Orientation of d-Tubocurarine in the Muscle Nicotinic Acetylcholine Receptor-binding Site

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...

Section: Resultsmentioning

confidence: 99%

“…Similarly weak interactions were observed in a double-mutant cycle analysis of the interaction of d-tubocurarine with the nicotinic acetylcholine receptor (Willcockson et al, 2002). Reeves et al (2003) developed a homology-based model of the 5-HT 3 R and carried out docking simulations using serotonin as the ligand.…”

mentioning

confidence: 99%

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

Dong¹,

White²

2005

“…An ⍀ value significantly different from 1 indicates an interaction between the functional group on the ligand and the residue in question on the receptor. Although initially used for analysis of the interaction of peptide toxins with K ϩ channels (Hildago and MacKinnon, 1995), this approach has also been applied to identify points of contact between AChRs and peptide toxins (Malany et al, 2000), AChRs and d-tubocurarine analogs (Willcockson et al, 2002) and granisetron and the 5-HT 3 R (Yan and White, 2005). Figure 3 shows two double-mutant cycles constructed for the interaction of dTC analogs with the receptor.…”

Section: Resultsmentioning

confidence: 99%

Mapping Residues in the Ligand-Binding Domain of the 5-HT₃Receptor ontod-Tubocurarine Structure

Dong¹,

Meyer²,

White³

2006

The serotonin 5-HT 3 receptor (5-HT 3 R) is a member of the cys-loop ligand-gated ion channel family. We have used the combination of site-directed mutagenesis, homology modeling of the 5-HT 3 R extracellular domain, and ligand docking simulations as a way to map the architecture of the 5-HT 3 R ligand binding domain. Mutation of Phe226 in loop C of the binding site to tyrosine (F226Y) has no effect on the apparent affinity of the competitive antagonist d-tubocurarine (dTC) for the receptor. On the other hand, replacement of Asn128 in loop A of the binding site with alanine (N128A) increases the apparent affinity of dTC by approximately 10-fold. Double-mutant cycle analysis employing a panel of dTC analogs with substitutions at various positions to identify specific points of interactions between the dTC analogs and Asn128 suggests that Asn128 makes a direct interaction with the 2ЈN of dTC. Molecular modeling of the 5-HT 3 R extracellular domain using the antagonist-bound conformation of the Aplysia californica acetylcholine binding protein as a template followed by ligand docking simulations produces two classes of structures of the 5-HT 3 R/dTC complex; only one of these has the 2ЈN of dTC positioned at Asn128 and thus is consistent with the data from this study and previously published data. The use of the rigid dTC analogs as "molecular rulers" in conjunction with double-mutant cycle analysis of mutant receptors can allow the spatial mapping of the position of various residues in 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 (AChR), the glycine receptor, and the GABA type A receptor (Connolly and Wafford, 2004;Lester et al., 2004). Two different subunits, termed 5-HT 3A and 5-HT 3B , have been shown to be present in functional 5-HT 3 Rs (Reeves and Lummis, 2002). In addition, several other putative 5-HT 3 R genes have been described, but whether or not they play a role in 5-HT 3 R-mediated processes is unknown at present (Karnovsky et al., 2003). The 5-HT 3A subunit alone can form functional receptors with the appropriate pharmacological properties when expressed in Xenopus laevis oocytes or mammalian cells. However, receptors comprising only the 5-HT 3A subunit have single-channel conductances in the subpicosiemens range, whereas those containing both the 5-HT 3A and 5-HT 3B subunits have conductances in the 10-to 30-pS range (Davies et al., 1999;Dubin et al., 1999;Hanna et al., 2000), similar to that observed in neurons from the peripheral nervous system (Derkach et al., 1989;Yang et al., 1992). The reduction in single-channel conductance of homomeric 5-HT 3A Rs is now know to be due to the presence of three arginine residues in the cytoplasmic domain, and removal of these three positive charges results in a single-channel conductance on the order of 25 pS (Kelley et al., 2003). Analysis of the expression patterns of the 5-HT 3A and 5-HT 3B su...

“…Mutations at residues ␣7-Asn111 and ␣7-Gln117 slightly decrease the potency for activation by ACh and pyrantel in a quantitatively similar manner, indicating that they do not govern pyrantel selectivity. The equivalent residues in the Torpedo AChR, ␥Tyr111 and ␥Tyr117, were reported to interact with d-tubocurarine (Chiara et al, 1999;Willcockson et al, 2002). Thus, it is possible that mutations at these residues affect only the affinity for agonists.…”

Section: Discussionmentioning

confidence: 99%

Molecular Determinants of Pyrantel Selectivity in Nicotinic Receptors

Bartos¹,

Rayes²,

Bouzat³

2006

Nicotinic receptors (acetylcholine receptors, AChRs) play key roles in synaptic transmission throughout the nervous system. AChRs mediate neuromuscular transmission in nematodes, and they are targets for antiparasitic drugs. The anthelmintic agents levamisole and pyrantel, which are potent agonists of nematode muscle AChRs, are partial agonists of mammalian muscle AChRs. To further explore the structural basis of the differential activation of AChR subtypes by anthelmintics, we studied the activation of ␣7 AChRs using the high-conductance form of the ␣7-5-hydroxytryptamine-3A receptor, which is a good model for pharmacological studies involving the extracellular region of ␣7. Macroscopic and single-channel current recordings show that levamisole is a weak agonist of ␣7. It is interesting that pyrantel is a more potent agonist of ␣7 than acetylcholine (ACh). To identify determinants of this differential activation, we replaced residues of the complementary face of the binding site by the homologous residues in the muscle subunit and evaluated changes in activation. The mutation Q57G does not affect the activation by either ACh or levamisole. However, it increases EC 50 values and decreases the maximal response to pyrantel. Kinetic analysis shows that gating of the mutant channel activated by pyrantel is profoundly impaired. The decreased sensitivity of ␣7-Q57G to pyrantel agrees with its weak action at muscle AChRs, indicating that when glycine occupies position 57, as in the mammalian muscle AChR, pyrantel behaves as a partial agonist. Thus, position 57 located at the complementary face of the binding site plays a key role in the selective activation of AChRs by pyrantel.Cysteine-loop receptors are pentameric ligand-gated ion channels that play key roles in chemical synapses throughout the nervous system. In vertebrates, the cysteine-loop receptor superfamily consists of two families of cation-selective channels: the nicotinic acetylcholine (AChR) and the 5-hydroxytryptamine type 3 receptors (5HT 3 Rs); and two families of anionic channels: the ␥-aminobutyric acid and the glycine receptors (Le Novére and Changeux, 2001;Lester et al., 2004). In invertebrates, this superfamily also includes ␥-aminobutyric acid-gated cationic channels and ACh-, serotonin-, glutamate-, and histamine-gated chloride channels (Jones and Sattelle, 2003;Putrenko et al., 2005).AChR subunits are classified as ␣, which contain a disulfide bridge involved in the recognition and binding of agonists, and non-␣ subunits, which lack this motif. To date, several ␣ and non-␣ subunits have been identified. Receptors can be either heteromeric, composed of ␣ and non-␣ subunits, or homomeric, composed of five identical ␣ subunits. Each subunit is divided into an N-terminal or ligand-binding domain and a transmembrane region. The binding sites are located at the interface between two subunits (Brejc et al., 2001;Sine, 2002). Results from affinity labeling and sitedirected mutagenesis studies on AChR, which were later supported by the structural...