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Query: UNIPROT:P46098 (5-HT3 receptor)
 2,290 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two splice variants of the ligand-gated 5-hydroxytryptamine or serotonin 5-HT3 receptor that differ in a six-amino-acid deletion were cloned by polymerase chain reaction from the hippocampus x neuroblastoma cell line HN9.10e. When expressed in Xenopus oocytes, both variants individually formed 5-HT3 receptors that revealed no significant differences in current responses to the agonists 5-HT and 1-phenylbiguanide and block by the specific antagonist LY-278, 584-maleate. For both receptors, the monovalent cations Na+, K+, Rb+ and Li+ showed the same relative permeability; NH4(+)permeated approximately 2.7 times better than Na+, and Tris+ was only poorly permeable. In contrast to other reports, the receptors were completely and reversibly blocked by extracellular Cs+ in both oocytes and native HN9.10 cells. Moreover, Ca2+ was not permeant and exhibited a concentration-dependent decrease (0.9-18 mM) of the 5-HT-induced currents without affecting the inward rectification of the current/voltage relation. The two receptors were reversibly inhibited by nanomolar concentrations of the specific inhibitor of protein kinase C (PKC) bisindolylmaleimide, but not by the equipotent and less specific inhibitor staurosporine. A regulatory effect on both 5-HT3 receptor subunits by PKC-mediated protein phosphorylation might be possible, however, a functional role of the two splice variants present in one cell remains to be determined.
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PMID:Functional characterization of two 5-HT3 receptor splice variants isolated from a mouse hippocampal cell line. 866 78

1. Using the whole-cell variation of the patch-clamp technique to record from mammalian NG108-15 cells, we have studied the ligand-gated ion channel current activated by a high concentration (100 microM) of local pressure-applied 5-hydroxytryptamine (5-HT). The response was induced at intervals of at least 90-120 s, which allowed the receptor to fully recover between activations. 2. The rapid inward current induced by pressure-applied 5-HT was reproducibly inhibited by the superfusion of low concentrations of 5-HT which evoked little or no detectable inward current alone (0.01-0.3 microM). This inhibitory effect was most likely to be due to a direct action on the 5-HT3 receptor as it could be recorded using intracellular solutions with or without adenosine triphosphate (ATP) and guanosine triphosphate (GTP). 3. The maximum inhibitory effect of a given concentration of 5-HT was not dependent on its superfusion time but on the number of activations of the receptor by pressure-applied 5-HT. This activation dependence was clearly evident, since the first inward current in the presence of 0.1 microM 5-HT was often unaffected in amplitude. 4. The inhibitory effect of 5-HT was evident at holding potentials of +60 and -60 mV; with the calcium chelator BAPTA in the recording pipette and with the nominal removal of extracellular calcium and magnesium ions. 5. The inhibitory effect was concentration dependent, with 50% inhibition of the inward current amplitude occurring at approximately 50 nM 5-HT. The slope factor of the inhibition curve was 1.3. The effect was mimicked by two other 5-HT3 receptor agonists, 2-methyl-5-HT and m-chlorophenylbiguanide (mCPBG) which gave 50% inhibition at approximately 600 nM and approximately 20 nM, respectively. These values are similar to the affinity values for these ligands determined in radioligand binding assays. 6. The 5-HT3 receptor "antagonists' (+)-tubocurarine and quipazine (both at 3 nM) reduced the inward current amplitude by approximately 50%. The rate of onset of the inhibitory effect of bath-applied 5-HT was slowed in the presence of (+)-tubocurarine but not in the presence of quipazine. This difference might be explained by the agonist properties seen only with quipazine. 7. The inhibition of the 5-HT3 receptor mediated inward current by low concentrations of bath-applied 5-HT3 receptor agonists is compatible with the cyclic model of receptor activation and desensitization. We conclude that we have been studying the high-affinity binding of agonists to the desensitized form of the 5-HT3 receptor.
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PMID:Electrophysiological consequences of ligand binding to the desensitized 5-HT3 receptor in mammalian NG108-15 cells. 868 67

Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT3 receptor, a ligand-gated ion channel. The 5-HT3 receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K+ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca2+ concentration ([Ca2+]i), K+-induced depolarization resulted in variable but rapid increases in apparent [Ca2+]i among the individual terminals, with some synaptosomes displaying large transient [Ca2+]i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca2+]i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT3 receptors induced slow increases in [Ca2+]i (rise time, 15-20 s) in a subset (approximately 5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca2+ entry and inhibited by millimolar external Mg2+. We conclude that significant increases in brain nerve terminal Ca2+, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.
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PMID:Direct observation of serotonin 5-HT3 receptor-induced increases in calcium levels in individual brain nerve terminals. 876 83

1. We have investigated the mechanism of regulation of 5-HT3 receptor channel sensitivity in voltage-clamped (-80 mV) NG108-15 neuroblastoma cells. 2. The 5-HT-induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, tau 1 (1.1 +/- 0.21s) and tau 2 (8.9 +/- 1.6s), respectively. Brief applications of 5-HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5-HT3 receptor-mediated peak inward currents. 3. Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5-HT-induced loss of responsiveness of 5-HT3 receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness. 4. Inclusion of the protein kinase inhibitor, staurosporine (1 microM) or of okadaic acid (1 microM), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5-HT3 receptor sensitivity. 5. Injection of cyclosporin A-cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5-HT response tau 1 (1.4 +/- 0.28s) and tau 2 (11.3 +/- 1.7s). The complex, however, prevented the loss of 5-HT3, receptor responsiveness upon repeated application of 5-HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 microM). 6. The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 microM) showed a half-life time (tau 1/2) of 2.6 +/- 0.12 min in control cells which was reduced to 1.6 +/- 0.09 min in cells treated with cyclosporin A-cyclophilin A (20 nM) complex. 7. We conclude that calcineurin does not affect the fast decay of the 5-HT3 receptor response but may be involved in a slower process which regulates channel activity.
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PMID:Modulation by calcineurin of 5-HT3 receptor function in NG108-15 neuroblastoma x glioma cells. 884 51

Pancreatic ganglia contain 5-hydroxytryptamine (5-HT)-immunoreactive axons, some of which are extensions of myenteric neurons located in the pyloric antrum and proximal duodenum. The present study investigated the effect of 5-HT on the membrane potential of cat pancreatic ganglion neurons by means of intracellular recordings in vitro. Pressure application of 5-HT evoked a fast depolarization in 29 of 147 neurons and a slow depolarization in 89 of 147 neurons. A biphasic response was observed in 10 of 108 neurons. The 5-HT-induced slow depolarizing response was not altered in a low Ca2+ (0.1 mM), high Mg2+ (15 mM) solution nor by hexamethonium (10(-4) M) or atropine (10(-6) M). The fast depolarizing response was associated with a decrease of membrane input resistance (-17.2%). The slow depolarizing response was associated with either a decrease (-19.6%) in 24, an increase (+25.0%) in 20, or without a detectable change of membrane input resistance in 10 out of 54 neurons tested. Conditioning hyperpolarization increased the amplitude of both fast and slow depolarizing responses. A low Na+ (68.5 mM) solution and a high K+ (23.5 mM) solution significantly reduced the amplitude of the slow depolarizing response. A low Cl- (9.6 mM) solution had no significant effect on the slow depolarization. The 5-HT3 receptor antagonist MDL 72222 (Bemesetron) blocked the 5-HT-evoked fast depolarizing response. BRL 24924 (Renzapride) and 5 HT-DP, antagonists for the putative 5-HT1P receptor, blocked the slow depolarizing response. The 5-HT3 receptor agonist 2-methyl-5-HT evoked a fast depolarizing response and MCPP, an agonist for the putative 5-HT1P receptor, evoked a slow depolarizing response. Spiperone (a 5-HT1A receptor antagonist) and mianserin (a 5-HT2 receptor antagonist) had no effect on either depolarizing response to 5-HT. The results show that pancreatic ganglion neurons responded to 5-HT with fast and slow depolarizing responses. The data suggest that these responses were mediated by the 5-HT3 receptor and the putative 5-HT1P receptor, respectively.
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PMID:5-Hydroxytryptamine depolarizes neurons of cat pancreatic ganglia. 886 89

Homopentameric complexes of either the A or As subunit of the 5-hydroxytryptamine3 receptor form Ca(2+)-permeable channels that can be activated by the selective agonist 1-(m-chlorophenyl)-biguanide (mCPBG). In both N1E-115 neuroblastoma cells and human embryonic kidney 293 cells stably expressing the 5-HT3 receptor As subunit, (+)-verapamil, (-)-verapamil, diltiazem, and nimodipine caused reversible and concentration-dependent (IC50 = 2.5-6.5 microM) inhibition of the increases in cytosolic [Ca2+] evoked by mCPBG. In voltage-clamped human embryonic kidney 293 cells stably expressing the 5-HT3 receptor As subunit, similar concentrations of the Ca2+ channel antagonists (IC50 = 3.0-6.8 microM) accelerated the rate at which 5-HT-evoked currents decayed without affecting the amplitude of the peak current. In equilibrium competition binding assays to membranes from Sf9 cells infected with the 5-HT3 receptor As subunit, [3H]mCPBG and [3H]granisetron were displaced by (+)-verapamil, (-)-verapamil, and diltiazem; (+)-verapamil was approximately 10-fold more potent than (-)-verapamil and approximately-30-fold more potent than diltiazem. Nimodipine neither displaced [3H]granisetron binding nor affected its displacement by diltiazem and (+)-verapamil. The stereoselectivity of verapamil binding, which contrasts with the similar potency of each isomer in functional assays, was maintained when the incubations were performed at 20 degrees or when an antagonist of the 5-HT3 receptor, [3H]granisetron, was used as the radioligand. The interaction between verapamil and either [3H]mCPBG or [3H]granisetron binding was not competitive. We conclude that the inhibition of [3H]mCPBG binding by diltiazem and verapamil is mediated by a site that is distinct from both the agonist-binding site and from the site through which nimodipine inhibits 5-HT3 receptor function. Our results provide evidence for allosteric regulation of agonist binding to 5-HT3 receptors and the first example of a ligandgated ion channel whose function is directly inhibited by members of all three major classes of L-type Ca2+ channel antagonists.
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PMID:Direct inhibition of 5-hydroxytryptamine3 receptors by antagonists of L-type Ca2+ channels. 891 60

The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N-terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7-V201-5HT3 chimera. Mutations E18Q or E44Q abolish calcium-enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161-172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161-172 domain in the calcium insensitive 5-hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160-174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161-172) domain may simultaneously contribute to calcium and agonist binding.
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PMID:Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor. 891 60

The effects of local application of the 5-HT3 receptor agonist, 1-(m-chlorophenyl)-biguanide (CPBG), and i.p. administration of ethanol on the extracellular levels of dopamine (DA) in the ventral tegmental area (VTA) were studied using in vivo microdialysis. Adult female Wistar rats were implanted with microdialysis probes in the VTA at least 24 h before each experiment. Stable extracellular levels of DA (101 +/- 9 fmol/20 min) were established before initiating the experiments. Application of 10-250 microM CPBG through the microdialysis probe dose-dependently enhanced the extracellular concentrations of DA but did not alter the levels of either 3,4-dihydroxyphenylacetic acid or homovanillic acid in the dialysate. The effects of CPBG were reversible and dependent upon Ca2+. Co-perfusion with the 5-HT3 receptor antagonist, 3-tropanyl-indole-3-carboxylate (ICS 205-930), inhibited the effects of CPBG on enhancing extracellular DA levels. The i.p. administration of 2 g/kg ethanol significantly (p < 0.005) enhanced the levels of DA to 150% of baseline values; this ethanol-induced increase was prevented by local perfusion with 100 microM ICS 205-930. These results suggest that 5-HT3 receptors in the VTA are involved in regulating the somatodendritic release of DA and in mediating the stimulatory effects of ethanol on this neuronal system.
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PMID:Serotonin-3 receptor and ethanol-stimulated somatodendritic dopamine release. 894 51

In the present study, we investigated the effects of chronic in vitro administration of amitriptyline, a tricyclic antidepressant, on cyclic GMP formation stimulated by 5-hydroxytryptamine (5-HT) in the neuroblastoma x glioma hybrid cell line, NG 108-15, 5-HT (0.01-100 microM)-stimulated cyclic GMP formation was concentration-dependent and was sensitive to ICS 205-930, a 5-HT3 receptor antagonist. Exposure of NG 108-15 cells to 5 microM amitriptyline for 3 days significantly reduced 5-HT-stimulated cyclic GMP formation. Acute treatment with amitriptyline had no effect on 5-HT-stimulated cyclic GMP formation. The reduction by chronic amitriptyline exposure of 10 microM 5-HT-stimulated cyclic GMP formation was concentration-dependent over the concentration range examined (0.5 to 10 microM). The IC50 of amitriptyline was 1.9 microM. In contrast, amitriptyline exposure, even at a concentration of 8 microM, failed to modify cyclic GMP formation stimulated by bradykinin, sodium nitroprusside, or atrial natriuretic peptide. Increases in intracellular Ca2+ concentration ([Ca2+]i) evoked by 10 microM 5-HT were attenuated in amitriptyline-exposed cells, while 100 nM bradykinin-induced [Ca2+]i increases were not affected. In addition, chronic exposure to 5 microM amitriptyline caused a decrease in affinity (Kd) of [3H]zacopride specific binding to 5-HT3 recognition sites. The Bmax for the labelled ligand remained unchanged. These results suggest that chronic amitriptyline exposure reduces 5-HT-stimulated cyclic GMP formation and [Ca2+]i increases, and this may reflect the functional changes of 5-HT3 receptors.
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PMID:Chronic amitriptyline exposure reduces 5-HT3 receptor-mediated cyclic GMP formation in NG 108-15 cells. 900 9

Two types of ligand-gated ion channels were expressed with the Semliki Forest virus (SFV) expression system. The cDNAs for mouse serotonin 5-HT3 receptor and rat and human purinoreceptor P2x subtypes were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper2 RNA into BHK cells, where in vivo packaging resulted in high titer SFV-5-HT3 and SFV-P2x virus stocks. Infection of BHK, CHO and RIN cells resulted in high-level expression of recombinant receptors. Saturation binding analysis indicated the presence of more than 3 x 10(6) 5-HT3 receptors per cell. Binding studies on isolated membranes yielded from 10 to 60 pmol of either 5-HT3 or P2x receptor per mg protein. Functional responses to the P2x receptors were demonstrated in SFV-infected CHO cells by Ca2+ mobilization or by 45Ca2+ influx. High amplitude electrophysiological responses were also detected for both SFV-5-HT3 and SFV-P2x infected CHO cells in whole-cell patch clamp recordings. To facilitate the purification procedure of SFV-expressed recombinant receptors a histidine tag was introduced at the C-terminus of the 5-HT3 receptor. This 5-HT3His receptor showed high levels of expression, specific binding and high amplitude electrophysiological responses. For large scale expression the BHK cells were adapted to suspension culture and were efficiently infected in a 11.5 liter fermentor culture with SFV-5-HT3His resulting in high-level expression, 52 pmol receptor per mg protein corresponding to 3.2 x 10(6) receptors per cell.
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PMID:Expression of ligand-gated ion channels with the Semliki Forest virus expression system. 902 84


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