Abstract
Cys-loop receptors are the site of action of many therapeutic drugs. One of these is the smoking cessation agent varenicline, which has its major therapeutic effects at nicotinic acetylcholine (nACh) receptors but also acts at 5-HT3 receptors. Here, we report the X-ray crystal structure of the 5-HT binding protein (5-HTBP) in complex with varenicline, and test the predicted interactions by probing the potency of varenicline in a range of mutant 5-HT3 receptors expressed in HEK293 cells and Xenopus oocytes. The structure reveals a range of interactions between varenicline and 5-HTBP. We identified residues within 5 Å of varenicline and substituted the equivalent residues in the 5-HT3 receptor with Ala or a residue with similar chemical properties. Functional characterization of these mutant 5-HT3 receptors, using a fluorescent membrane potential dye in HEK cells and voltage clamp in oocytes, supports interactions between varenicline and the receptor that are similar to those in 5-HTBP. The structure also revealed C-loop closure that was less than in the 5-HT-bound 5-HTBP, and hydrogen bonding between varenicline and the complementary face of the binding pocket via a water molecule, which are characteristics consistent with partial agonist behavior of varenicline in the 5-HT3 receptor. Together, these data reveal detailed insights into the molecular interaction of varenicline in the 5-HT3 receptor.
Highlights
The 5-HT3 receptors belong to the Cys-loop family of pentameric ligand-gated ion channels, a family that includes nicotinic acetylcholine, GABA, and glycine receptors.[1]
There is considerable overlap between compounds that act at α7 nicotinic acetylcholine (nACh) and 5-HT3 receptors,[6] and recently, we have shown that varenicline is a high affinity agonist at the human 5-HT3 receptor.7 5HT3 receptors are located in the chemoreceptor trigger zone in the area postrema and in the gastrointestinal tract, where they have roles in regulating gut motility and the emesis reflex.[8]
We present the structure of varenicline bound to 5-HT binding protein (5-HTBP), an acetylcholine binding protein (AChBP) from Aplysia californica (Ac) engineered to bind 5HT with high affinity.[10]
Summary
The 5-HT3 receptors belong to the Cys-loop family of pentameric ligand-gated ion channels, a family that includes nicotinic acetylcholine (nACh), GABA, and glycine receptors.[1]. Subunits of 5-HT3A can assemble to form homopentamers, but the other subtypes must coassemble with 5-HT3A subunits to form functional receptors It is as yet unclear what effect subunits 5HT3C-5-HT3E have on the receptor function.[2,3]. The design of improved smoking cessation drugs that do not have such cross-reactivity at the 5-HT3 receptor would clearly be of benefit. For this aim to be achieved, further information is needed about the structure−activity relationships of varenicline binding to different receptors. The corresponding 5-HT3 receptor residues are in parentheses. (D) Hydrogen bonds are present between varenicline and residues Y91, Y193, and W145 on the principal face and residues I104 and I116 on the complementary face via a water molecule
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