Abstract

Bilobalide (BB), ginkgolide B (GB), diltiazem (DTZ), and picrotoxinin (PXN) are 5-hydroxytryptamine type 3 (5-HT₃) receptor antagonists in which the principal sites of action are in the channel. To probe their exact binding locations, 5-HT₃ receptors with substitutions in their pore lining residues were constructed (N−4′Q, E−1′D, S2′A, T6′S, L7′T, L9′V, S12′A, I16′V, D20′E), expressed in Xenopus laevis oocytes, and the effects of the compounds on 5-HT-induced currents were examined. EC₅₀ values at mutant receptors were less than 6-fold different from those of wild type, indicating that the mutations were well tolerated. BB, GB, DTZ, and PXN had pIC₅₀ values of 3.33, 3.14, 4.67, and 4.97, respectively. Inhibition by BB and GB was abolished in mutant receptors containing T6′S and S12′A substitutions, but their potencies were enhanced (42- and 125-fold, respectively) in S2′A mutant receptors. S2′A substitution also caused GB ligand trap. PXN potency was modestly enhanced (5-fold) in S2′A, abolished in T6′S, and reduced in L9′V (40-fold) and S12′A (7-fold) receptors. DTZ potency was reduced in L7′T and S12′A receptors (5-fold), and DTZ also displaced [³H]granisetron binding, indicating mixed competitive/noncompetitive inhibition. We conclude that regions close to the hydrophobic gate of M2 are important for the inhibitory effects of BB, GB, DTZ, and PXN at the 5-HT₃ receptor; for BB, GB, and PXN, the data show that the 6′ channel lining residue is their major site of action, with minor roles for 2′, 9′, and 12′ residues, whereas for DTZ, the 7′ and 12′ sites are important.