Molecular determinants of d-tubocurarine binding at recombinant 5-HT3A receptors
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The 5-hydroxytryptamines (5-HT3) receptor is a member of a superfamily of ligand-gated ion channels. Other members of this family include the nicotinic acetylcholine receptor, GABAA receptor, and glycine receptor. Each receptor shares a lot of structural and pharmacological properties. To date, two 5-HT3 receptor subunits, 5- HT3A and 5-HT3B, have been identified and cloned. Until recently, research has focused on 5-HT3 receptors has been done with the recombinant 5-HT3A receptors. This receptor displays a species-dependent pharmacology exemplified by a 140-fold difference in the potency of d-tubocurarine, which is a competitive antagonist, at human and mouse 5- HT3A receptors. In the present study, by constructing a series of chimeras and point mutants, we attempt to identify the amino acids contributing to the interspecies differential potency of d-tubocurarine at human and mouse 5-HT3A receptors. Our study shows that the distal one-third of the extracellular N-terminal domain is completely responsible for the differential potency of d-tubocurarine at human and mouse 5-HT3A receptors. This region includes loop C and loop G binding domains. Both of the loops contribute to the difference. Within loop G, Lysl95 and Val202 in the human wild-type 5-HT3A receptor and their corresponding residues in mouse wild-type 5-HT3A receptor contribute significantly to the difference, but Leul92 and its corresponding ortholog in the mouse receptor do not. Residues outside loop G, but within the distal one-third of the N-terminus, were also examined. Mutation of residues Serl88, Gly213, and Leu215 and the mouse orthologs did not change the IC50 value of J-tubocurarine, suggesting that they do not play a role in the differential potency of <i-tubocurarine.