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)

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

5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.
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PMID:Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel. 1628 Oct 19

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A-F. Here we examine the 5-HT3 receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [3H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC50 when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in Kd and EC50 values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT3 receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT3 receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.
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PMID:Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor. 1706 Jan 61

Calcium modulates the 5-HT3 receptor response by reducing peak current amplitude and increasing rates of activation, deactivation and desensitisation, but the binding site(s) and mechanism(s) of this modulation are unknown. Here we study residues that may be involved in calcium binding in two partially overlapping regions of the extracellular domain (E213-E215-E218 and D204-E218-V219). The modulatory effects of calcium were assessed by radioligand binding and whole-cell patch-clamp. Comparisons of [3H]granisetron binding showed an increase in Kd in 10mM calcium that was abolished by the substitutions E213Q, E215Q, D204N and V219L. E218Q mutant receptors displayed no specific binding or function, and immunofluorescence showed that they did not reach the cell surface. E213Q increased inherent rates of desensitisation, but the relative effects of calcium on these rates, and on the reduction in current amplitude, were similar to wild type receptors. Current responses and calcium-mediated effects at E215Q mutant receptors were indistinguishable from wild type. D204N and V219L mutants were non-functional. A calcium impermeable mutant (E277A/S297R) revealed no changes in peak amplitude or kinetics with increased calcium. Our results are consistent with residues D204, E218 and V219 participating in receptor assembly, structure and/or trafficking to the plasma membrane, and we speculate that this might rely upon the stabilising effect of bound calcium. E213, E215, D204 and V219 may contribute to a calcium binding site that is responsible for the calcium-mediated effects on ligand binding. However, the major site for calcium-dependent modulation of the 5-HT3 current is located within the ion channel or cell interior.
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PMID:Calcium modulation of 5-HT3 receptor binding and function. 1867 30

5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4) and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-A(n = 2-7)). Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC(50)'s were at most 5-fold greater than wild-type (WT). The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A(2), 5-HT3A-A(4), 5-HT3A-A(6), and 5-HT3A-A(7) were similar to WT. In contrast, 5-HT3A-A(3) and 5-HT3A-A(5) had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A(1), entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function.
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PMID:Length and amino acid sequence of peptides substituted for the 5-HT3A receptor M3M4 loop may affect channel expression and desensitization. 2253 82