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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    • "| of the acid on homomeric α1 GlyRs . To cells expressing human α1 GlyR , pulses of glycine of different concentrations and 2 - s duration were applied before , during and after addition of ginkgolic acid . In contrast to the previously described inhibitory action of ginkgolides ( Kondratskaya et al . , 2002 , 2004 ; Hawthorne et al . , 2006 ) , ginkgolic acid at relatively low concentrations ( 30 nM – 3 μM ) strongly enhanced whole - cell currents induced by sub - saturating ( EC 10 – EC 50 ) glycine concentrations ( Figures 1 and 2 ) . In different cells the degree of potentiation induced by pre - application of 100 nM ginkgolic acid during 2 min varied from 30 to 100% ( "

    Full-text · Article · Jun 2015 · SpringerPlus
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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.
    Full-text · Article · Nov 2012 · Neuropharmacology
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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.
    Preview · Article · Jul 2011 · Molecular pharmacology
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