A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

Abstract

Cys-loop receptors are pentameric ligandgated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cysloop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT 3A receptors (5-HT 3A Rs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT 3A R can be exchanged between the homomeric 5-HT 3A R and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Further, nAChR-type mutations of the 5-HT 3A R affect the affinity of nicotine, a competitive antagonist of 5-HT 3A Rs, in a cooperative manner. Using mutant cycle analyses between the 5-HT 3A triad, loop A residues Asn-101 and Glu-102, ß9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

The Binding of Palonosetron and Other Antiemetic Drugs to the Serotonin 5-HT3 Receptor

“…In addition, E102 creates an electronegative patch next to the binding site entrance, which might participate in ligand attraction, in line with the observation that the deleterious effect of introducing a net positive charge in loop A (e.g. N101K, E102Q) can be assuaged by removal of the positive charge at K211(regardless of H-bonding ability at position 211) 24 . At position 103, granisetron binding appears only marginally affected by F103Y/N substitutions 25 , while it was decreased ~5-fold by F103A/W mutations 21 .…”