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Selective potentiation of alpha 1 glycine receptors by ginkgolic acid

View Article: PubMed Central

ABSTRACT

Glycine receptors (GlyRs) belong to the superfamily of pentameric cys-loop receptor-operated channels and are involved in numerous physiological functions, including movement, vision, and pain. In search for compounds performing subunit-specific modulation of GlyRs we studied action of ginkgolic acid, an abundant Ginkgo biloba product. Using patch-clamp recordings, we analyzed the effects of ginkgolic acid in concentrations from 30 nM to 25 μM on α1–α3 and α1/β, α2/β configurations of GlyR and on GABAARs expressed in cultured CHO-K1 cells and mouse neuroblastoma (N2a) cells. Ginkgolic acid caused an increase in the amplitude of currents mediated by homomeric α1 and heteromeric α1/β GlyRs and provoked a left-shift of the concentration-dependent curves for glycine. Even at high concentrations (10–25 μM) ginkgolic acid was not able to augment ionic currents mediated by α2, α2/β, and α3 GlyRs, or by GABAAR consisting of α1/β2/γ2 subunits. Mutation of three residues (T59A/A261G/A303S) in the α2 GlyR subunit to the corresponding ones from the α1 converted the action of ginkgolic acid to potentiation with a distinct decrease in EC50 for glycine, suggesting an important role for these residues in modulation by ginkgolic acid. Our results suggest that ginkgolic acid is a novel selective enhancer of α1 GlyRs.

No MeSH data available.


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Effect of ginkgolic acid on Igly mediated by homomeric α2 and α3 GlyRs and heteromeric α2/β GlyRs.(A) Cumulative data. Mean percentage of the effect of 25 μM ginkgolic acid on homomeric α2 GlyRs (light gray), heteromeric α2/β GlyRs (white colomn), and 10 μM of ginkgolic acid on α2 GlyRs (striped). Glycine 30 μM was applied. Data from 7 to 12 cells for each case. (B) Superimposed traces of glycine-evoked currents induced by low for this subunit (100 μM; left) and high (300 μM; right) concentrations of glycine, in control (black), after ginkgolic acid application (red) and after washout (green). Symmetrical ‘CsCl’ pipette solution, Vh = –30 mV. (C) Summary of the data on the effect of ginkgolic acid on the α3-long subunit of GlyR. Mean amplitudes of currents (pA) ± SEM induced by 100 μM glycine from six cells in control (black), during ginkgolic acid application (white), and after washout (striped).
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Figure 4: Effect of ginkgolic acid on Igly mediated by homomeric α2 and α3 GlyRs and heteromeric α2/β GlyRs.(A) Cumulative data. Mean percentage of the effect of 25 μM ginkgolic acid on homomeric α2 GlyRs (light gray), heteromeric α2/β GlyRs (white colomn), and 10 μM of ginkgolic acid on α2 GlyRs (striped). Glycine 30 μM was applied. Data from 7 to 12 cells for each case. (B) Superimposed traces of glycine-evoked currents induced by low for this subunit (100 μM; left) and high (300 μM; right) concentrations of glycine, in control (black), after ginkgolic acid application (red) and after washout (green). Symmetrical ‘CsCl’ pipette solution, Vh = –30 mV. (C) Summary of the data on the effect of ginkgolic acid on the α3-long subunit of GlyR. Mean amplitudes of currents (pA) ± SEM induced by 100 μM glycine from six cells in control (black), during ginkgolic acid application (white), and after washout (striped).

Mentions: In contrast to the action on α1 GlyRs, low concentrations of ginkgolic acid (<10 μM) had no effect on the amplitude of Igly. At ginkgolic acid concentrations of 10 μM or higher, a small inhibition of currents was observed. Thus, 10 and 25 μM of ginkgolic acid inhibited α2 GlyRs by about –10 ± 3% (n = 8) and –20 ± 5% (n = 11), respectively (Figure 4A). However, in many cells high doses of ginkgolic acid stimulated non-reversible run-down, which could be an additional reason for this small inhibition. At low concentrations (1 μM) the effect of ginkgolic acid was not detectable; with a long application (5–6 mins) even the tendency to weak elevation of Igly was observed. This may result partially from a spontaneous run-up of responses during long-lasting whole cell recordings (data not shown, but see Fucile et al., 2000).


Selective potentiation of alpha 1 glycine receptors by ginkgolic acid
Effect of ginkgolic acid on Igly mediated by homomeric α2 and α3 GlyRs and heteromeric α2/β GlyRs.(A) Cumulative data. Mean percentage of the effect of 25 μM ginkgolic acid on homomeric α2 GlyRs (light gray), heteromeric α2/β GlyRs (white colomn), and 10 μM of ginkgolic acid on α2 GlyRs (striped). Glycine 30 μM was applied. Data from 7 to 12 cells for each case. (B) Superimposed traces of glycine-evoked currents induced by low for this subunit (100 μM; left) and high (300 μM; right) concentrations of glycine, in control (black), after ginkgolic acid application (red) and after washout (green). Symmetrical ‘CsCl’ pipette solution, Vh = –30 mV. (C) Summary of the data on the effect of ginkgolic acid on the α3-long subunit of GlyR. Mean amplitudes of currents (pA) ± SEM induced by 100 μM glycine from six cells in control (black), during ginkgolic acid application (white), and after washout (striped).
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Figure 4: Effect of ginkgolic acid on Igly mediated by homomeric α2 and α3 GlyRs and heteromeric α2/β GlyRs.(A) Cumulative data. Mean percentage of the effect of 25 μM ginkgolic acid on homomeric α2 GlyRs (light gray), heteromeric α2/β GlyRs (white colomn), and 10 μM of ginkgolic acid on α2 GlyRs (striped). Glycine 30 μM was applied. Data from 7 to 12 cells for each case. (B) Superimposed traces of glycine-evoked currents induced by low for this subunit (100 μM; left) and high (300 μM; right) concentrations of glycine, in control (black), after ginkgolic acid application (red) and after washout (green). Symmetrical ‘CsCl’ pipette solution, Vh = –30 mV. (C) Summary of the data on the effect of ginkgolic acid on the α3-long subunit of GlyR. Mean amplitudes of currents (pA) ± SEM induced by 100 μM glycine from six cells in control (black), during ginkgolic acid application (white), and after washout (striped).
Mentions: In contrast to the action on α1 GlyRs, low concentrations of ginkgolic acid (<10 μM) had no effect on the amplitude of Igly. At ginkgolic acid concentrations of 10 μM or higher, a small inhibition of currents was observed. Thus, 10 and 25 μM of ginkgolic acid inhibited α2 GlyRs by about –10 ± 3% (n = 8) and –20 ± 5% (n = 11), respectively (Figure 4A). However, in many cells high doses of ginkgolic acid stimulated non-reversible run-down, which could be an additional reason for this small inhibition. At low concentrations (1 μM) the effect of ginkgolic acid was not detectable; with a long application (5–6 mins) even the tendency to weak elevation of Igly was observed. This may result partially from a spontaneous run-up of responses during long-lasting whole cell recordings (data not shown, but see Fucile et al., 2000).

View Article: PubMed Central

ABSTRACT

Glycine receptors (GlyRs) belong to the superfamily of pentameric cys-loop receptor-operated channels and are involved in numerous physiological functions, including movement, vision, and pain. In search for compounds performing subunit-specific modulation of GlyRs we studied action of ginkgolic acid, an abundant Ginkgo biloba product. Using patch-clamp recordings, we analyzed the effects of ginkgolic acid in concentrations from 30 nM to 25 &mu;M on &alpha;1&ndash;&alpha;3 and &alpha;1/&beta;, &alpha;2/&beta; configurations of GlyR and on GABAARs expressed in cultured CHO-K1 cells and mouse neuroblastoma (N2a) cells. Ginkgolic acid caused an increase in the amplitude of currents mediated by homomeric &alpha;1 and heteromeric &alpha;1/&beta; GlyRs and provoked a left-shift of the concentration-dependent curves for glycine. Even at high concentrations (10&ndash;25 &mu;M) ginkgolic acid was not able to augment ionic currents mediated by &alpha;2, &alpha;2/&beta;, and &alpha;3 GlyRs, or by GABAAR consisting of &alpha;1/&beta;2/&gamma;2 subunits. Mutation of three residues (T59A/A261G/A303S) in the &alpha;2 GlyR subunit to the corresponding ones from the &alpha;1 converted the action of ginkgolic acid to potentiation with a distinct decrease in EC50 for glycine, suggesting an important role for these residues in modulation by ginkgolic acid. Our results suggest that ginkgolic acid is a novel selective enhancer of &alpha;1 GlyRs.

No MeSH data available.


Related in: MedlinePlus