Huang SH , Lewis TM , Lummis SC , Thompson AJ , Chebib M , Johnston GA , Duke RK .

Wellcome Trust

	Fig. 1. Structures of ginkgolides A, B and C (GA, GB and GC) (20 carbon atoms), bilobalide (15 carbon atoms) and picrotoxinin (PTX) (15 carbon atoms). These compounds have cavity-like structures made up of a highly oxygenated carbon skeleton, including two lactone rings and an epoxy group in PTX, and three lactone rings in bilobalide and ginkgolides. The lipophilic side chain (isopropenyl group in PTX and t-butyl group in bilobalide and ginkgolides) is attached to the underside of the cavity. Ginkgolides A, B and C have two common hydroxyl groups at C3 and C10 as shown in ginkgolide A. The hydroxyl group at C10 is located at the top and the C3 hydroxyl underneath the main cage comprising rings F, A, D and C. Two additional rings E and B project outward from the main cage. The C3 hydroxyl of the ginkgolides is next to the polar lactone ring E whereas the only hydroxyl group in PTX is located at the nonpolar underside of the cage. Note that ginkgolide A lacks the hydroxyl at C1 which acts as an H-bond donor to the C10 hydroxyl in ginkgolides B and C. The C7 in ginkgolide C projects upwards to the oxolane ring D.
	Fig. 2. Current traces produced by 1.2 μM GABA (EC50, solid bar) in the presence of (A) ginkgolide A, (B) ginkgolide B and (C) ginkgolide C at various concentrations from human ρ1 GABAC receptors expressed in Xenopus oocytes. The bars indicate duration of drug application. The ginkgolides did not have any effect on their own when tested at 100 μM.
	Fig. 3. Agonist concentration–response curves for GABA, in the presence and absence of (A) ginkgolide A, (B) ginkgolide B and (C) ginkgolide C from recombinant human ρ1 GABAC receptors. For each panel, the control concentration–response curve (●) is shown along with the subsequent curves obtained in the presence of 10 μM (■), 30 μM (▴) and 100 μM (▾) ginkgolide. Note the approximately parallel rightward shifts in the concentration–response curves, along with an inhibition of the maximum response with an increase in ginkgolide concentration, which is characteristic of a mixed-antagonism. The inhibition of the maximum response by ginkgolides exhibits some degree of ‘saturation’ that is most obvious for ginkgolide C. That is, there is no further inhibition of the maximum response (or a small change) with a further increase in ginkgolide concentration from 30 to 100 μM. The data are fitted with the Hill equation and the average EC50, Hill coefficients and normalized maximum responses estimated from these data are shown in Table 1. Data are mean ± S.E.M. (n = 4–6). Where the error bar is not obvious, it is entirely within the plotted symbol.
	Fig. 4. Inhibition concentration–response curves recorded from recombinant human ρ1 GABAC receptors for (A) ginkgolide A, (B) ginkgolide B and (C) ginkgolide C, in the presence of 0.5 μM (●), 1.2 μM (■), 3 μM (▴) and 10 μM () GABA. The data are fitted with the Hill equation and the average IC50 and Hill coefficients estimated from these data are shown in Table 2. Data are mean ± S.E.M. (n = 5–7 oocytes). Where the error bar is not obvious, it is entirely within the plotted symbol. Note that antagonism appears to saturate at the highest concentration of ginkgolide B.
	Fig. 5. Use-dependent effect in the antagonism of ρ1 GABAC receptors by ginkgolides. A. Examples of currents recorded during repeated co-applications of GABA and ginkgolides. The ginkgolides were applied 40 s prior to the first co-application with GABA and were continuously present until the fifth co-application. Each application was separated by a 3-min interval. B. Histogram representing the mean data of consecutive co-applications of GABA and ginkgolide A (n = 4), ginkgolide B (n = 6) and ginkgolide C (n = 4). The small increase in inhibition with the second application of ginkgolide B and C was not significant. Overall, there was no obvious evidence for use-dependent inhibition by the ginkgolides.
	Fig. 6. Recovery time from ginkgolide inhibition. Examples of current traces produced by the co-application of 5 or 50 μM ginkgolide A, B and C during the plateau phase of the response to 1 or 3 μM GABA. Note the faster recovery from block at higher GABA concentrations. The recovery times (10–90% rise times) are shown in Table 3.
	Fig. 7. Macroscopic kinetic schemes fitted to equilibrium concentration–response curve data. Scheme 1 represents the sequential mechanism of Amin and Weiss (1996), which adequately describes the homomeric ρ1 GABAC receptor function. This assumes five equivalent agonist binding sites but only three are required to be occupied to activate (open) the channel pore. All subsequent kinetic schemes are derivatives of the linear scheme, by incorporating different possible mechanisms for antagonism by ginkgolides. These are drawn from the cyclic mixed-antagonism mechanism described by Smart and Constanti (1986). Scheme 2 and scheme 3 both assume that ginkgolide binds to a single site in the receptor complex. The only difference between these schemes is that ginkgolide bound to the open state does not allow ion conduction in scheme 3 (BA3R′), while in scheme 2 the open state remains conducting (BA3R*). For scheme 4, the equilibrium between the fully-liganded closed and open states is removed, which allows for the possibility that the ginkgolide binds to a separate site in the closed state compared to the open state. In each case: R represents the receptor complex; A represents the agonist and the subscript the number of agonist molecules bound to the receptor; B represents the antagonist (blocker); the asterisk denotes the open (conducting) state of the receptor; Ka is the microscopic equilibrium constant for the agonist (GABA) and Ka′ is the microscopic agonist equilibrium constant when ginkgolide is bound to the receptor (the macroscopic equilibrium constants are shown in each of the schemes); Kb0 to Kb4 are the equilibrium constants for the ginkgolide for each state of the receptor; E is the equilibrium constant for the opening reaction of the channel pore, where E = β/α, and β is the forward rate constant and α is the reverse rate constant; and similarly E′ is the equilibrium constant when ginkgolide is bound.
	Fig. 8. Results from scheme 2 (Fig. 7). Equilibrium concentration–response curves fitted with scheme 2 for (A) ginkgolide A, (B) ginkgolide B and (C) ginkgolide C. In each case, the parameters for the response to GABA alone were fixed (Ka = 3.2 μM and E = 11) to improve the precision on the remaining parameters estimated. All parameters for the fit were shared across the data sets for each ginkgolide with the exception of the concentration of the ginkgolide (0, 10, 30 and 100 μM). The fit of scheme 2 describes well the mixed-antagonism of the data sets for ginkgolide A, B and C. Panels D–F show the predicted inhibition curves generated with the parameters obtained from the fit to the concentration–response curves exhibit saturation of inhibition, where the bottom of the curve reaches a plateau rather than a full inhibition of the response. The plotted symbols in each panel are as for Fig. 3; equilibrium concentration–response data for GABA in the absence of ginkgolide (●) and in the presence of 10 μM (■), 30 μM (▴) and 100 μM (▾) ginkgolide.
	Fig. 9. Results from scheme 3 (Fig. 7). Equilibrium concentration–response curves fitted with scheme 3 for (A) ginkgolide A, (B) ginkgolide B and (C) ginkgolide C. As for scheme 2, the parameters for the response to GABA alone were fixed (Ka = 3.2 μM and E = 11) and all other parameters were shared across the data sets for each ginkgolide with the exception of the concentration of the ginkgolide (0, 10, 30 and 100 μM). The fit with scheme 2 was noticeably worse, with the curve failing to follow the data points at the top of the concentration–response curve and consequently an increase in the standard deviation of the residuals for the fit. The predicted inhibition curves (panels D–F) exhibit complete inhibition at each of the GABA concentrations (numbers indicate 0.5, 1.2, 3.0 and 10 μM GABA). The plotted symbols in each panel are as for Fig. 3; equilibrium concentration–response data for GABA in the absence of ginkgolide (●) and in the presence of 10 μM (■), 30 μM (▴) and 100 μM (▾) ginkgolide.
	Fig. 10. A comparison of the channel from 3EAM and the ρ1 GABAC receptor homology model used in this study. The profile is plotted as the average pore diameter for the whole pentamer (solid line, ρ1 GABAC receptor homology model; dotted line, 3EAM).
	Fig. 11. Examples of the two main docked poses for ginkgolide B in the ρ1 GABAC receptor homology model. M2 helices are shown as ribbons, and apart from the side chains of residues P2′ and T6′, all others have been removed for clarity. For ginkgolide A, ginkgolide B (GB) and ginkgolide C, the orientations of the ligands were the same, and so only ginkgolide B (the most potent ligand) is shown. Panels A and D show the docked pose as seen looking from the extracellular domain, down through the receptor pore. Panels B and E are the same poses viewed from the side. Ginkgolide B is shown in stick representation. Panel C is in the same orientation as panel A, but shows an overlay of two poses, highlighting the rotation of the ginkgolide B ligand.

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