Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P46098 (5-HT3 receptor)
 2,290 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 5-HT3 receptor is a ligand-gated ion channel with significant structural similarity to the nicotinic acetylcholine receptor. Several regions that form the ligand binding site in the nicotinic acetylcholine receptor are partially conserved in the 5-HT3 receptor, presumably reflecting the conserved signal transduction mechanism. Specific amino acid differences in these regions may account for their distinct ligand recognition properties. Using site-directed mutagenesis, we have replaced one of these residues, glutamate 106 (E106), with aspartate (D), asparagine (N), alanine (A) or glutamine (Q) and characterized the ligand-binding and electrophysiological properties of the mutant receptors after transient expression in HEK-293 cells. The affinity for the selective 5-HT3 receptor antagonist [3H]GR65630 was decreased 14-fold in the mutant E106D (Kd = 3.69 +/- 0.32 nM) when compared to wildtype (WT, E106) 5-HT3 receptor (0.27 +/- 0.03 nM), while the affinity for E106N was unchanged (0.42 +/- 0.07 nM, means +/- SEM, n = 3-10). Decreased affinities for both E106D and E106N were observed for the antagonists granisetron, ondansetron and renzapride and for the agonists 5-HT (130- and 30-fold) and 2-methyl-5-HT (250- and 20-fold), respectively. Both mutants still formed 5-HT-activatable ion channels, but the high Hill coefficient of the concentration effect curves in wildtype (2.0) was decreased to unity in both cases. The EC50 of 5-HT was increased seven-fold in E106N (8.7 microM) when compared to wildtype (1.2 microM), but unchanged in E106D, and the potency of the antagonist ondansetron for both mutants was decreased. E106A and E106Q expressed poorly preventing a detailed characterization. These data suggest that E106 contributes to the ligand-binding site of the 5-HT3 receptor and may form an ionic or hydrogen bond interaction with the primary ammonium group of 5-HT.
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PMID:Analysis of the ligand binding site of the 5-HT3 receptor using site directed mutagenesis: importance of glutamate 106. 922 89

1. Whole cell voltage clamp electrophysiology and radioligand binding were used to examine the agonist characteristics of the two splice variants of the 5-HT3 receptor which have been cloned from neuronal cell lines. Homo-oligomeric 5-HT3 receptors were examined in HEK 293 cells stably transfected with either long (5-HT3-L) or short (5-HT3-S) receptor subunit DNAs. 2. Functional homo-oligomeric receptors were formed from both subunits, and responses to 5-HT3 receptor agonists (5-hydroxytryptamine (5-HT), 2-methyl 5-HT and m-chlorophenylbiguanide) were qualitatively similar. 3. Maximum currents (Rmax) elicited by the 5-HT3 receptor agonists m-chlorophenylbiguanide (mCPBG) and 2-methyl-5-HT (2-Me-5-HT), as compared to 5-HT, differed in the two splice variants: Rmax mCPBG/Rmax 5-HT values were 0.68+/-0.04 and 0.91+/-0.01 in 5-HT3-L and 5-HT3-S receptors, respectively. Comparable values for 2-Me-5-HT were 0.30+/-0.02 and 0.23+/-0.02. 4. Radioligand binding data showed no difference in affinity of agonist or antagonist binding sites; thus the six amino acid deletion appears to cause differences in agonist efficacy. 5. The role of the 6 amino acid insertion was further investigated by use of site-directed mutagenesis to create two mutant receptors, one where serine 286 was replaced with alanine, and the second where all 6 amino acids were replaced with alanines. 6. Examination of the mutant receptors when stably expressed in HEK 293 cells revealed agonist properties resembling long and not short 5-HT3 receptors. Thus specific amino acids in this region are not responsible for the observed differences. 7. The data show intracellular structure can have significant effects on ligand-gated ion channel function, and suggest that minor changes in structure may be responsible for differences in function observed when ligand-gated ion channel proteins are modulated intracellularly.
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PMID:Different efficacy of specific agonists at 5-HT3 receptor splice variants: the role of the extra six amino acid segment. 951 85

The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT3R) are members of the ligand-gated ion channel gene family. Both receptors are inhibited by nanomolar concentrations of d-tubocurarine (curare) in a competitive fashion. Chemical labeling studies on the AChR have identified tryptophan residues on the gamma (gammaTrp-55) and delta (deltaTrp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT3R shows that a tryptophan residue is found in the homologous position in the 5HT3R (Trp-89), suggesting that this residue may be involved in curare-5HT3R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT3R for the antagonists curare and granisetron but has little effect on the affinity for the agonist serotonin. To further examine the role of this region of the receptor in ligand-receptor interactions, alanine-scanning mutagenesis analysis of the region centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only at Trp-89, whereas serotonin affinity is reduced only by substitution at Arg-91. On the other hand, granisetron affinity is reduced by substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect of substitutions on ligand affinity suggests that different ligands may have different points of interaction within the ligand-binding pocket. In addition, the every-other-residue periodicity of the effects on granisetron affinity strongly suggests that this region of the ligand-binding site of the 5HT3R (and by inference, other members of the ligand-gated ion channel family) is in a beta-strand conformation.
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PMID:Structural features of the ligand-binding domain of the serotonin 5HT3 receptor. 1002 68

5-HT3 receptors possess a number of highly conserved proline residues. We changed each of these to alanine, expressed the mutants as homomeric 5-HT3A receptors in HEK293 cells, and analyzed them with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Pro56, Pro104, Pro123, and Pro170 resulted in ablation of radioligand binding, whereas mutation of Pro257 and Pro301 did not. Only the latter were expressed at the plasma membrane but were non-functional. Thus the former, which are in the N-terminal domain, may be involved in forming correct receptor structure, while those in the transmembrane region (Pro257 and Pro301) are necessary for the function of the protein. To explore the conformational preference (propensity) of these residues we examined the proportion of cis-prolines and the influence of adjacent residues in known protein structures. 4.7% of prolines in the protein data base were in the cis conformation, and the distribution of amino acids adjacent to cis-prolines was not randomly distributed. Comparison of the proportion of each amino acid residue adjacent to a cis-proline revealed that aromatic and bend-facilitating residues were favored while those with beta-branched chains were not. Thus five residues (Gly, Pro, Tyr, Trp, Phe) and three residues (Pro, Tyr, Phe) were found more frequently than expected before and after cis-prolines respectively, whereas five residues (Val, Ile, Leu, Asp, Thr) and two residues (Asp, Glu) were found less frequently. Of the 20 proline residues in the 5-HT3A receptor subunit only Pro170 has adjacent residues that are favorable. Mutating these to non-favorable residues resulted in ablation of ligand binding, whereas replacement with alternative favorable residues did not. We therefore propose that Pro170, which is part of the characteristic cys-loop found in this family of proteins, may be in the cis conformation.
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PMID:The role and predicted propensity of conserved proline residues in the 5-HT3 receptor. 1149 6

The effects of phorbol 12-myristate, 13-acetate (PMA) on 5-hydroxytryptamine (5-HT)-evoked ion currents in the mouse 5-HT3A receptor were examined. Perfusion with PMA caused a concentration dependent potentiation of 5-HT mediated currents and increased both potency and efficacy of 5-HT at the 5-HT3A receptor expressed in Xenopus oocytes. Enhancement of receptor function was partially blocked by injection of oocytes with PKCI, the peptide inhibitor of protein kinase C (PKC). Mutation of all 12 intracellular serine and threonine residues to alanine was without effect on PMA-induced potentiation of 5-HT elicited currents. Mutation of tyrosine 458 in the 5-HT3A receptor lacking intracellular serines and threonines reduced the PMA-induced potentiation of 5-HT evoked currents by approximately 55%. In contrast, mutation of tyrosine 458 in the wild-type receptor did not alter PMA-induced enhancement. The tyrosine kinase inhibitor, lavendustin A, reduced the enhancement of 5-HT3A receptor mediated currents by PMA in the mutant 5-HTA3A receptor containing no intracellular serine or threonine residues, but not in the wild-type receptor. Thus, the role of intracellular serines and threonines is redundant with that of tyrosine, suggesting that these two components act through a similar pathway in response to PMA treatment.
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PMID:Enhancement of 5-hydroxytryptamine3A receptor function by phorbol 12-myristate, 13-acetate is mediated by protein kinase C and tyrosine kinase activity. 1244 87

Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT3A receptors and alpha7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wild-type and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5- and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT3 receptor antagonist/very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT3A receptors.
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PMID:Arginine 222 in the pre-transmembrane domain 1 of 5-HT3A receptors links agonist binding to channel gating. 1297 Mar 51

Aromatic residues play an important role in the ligand-binding domain of Cys loop receptors. Here we examine the role of the 11 tyrosines in this domain of the 5-HT3 receptor in ligand binding and receptor function by substituting them for alanine, for serine, and, for some residues, also for phenylalanine. The mutant receptors were expressed in HEK293 cells and Xenopus oocytes and examined using radioligand binding, Ca2+ imaging, electrophysiology, and immunochemistry. The data suggest that Tyr50 and Tyr91 are critical for receptor assembly and/or structure, Tyr141 is important for antagonist binding and/or the structure of the binding pocket, Tyr143 plays a critical role in receptor gating and/or agonist binding, and Tyr153 and Tyr234 are involved in ligand binding and/or receptor gating. Tyr73, Tyr88, Tyr94, Tyr167, and Tyr240 do not appear to play major roles either in the structure of the extracellular domain or in ligand binding. The data support the location of these residues on a model of 5-HT docked into the ligand-binding domain and also provide evidence for the structural similarity of the extracellular domain to AChBP and the homologous regions of other Cys loop ligand-gated ion channels.
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PMID:The role of tyrosine residues in the extracellular domain of the 5-hydroxytryptamine3 receptor. 1499 95

Previously, we reported that the GABA(A) receptor antagonist picrotoxin also antagonizes serotonin (5-HT)3 receptors and that its effects are subunit-dependent. Here, we sought to identify amino acids involved in picrotoxin inhibition of 5-HT3 receptors. Mutation of serine to alanine at the transmembrane domain 2 (TM2) 2' position did not affect picrotoxin (PTX) sensitivity in murine 5-HT3A receptors. However, mutation of the 6' TM2 threonine to phenylalanine dramatically reduced PTX sensitivity. Mutation of 6' asparagine to threonine in the 5-HT3B subunit enhanced PTX sensitivity in heteromeric 5-HT3A/3B receptors. Introduction of serine (native to the human 3B subunit) at the 6' position also increased PTX sensitivity, suggesting a species-specific effect. Mutation of the 7' leucine to threonine in 5-HT3A receptors increased PTX sensitivity roughly 10-fold, comparable with that observed in GABA(A) receptors, and also conferred distinct gating kinetics. The equivalent mutation in the 3B subunit (i.e., 7' valine to threonine) had no impact on PTX sensitivity in 5-HT3A/3B receptors. Interestingly, [3H]ethynylbicycloorthobenzoate ([3H]EBOB), a high-affinity ligand to the convulsant site in GABA(A) receptors, did not exhibit specific binding in 5-HT3A receptors. The structurally related compound, tert-butylbicyclophosphorothionate (TBPS), which potently inhibits GABA(A) receptors, did not inhibit 5-HT3 currents. Our results indicate that the TM2 6' residue is a common determinant of PTX inhibition of both 5-HT3 and GABA(A) receptors and demonstrate a role of the 7' residue in PTX inhibition. However, lack of effects of EBOB and TBPS in 5-HT3A receptors suggests that the functional domains in the two receptors are not equivalent and underscores the complexity of PTX modulation of LGICs.
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PMID:Molecular determinants of picrotoxin inhibition of 5-hydroxytryptamine type 3 receptors. 1581 70

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (gamma) below the resolution of single channel recording (966 +/- 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range gamma values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 (-4'), 436 (0'), and 440 (4') residues by 5-HT3B (-4'Gln, 0'Asp, and 4'Ala) residues increases gamma to 36.5 +/- 1.0 pS. The 0' residue makes the most substantial contribution to gamma of the 5-HT3AR. Replacement of 0'Arg by aspartate, glutamate (alpha7 nAChR subunit MA 0'), or glutamine (beta2 subunit MA 0') increases gamma to the resolvable range (>6 pS). By contrast, replacement of 0'Arg by phenylalanine (alpha4 subunit MA 0') reduced gamma to 416 +/- 107 fS. In reciprocal experiments with alpha4beta2 nAChRs (gamma = 31.3 +/- 0.8 pS), replacement of MA 0' residues by arginine in alpha4beta2(Q443R) and alpha4(F588R)beta2 reduced gamma slightly. By contrast, the gamma of double mutant alpha4(F588R)beta2(Q443R) was halved. The MA -4' and 4' residues also influenced gamma of 5-HT3ARs. Replacement of nAChR alpha4 or beta2 MA 4' residues by arginine made current density negligible. By contrast, replacement of both -4' residues by arginine produced functional nAChRs with substantially reduced gamma (11.4 +/- 0.5 pS). Homology models of the 5-HT3A and alpha4beta2 nAChRs against Torpedo nAChR revealed MA -4', 0', and 4' residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family.
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PMID:Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and alpha4beta2 nicotinic acetylcholine receptors. 1640 31

Amongst the family members of Cys-loop LGICs, the atypical ability of the 5-HT3A subunit to form functional homomeric receptors allowed a direct investigation of the role of the C-terminus. Deletion of the three C-terminal amino acids (DeltaGln453-DeltaTyr454-DeltaAla455) from the h5-HT3A subunit prevented formation of a specific radioligand binding site as well as expression within the cell membrane. Removal of merely the C-terminal residue (DeltaAla455) reduced specific radioligand binding (to 4+/-1% relative to the wild-type; cells grown at 37 degrees C) and also cell membrane expression; these reductions were less evident when the DeltaAla455 expressing cells were grown at 27 degrees C (specific radioligand binding levels 27+/-5% relative to wild-type also grown at 27 degrees C). Mutation of the h5-HT3A C-terminal amino acid, alanine, for either glycine (Ala455Gly), valine (Ala455Val) or leucine (Ala455Leu) reduced specific radioligand binding levels by 24+/-23%, 32+/-12% and 88+/-1%, respectively; the latter mutant also displaying reduced membrane expression. In contrast, mutation to alanine of the two amino acids preceding the C-terminal alanine (Gln453Ala and Tyr454Ala) had no detrimental effects on specific radioligand binding or cell membrane expression levels. The present study demonstrates an important role for the C-terminus in the formation of the functional h5-HT3A receptor. The partial restoration of 5-HT3 receptor binding and cell membrane expression when cells expressing C-terminal mutant 5-HT3A subunits were grown at a lower temperature (27 degrees C) suggests that the C-terminus stabilises the 5-HT3 receptor allowing subunit folding and subsequent maturation.
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PMID:Importance of the C-terminus of the human 5-HT3A receptor subunit. 1878 52


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