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

Abstract

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...

A Structural and Mutagenic Blueprint for Molecular Recognition of Strychnine and d-Tubocurarine by Different Cys-Loop Receptors

“…For d-TC , previous mutagenesis studies have been carried out either on the muscle-type nAChR [1] or 5-HT 3 R [5],[12]. The heteropentameric muscle-type nAChR contains two different binding sites for d-TC with a 100-fold difference in affinity, whereas differences between human and mouse 5-HT 3 R yield a more than 1,000-fold difference in affinity.…”

Topological Mapping of the Asymmetric Drug Binding to the Human Ether-à-go-go-Related Gene Product (HERG) Potassium Channel by Use of Tandem Dimers

“…The contribution of residues to the binding and the interactions between residues upon binding can be evaluated quantitatively by examining the coupling energies (⌬⌬G int ) in the thermodynamic doublemutant cycle analysis (see Materials and Methods; Schreiber and Fersht, 1995;Ranganathan et al, 1996;Fersht, 1999;Yan et al, 2006). For each of two double mutants, a cyclic diagram was drawn (Fig.…”

“…Thermodynamic Double-Mutant Cycle. To estimate the interaction free energy upon binding, thermodynamic double-mutant cycle analysis was applied (Schreiber and Fersht, 1995;Ranganathan et al, 1996;Fersht, 1999;Yan et al, 2006). First, the binding free energy (G) was evaluated from the inhibition constant.…”

Unbalanced Peptidergic Inhibition in Superficial Neocortex Underlies Spike and Wave Seizure Activity

“…4). D-tubocurarine itself has antagonist actions at both of these receptor subtypes (Yan et al, 2006) so its ability to generate SpW was used as a reference for other, more selective agents. Bath application of MG624 or ACV1 (blockade of ␣7-or ␣9/10-containing nicotinic receptors generated SpWs with incidence not significantly different from dTC ( p Ͼ 0.1, n ϭ 5).…”