Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT₃ Receptor

Abstract

Bilobalide (BB), ginkgolide B (GB), diltiazem (DTZ), and picrotoxinin (PXN) are 5-hydroxytryptamine type 3 (5-HT 3 ) receptor antagonists in which the principal sites of action are in the channel. To probe their exact binding locations, 5-HT 3 receptors with substitutions in their pore lining residues were constructed (NϪ4ЈQ, EϪ1ЈD, S2ЈA, T6ЈS, L7ЈT, L9ЈV, S12ЈA, I16ЈV, D20ЈE), expressed in Xenopus laevis oocytes, and the effects of the compounds on 5-HT-induced currents were examined. EC 50 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 pIC 50 values of 3.33, 3.14, 4.67, and 4.97, respectively. Inhibition by BB and GB was abolished in mutant receptors containing T6ЈS and S12ЈA substitutions, but their potencies were enhanced (42-and 125-fold, respectively) in S2ЈA mutant receptors. S2ЈA substitution also caused GB ligand trap. PXN potency was modestly enhanced (5-fold) in S2ЈA, abolished in T6ЈS, and reduced in L9ЈV (40-fold) and S12ЈA (7-fold) receptors. DTZ potency was reduced in L7ЈT and S12ЈA receptors (5-fold), and DTZ also displaced [3 H]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-HT 3 receptor; for BB, GB, and PXN, the data show that the 6Ј channel lining residue is their major site of action, with minor roles for 2Ј, 9Ј, and 12Ј residues, whereas for DTZ, the 7Ј and 12Ј sites are important.

VUF10166, a Novel Compound with Differing Activities at 5-HT3A and 5-HT3AB Receptors

“…VUF10166 is unlikely to bind in the pore because the partial agonist response at high concentrations shows that the channel is not blocked, and its effects are not voltage-dependent. To confirm this, we exchanged the 6Ј channel lining residues in 5-HT 3 A and 5-HT 3 B subunits, because this residue interacts with all of the channel-binding compounds described to date (Das and Dillon, 2005;Thompson et al, 2011a). The properties of VUF10166 were not altered by these mutations, providing further evidence that it does not act within the pore.…”

“…The pharmacology of these competitive ligands is almost identical at 5-HT 3 A and 5-HT 3 AB receptors, and to date none can be used to distinguish homomeric from heteromeric receptors (Brady et al, 2001). In contrast, the potencies of noncompetitive antagonists that bind to the channels of 5-HT 3 A and 5-HT 3 AB receptors are different (Das and Dillon, 2005;Thompson and Lummis, 2008;Thompson et al, 2011a). In other Cys-loop receptors, the identification of li-gands with subtype specificity has been pharmacologically and therapeutically important (e.g., benzodiazepines and pentobarbital), and the identification of comparable 5-HT 3 receptor ligands has the potential to be similarly useful (for review, see Jensen et al, 2008;Walstab et al, 2010).…”

“…To date the only ligands that can distinguish 5-HT 3 A from 5-HT 3 AB receptors are those that bind in the receptor pore. To test whether VUF10166 binds at this location, we examined its effects in a receptor modified at the 6Ј channel lining residue, which is critical for ligand binding in the pore (Das and Dillon, 2005;Thompson et al, 2011a). Receptors containing a T6ЈS substitution in the 5-HT 3 A subunit had 5-HT concentration-response curves similar to those of wild-type receptors, as did heteromeric receptors with S6ЈT 5-HT 3 B subunits (Fig.…”

Noncompetitive Inhibition of 5-HT3 Receptors by Citral, Linalool, and Eucalyptol Revealed by Nonlinear Mixed-Effects Modeling

“…For each test compound, inhibition of a supramaximal 5-HT–induced (100 µ M) response was measured alone and in combination with bilobalide (BB) and diltiazem (DTZ), both well characterized 5-HT 3 blockers (Fig. 1; Thompson et al, 2011a). Concentrations of the test compounds, BB and DTZ, were selected such that they caused approximately 62% inhibition when used alone.…”

Structure–Activity Relationships of Quinoxaline‐Based 5‐HT3A and 5‐HT3AB Receptor‐Selective Ligands

“…Pharmacologically distinguishing 5-HT 3 A from 5-HT 3 AB receptors has historically required the use of compounds that bind in the pore, such as bilobalide, ginkgolide, and picrotoxinin 6. In contrast, competitive ligands usually have very similar affinities at 5-HT 3 A and 5-HT 3 AB receptors.…”