Analysis of the Ligand Binding Site of the 5-HT3 Receptor Using Site Directed Mutagenesis: Importance of Glutamate 106
Section snippets
Site-directed mutagenesis of the 5-HT3-AL cDNA
A cDNA isolated from NG108-15 cells encoding the mouse 5-HT3-AL receptor (Werner et al., 1994) was generously provided by Dr Eric Kawashima (Glaxo Molecular Biology Institute, Geneva). The cDNA sequence was subcloned into the phagemid pAlter (Promega, Madison, WI, U.S.A.) and the eukaryotic expression vector pRC/CMV (Promega). Mutagenesis was performed using the Altered Sites Mutagenesis Kit (Promega). Glutamate (E) 106 was mutated to aspartate (D), asparagine (N), glutamine (Q), or alanine (A)
Radioligand binding characterization of mutant 5-HT3 receptors E106D and E106N
In saturation studies with membranes obtained from HEK 293 cells transiently expressing wildtype (WT), E106D or E106N mutant 5-HT3 receptors, [3H]GR65630 labelled a homogeneous population of binding sites (Fig. 2). The binding site density varied with the efficiency of both transfection and expression. Therefore, for ease of comparison, the saturation data are normalized to Bmax in the Scatchard transformations (Fig. 2B). The affinity of [3H]GR65630 was decreased by 14-fold for the mutant E106D
DISCUSSION
In the nicotinic acetylcholine receptor, amino acids involved in ligand recognition are concentrated in three “loops” of the α subunits and additional sites are present on the γ and δ subunits (reviewed in Changeux et al., 1992; Karlin and Akabas, 1995). A comparison of the amino acid sequence of the 5-HT3 receptor and nAChR α subunits shows that there is significant sequence conservation in the first of these recognition loops (Fig. 1). However, there are also major differences, for example,
Acknowledgements
The authors would like to thank Drs Tom Blackburn (SmithKline Beecham) and Gavin Kilpatrick (Glaxo) for their kind gifts of drugs. Supported by Glaxo Canada and MRC Canada. L.J.S. holds a MRC/PMAC Pfizer (Canada) Fellowship.
References (44)
- et al.
Ligand-receptor interactions in the nicotinic acetylcholine receptor probed using multiple substitutions at conserved tyrosines on the α subunit
FEBS Letters
(1994) - et al.
5-HT3 receptors in NG108–15 neuroblastoma × glioma cells: effect of the novel agonist 1-(m-chlorophenyl)-biguanide
Neuropharmacology
(1992) - et al.
Molecular biology of 5-HT receptors
Neuropharmacology
(1994) - et al.
Structure of the agonist-binding site of the nicotinic acetylcholine receptor
Journal of Biological Chemistry
(1991) The Cheng-Prusoff relationship: something lost in the translation
Trends in Pharmacological Sciences
(1993)- et al.
Molecular neurobiology of the GABAA receptor
International Review of Nèurobiology
(1994) - et al.
Identification of a novel amino-acid α-tyrosine 93 within the cholinergic ligand binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligand-binding sites
Journal of Biological Chemistry
(1990) - et al.
Neuronal nicotinic receptors: Molecular organization and regulations
Neuropharmacology
(1995) - et al.
Expression of recombinant homo-oligomeric 5-hydroxytryptamine3 receptors provides new insights into their maturation and structure
Journal of Biological Chemistry
(1995) - et al.
Cloning and functional expression of an apparent splice variant of the murine 5-HT3 receptor A subunit
European Journal of Pharmacology (Molecular Pharmacology Section)
(1993)
Single channel currents in the nicotinic acetylcholine receptor: a direct demonstration of allosteric transitions
Trends in Biochemical Sciences
The inhibitory glycine receptor: architecture, synaptic localization and molecular pathology of a postsynaptic ion-channel complex
Current Opinion in Neurobiology
Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells
Journal of Biological Chemistry
Conserved tyrosines in the α subunit of the nicotinic acetylcholine receptor stabilize quaternary ammonium groups of agonists and curariform antagonists
Journal of Biological Chemistry
Organisation of the murine 5-HT3 receptor gene and assignment to human chromosome 11
FEBS Letters
Nicotinic acetylcholine receptor at 9 Å resolution
Journal of Molecular Biology
Organisation of the mouse 5-HT3 receptor gene and the functional expression of two splice variants
Molecular Brain Research
Electrophysiological consequences of ligand binding to the desensitized 5-HT3 receptor in mammalian NG108–15 cells
Journal of Physiology
Cloning and functional expression of a human 5-hydroxytryptamine type 3As receptor subunit
Molecular Pharmacology
Molecular properties of 5-hydroxytryptamine3 receptor-type binding sites purified from NG108–15 cells
Journal of Neurochemistry
Ultra-structure of the 5-HT3 receptor
Journal of Neurochemistry
Kinetics and selectivity of a low-voltage-activated calcium current in chick and rat sensory neurones
Journal of Physiology
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2020, StructureCitation Excerpt :Accordingly, simulations show an absence of H bond with the drug at this residue. [3H]Granisetron-binding experiments illuminate a drastic effect of mutations of E102, with a complete loss of binding at the subnanomolar range for A, D, G, K, and N (data obtained on oocytes) (Price et al., 2008); or 60- to 80-fold decrease in Kd for E102D/N (data obtained on HEK cells) (Boess et al., 1997). E102 is not oriented toward the binding site and interacts with S150 and T152 (β7), while its main chain carboxyl forms an H bond with A134 (β6) (Figure 6A).
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2017, NeuropharmacologyCitation Excerpt :From the crystal structure 4PIR it is clear that a hydrogen bond between E129 and T179 is structural (Hassaine et al., 2014). Such an interaction is supported by our finding that E129C abolishes binding of both ligands and by other reports that describe similar effects on granisetron, GR65630 and VUF10166 (Boess et al., 1997; Price et al., 2008; Thompson et al., 2005, 2014). For T179C the effects on the two ligands were not as pronounced, but as both threonine and cysteine can act as hydrogen bond donors this property may be retained following substitution; consistent with this, substitution to serine also preserves granisetron binding (Thompson et al., 2005).
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Present address: F. Hoffman-La Roche AG, Pharma Division, Preclinical Research, P.O. Box, CH-4070 Basel, Switzerland.
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Present address: Institute of Biomedical and Life Sciences, Division of Molecular Genetics, Robertson Building, University of Glasgow, Glasgow, G11 6NU, U.K.