Abstract
Palonosetron (Aloxi) is a potent second generation 5-HT3 receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT3 receptor binding site but recent computational studies indicated other possible sites of action in the extracellular domain. To test this hypothesis we mutated a series of residues in the 5-HT3A receptor subunit (Tyr73, Phe130, Ser163, and Asp165) and in the 5-HT3B receptor subunit (His73, Phe130, Glu170, and Tyr143) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT3A) and heteromeric (5-HT3AB) receptors were then expressed in HEK293 cells to determine the potency of palonosetron using both fluorimetric and radioligand methods to test function and ligand binding, respectively. The data show that the substitutions have little or no effect on palonosetron inhibition of 5-HT-evoked responses or binding. In contrast, substitutions in the orthosteric binding site abolish palonosetron binding. Overall, the data support a binding site for palonosetron at the classic orthosteric binding pocket between two 5-HT3A receptor subunits but not at allosteric sites previously identified by in silico modelling and docking.
Highlights
Cys-loop receptors constitute one of the largest families of ligand-gated ion channels responsible for the transmission of fast synaptic impulses in the CNS and PNS
Abbreviations: 5-HT, 5-hydroxytryptamine; EC50, concentration of agonist required for half-maximal response; Human embryonic kidney (HEK), human embryonic kidney; IC50, concentration of antagonist required for half-maximal inhibition; Kd, affinity constant
Our substitutions here caused a small increase in the Kd for palonosetron binding and the IC50 for inhibition of 5-HT-induced responses, suggesting that Y73 does not have a major interaction with palonosetron, but may influence the orthosteric site above, or may form part of a temporary binding location on route to the ‘classic’ binding pocket; a simulation study showing the trajectory of granisetron as it unbinds from the receptor indicates that ligands exits below the binding site close to Y73,19 altering the equivalent residue in mouse 5-HT3 receptors has no effect on [3H]granisetron binding.[20]
Summary
Cys-loop receptors constitute one of the largest families of ligand-gated ion channels responsible for the transmission of fast synaptic impulses in the CNS and PNS. The nicotinic acetylcholine, glycine, GABAA and 5-HT3 receptors, all belong to this family, sharing homologous amino acid sequences and similar arrangements of subunits.[1,2] The 5-HT3 receptor is formed by a pentameric assembly of identical (homomeric) or different (heteromeric) subunits surrounding a central ion-conducting pore. The binding pocket is formed between two adjacent subunits, and is constituted of the + (or principle) face of one subunit and the À (or complementary) face of the adjacent subunit (e.g., A+AÀ when between two 5-HT3A receptor subunits).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
