Michael M. White

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

The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT3R) 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 γ (γTrp-55) and δ (δTrp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT3R shows that a tryptophan residue is found in the homologous position in the 5HT3R (Trp-89), suggesting that this residue may be involved in curare-5HT3R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT3R 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 5HT3R (and by inference, other members of the ligand-gated ion channel family) is in a β-strand conformation.

Ligand—receptor interactions in the nicotinic acetylcholine receptor probed using multiple substitutions at conserved tyrosines on the α subunit

Affinity labeling studies have identified several conserved tyrosine residues in the α subunit of the nicotinic acetylcholine receptor (αY93, αY190, and αY198) as being in or near the ligand binding site. Mutagenesis studies from several laboratories have shown that substitution of phenylalanine for tyrosine at these positions reduces the apparent affinity for ACh. We have examined this apparent reduction in affinity further through the use of multiple substitutions at each position. Substitution of either phenylalanine, tryptophan, or serine resulted in an apparent decrease in agonist affinity, but the degree of reduction depended on both the position and the nature of the substitution. Analysis of the effects of each substitution suggests that each residue interacts with the quaternary N of ACh, and that each residue may make a different type of interaction with the agonist.

Acetylcholine Receptor Gating is Influenced by the Polarity of Amino Acids at Position 9′ in the M2 Domain

Abstract. Ligand-gated ion channels contain a conserved leucine at position 9′ (L9′) in the M2 transmembrane domain. We used multiple substitutions at this position in the γ subunit of the mouse acetylcholine receptor (AChR) (γL9′) to examine the role of residue polarity at this position in the gating process at both the macroscopic and single-channel levels. The midpoint of the macroscopic dose-response relationship (EC50) and the channel closing rate constant, α, decreased as the polarity of the residue at that position increased, suggesting a stabilization of the open state of the channel. Both parameters showed similar dependencies on the polarity of the substituted residue. These data support the notion that during AChR gating, the amino acid at the 9′ position moves into a polar environment, and that interactions between this residue and the polar environment determine the stability of the open state. Since this residue is conserved in all other members of the ligand-gated ion channel family, we suggest that a similar mechanism applies to the other members of the family.

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

Soluble β-amyloid has been shown to regulate presynaptic Ca2+ and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimers patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca2+ imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator.

Mapping Residues in the Ligand-Binding Domain of the 5-HT3 Receptor onto d-Tubocurarine Structure

The serotonin 5-HT3 receptor (5-HT3R) 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-HT3R extracellular domain, and ligand docking simulations as a way to map the architecture of the 5-HT3R 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 2′N of dTC. Molecular modeling of the 5-HT3R 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-HT3R/dTC complex; only one of these has the 2′N 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.

Expression of a biologically-active conotoxin PrIIIE in Escherichia coli

Conotoxin PrIIIE is a 22-amino acid peptide containing three disulfide bonds isolated from the venom of Conus parius Reeve. It is a non-competitive antagonist of the mammalian muscle nicotinic acetylcholine receptor (nAChR). We fused the PrIIIE to small ubiquitin-like modifier (SUMO) and expressed the fusion protein in an Escherichia coli strain with an oxidizing cytoplasm. We purified the fusion protein by immobilized metal affinity chromatography and further purified PrIIIE from cleaved SUMO using cation exchange chromatography. The yield of peptide was 1.5mg/L of culture. The recombinant peptide is functional, as demonstrated by two-electrode voltage clamp experiments. This system may prove valuable for future structure-function studies.

Mapping Spatial Relationships between Residues in the Ligand-Binding Domain of the 5-Ht3 Receptor Using a Molecular Ruler

The serotonin 5-HT(3) receptor (5-HT(3)R) 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.

Interaction of d-tubocurarine analogs with mutant 5-HT3 receptors

d-Tubocurarine is a potent competitive antagonist of both the muscle-type nicotinic acetylcholine receptor (AChR) and the serotonin type-3 receptor (5HT(3)R). 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 5HT(3)Rs 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.

Forskolin modulates acetylcholine receptor gating by interacting with the small extracellular loop between the M2 and M3 transmembrane domains.

Abstract1. Forskolin acts as an allosteric modulator of muscle-type nicotinic acetylcholine receptors. Receptors from mouse muscle and Torpedo electroplax demonstrate differential sensitivity to inhibition by forskolin. Previous work from this laboratory suggested that the γ subunit is responsible for this differential sensitivity.2. We have used a series of mouse/Torpedo species-chimeric γ subunits to further define the site of forskolin interaction with the γ subunit. Analysis of the patterns of forskolin inhibition of receptors containing mouse/Torpedo chimeric γ subunits along with the mouse α, β, and δ subunits suggests that forskolin interacts with the small extracellular domain that links the M2 and M3 transmembrane domains (the M2–M3 linker).3. We suggest that the M2–M3 linker domain plays an important role in the transduction of ligand binding to the conformational changes that result in channel opening.