Spatial Orientation of the Antagonist Granisetron in the Ligand-Binding Site of the 5-HT₃ Receptor

Abstract

The serotonin type 3 receptor (5-HT 3 R) is a member of the cys-loop ligand-gated ion channel (LGIC) superfamily. Like almost all membrane proteins, high-resolution structural data are unavailable for this class of receptors. We have taken advantage of the high degree of homology between LGICs and the acetylcholine binding protein (AChBP) from the freshwater snail Lymnea stagnalis, for which high-resolution structural data are available, to create a structural model for the extracellular (i.e., ligand-binding) domain of the 5-HT 3 R and to perform a series of ligand docking experiments to delineate the architecture of the ligand-binding site. Structural models were created using homology modeling with the AChBP as a template. Docking of the antagonist granisetron was carried out using a Lamarckian genetic algorithm to produce models of ligand-receptor complexes. Two energetically similar conformations of granisetron in the binding site were obtained from the docking simulations. In one model, the indazole ring of granisetron is near Trp90 and the tropane ring is near Arg92; in the other, the orientation is reversed. We used double-mutant cycle analysis to determine which of the two orientations is consistent with experimental data and found that the data are consistent with the model in which the indazole ring of granisetron interacts with Arg92 and the tropane ring interacts with Trp90. The combination of molecular modeling with double-mutant cycle analysis offers a powerful approach for the delineation of the architecture of the ligand-binding site.The serotonin type 3 receptor (5-HT 3 R) is a member of the cys-loop ligand-gated ion channel gene family, which includes the muscle and neuronal nicotinic acetylcholine receptors, the glycine receptor, and the GABA A receptor (Connolly and Wafford, 2004;Lester et al., 2004). Two different subunits, termed 5-HT 3A and 5-HT 3B , have been described previously (Reeves and Lummis, 2002). The 5-HT 3A subunit forms functional receptors with the appropriate pharmacological properties when expressed in Xenopus laevis oocytes or mammalian cells. However, there are some differences between the properties of the expressed homomeric receptors and 5-HT 3 Rs in some, but not all, neurons. Perhaps the most significant difference is that the single-channel conductance of the expressed receptors is in the subpicosiemen range, whereas that of the receptors in many (but not all) neurons is in the range of 10 to 20 pS (Yang et al., 1992;Hussy et al., 1994). This difference, along with the fact that other members of the cys-loop LGIC family are composed of several different subunits, led to the search and subsequent discovery of an additional 5-HT 3 R subunit, now termed the 5-HT 3B subunit (Davies et al., 1999;Dubin et al., 1999).When expressed by itself, the 5-HT 3B subunit does not form functional receptors. When the 5-HT 3B subunit is coexpressed with the 5-HT 3A subunit, the ligand-binding properties of the expressed receptors are identical to those resulting from expressi...

Molecular actions of smoking cessation drugs at α4β2 nicotinic receptors defined in crystal structures of a homologous binding protein

“…In the crystal structure of Ct-AChBP in complex with varenicline, a water molecule that occupies a common position in nicotine and lobeline complexes with AChBP plays a key role in establishing hydrogen bonds with the backbone atoms of hydrophobic residues of loop E. In addition, a highly conserved aromatic residue of loop D, W57 in α4β2, also plays a key role in defining the architecture of the complementary subunit. The critical contribution of this residue has been demonstrated in other Cysloop receptors, including α7 nicotinic receptors (34), GABA A receptors (35), 5-HT 3 receptors (36)(37)(38), and glycine receptors (39). Consistent with earlier observations (26), we took the assumption that these specific contacts of ligand and binding site residues might be of key importance for discriminating agonists from partial agonists.…”

The structural basis of function in Cys-loop receptors

“…The orientation that is best supported by the experimental evidence, however, is A1, where the orientation of granisetron is reversed so that the indazole ring is located between W183 and Y234 with the azabicyclic ring orientated towards the transmembrane region, between residues E129 and W90. Double-mutant cycle analysis shows that the azabicyclic ring of granisetron is close to W90 and the indazole ring is orientated away from the membrane (Yan & White, 2005), and this orientation is also supported by experimental evidence as described by both Joshi et al (2006) and Thompson et al (2005). In both orientations A1 and A2, there is an interaction with W183, a residue that is important for both agonist and antagonist binding (Beene et al 2002 ;Spier & Lummis, 2000), and with Y234, which also contributes to the binding site ; substitutions of Y234 to Ala or Ser severely compromise granisetron binding, although Y234F mutants have similar binding affinities to wild-type receptors (Spier & Lummis, 2000 ;Suryanarayanan et al 2005).…”

“…Nevertheless, homology models have been produced for many receptors, and a range of ligands docked into their binding sites (e.g. Abdel-Halim et al 2008 ;Cromer et al 2002 ;Le Novere et al 2002 ;Maksay et al 2003 ;Reeves et al 2003 ;Schapira et al 2002 ;Trudell & Bertaccini, 2004 ;Yan & White, 2005). The ability of 5-HT 3 Rs to form homomeric receptors means that they are a relatively simple system for molecular modelling, and they have the considerable advantage that the experimental determination of the effects of amino acid substitutions on the properties of the receptor is straightforward.…”

“…The 5-HT 3 R is a typical Cys-loop receptor, and has the advantage that it functions as a homomeric receptor, which simplifies the interpretation of experimental data. This protein has also been used extensively in homology modelling and ligand docking (Maksay et al 2003 ;Reeves et al 2003 ;Thompson et al 2005 ;Yan & White, 2005). As this technique is becoming an accepted route to understanding the structural details of the proteins, we use the new homology models and docked ligands to explore the validity of these techniques to define specific molecular interactions with agonists and antagonists in the 5-HT 3 ligand-binding site.…”

Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A

“…The selective 5-HT3A receptor antagonist granisetron was compared with the supposed binding of azelastine. Granisetron is a well-known antagonist that exerts its action by competition with 5-HT for binding to the orthosteric ligand-binding site [27]. The blocking properties were evaluated by recording responses upon application of 5-HT at a concentration closed to EC 80 (7 µM) determined previously ( (Fig.3C) after preincubation (2-5 min) with increasing concentrations of granisetron or azelastine (Fig.8B).…”