Interaction of d-tubocurarine analogs with the 5HT3 receptor

doi:10.1016/S0028-3908(98)00010-0

Neuropharmacology

Volume 37, Issue 2, February 1998, Pages 251-257

https://doi.org/10.1016/S0028-3908(98)00010-0 Get rights and content

Abstract

d-Tubocurarine is a potent competitive antagonist of two members of the ligand-gated ion channel family, the muscle-type nicotinic acetylcholine receptor (AChR) and serotonin type-3 receptor (5HT₃R). We have used a series of analogs of d-tubocurarine to determine the effects of methylation, stereoisomerization and halogenation on the interaction of d-tubocurarine with the 5HT₃R. The affinities of the analogs for the 5HT₃R span a 200-fold concentration range and fall into three broad groups. The first group, with affinity constants (K_i) <150 nM, consists of d-tubocurarine and analogs modified at the nitrogens or 7′ hydroxyl. The fact that these compounds all have high affinity for the 5HT₃R suggests that these portions of the ligand do not make interactions with the receptor that are critical for high-affinity binding. The second group, with K_i's in the 1–5 μM range, consists of analogs modified at the 12′-hydroxyl or the adjacent 13′-carbon, which suggests that this portion of the ligand makes interactions that are important for high-affinity binding. The third, very low affinity, group is a compound with altered stereoconfiguration at the 1 carbon, demonstrating the importance of proper configuration of the antagonist in ligand-receptor interactions. For the most part, this pattern of selectivity is similar to that for the AChR, suggesting that the structures of the ligand-binding sites of these two receptors share common structural features.

Introduction

The serotonin type 3 receptor (5HT₃R) is a member of the ligand-gated ion channel gene family, which includes the muscle and neuronal nicotinic acetylcholine receptors (AChR), the glycine receptor and the γ-aminobutyric acid type A receptor (Unwin, 1993; Karlin and Akabas, 1995; Ortells and Lunt, 1995). Like all members of the family, the 5HT₃R exhibits a large degree of sequence similarity to the AChR (Maricq et al., 1991). Chimeras, consisting of the N-terminal domain of the α7 neuronal AChR and the C-terminal region of the 5HT₃R, form functional ligand-gated ion channels with pharmacological specificity of the AChR and permeability properties of the 5HT₃R (Eisele et al., 1993), suggesting that the two receptors also have quite similar structures and signal transduction properties.

A large number of chemical labeling and mutagenesis studies have been carried out to determine the structural features of the ligand-binding site of the AChR and some details of the site(s) for agonists and competitive antagonists are beginning to appear (for reviews, see Devillers-Thiery et al., 1993; Karlin and Akabas, 1995). However, little is known about the types of interactions between ligands and the 5HT₃R. We are interested in using information obtained from the (relatively) well-studied AChR, to guide structure-function studies on the 5HT₃R. One ligand that the two receptors have in common is the competitive antagonist d-tubocurarine (Bernard, 1857), which has nanomolar affinity for both receptors (Peters et al., 1990). d-Tubocurarine binds to the α-γ and α-δ subunit interfaces of the AChR (Pedersen and Cohen, 1990). Several residues on the γ and δ subunits have been shown, by the use of chimeras and site-directed mutagenesis, to be important for the interaction of d-tubocurarine with the receptor (Sine, 1993; Fu and Sine, 1994; O'Leary et al., 1994).

If the 5HT₃R has a structure similar to that of the AChR, one might expect that the interactions between d-tubocurarine and the binding sites of both receptors may be quite similar. One way to approach this is to examine the interaction of a series of structural analogs of d-tubocurarine with both receptors and to look for similarities and differences in the affinities and energies of interaction. A series of d-tubocurarine analogs have been synthesized and their interactions with the Torpedo (Pedersen and Papineni, 1995) and mouse (Papineni and Pedersen, 1997) AChR have been characterized. Several structural features in the ligand have been identified as playing an important role in both ligand affinity as well as α-γ and α-δ interface site discrimination. In this report, we extend these studies to the 5HT₃R and identify some structural features of d-tubocurarine that are important for the interaction of d-tubocurarine with the 5HT₃R.

Section snippets

Transfection

A full length cDNA clone, corresponding to the 5HT_3A(b) form (Hope et al., 1993) of the receptor, was isolated from a neuroblastoma N1E-115 cell line cDNA library and subcloned into vector pCI (Promega, Madison, WI). Cultures of tsA201 cells, a derivative of the widely-used HEK 293 cell line, were maintained in DMEM medium containing 10% fetal bovine serum, 100 units/ml penicillin and 100 units/ml streptomycin. Cultures at 30–40% confluence were transfected with 20 μg receptor cDNA per 100 mm

Results

The affinities of d-tubocurarine and a series of nine analogs (Fig. 1) for the 5HT₃R were determined by competitive inhibition of [³H] granisetron binding (Table 1). Detailed examination of the effects of the various substitutions in the analogs can provide some insight into the structural features of the antagonist that are important for antagonist-5HT₃R interactions.

Discussion

We have measured the affinity of d-tubocurarine and a number of analogs for the 5HT₃R as a means of identifying important interactions that take place at the ligand-binding site of the receptor. The analogs used in this study have substitutions at several different positions and the use of pairwise comparisons allows the determination of structural features that are important for antagonist-receptor interactions.

The affinities of the compounds for the 5HT₃R fall into three broad groups: high

Acknowledgements

This work was supported by NIH grants NS23885 (MMW), NS28879 (SEP) and NS35212 (SEP). SEP is the recipient of NIH Research Career Development Award NS01618.

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We subsequently showed that a major determinant for this difference is located in loop F of the binding site (22). Using a series of dTC analogs, we demonstrated that the same regions of dTC that are important for high-affinity binding to the AChR (23,24) are also important for binding to the 5-HT3R (25). In a subsequent study, we showed that N128 in the 5-HT3R interacted with the 2′N of dTC, and that R92 most likely interacted with the 2N of dTC (15).
The serotonin 5-HT₃ receptor (5-HT₃R) is a member of the Cys-loop ligand-gated ion channel family. We used a combination of site-directed mutagenesis, homology modeling, and ligand-docking simulations to analyze antagonist-receptor interactions. Mutation of E236, which is near loop C of the binding site, to aspartate prevents expression of the receptor on the cell surface, and no specific ligand binding can be detected. On the other hand, mutation to glutamine, asparagine, or alanine produces receptors that are expressed on the cell surface, but decreases receptor affinity for the competitive antagonist d-tubocurarine (dTC) 5-35-fold. The results of a double-mutant cycle analysis employing a panel of dTC analogs to identify specific points of interactions between the dTC analogs and E236 are consistent with E236 making a direct physical interaction with the 12 –OH of dTC. dTC is a rigid molecule of known three-dimensional structure. Together with previous studies linking other regions of dTC to specific residues in the binding site, these data allow us to define the relative spatial arrangement of three different residues in the ligand-binding site: R92 (loop D), N128 (loop A), and E236 (near loop C). Molecular modeling employing these distance constraints followed by molecular-dynamics simulations produced a dTC/receptor complex consistent with the experimental data. The use of the rigid ligands as molecular rulers in conjunction with double-mutant cycle analysis provides a means of mapping the relative positions of various residues in the ligand-binding site of any ligand-receptor complex, and thus is a useful tool for delineating the architecture of the binding site.
A comprehensive study on the 5-hydroxytryptamine<inf>3A</inf> receptor binding of agonists serotonin and m-chlorophenylbiguanidine
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Serotonin type 3 receptors (5-HT₃R) are members of the ligand gated ion channel receptor family. In this study, the interactions of the agonists serotonin (5-HT) and m-chlorophenylbiguanidine (mCPBG) at the binding site of the 5-HT_3AR were investigated at an atomic level. Site-directed mutagenesis studies in Loop B and E along with our earlier published results from mutations within Loops A, C, and D provide comprehensive data on the interaction of 5-HT and mCPBG with 5-HT_3ARs. Using this data we have constructed a refined homology model of the 5-HT_3AR that considers all of the available experimental data. 5-HT and mCPBG were docked into the newly constructed homology model and the amino acid residues critical in binding of these agonists were compared and analyzed. Our docking results reveal many similar binding interactions for 5-HT and mCPBG. Namely, residues THR181, TRP183, PHE226, ILE228, TYR234 and GLU129 were all found to play key roles in binding of both 5-HT and mCPBG. However, the results also revealed two important differences that exist between the interactions of the two agonists. In our model, a hydrogen bond is formed between the indole hydrogen of 5-HT and the residue TYR153. This interaction is not present in the case of mCPBG. Conversely, a hydrogen bond exists between SER182 and a protonated nitrogen of mCPBG, which does not exist in 5-HT. Our modeling results were found to be in accordance with experimental data.
Locating an antagonist in the 5-HT<inf>3</inf> receptor binding site using modeling and radioligand binding
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Nevertheless our data indicate that Phe-226 does not play a role in granisetron binding. The Roles of Non-aromatic Residues That May Interact with Granisetron (Arg-92, Thr-179, Ser-182, Leu-184, Ser-203, Ser-206, Ile-228, Asp-229, and Ile-230)—Arg-92 has the potential to hydrogen bond with granisetron in our model and previous data support its role in antagonist binding (38). It has also been suggested that Arg-92 forms a salt bridge with Asp-229 and/or Glu-200 (10), which would give it a role in the local structure of the binding pocket.
We have used a homology model of the extracellular domain of the 5-HT₃ receptor to dock granisetron, a 5-HT₃ receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 Å from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [³H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.
PSAB-OFP, a selective α7 nicotinic receptor agonist, is also a potent agonist of the 5-HT<inf>3</inf> receptor
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5-Hydroxytryptamine 3 (5-HT₃) and α7 nicotinic receptors share high sequence homology and pharmacological cross-reactivity. An assessment of the potential role of α7 receptors in many neurophysiological processes, and hence their therapeutic value, requires the development of selective α7 receptor agonists. We used a recently reported selective α7 receptor agonist, (R)-(−)-5′Phenylspiro[1-azabicyclo[2.2.2] octane-3,2′-(3′H)furo[2,3-b]pyridine (PSAB-OFP) and confirmed its activity on human recombinant α7 receptors. However, PSAB-OFP also displayed high affinity binding to 5-HT₃ receptors. To assess the functional activity of PSAB-OFP on 5-HT₃ receptors we studied recombinant human 5-HT₃ receptors expressed in Xenopus oocytes, as well as native mouse 5-HT₃ receptors expressed in N1E-115 neuroblastoma cells, using whole-cell patch clamp and Ca²⁺ imaging. Our results show that PSAB-OFP is an equipotent, partial agonist of both α7 and 5-HT₃ receptors. We conclude that it will be necessary to identify the determinant of this overlapping pharmacology in order to develop more selective α7 receptor ligands.
Interaction of d-tubocurarine analogs with mutant 5-HT<inf>3</inf> receptors
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d-Tubocurarine is a potent competitive antagonist of both the muscle-type nicotinic acetylcholine receptor (AChR) and the serotonin type-3 receptor (5HT₃R). We have previously used a series of structural analogs of d-tubocurarine to demonstrate that the ligand-binding domains of both receptors share common structural features. We have now extended these studies to examine the interaction of a series of d-tubocurarine analogs with 5HT₃Rs containing mutations at either of two residues within the ligand-binding domain of the receptor (W90F and R92A). The W90F mutation results in an approximately 2–4-fold decrease in the affinity of the analogs relative to wild-type receptors, while the R92A results in an approximately 8–10-fold increase in affinity. However, since the effect of a given mutation is more or less equivalent for all analogs, neither residue W90 nor R92 is likely to make a specific interaction with d-tubocurarine itself. Rather, these two residues are likely to play a role in determining both the geometry of the binding site, as well as the overall environment that a ligand encounters in the binding site.
Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors
2000, Neurochemistry International
Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both α subunits in a pocket approximately 30–35 Å above the surface membrane. Since both α subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the α subunit with other non-α subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the αδ subunit interface, whereas the low-affinity ACh binding site is located on the αγ subunit interface. Regarding homomeric AChRs (e.g. α7, α8, and α9), up to five binding sites may be located on the αα subunit interfaces. From the point of view of subunit arrangement, the γ subunit is in between both α subunits and the δ subunit follows the α aligned in a clockwise manner from the γ. Although some competitive antagonists such as lophotoxin and α-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the αγ and the αδ subunit interface, respectively. The case of α-conotoxins (α-CoTxs) is unique since each α-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of α-CoTxs for each subunit interface is species-dependent.
In general terms we may state that both α subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-α subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the α subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A–C). Loop A (from mouse sequence) is mainly formed by residue Y⁹³, loop B is molded by amino acids W¹⁴⁹, Y¹⁵², and probably G¹⁵³, while loop C is shaped by residues Y¹⁹⁰, C¹⁹², C¹⁹³, and Y¹⁹⁸. The complementary component corresponding to each non-α subunit probably contributes with at least four loops. More specifically, the loops at the γ subunit are: loop D which is formed by residue K³⁴, loop E that is designed by W⁵⁵ and E⁵⁷, loop F which is built by a stretch of amino acids comprising L¹⁰⁹, S¹¹¹, C¹¹⁵, I¹¹⁶, and Y¹¹⁷, and finally loop G that is shaped by F¹⁷² and by the negatively-charged amino acids D¹⁷⁴ and E¹⁸³. The complementary component on the δ subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding α-neurotoxins, several snake and α-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake α-neurotoxins is located on the residue sequence α1W¹⁸⁴–D²⁰⁰, which includes loop C. In addition, amino acid sequence 55–74 from the α1 subunit (which includes loop E), and residues γL¹¹⁹ (close to loop F) and γE¹⁷⁶ (close to loop G) at the low-affinity binding site, or δL¹²¹ (close to the homologous region of loop G) at the high-affinity binding site, are involved in snake α-neurotoxin binding. The above expounded evidence indicates that each cholinergic molecule binds to specific residues which form overlapping binding sites on the AChR.
Monoclonal antibodies have been of fundamental importance in the elucidation of several aspects of the biology of the AChR. Interestingly, certain antibodies partially overlap with the agonist/competitive antagonist binding sites at multiple points of contact. In this regard, a monoclonal antibody directed against the high-affinity ACh binding site (αδ subunit interface) induced a structural change on the AChR where the low-affinity ACh locus (αγ subunit interface) approached to the lipid membrane.
The α subunits also carry the binding site for noncompetitive agonists. Noncompetitive agonists such as the acetylcholinesterase inhibitor (−)-physostigmine, the alkaloid galanthamine, and the opioid derivative codeine are molecules that weakly activate the receptor without interacting with the classical agonist binding sites. This binding site was found to be located at K¹²⁵ in an amphipathic domain of the extracellular portion of the α1 subunit. Interestingly, the neurotransmitter 5-hydroxytryptamine (5-HT) also binds to this site and enhances the agonist-induced ion flux activity. This suggests that 5-HT may act as an endogenous modulator (probably as co-agonist) of neuronal-type AChRs. The enhancement of the agonist-evoked currents elicited by noncompetitive agonists seems to be physiologically more important than their weak agonist properties.

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Neuropharmacology

Abstract

Introduction

Section snippets

Transfection

Results

Discussion

Acknowledgements

References (20)

Competitive antagonists bridge the α-γ subunit interface of the acetylcholine receptor through quaternary ammonium-aromatic interactions

J. Biol. Chem.

Cloning and functional expression of an apparent splice variant of the murine 5-HT₃ receptor A subunit

Eur. J. Pharmacol.

Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins

Neuron

Evolutionary history of the ligand-gated ion-channel superfamily of receptors

Trends Neurosci.

Interaction of d-tubocurarine with the mouse nicotinic acetylcholine receptor: Ligand orientation at the bindng site

J. Biol. Chem.

Interaction of d-tubocurarine analogs with the Torpedo nicotinic acetylcholine receptor. Methylation and stereoisomerization affect site-selective competitive binding and binding to the noncompetitive site

J. Biol. Chem.

Antagonism of 5HT₃ receptor-mediated currents in murine N1E-115 neuroblastoma cells by (+)-tubocurarine

Neurosci. Lett.

Neurotransmitter action: Opening of ligand-gated ion channels

Neuron

Relationship between inhibition constant (K_i) and the concentration of inhibitor which causes 50 percent inhibition (IC₅₀) of an enzymatic reaction

Biochem. Pharmacol.

Cited by (26)

Mapping spatial relationships between residues in the ligand-binding domain of the 5-Ht<inf>3</inf> receptor using a molecular ruler

A comprehensive study on the 5-hydroxytryptamine<inf>3A</inf> receptor binding of agonists serotonin and m-chlorophenylbiguanidine

Locating an antagonist in the 5-HT<inf>3</inf> receptor binding site using modeling and radioligand binding

PSAB-OFP, a selective α7 nicotinic receptor agonist, is also a potent agonist of the 5-HT<inf>3</inf> receptor

Interaction of d-tubocurarine analogs with mutant 5-HT<inf>3</inf> receptors

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Interaction of d-tubocurarine analogs with the 5HT3 receptor

Abstract

Introduction

Section snippets

Transfection

Results

Discussion

Acknowledgements

J. Biol. Chem.

Eur. J. Pharmacol.

Neuron

Trends Neurosci.

J. Biol. Chem.

J. Biol. Chem.

Neurosci. Lett.

Neuron

Relationship between inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction

Biochem. Pharmacol.

Interaction of d-tubocurarine analogs with the 5HT₃ receptor

Relationship between inhibition constant (K_i) and the concentration of inhibitor which causes 50 percent inhibition (IC₅₀) of an enzymatic reaction