The agonist properties of <i>m</i>‐chlorophenylbiguanide and 2‐methyl‐5‐hydroxytryptamine on 5‐HT<sub>3</sub> receptors in N1E‐115 neuroblastoma cells

Sepúlveda, M.-I.; Lummis, Sarah C. R.; Martin, Ian L.

doi:10.1111/j.1476-5381.1991.tb12464.x

Cited by 67 publications

(39 citation statements)

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“…Series resistance was less than 10 M⍀. Cells were voltage clamped at Ϫ60 mV in all experiments, and drugs were applied via a U-tube delivery system capable of a complete change of solution within 100 ms (31). Data acquisition, monitoring, storage, and analysis were performed using HEKA software.…”

Section: Methodsmentioning

confidence: 99%

The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain

Spier¹,

Lummis²

2000

Journal of Biological Chemistry

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Aromatic amino acids are important components of the ligand binding site in the Cys loop family of ligandgated ion channels. To examine the role of tryptophan residues in the ligand binding domain of the 5-hydroxytryptamine 3 (5-HT 3 ) receptor, we used site-directed mutagenesis to change each of the eight N-terminal tryptophan residues in the 5-HT 3A receptor subunit to tyrosine or serine. The mutants were expressed as homomeric 5-HT 3A The 5-HT 3 1 receptor is a member of the Cys loop family of ligand-gated ion channels, which includes nicotinic acetylcholine (nACh), ␥-aminobutyric acid (GABA), and glycine receptors. The predicted 5-HT 3 receptor subunit structure shows the expected characteristics of a nACh receptor-type subunit, including a large extracellular N-terminal region, and four putative transmembrane domains (TM1-TM4) with a bulky intracellular loop between TM3 and TM4. 5-HT 3A receptor subunits have been cloned from mouse, rat, human, and guinea pig (1-9), and a 5-HT 3B receptor subunit, which appears to have a structural role, has been cloned from human (10).The nACh receptor is the receptor most closely related to the 5-HT 3 receptor with up to 30% amino acid sequence identity (5). Evidence from biochemical and mutagenesis studies on nACh receptors indicate that the ligand binding site is located in discontiguous regions of the extracellular N-terminal domain, and this has been further confirmed by the construction of a chimeric protein consisting of the N-terminal domain of the ␣7 neuronal nACh receptor subunit linked to the C-terminal portion of the 5-HT 3A receptor subunit, which showed nACh receptor pharmacological properties and 5-HT 3 receptor channel properties (11 in the 5-HT 3A receptor subunit. Biochemical evidence also indicates that tryptophan residues are involved in 5-HT 3 receptor ligand binding; modifying these residues with N-bromosuccinimide inhibits radiolabeled antagonist binding to the 5-HT 3 receptor in a ligand-protectable manner (26). To examine the role of tryptophan residues in the N-terminal ligand binding domain of the 5-HT 3A receptor, we have changed the eight tryptophans in this region (Fig. 1) using site-directed mutagenesis. The resulting mutants have been expressed in HEK293 cells and characterized with radioligand binding, electrophysiology, and immunocytochemistry. EXPERIMENTAL PROCEDURESCell Culture and Transient/Stable DNA Transfection-Human embryonic kidney (HEK293) cells were cultured on 90-mm tissue culture plates in Dulbecco's modified Eagles medium/F-12 medium (1:1) medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Sigma) at 37°C and 7% CO 2 in a humidified atmosphere. HEK293 cells stably expressing 5-HT 3A receptors were developed using the eukaryotic expression vector pRc/CMV (In Vitrogen, Abingdon, UK) containing the complete coding sequence for the 5-HT 3A(b) subunit from NIE-115 cells as described previously (27). Mutagenesis reactions were performed using the Kunkel method (28) and confirmed by DNA sequencing. For transien...

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Section: Methodsmentioning

confidence: 99%

The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain

Spier¹,

Lummis²

2000

Journal of Biological Chemistry

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“…Other data consistent with this hypothesis include the agonist dependence of the changes, the lack of effect of 5-HT 3B on the rate of recovery from desensitization, 2 and the shallower slope of agonist dose-response relationships. 5-HT 3 receptors reveal very strong cooperativity; Hill coefficients have been determined from functional studies to be near 2 or 3 (47,73,74). 5-HT 3B subunits, which apparently have no functional agonist binding sites, may disrupt cooperativity between 5-HT 3A subunits.…”

Section: -Ht 3b Specifically Modifies 5-ht 3a Receptor Functionmentioning

confidence: 99%

The Pharmacological and Functional Characteristics of the Serotonin 5-HT3A Receptor Are Specifically Modified by a 5-HT3B Receptor Subunit

Dubin¹,

Huvar²,

D’Andrea³

et al. 1999

Journal of Biological Chemistry

264

232

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(9), and Ca 2ϩ influx into lymphocytes (10). 5-HT 3 receptor activation mediates emetic and inflammatory responses (11) and may contribute to pain reception, anxiety, cognition, cranial motor neuron activity, modulation of affect, and the behavioral consequences of drug abuse (Refs. 12 and 13; but see Ref. 14).The 5-HT 3A receptor subunit shares structural similarities with members of the superfamily of ligand-gated ion channels (15) and is thought to be a pentameric protein (16, 17) with multiple agonist and allosteric ligand binding sites (2, 11). Both native and recombinant 5-HT 3 receptors reveal rapid and cooperative activation by agonists and desensitization to prolonged application of 5-HT (reviewed in Refs. 1, 2, and 6). With a few exceptions, ligand-gated channels require the association of more than one kind of homologous subunit for function, and subunit composition determines the pharmacological (18) and kinetic (e.g. desensitization (19,20)) profile of heteromeric receptors. While the 5-HT 3A subunit expressed in heterologous systems functions efficiently as homomers, different voltagedependence, desensitization, and pharmacological properties between recombinant and native 5-HT 3 receptors suggest that native 5-HT 3 receptors may exist as heteromers (3,(21)(22)(23)(24)(25)(26)(27). Although the 5-HT 3A gene encodes splice variants in mouse (28, 29) and guinea pig (30), most of the characteristics of these variants are similar (29,31,32), and the subtle pharmacological differences (22, 31, 32) cannot completely account for the differences observed between recombinant homomers and native 5-HT 3 receptors. In fact, co-expression of both splice variants in oocytes could not reproduce the responses observed in the cell line from which the splice variants were cloned (22). Biochemical studies on porcine brain have revealed the existence of at least four proteins (52-71 kDa) closely associated with the 5-HT 3A subunit that may represent antigenically distinct channel subunits (33).The cloning of a 5-HT 3B subunit that modified the pharmacological and single channel characteristics of the 5-HT 3A subunit when co-expressed in heterologous expression systems was recently reported (34). We extend this report and provide evidence for the co-existence of both 5-HT 3B and 5-HT 3A receptor subunits in native cells, a requirement for heteromeric association. We show that 5-HT 3B has no effect on the function of ␣2␤2, ␣3␤4, ␣4␤2, and ␣7 nicotinic ACh receptors. Furthermore, the characterization of the pharmacology and function of heteromeric receptors in Xenopus oocyte and mammalian expression systems reported here differs from that described (34), and these differences have important consequences for the function of heteromers in vivo.* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s) 1 The abbreviations used...

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“…Similar transient ion currents are evoked by the application of other 5-HT3 receptor agonists, e.g., dopamine (Neijt et al, 1986), 2-methyl-5-HT and metachlorophenylbiguanide (mCPBG) (Sepulveda et al, 1991). Dopamine acts as a low-affinity, partial agonist on the 5-HT3 receptor, with an efficacy of approximately 40% of that of 5-HT, as measured by the amplitude ratio of the maximum in-1 Author for correspondence.…”

Section: Introductionmentioning

confidence: 97%

“…the number of steps between the desensitized and resting states, depends on the agonist used to induce desensitization. and mCPBG (IC50 = 2 nM; Sepulveda et al, 1991) Figure 5a). 1992).…”

Section: Dynamic Equilibria Between Receptor Statesmentioning

confidence: 99%

Selection of distinct conformational states of the 5‐HT₃ receptor by full and partial agonists

Hooft

Vijverberg

1996

British J Pharmacology

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2 The concentration-dependent activation and desensitization of the ion currents evoked by the agonists yield the potency order: mCPBG > 5-HTQ % 5-HT > > tryptamine > dopamine, and the efficacy order: 5-HT mCPBG 5-HTQ> > dopamine tryptamine. Thus, 5-HT, 5-HTQ and mCPBG are full agonists, whereas dopamine and tryptamine are partial agonists at the 5-HT3 receptor. 3 Full and partial agonists cause complete cross-desensitization and activate single channels with similar conductances and open lifetimes. This shows that full and partial agonists act on the same population of 5-HT3 receptors. 4 The time course of recovery from desensitization depends on the agonist used. Recovery from partial agonist-induced desensitization is single exponential, whereas the desensitization induced by full agonists recovers with sigmoid kinetics, suggesting at least 3 steps between 4 states. 5 During the process of recovery from cross-desensitization, the full agonists activate a larger fraction of the 5-HT3 receptors than the partial agonists, irrespective of the agonist used to induce desensitization. 6 It is concluded that full and partial agonists induce distinct desensitized states and, during recovery from desensitization, recognize distinct conformations of unoccupied 5-HT3 receptors. This conformational selection is likely to account for the different efficacies of full and partial 5-HT3, receptor agonists.

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The agonist properties of m‐chlorophenylbiguanide and 2‐methyl‐5‐hydroxytryptamine on 5‐HT₃ receptors in N1E‐115 neuroblastoma cells

Cited by 67 publications

References 23 publications

The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain

The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain

The Pharmacological and Functional Characteristics of the Serotonin 5-HT3A Receptor Are Specifically Modified by a 5-HT3B Receptor Subunit

Selection of distinct conformational states of the 5‐HT₃ receptor by full and partial agonists

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The agonist properties of m‐chlorophenylbiguanide and 2‐methyl‐5‐hydroxytryptamine on 5‐HT3 receptors in N1E‐115 neuroblastoma cells

Cited by 67 publications

References 23 publications

The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain

The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain

The Pharmacological and Functional Characteristics of the Serotonin 5-HT3A Receptor Are Specifically Modified by a 5-HT3B Receptor Subunit

Selection of distinct conformational states of the 5‐HT3 receptor by full and partial agonists

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The agonist properties of m‐chlorophenylbiguanide and 2‐methyl‐5‐hydroxytryptamine on 5‐HT₃ receptors in N1E‐115 neuroblastoma cells

Selection of distinct conformational states of the 5‐HT₃ receptor by full and partial agonists