Molecular determinants of ginkgolide binding in the glycine receptor pore

School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia.
Journal of Neurochemistry (Impact Factor: 4.28). 08/2006; 98(2):395-407. DOI: 10.1111/j.1471-4159.2006.03875.x
Source: PubMed


Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha1, alpha2, alpha1beta and alpha2beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha2beta GlyR relative to the alpha2 GlyR but not in the alpha1beta GlyR relative to the alpha1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha2beta GlyR was transferred to the alpha1beta GlyR by the G2'A (alpha1 to alpha2 subunit) substitution. In addition, the alpha1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.

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Available from: Brett A Cromer, Jan 15, 2015
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    • "The binding site was defined by the C a atom of T6 0 on chain A and constrained to be within 10 Å of the T6 0 residue of chains A and C (a region that encompassed the positions between 2 0 and 9 0 ). These amino acids were chosen based on the binding of similar compounds at other Cys-loop receptors (Enz and Bormann, 1995; Xu et al., 1995; Zhang et al., 1995; Perret et al., 1999; Zhorov and Bregestovski, 2000; Das and Dillon, 2005; Hawthorne et al., 2006; Chen et al., Table 1 Parameters estimated from the fit of the Hill equation to concentrationeresponse curves in the presence and absence of ginkgolides. "
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    ABSTRACT: The diterpene lactones of Ginkgo biloba, ginkgolides A, B and C are antagonists at a range of Cys-loop receptors. This study examined the effects of the ginkgolides at recombinant human ρ1 GABAC receptors expressed in Xenopus oocytes using two-electrode voltage clamp. The ginkgolides were moderately potent antagonists with IC50s in the μM range. At 10 μM, 30 μM and 100 μM, the ginkgolides caused rightward shifts of GABA dose–response curves and reduced maximal GABA responses, characteristic of noncompetitive antagonists, while the potencies showed a clear dependence on GABA concentration, indicating apparent competitive antagonism. This suggests that the ginkgolides exert a mixed-type antagonism at the ρ1 GABAC receptors. The ginkgolides did not exhibit any obvious use-dependent inhibition. Fitting of the data to a number of kinetic schemes suggests an allosteric inhibition as a possible mechanism of action of the ginkgolides which accounts for their inhibition of the responses without channel block or use-dependent inhibition. Kinetic modelling predicts that the ginkgolides exhibit saturation of antagonism at high concentrations of GABA, but this was only partially observed for ginkgolide B. It also suggests that there may be different binding sites in the closed and open states of the receptor, with a higher affinity for the receptor in the closed state.
    Neuropharmacology 11/2012; 63(6):1127-1139. DOI:10.1016/j.neuropharm.2012.06.067 · 5.11 Impact Factor
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    • "Sensitivity to BB and GB was abolished by substitutions at the 6Ј residue, similar to observations at the glycine receptor (Hawthorne et al., 2006). Inhibition was also affected by 2Ј substitutions in this receptor, and, in combination with evidence from mutant cycle analysis, suggests that ginkgolides bind close to both positions 2Ј and 6Ј (Hawthorne et al., 2006). Similar 2Ј sensitivity was shown in the present study, with an enhancement of 42-fold for BB and 125-fold for GB. "
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    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 (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. 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 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 [(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 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.
    Molecular pharmacology 07/2011; 80(1):183-90. DOI:10.1124/mol.111.071415 · 4.13 Impact Factor
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    • "Most of the work on ginkgolide action at Cys loop receptors has been performed on GABA and glycine receptors, where BB, GB and PTX at low micromolar concentrations have been shown to bind to and block the receptor channel: In the GABA A receptor, for example, PTX protects against MTS modification of residues at the cytoplasmic end of M2, and mutations in this region effect PTX inhibition , but not GABA EC 50 or benzodiazepine modulatory effects (Sedelnikova et al., 2006; Ticku et al., 1978; Xu et al., 1995). In silico docking into GABA A and glycine receptors shows that ginkgolides and PTX can dock in the pore, and has provided plausible binding locations and ligand orientations (Hawthorne and Lynch, 2005; Erkkila et al., 2008; Hawthorne et al., 2006; Jensen et al., 2008; Yang et al., 2007). Our data show that the 5-HT 3 A receptor channel is also the most likely site of action as BB and GB do not displace the competitive antagonist [ 3 H]granisetron, the effects of PXN on 5-HT-evoked currents were insurmountable, and there is overlap between the BB and PXN, and BB and GB binding sites. "
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    ABSTRACT: Extracts from the Ginkgo biloba tree are widely used as herbal medicines, and include bilobalide (BB) and ginkgolides A and B (GA and GB). Here we examine their effects on human 5-HT(3)A and 5-HT(3)AB receptors, and compare these to the effects of the structurally related compounds picrotin (PTN) and picrotoxinin (PXN), the two components of picrotoxin (PTX), a known channel blocker of 5-HT(3), nACh and GABA(A) receptors. The compounds inhibited 5-HT-induced responses of 5-HT(3) receptors expressed in Xenopus oocytes, with IC(50) values of 470 μM (BB), 730 μM (GB), 470 μM (PTN), 11 μM (PXN) and >1mM (GA) in 5-HT(3)A receptors, and 3.1mM (BB), 3.9 mM (GB), 2.7 mM (PTN), 62 μM (PXN) and >1mM (GA) in 5-HT(3)AB receptors. Radioligand binding on receptors expressed in HEK 293 cells showed none of the compounds displaced the specific 5-HT(3) receptor antagonist [(3)H]granisetron, confirming that they do not act at the agonist binding site. Inhibition by GB at 5-HT(3)A receptors is weakly use-dependent, and recovery is activity dependent, indicating channel block. To further probe their site of action at 5-HT(3)A receptors, BB and GB were applied alone or in combination with PXN, and the results fitted to a mathematical model; the data revealed partially overlapping sites of action. We conclude that BB and GB block the channel of the 5-HT(3)A receptor. Thus these compounds have comparable, although less potent, behaviour than at some other Cys-loop receptors, demonstrating their actions are conserved across the family.
    Neuropharmacology 11/2010; 60(2-3):488-95. DOI:10.1016/j.neuropharm.2010.11.003 · 5.11 Impact Factor
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