONCLUSIONS: LSD produced mental imagery similar to dreaming, primarily via activation of the 5-HT2A receptor and in relation to loss of self-boundaries and cognitive control. Future psychopharmacological studies should assess the differential contribution of the D2/D1 and 5-HT1A receptors to cognitive bizarreness.

Activity of etv5a and etv5b genes in the hypothalamus of fasted zebrafish is influenced by serotonin.

Serotonin has been implicated in the inhibition of food intake in vertebrates. However, the mechanisms through which serotonin acts has yet to be elucidated. Recently, ETV5 (ets variant gene 5) has been associated with obesity and food intake control mechanisms in mammals. We have analyzed a putative physiological function of the two etv5 paralogous genes (etv5a and etv5b) in neuronal food intake control in adult zebrafish that have been exposed to different nutritional conditions. A feeding assay was established and fluoxetine, a selective serotonin re-uptake inhibitor (SSRI), was applied. Gene expression changes in the hypothalamus were determined using real-time PCR. Fasting induced an up-regulation of etv5a and etv5b in the hypothalamus, whereas increased serotonin levels in the fasted fish counteracted the increase in expression. To investigate potential mechanisms the expression of further food intake control genes was determined. The results show that an increase of serotonin in fasting fish causes a reduction in the activity of genes stimulating food intake. This is in line with a previously demonstrated anorexigenic function of serotonin. Our results suggest that obesity-associated ETV5 has a food intake stimulating function and that this function is modulated through serotonin.

Synthesis and evaluation of antidepressant-like activity of some 4-substituted 1-(2-methoxyphenyl)piperazine derivatives.

A series of new derivatives of N-(2-methoxyphenyl)piperazine have been synthesized for their affinity toward serotonergic receptors and for their potential antidepressant-like activity. They have been evaluated toward receptors 5-HT1A , 5-HT6 , and 5-HT7 , as well as in vivo in the tail suspension, locomotor activity, and motor co-ordination tests. All the tested compounds proved very good affinities toward 5-HT1A and 5-HT7 receptors. The most promising compound was 1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride, exhibiting affinity toward receptors Ki <1 nm (5-HT1A ) and Ki = 34 nm (5-HT7 ). Antidepressant-like activity (tail suspension test) was observed at 2.5 mg/kg b.w. (mice, i.p.), and the effect was stronger than that observed for imipramine (5 mg/kg b.w.). Sedative activity was observed at ED50 (locomotor test, mice, i.p.) = 17.5 mg/kg b.w. and neurotoxicity was observed at TD50 (rotarod, mice, i.p.) = 53.2 mg/kg b.w.

Synthesis and evaluation of pharmacological properties of some new xanthone derivatives with piperazine moiety.

A series of new xanthone derivatives with piperazine moiety [1-7] was synthesized and evaluated for their pharmacological properties. They were subject to binding assays for α1 and ß1 adrenergic as well as 5-HT1A, 5-HT6 and 5-HT7b serotoninergic receptors. Five of the tested compounds were also evaluated for their anticonvulsant properties. The compound 3a 3-methoxy-5-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-9H-xanthen-9-one hydrochloride exhibited significantly higher affinity for serotoninergic 5-HT1A receptors (Ki=24 nM) than other substances. In terms of anticonvulsant activity, 6-methoxy-2-{[4-(benzyl)piperazin-1-yl]methyl}-9H-xanthen-9-one (5) proved best properties. Its ED50 determined in maximal electroshock (MES) seizure assay was 105 mg/kg b.w. (rats, p.o.). Combining of xanthone with piperazine moiety resulted in obtaining of compounds with increased bioavailability after oral administration.

Synthesis and evaluation of a series of 3,4,5-trimethoxycinnamic acid derivatives as potential antinarcotic agents.

A series of 3,4,5-trimethoxycinnamic acid derivatives was prepared and evaluated for antinarcotic effects on morphine dependence in mice and binding affinities on serotonergic receptors. The key synthetic strategies involve generation of ketones 6-7, esters 9-12 through condensation reaction, and amides 13-19 via coupling reaction using 1-hydroxybenzotriazole/ethyl(dimethylaminopropryl)carbodiimide system in high yield. We found that the naloxone-induced morphine withdrawal syndrome was significantly suppressed by new synthetic 3,4,5-trimethoxycinnamic acid derivatives (20 mg/kg/day). Most of 3,4,5-trimethoxycinnamic acid derivatives were found to have high affinity to 5-HT(1A) receptor. The naloxone-induced morphine withdrawal syndrome was attenuated by (+)8-OH-DPAT (0.1 mg/kg/day, i.p.), a 5-HT(1A) receptor agonist. In cortical neuronal cells, (+)8-OH-DPAT (1 µM) produced an elevation of the pERK 1/2 expression, and the elevated pERK levels were inhibited by WAY 100635, a 5-HT(1A) receptor-specific antagonist. Interestingly, the pERK levels were increased by the 3,4,5-trimethoxycinnamic acid derivatives and the derivatives-mediated changes in pERK levels were blocked by the WAY 100635. These results suggested that new synthetic 3,4,5-trimethoxycinnamic acid derivatives have a potential antinarcotic effect through acting as a 5-HT(1A) receptor agonist in mice.

Functional reconstitution of the human serotonin receptor 5-HT(6) using synthetic transmembrane peptides.

Seven transmembrane (7TM) synthetic peptides mimicking the alpha-helical TM domains of the human serotonin receptor subtype-6 (5-HT(6)) were autonomously reconstituted in detergent micelle and liposome environments. The degree of assembly of the 7TM peptides was characterized by monitoring the fluorescence resonance energy transfer (FRET) between donor and acceptor probes labeled at the amino termini of the second and fourth TM-peptides, respectively. The FRET efficiency of these peptides significantly increased when the 7TM peptides were reconstituted in liposome compare to detergent micelles. Furthermore, the 7TM peptides reconstituted in liposomes selectively bound to free serotonin and serotonin-conjugated magnetic beads, yielding a dissociation constant of 0.84 microM. These results show that the seven individual TM domains of 5-HT(6) can spontaneously assemble into liposomes in a conformation that mimics a native structure, and further demonstrate that specific interactions between TM helices play a critical role in the folding and stabilizing of GPCRs. The autonomous assembly of 7TM-peptides can be applied to the screening of agonists for GPCRs that are difficult to manipulate.

Naturally occurring variants in the HTR3B gene significantly alter properties of human heteromeric 5-hydroxytryptamine-3A/B receptors.

OBJECTIVES: The 5-hydroxytryptamine-3 (5-HT3) receptor, a ligand-gated ion channel, is known to be involved in gut motility and peristalsis, the mediation of pain and psychiatric diseases. 5-HT3 receptor antagonists are effectively used to treat chemotherapy-induced emesis and irritable bowel syndrome. We have characterized the impact of four naturally occurring variants in the HTR3B gene leading to amino acid exchanges within the respective subunit of heteromeric 5-HT3A/B receptors on a functional and expressional level. METHODS AND RESULTS: For functional characterization, a Ca influx assay based on aequorin bioluminescence was used. Radioligand-binding studies with the 5-HT3 receptor antagonist [H]GR65630 were carried out to determine expression levels of heteromeric 5-HT3A/B receptors. Transiently transfected human embryonic kidney 293 cells using 5-HT3A and 5-HT3B complementary DNA constructs were shown to coexpress homopentameric 5-HT3A next to heteromeric 5-HT3A/B receptors. The variant p.V183I decreased surface expression, whereas p.Y129S and p.S156R led to pronounced increases of 5-HT maximum responses, despite nearly unaltered surface expression levels of heteromeric 5-HT3A/B receptors. CONCLUSION: These results may help to explain earlier reported association findings of the frequent p.Y129S and p.V183I variants with psychiatric diseases. Replication studies with larger sample pools, especially regarding the rare p.S156R variant would be useful, to obtain an idea about the predisposing role of these single nucleotide polymorphisms as susceptibility variants.

Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system.

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination.

Modulation of 5-HT3 receptor-mediated response and trafficking by activation of protein kinase C.

Modulation of neurotransmitter-gated membrane ion channels by protein kinase C (PKC) has been the subject of a number of studies. However, less is known about PKC modulation of the serotonin type 3 (5-HT3) receptor, a ligand-gated membrane ion channel that can mediate fast synaptic transmission in the central and peripheral nervous system. Here, we show that PKC potentiated 5-HT3 receptor-mediated current in Xenopus oocytes expressing 5-HT3A receptors and mouse N1E-115 neuroblastoma cells. In addition, using a specific antibody directed to the extracellular N-terminal domain of the 5-HT3A receptor, treatment with the PKC activator, 4 beta-phorbol 12-myristate 13-acetate (PMA), significantly increased surface immunofluorescence. PKC also increased the amount of 5-HT3A receptor protein in the cell membrane without affecting the amount receptor protein in the total cell extract. The magnitude of PMA potentiation of 5-HT3A receptor-mediated responses is correlated with the magnitude of PMA enhancement of the receptor abundance in the cell surface membrane. PMA potentiation is unlikely to occur via direct phosphorylation of the 5-HT3A receptor protein since the potentiation was not affected by point mutation of each of the putative sites for PKC phosphorylation. However, preapplication of phalloidin, which stabilizes the actin polymerization, significantly inhibited PMA potentiation of 5-HT-activated responses in both N1E-115 cells and oocytes expressing 5-HT3A receptors. On the other hand, latrunculin-A, which destabilizes actin cytoskeleton, enhanced the PMA potentiation of 5-HT3A receptors. The observations suggest that PKC can modulate 5-HT3A receptor function and trafficking through an F-actin-dependent mechanism.

A direct interaction of PSD-95 with 5-HT2A serotonin receptors regulates receptor trafficking and signal transduction.

The serotonin (5-hydroxytryptamine) 2A receptor (5-HT2A) is an important G protein-coupled receptor (GPCR) that mediates the effects of hallucinogens and is the target of a number of commonly prescribed medications including atypical antipsychotics, antidepressants, and anxiolytics. The 5-HT2A receptor possesses a canonical Type I PDZ-binding domain (X-Ser/Thr-X-Phi) at the carboxyl terminus and has been predicted, but never demonstrated, to interact with PDZ domain-containing proteins. We discovered that PSD-95, a prototypic PDZ domain-containing protein, directly associates with the 5-HT2A receptor and regulates 5-HT2A receptor-mediated signaling and trafficking in HEK-293 cells. Co-immunoprecipitation studies revealed that the native 5-HT2A receptor, but not a mutant lacking the PDZ-binding domain, interacted directly with PSD-95. The association with PSD-95 enhanced 5-HT2A receptor-mediated signal transduction, a novel action of PSD-95 on GPCRs. The augmentation of 5-HT2A receptor signaling by PSD-95 was not accompanied by alteration in the kinetics of 5-HT2A receptor desensitization but was associated with the inhibition of agonist-induced 5-HT2A receptor internalization. Additional studies demonstrated that 5-HT2A receptor and PSD-95 were co-localized in clusters on the cell surface of HEK-293 cells. Taken together, the present work elucidates novel roles for PSD-95 in regulating the functional activity and intracellular trafficking of 5-HT2A receptors and possibly other GPCRs.

Conserved helix 7 tyrosine acts as a multistate conformational switch in the 5HT2C receptor. Identification of a novel "locked-on" phenotype and double revertant mutations.

Studies in many rhodopsin-like G-protein-coupled receptors are providing a general scheme of the structural processes underlying receptor activation. Microdomains in several receptors have been identified that appear to function as activation switches. However, evidence is emerging that these receptor proteins exist in multiple conformational states. To study the molecular control of this switching process, we investigated the function of a microdomain involving the conserved helix 7 tyrosine in the serotonin 5HT2C receptor. This tyrosine of the NPXXY motif was substituted for all naturally occurring amino acids. Three distinct constitutively active receptor phenotypes were found: moderate, high, and "locked-on" constitutive activity. In contrast to the activity of the other receptor mutants, the high basal signaling of the locked-on Y7.53N mutant was neither increased by agonists nor decreased by inverse agonists. The Y7.53F mutant was uncoupled. Computational modeling based on the rhodopsin crystal structure suggested that Y7.53 interacts with the conserved aromatic ring at position 7.60 in the recently identified helix 8 domain. This provided a basis for seeking revertant mutations to correct the defective function of the Y7.53F receptor. When the Y7.53F receptor was mutated at position 7.60, the wild-type phenotype was restored. These results suggest that Y7.53 and Y7.60 contribute to a common functional microdomain connecting helices 7 and 8 that influences the switching of the 5HT2C receptor among multiple active and inactive conformations.

Constitutively active mutants of 5-HT4 receptors are they in unique active states?

Somatic mutations leading to constitutively active G-protein coupled receptors (GPCRs) are responsible for certain human diseases. A consistent structural description of the molecular change underlying the conversion of GPCRs from an inactive R state to an active R* state is lacking. Here, we show that a series of constitutively active 5-HT4 receptors (mutated or truncated in the C-terminal and the third intracellular loop) were characterized by an increase in their denaturation rate at 55 degrees C. The thermal denaturation kinetics were monophasic, suggesting that we were measuring mainly the denaturation rate of R*. Analysis of these kinetics revealed that constitutively active C-terminal domain mutants, were due to a change in the J constant governing the R/R* equilibrium. However, the constitutive activity of the receptor mutated within the third intracellular loop was the result of both a change in the allosteric J constant and a change in the R* conformation.

First pharmacophoric hypothesis for 5-HT7 antagonism.

In order to make the first contribution to the elucidation of essential structural features for 5-HT7 antagonism, a set of thirty 5-HT7 antagonists were selected from the literature. A pharmacophore model was built using Molecular Modeling studies with Catalyst program. The information contained in this model was validated with new synthesized compounds.

Modified ligand binding to the naturally occurring Cys-124 variant of the human serotonin 5-HT1B receptor.

A substitution of phenylalanine by cysteine in position 124 is the only known naturally occurring variant of the human 5-HT1B (h5-HT1B) receptor. The present study was designed to evaluate the potential influence of the Cys-124 variant on pharmacological properties of the receptor and to test for an involvement of the mutation in the genetic predisposition to schizophrenia or bipolar affective disorder. Binding of [3H]5-carboxamidotryptamine ([3H]5-CT) and its competition with serotonin receptor agonists and antagonists were determined in COS-7 cells transfected with the wild-type or the variant h5-HT1B receptor cDNA. In saturation experiments with [3H]5-CT, the maximum binding (Bmax) of the variant receptor was approximately 30% of the wild-type receptor. In competition experiments with 1 nM [3H]5-CT, the following serotonin receptor ligands exhibited a two to three times higher affinity for the mutant than for the wild-type receptor: dihydroergotamine, L-694,247, SB-216641, 5-CT, 5-HT, sumatriptan, RU24969 and methysergide (compounds listed at decreasing order of potency at the wild-type receptor). In contrast, the serotonin receptor antagonist ketanserin exhibited higher binding affinity for the wild-type than for the mutant h5-HT1B receptor and GR127939, (-)propranolol and BRL-15572 showed equal affinity for both types of receptor. Mutation screening of schizophrenic and bipolar patients revealed no relationship between the variant receptor and development of disease. In conclusion, our data suggest that the Cys-124 variant significantly affects the pharmacological properties of the h5-HT1B receptor. Carriers of the variant may exhibit differences in response to drugs acting on the h5-HT1B receptor or may develop side-effects to such agents.

Nicotinic agonists competitively antagonize serotonin at mouse 5-HT3 receptors expressed in Xenopus oocytes.

The 5-HT3 receptor (5-HT3R) is part of a superfamily of ligand-gated ion channels which includes nicotinic acetylcholine receptors (nAChR). cRNA derived from the long isoform cloned mouse 5-HT3R was used to drive expression of 5-HT3Rs in Xenopus oocytes. 5-HT-induced currents were monitored using two-electrode voltage-clamp. Eight nicotinic agonists, including ACh and nicotine, but not alpha-anatoxin, were found to antagonize 5-HT-induced currents. With the exception of 3-(2,4)-dimethoxybenzylidene-anabaseine (DMXB-anabaseine; GTS-21) this antagonism appeared to be competitive since it could be overcome by increasing concentrations of 5-HT. Potency of 5-HT3 antagonism was comparable to reported values for nAChR alpha7 activation. These results confirm the notion of families of receptors and further indicate that strong similarities can exist in some critical binding domains.