Structural Features of the Ligand-binding Domain of the Serotonin 5HT3 Receptor

Abstract

The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT 3 R) 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 ␥ (␥Trp-55) and ␦ (␦Trp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT 3 R shows that a tryptophan residue is found in the homologous position in the 5HT 3 R (Trp-89), suggesting that this residue may be involved in curare-5HT 3 R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT 3 R 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 5HT 3 R (and by inference, other members of the ligand-gated ion channel family) is in a ␤-strand conformation.The serotonin type 3 receptor (5HT 3 R) 1 is a member of a superfamily of ligand-gated ion channels, which includes the muscle and neuronal nicotinic acetylcholine receptor (AChR), the glycine receptor, and the ␥-aminobutyric acid type A receptor (1-3). Like the other members of the gene superfamily, the 5HT 3 R exhibits a large degree of sequence similarity, and thus presumably structural homology, with the AChR (4). Chimeras containing the amino-terminal domain of the ␣7 neuronal AChR and the carboxyl-terminal domain of the 5HT 3 R form functional ligand-gated channels with ligand specificities characteristic of AChR and permeability properties of 5HT 3 Rs (5). These experiments suggest that the AChR and 5HT 3 R have quite similar structures and signal transduction mechanisms.A large number of studies have been conducted to elucidate the structure of the ligand-binding site for AChRs (6, 7); however, very little is known about the interactions between ligands and the 5HT 3 R. A common competitive antagonist of the AChR and 5HT 3 R is d-tubocurarine (curare), which inhibits both receptors at nanomolar concentrations (8, 9). If both receptors are similar in structure, it seems reasonable to assume that similar regions of the receptors may...

The Role of Tyrosine Residues in the Extracellular Domain of the 5-Hydroxytryptamine3 Receptor

“…Thus, Y141A and Y141S receptors did not function when expressed in HEK293 cells, as previously observed for Y141A receptors (9). Tyr 141 is proposed to be more than 5 Å from 5-HT and located just outside the binding pocket; it would therefore be unlikely to participate directly in agonist binding, although it could still bind larger antagonists.…”

“…The model of the binding site places Tyr 91 at more than 5 Å from 5-HT, and thus it is unlikely that it would participate directly in ligand binding. However, there is evidence that the adjacent residues, Trp 90 and Arg 92 , which are within 5 Å of 5-HT, are involved in such an interaction (9,22). Previous data suggest that this region has a ␤-sheet composition (9) H]granisetron binding affinity for Y141A (Y142A using their numbering) and Y153A (Y152A) mutant receptors.…”

“…In particular a role for tyrosine residues has been demonstrated (8): pretreatment of 5-HT 3 receptors in NG108 -15 cells with the tyrosine-modifying reagent tetranitromethane caused a 30% reduction in specific binding of the 5-HT 3 receptor antagonist [ 3 H]zacopride. In addition, mutation of Tyr 94 to alanine caused a small shift in [ 3 H]granisetron binding affinity (9), and alanine substitutions of Tyr 141 , Tyr 143 , and Tyr 153 have been shown to affect both agonist and antagonist affinities and 5-HT-induced currents (10). Here we extend these studies to examine the radioligand binding, electrophysiological, and cell surface expression properties of mutants of each of the 11 tyrosine residues in the mouse 5-HT 3 receptor extracellular domain.…”

Contributions of Torpedo Nicotinic Acetylcholine Receptor γTrp-55 and δTrp-57 to Agonist and Competitive Antagonist Function

“…In contrast, substitution at the equivalent position in ␥2 subunit (␥2Phe-77) selectively alters binding of ligands at the benzodiazepine site (39). The serotonin 5-HT 3 receptor also contains a tryptophan (Trp-89) at the equivalent position, and substitutions at the position reduce antagonist but not agonist affinity (40).…”

Different Residues in the GABAA Receptor α1T60-α1K70 Region Mediate GABA and SR-95531 Actions

“…In the homologous region of the serotonin-type 3 receptor, White and colleagues (20) used alanine-scanning mutagenesis and determined that different amino acid residues contribute to the binding of agonists and antagonists. We hypothesized that the region surrounding ␣ 1 F64 of the GABA-binding site also contains unique residues important for agonist and antagonist binding, and we tested this hypothesis by using the substituted cysteine accessibility method (SCAM).…”