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NEUROPHARMACOLOGY

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

Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas

Previously, we reported that the GABA_A 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-HT_3A receptors. However, mutation of the 6' TM2 threonine to phenylalanine dramatically reduced PTX sensitivity. Mutation of 6' asparagine to threonine in the 5-HT_3B subunit enhanced PTX sensitivity in heteromeric 5-HT_3A/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-HT_3A receptors increased PTX sensitivity roughly 10-fold, comparable with that observed in GABA_A 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-HT_3A/3B receptors. Interestingly, [³H]ethynylbicycloorthobenzoate ([³H]EBOB), a high-affinity ligand to the convulsant site in GABA_A receptors, did not exhibit specific binding in 5-HT_3A receptors. The structurally related compound, tert-butylbicyclophosphorothionate (TBPS), which potently inhibits GABA_A 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 GABA_A receptors and demonstrate a role of the 7' residue in PTX inhibition. However, lack of effects of EBOB and TBPS in 5-HT_3A receptors suggests that the functional domains in the two receptors are not equivalent and underscores the complexity of PTX modulation of LGICs.

Address correspondence to: Dr. Glenn H. Dillon, Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. E-mail: gdillon{at}hsc.unt.edu

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