Probing the architecture of the serotonin 5-HT₃ receptor ligand-binding domain: importance of select amino acids in antagonist-receptor interactions

Heather L. Nyce

doi:10.17918/00010098

The serotonin 5-HT₃ receptor (5-HT₃R) is a member of the cys-loop family of ligand-gated ion channels. Functional receptors are pentamers and the ligand-binding domain is composed of contributions from six non-contiguous loops (A-F) that converge at subunit-subunit interfaces. 5-HT₃R's are found in bath the central and peripheral nervous systems and receptor antagonists have proven effective in the treatment of irritable bowel syndrome (IBS) and emesis due to cancer chemotherapy or anesthesia. This dissertation employs the combination of site-directed mutagenesis, double-mutant cycle analysis and homology modeling as a way to map the functional architecture of the 5-HT₃R ligand-binding domain. Two regions are examined: Loop A residues (I127, E129, F130, V131, D132, V133, K135 and S136) and four residues located within 6 A of the bound antagonist in a ligand-receptor model (T181, S182, H185 and E236). Antagonists of differing structure were used to determine the spatial orientation of residues in the binding site and double-mutant cycle analysis indicated residue involvement in ligand-binding. Mutation of E129 and H185 to alanine and E236 to aspartate abolished surface expression and prevented ligand binding. Mutation of the other residues resulted in specific binding of radioligand and small changes in affinity of antagonists for the receptor; however, double-mutant cycle analysis is only consistent with residues E236 and T181 making a direct physical contact with the antagonists d-tubocurarine (dTC) and granisetron, respectively. When combining previous data linking regions of dTC to 5-HT₃R residues R92 and N128 with the knowledge that dTC is a rigid molecule of known three-dimensional structure, the present data allow for the addition of a third spatial constraint (E236) to the arrangement of residues in the 5-HT₃R ligand-binding domain and permit the creation of a more accurate homology model. The use of antagonists as "molecular rulers", in conjunction with double-mutant cycle analysis of mutant receptors, permits the spatial mapping of residues in the ligand-binding domain. The identification of specific residues involved in ligand-binding, as well as classification of residues near the binding site but not directly involved in binding, is essential for building a more accurate model of the 5-HT₃R ligand-binding domain that may aid in the future development of 5-HT₃R-selective therapeutic agents.

Probing the architecture of the serotonin 5-HT₃ receptor ligand-binding domain: importance of select amino acids in antagonist-receptor interactions

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Probing the architecture of the serotonin 5-HT₃ receptor ligand-binding domain: importance of select amino acids in antagonist-receptor interactions

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