5-HT3AB Receptors Research Articles

Ginger and its pungent constituents non-competitively inhibit activation of human recombinant and native 5-HT3 receptors of enteric neurons.

Beneficial effects of ginger in the treatment of gastrointestinal (GI) problems and chemotherapy-induced nausea and vomiting are well accepted. In rodents, the action of ginger seems to be mediated by the inhibition of 5-HT3 receptors, which are established targets to combat emesis and irritable bowel syndrome. Heterologously expressed human 5-HT3 A or 5-HT3 AB receptors were characterized by means of Ca(2+) influx studies using HEK293 cells. Complementing Ca(2+) measurements in Fluo-4-AM-stained whole-mount preparations of the human submucous plexus were carried out. Furthermore, [3H]GR65630 binding assays were performed to reveal the mode of action of ginger and its pungent compounds. We show for the first time that ginger extracts and its pungent arylalkane constituents concentration-dependently inhibit activation of human 5-HT3 receptors. Ginger extracts inhibited both receptors with increasing content of pungent compounds, confirming that these are part of ginger's active principle. Inhibition potencies of the arylalkanes 6-gingerol and 6-shogaol on both receptors were in the low micromolar range. A lipophilic ginger extract and 6-gingerol had no influence on 5-HT potency, but reduced the 5-HT maximum effect, indicating non-competitive inhibition. The non-competitive action was confirmed by [(3) H]GR65630 binding, showing that the ginger extract did not displace the radioligand from 5-HT3 A and 5-HT3 AB receptors. The potential relevance of the inhibitory action of ginger on native 5-HT3 receptors in the gut was confirmed in whole-mount preparations of the human submucous plexus. While a general neurotoxic effect of 6-gingerol was ruled out, it inhibited the 2-methyl-5-HT-mediated activation of 5-HT3 receptors residing on enteric neurons. Our findings may encourage the use of ginger extracts to alleviate nausea in cancer patients receiving chemotherapy and to treat functional GI disorders.

Subunit‐dependent inhibition and potentiation of 5‐HT3 receptor by the anticancer drug, topotecan

The 5-hydroxytryptamine (serotonin, 5-HT) type 3 (5-HT3) receptor belongs to the superfamily of Cys-loop ligand-gated ion channels, and can be either homopentameric (5-HT3A) or heteropentameric (5-HT3AB) receptor. Several modulators are known, which either inhibit or potentiate this channel, but few have any appreciable selectivity between the two subtypes or can modulate one receptor differently to the other. In this study, we show that the anticancer drug, topotecan, bidirectionally modulates the 5-HT3 receptor using a two-electrode voltage clamp technique. Topotecan inhibited 5-HT-gated current through homomeric 5-HT3A receptors. Interestingly, however, additional expression of the 5-HT3B subunit changed the response to topotecan dramatically from an inhibitory to a potentiatory one. This effect was dependent on the level of 5-HT3B subunit expression. Moreover, the effect was reduced in the receptors containing the 5-HT3B(Y129S) polymorphic variant. These finding could explain individual differences in the sensitivity to topotecan-induced nausea and vomiting.

VUF10166, a novel compound with differing activities at 5-HT₃A and 5-HT₃AB receptors.

The actions of a novel, potent 5-HT₃ receptor ligand, [2-chloro-(4-methylpiperazine-1-yl)quinoxaline (VUF10166)], were examined at heterologously expressed human 5-HT₃A and 5-HT₃AB receptors. VUF10166 displaced [³H]granisetron binding to 5-HT₃A receptors expressed in human embryonic kidney cells with high affinity (K(i) = 0.04 nM) but was less potent at 5-HT₃AB receptors (K(i) = 22 nM). Dissociation of [³H]granisetron in the presence of VUF10166 was best fit with a single time constant (t(1/2) = 53 min) at 5-HT₃A receptors, but with two time constants (t(1/2) = 55 and 2.4 min) at 5-HT₃AB receptors. Electrophysiological studies in oocytes revealed that VUF10166 inhibited 5-HT-induced responses at 5-HT₃A receptors at nanomolar concentrations, but inhibition and recovery were too slow to determine an IC₅₀. At 5-HT₃AB receptors, inhibition and recovery were faster, yielding an IC₅₀ of 40 nM. Cysteine substitutions in the complementary (-), but not the principal (+), face of the 5-HT₃B subunit produced heteromeric receptors in which the actions of VUF10166 resembled those at homomeric receptors. At 5-HT₃A receptors, VUF10166 at higher concentrations also behaved as a partial agonist (EC₅₀ = 5.2 μM; R(max) = 0.24) but did not elicit significant responses at 5-HT₃AB receptors at ≤100 μM. Thus, we propose that VUF10166 binds to the common A+A- site of both receptor types and to a second A+B- modulatory site in the heteromeric receptor. The ability of VUF10166 to distinguish between 5-HT₃A and 5-HT₃AB receptors could help evaluate differences between these receptor types and has potential therapeutic value.

Anticancer drug irinotecan inhibits homomeric 5-HT3A and heteromeric 5-HT3AB receptor responses

It has been shown that anti-cancer drug induces secretion of serotonin (5-HT) from small intestine which activates serotonin type 3 (5-HT3) receptor to cause nausea and vomiting. In general, antagonist for 5-HT3 receptor is used as anti-emetics during chemotherapy. However, we found that anti-cancer drug irinotecan itself inhibits 5-HT-gated current through the homomeric 5-HT3A and heteromeric 5-HT3AB receptor in a concentration-dependent manner. The inhibitory effect of irinotecan on 5-HT3A receptor was more potent than that on 5-HT3AB receptor. On the other hand, SN-38, a metabolite of irinotecan, had no effect on the responsiveness. Our findings suggest that irinotecan itself could have anti-emetic activities through inhibition of the 5-HT3A and 5-HT3AB receptor.

Cysteine modification reveals which subunits form the ligand binding site in human heteromeric 5-HT3AB receptors.

The ligand binding site of Cys-loop receptors is formed by residues on the principal (+) and complementary (-) faces of adjacent subunits, but the subunits that constitute the binding pocket in many heteromeric receptors are not yet clear. To probe the subunits involved in ligand binding in heteromeric human 5-HT(3)AB receptors, we made cysteine substitutions to the + and - faces of A and B subunits, and measured their functional consequences in receptors expressed in Xenopus oocytes. All A subunit mutations altered or eliminated function. The same pattern of changes was seen at homomeric and heteromeric receptors containing cysteine substitutions at A(R92) (- face), A(L126)(+), A(N128)(+), A(I139)(-), A(Q151)(-) and A(T181)(+), and these receptors displayed further changes when the sulphydryl modifying reagent methanethiosulfonate-ethylammonium (MTSEA) was applied. Modifications of A(R92C)(-)- and A(T181C)(+)-containing receptors were protected by the presence of agonist (5-HT) or antagonist (d-tubocurarine). In contrast modifications of the equivalent B subunit residues did not alter heteromeric receptor function. In addition a double mutant, A(S206C)(-)(/E229C)(+), only responded to 5-HT following DTT treatment in both homomeric and heteromeric receptors, indicating receptor function was inhibited by a disulphide bond between an A+ and an A- interface in both receptor types. Our results are consistent with binding to an A+A- interface at both homomeric and heteromeric human 5-HT(3) receptors, and explain why the competitive pharmacologies of these two receptors are identical.

Ginkgolide B and bilobalide block the pore of the 5-HT3 receptor at a location that overlaps the picrotoxin binding site

Extracts from the Ginkgo biloba tree are widely used as herbal medicines, and include bilobalide (BB) and ginkgolides A and B (GA and GB). Here we examine their effects on human 5-HT3A and 5-HT3AB receptors, and compare these to the effects of the structurally related compounds picrotin (PTN) and picrotoxinin (PXN), the two components of picrotoxin (PTX), a known channel blocker of 5-HT3, nACh and GABAA receptors. The compounds inhibited 5-HT-induced responses of 5-HT3 receptors expressed in Xenopus oocytes, with IC50 values of 470μM (BB), 730μM (GB), 470μM (PTN), 11μM (PXN) and >1mM (GA) in 5-HT3A receptors, and 3.1mM (BB), 3.9mM (GB), 2.7mM (PTN), 62 μM (PXN) and >1mM (GA) in 5-HT3AB receptors. Radioligand binding on receptors expressed in HEK 293 cells showed none of the compounds displaced the specific 5-HT3 receptor antagonist [3H]granisetron, confirming that they do not act at the agonist binding site. Inhibition by GB at 5-HT3A receptors is weakly use-dependent, and recovery is activity dependent, indicating channel block. To further probe their site of action at 5-HT3A receptors, BB and GB were applied alone or in combination with PXN, and the results fitted to a mathematical model; the data revealed partially overlapping sites of action. We conclude that BB and GB block the channel of the 5-HT3A receptor. Thus these compounds have comparable, although less potent, behaviour than at some other Cys-loop receptors, demonstrating their actions are conserved across the family.

Agonists and Antagonists Bind to an A-A Interface in the Heteromeric 5-HT3AB Receptor

The 5-HT3 receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT3AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT3AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [3H]granisetron binding affinity (Kd) and 5-HT EC50 were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC50 values were increased. In B-to-A mutant receptors there were no changes in Kd, although EC50 values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT3A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT3AB receptor.

LGIC

LGIC

Characterization of the effects of four HTR3B polymorphisms on human 5-HT3AB receptor expression and signalling

5-Hydroxytryptamine 3 (5-HT3) receptors mediate the fast excitatory neurotransmission of serotonin. In this study, we have characterized the effects of four naturally occurring, nonsynonymous variants of the human 5-HT3B subunit on expression and signalling properties of heteromeric 5-HT3AB receptors. 5-HT3AB receptor signalling was studied in a fluorescence-based cell membrane potential assay and by electrophysiology. Expression levels of cotransfected epitope-tagged 5-HT3A and 5-HT3B subunits were determined using enzyme-linked immunosorbent assay and immunocytochemistry. In cells coexpressing 5-HT3A and 5-HT3B(I143T) subunits, cell surface expression levels of 5-HT3B in particular, and also 5-HT3A were markedly reduced compared with those of wild-type (WT) 5-HT3AB receptor-expressing cells. Electrophysiological recordings on cells coexpressing 5-HT3A and 5-HT3B(I143T) indicated cell surface expression of 5-HT3AB(I143T) receptors with macroscopic current kinetics similar to those of WT 5-HT3AB receptors but with 3-fold lower current densities. In the membrane potential assay, 5-HT3AB(I143T)-transfected cells exhibited signalling properties intermediate to those of WT 5-HT3AB and 5-HT3A receptors. Cotransfection of 5-HT3A, 5-HT3AB(I143T) and WT 5-HT3AB subunit cDNAs did not increase cell surface expression of the variant subunit nor did it restore WT 5-HT3AB receptor signalling completely in the membrane potential assay. In contrast to 5-HT3B(I143T), the 5-HT3B variants S156R, V183I and A223T did not give rise to significant changes in 5-HT3AB receptor expression or signalling properties. 5-HT3B(I143T)-containing 5-HT3AB receptors display significantly reduced cell surface expression and different signalling properties compared with WT 5-HT3AB receptors. In contrast, three other 5-HT3B variants, S156R, V183I and A223T, do not appear to alter 5-HT3AB receptor expression or signalling.

The 5-HT3B Subunit Confers Spontaneous Channel Opening and Altered Ligand Properties of the 5-HT3 Receptor

Current receptor theory suggests that there is an equilibrium between the inactive (R) and active (R*) conformations of ligand-gated ion channels and G protein-coupled receptors. The actions of ligands in both receptor types could be appropriately explained by this two-state model. Ligands such as agonists and antagonists affect receptor function by stabilizing one or both conformations. The 5-HT3 receptor is a member of the Cys-loop ligand-gated ion channel superfamily participating in synaptic transmission. Here we show that co-expression of the 5-HT3A and 5-HT3B receptor subunits in the human embryonic kidney (HEK) 293 cells results in a receptor that displays a low level of constitutive (or agonist-independent) activity. Furthermore, we also demonstrate that the properties of ligands can be modified by receptor composition. Whereas the 5-hydroxytryptamine (5-HT) analog 5-methoxyindole is a partial agonist at the 5-HT3A receptor, it becomes a "protean agonist" (functioning as an agonist and an inverse agonist at the same receptor) at the 5-HT3AB receptor (after the Greek god Proteus, who was able to change his shape and appearance at will). In addition, the 5-HT analog 5-hydroxyindole is a positive allosteric modulator for the liganded active (AR*) conformation of the 5-HT3A and 5-HT3AB receptors and a negative allosteric modulator for the spontaneously active (R*) conformation of the 5-HT3AB receptor, suggesting that the spontaneously active (R*) and liganded active (AR*) conformations are differentially modulated by 5-hydroxyindole. Thus, the incorporation of the 5-HT3B subunit leads to spontaneous channel opening and altered ligand properties.

High-frequency HTR3B variant associated with major depression dramatically augments the signaling of the human 5-HT 3AB receptor

The 5-hydroxytryptamine-3 (5-HT3) receptor mediates the fast excitatory neurotransmission of serotonin and is known to mediate the nausea/emesis induced by radio/chemotherapy and anesthetics. A polymorphism encoding the variation Y129S in the 5-HT3B subunit exists in high frequency in the general population and has been shown to be inversely correlated to the incidence of major depression in women. We show that 5-HT3AB(Y129S) receptors exhibit a substantially increased maximal response to serotonin compared with WT receptors in two fluorescence-based cellular assays. In electrophysiological recordings, the deactivation and desensitization kinetics of the 5-HT3AB(Y129S) receptor are 20- and 10-fold slower, respectively, than those of the WT receptor. Single-channel measurements reveal a 7-fold-increased mean open time of 5-HT3AB(Y129S) receptors compared with WT receptors. The augmented signaling displayed by 5-HT3AB(Y129S) receptors may confer protection against the development of depression. The variant also may influence the development and/or treatment of nausea and other disorders involving 5-HT3 receptors. Thus, the impact of the high-frequency variant 5-HT3B(Y129S) on 5-HT3AB receptor signaling calls for a search for additional phenotypes, and the variant may thus aid in establishing the role of the 5-HT3AB receptor in pathophysiology.

5-Fluorotryptamine is a partial agonist at 5-HT3 receptors, and reveals that size and electronegativity at the 5 position of tryptamine are critical for efficient receptor function

Antagonists, but not agonists, of the 5-HT3 receptor are useful therapeutic agents, and it is possible that partial agonists may also be potentially useful in the clinic. Here we show that 5-fluorotryptamine (5-FT) is a partial agonist at both 5-HT3A and 5-HT3AB receptors with an Rmax (Imax/Imax5-HT) of 0.64 and 0.45 respectively. It is about 10 fold less potent than 5-HT: EC50=16 and 27 μM, and Ki for displacement of [3H]granisetron binding=0.8 and 1.8 μM for 5-HT3A and 5-HT3AB receptors respectively. We have also explored the potencies and efficacies of tryptamine and a range of 5-substituted tryptamine derivatives. At 5-HT3A receptors tryptamine is a weak (Rmax=0.15), low affinity (EC50=113 μM; Ki=4.8 μM) partial agonist, while 5-chlorotryptamine has a similar affinity to 5-FT (EC50=8.1 μM; Ki=2.7 μM) but is a very weak partial agonist (Rmax=0. 0037). These, and data from 5-methyltryptamine and 5-methoxytryptamine, reveal the importance of size and electronegativity at this location for efficient channel opening.

Inhibition of human 5-HT3A and 5-HT3AB receptors by etomidate, propofol and pentobarbital

The actions of intravenous anaesthetics on 5-HT(3AB) receptors have not been studied. Using oocyte electrophysiology, the effects of etomidate, propofol, and pentobarbital on human 5-HT(3A) and 5-HT(3AB) receptors were studied and compared. Inhibition of peak currents by all three compounds in both receptor subtypes was anaesthetic concentration-dependant and non-competitive. Because the half-maximal inhibitory concentrations for etomidate, propofol and pentobarbital in 5-HT(3A) and 5-HT(3AB) receptors were all above their respective anaesthetic concentrations, the results of our study suggest that neither 5-HT(3) receptor subtype contributes to the anaesthetic actions of etomidate, propofol or pentobarbital.

Potentiation of 5-HT3AB receptors by small n-alcohols is caused by shifting the open-close equilibrium to the open state

Potentiation of 5-HT3AB receptors by small n-alcohols is caused by shifting the open-close equilibrium to the open state

General anesthetic action on 5-HT3 receptors: Influence of subunit composition

The 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptor is one member of an anesthetic-sensitive superfamily of Cys-loop ligand-gated ion channels that is thought to be an important modulator of post-operative nausea and vomiting. Electrophysiological techniques were used to define and compare the actions of volatile and alcohol anesthetics on homomeric 5-HT3A and heteromeric 5-HT3AB receptors. We found that 5-HT3AB receptors were significantly less sensitive than 5-HT3A receptors to anesthetic-induced current potentiation by physically small anesthetics, but the degree of current inhibition induced by large anesthetics was similar in both receptor subtypes. We then used dopamine (DA), an inefficacious (i.e. partial) agonist of the 5-HT3 receptor, to determine whether anesthetic-induced effects were due to changes in agonist affinity or channel gating. The small anesthetics chloroform and halothane elicited dramatic increases in DA-evoked currents at all concentrations of DA for the 5-HT3A receptor, but only modest increases in DA-evoked currents were elicited for the 5-HT3AB receptor. Neither anesthetic had a significant effect on the EC50 of the DA concentration–response relationship. These results suggest that small anesthetics potentiate agonist-evoked currents for the 5-HT3 receptor by enhancing channel gating, and not by increasing agonist affinity of the receptor.

Modulation of Human 5-Hydroxytryptamine Type 3AB Receptors by Volatile Anesthetics and n-Alcohols

Functional 5-hydroxytryptamine type 3 (5-HT3) receptors can be formed by 5-HT3A subunits alone or in combination with the 5-HT3B subunit, but only the 5-HT3A receptor has been previously studied with respect to the modulation by volatile anesthetics and n-alcohols. Using two-electrode voltage-clamp, we show for the first time the modulation of heteromeric human (h)5-HT3AB receptors, expressed in Xenopus oocytes, by a series of n-alcohols and halogenated volatile anesthetics. At twice their anesthetic concentration, compounds having a molecular volume of less than 110 A3 enhanced submaximal 5-HT-evoked current. Compounds larger than 110 A3 inhibited submaximal 5-HT-evoked current. In experiments examining 5-HT concentration-response relationships, chloroform and butanol caused a slight decrease in the 5-HT EC50. Sevoflurane and octanol inhibited 5-HT-evoked current at all 5-HT concentrations tested but had no effect upon the 5-HT EC50. Compared with previous data on homomeric h5-HT3A receptors, the presence of the h5-HT3B subunit reduces the enhancement of h5-HT3 receptors by smaller halogenated volatile anesthetics and n-alcohols. In summary, these results suggest that heteromeric h5-HT3AB receptors are modulated by halogenated volatile anesthetics at clinically relevant concentrations, in addition to n-alcohols, suggesting that these receptors may be another physiological target for these compounds. The modulation is dependent upon the molecular volume of the compound, further supporting the concept of an anesthetic binding pocket of limited volume common on other Cys-loop ligand-gated ion channels. Incorporation of the 5-HT3B subunit alters either the anesthetic binding site or the allosteric interactions between anesthetic binding and channel opening.

Subunit rotation models activation of serotonin 5-HT3AB receptors by agonists

The N-terminal extracellular regions of heterooligomeric 3AB-type human 5-hydroxytryptamine receptors (5-HT3ABR) were modelled based on the crystal structure of snail acetylcholine binding protein AChBP. Stepwise rotation of subunit A by 5 degrees was performed between -10 degrees and 15 degrees to mimic agonist binding and receptor activation. Anticlockwise rotation reduced the size of the binding cavity in interface AB and reorganised the network of hydrogen bonds along the interface. AB subunit dimers with different rotations were applied for docking of ligands with different efficacies: 5-HT, m-chlorophenylbiguanide, SR 57227, quinolinyl piperazine and lerisetron derivatives. All ligands were docked into the dimer with -10 degrees rotation representing ligand-free, open binding cavities similarly, without pharmacological discrimination. Their ammonium ions were in hydrogen bonding distance to the backbone carbonyl of W183. Anticlockwise rotation and contraction of the binding cavity led to distinctive docking interactions of agonists with E129 and cation-pi interactions of their ammonium ions. Side chains of several further amino acids participating in docking (Y143, Y153, Y234 and E236) are in agreement with the effects of point mutations in the binding loops. Our model postulates that 5-HT binds to W183 in a hydrophobic cleft as well as to E236 in a hydrophilic vestibule. Then it elicits anticlockwise rotation to draw in loop C via pi-cation-pi interactions of its ammonium ion with W183 and Y234. Finally, closure of the binding cavity might end in rebinding of 5-HT to E129 in the hydrophilic vestibule.

The importance of receptor subunit composition and anaesthetic molecular volume in defining modulation of 5-HT3AB receptors by anaesthetics

The importance of receptor subunit composition and anaesthetic molecular volume in defining modulation of 5-HT3AB receptors by anaesthetics