Molecular Determinants of Picrotoxin Inhibition of 5-Hydroxytryptamine Type 3 Receptors

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Abstract
Previously, we reported that the GABAA receptor antagonist picrotoxin also antagonizes serotonin (5-HT)3 receptors and that its effects are subunit-dependent. Here, we sought to identify amino acids involved in picrotoxin inhibition of 5-HT3 receptors. Mutation of serine to alanine at the transmembrane domain 2 (TM2) 2′ position did not affect picrotoxin (PTX) sensitivity in murine 5-HT3A receptors. However, mutation of the 6′ TM2 threonine to phenylalanine dramatically reduced PTX sensitivity. Mutation of 6′ asparagine to threonine in the 5-HT3B subunit enhanced PTX sensitivity in heteromeric 5-HT3A/3B receptors. Introduction of serine (native to the human 3B subunit) at the 6′ position also increased PTX sensitivity, suggesting a species-specific effect. Mutation of the 7′ leucine to threonine in 5-HT3A receptors increased PTX sensitivity roughly 10-fold, comparable with that observed in GABAA receptors, and also conferred distinct gating kinetics. The equivalent mutation in the 3B subunit (i.e., 7′ valine to threonine) had no impact on PTX sensitivity in 5-HT3A/3B receptors. Interestingly, [3H]ethynylbicycloorthobenzoate ([3H]EBOB), a high-affinity ligand to the convulsant site in GABAA receptors, did not exhibit specific binding in 5-HT3A receptors. The structurally related compound, tert-butylbicyclophosphorothionate (TBPS), which potently inhibits GABAA receptors, did not inhibit 5-HT3 currents. Our results indicate that the TM2 6′ residue is a common determinant of PTX inhibition of both 5-HT3 and GABAA receptors and demonstrate a role of the 7′ residue in PTX inhibition. However, lack of effects of EBOB and TBPS in 5-HT3A receptors suggests that the functional domains in the two receptors are not equivalent and underscores the complexity of PTX modulation of LGICs.