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Articles, Cellular/Molecular

Allosteric Modulation of Retinal GABA Receptors by Ascorbic Acid

Cecilia I. Calero, Evan Vickers, Gustavo Moraga Cid, Luis G. Aguayo, Henrique von Gersdorff and Daniel J. Calvo
Journal of Neuroscience 29 June 2011, 31 (26) 9672-9682; https://doi.org/10.1523/JNEUROSCI.5157-10.2011
Cecilia I. Calero
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Evan Vickers
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Gustavo Moraga Cid
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Luis G. Aguayo
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Henrique von Gersdorff
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Daniel J. Calvo
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  • Figure 1.
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    Figure 1.

    Ascorbic acid reversibly enhances GABAC-mediated standing leak current in axotomized Mb bipolar cell presynaptic terminals. A, GABAC leak current is enhanced by bath application of Asc. Whole-cell voltage-clamp recording of an Mb terminal in the presence of 25 μm SR-95531 shows a GABAC-mediated leak current of −52.5 pA at −60 mV (left). The leak current was enhanced by 8 pA following bath application of 3 mm ascorbic acid. During wash (0 mm Asc), the leak current was reduced nearly to its original level. Ascorbic acid significantly and reversibly enhanced GABAC-mediated leak current in the absence of NO-711 (n = 11; right). The mean for all cells with SE is shown in dark gray (circles) and is connected by a solid line; data from each single cell are shown in light gray (“x”) and are connected by a dashed line. B, Whole-cell voltage-clamp recording of an Mb terminal in the presence of 25 μm SR-95531 and 3 μm NO-711 shows a GABAC-mediated leak current of −400.6 pA at −60 mV (left). The leak current was enhanced by 55 pA following bath application of 3 mm Asc. During wash (0 mm Asc, 3 μm NO-711), the leak current was reduced to its original level. Ascorbic acid significantly enhanced GABAC-mediated leak current in the presence of 1–3 μm NO-711 (n = 4; right). Washout was nearly significant. C, Standing leak current consists of both TPMPA-sensitive and TPMPA-insensitive components. Whole-cell recording (left) of an axotomized Mb1 terminal voltage-clamped at −60 mV shows a leak current of −34.7 pA that is stably reduced to −16.4 pA by bath application of 300 μm TPMPA in the presence of 0 mm ascorbic acid. Addition of 300 μm TPMPA in the presence of 25 μm SR95531 and 0 mm ascorbic acid produced a statistically significant reduction in leak current (n = 4; right). *p < 0.05; **p < 0.01; ***p < 0.001; n.s., p > 0.05; all statistical tests reflect a repeated-measures design; all errors are expressed as ±SE, unless otherwise noted.

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    Figure 2.

    Rundown of GABAC-mediated responses to puff application of GABA in axotomized Mb bipolar cell presynaptic terminals is significantly slowed and/or prevented by the presence of intracellular and extracellular ascorbic acid. A, Puff application of GABA (25 ms duration, indicated by black bar) was performed every 30 s for 15 min to assess the degree of rundown of the resulting GABAC-mediated currents. Perfusion solution contained 25 μm SR-95531 (a GABAA antagonist) in all cases. Some experiments were performed with 25 μm SR-95531 in the puff pipette, with no change in rundown kinetics (data not shown). An individual example of GABAC current rundown shows that, in the absence of ascorbic acid either in the puff pipette, patch-clamp pipette, or perfusion solution, 25 ms puff application of 200 μm GABA with a 30 s (ISI) resulted in a 27.3% rundown in current amplitude over 14.5 min (left). Right, Same as left, except that in this example 3 mm ascorbic acid was included in the puff pipette, patch-clamp pipette, and perfusion solution. Under these conditions, rundown of GABAC current amplitude over 14.5 min was 8.7%. Over the first 5 min, GABAC current amplitude increased by 3.7%. B, The GABA puff experiments from A were repeated in the presence (n = 7) or absence (n = 7) of 3 mm ascorbic acid in the puff pipette, patch pipette, and perfusion solution, with 25 μm SR-95531 in the perfusion solution (and, in some cases, in the puff pipette). Shown are the mean normalized GABAC current amplitudes for each 30 s interval at which GABA was puff applied for 25 ms. Error bars indicate mean ± SEM. The slope of rundown for each condition was fit with linear regression (solid lines). Dashed lines indicate 95% confidence interval boundaries for each condition. The effect of ascorbic acid on rundown was highly significant at 5 and 10 min. **p < 0.01; all errors expressed as ±SE, unless otherwise noted.

  • Figure 3.
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    Figure 3.

    Response to puffing GABA directly onto axotomized Mb bipolar cell presynaptic terminals is enhanced by bath application of ascorbic acid. A, Puff application of 1 mm GABA (25 ms duration, indicated by black bar) directly onto an axotomized Mb bipolar cell presynaptic terminal in the presence of 25 μm SR-95531 produced (upper) a large GABAC-mediated inward current (black trace). Bath application of 3 mm ascorbic acid (red trace) increased the amplitude of this current in a reversible manner (washout; blue trace). Each trace is the average of three successive stimulations with an intertrace interval of 20 s. Summary data (bottom; n = 8) showed a significant and reversible increase in puff response amplitude caused by 3 mm ascorbic acid. Mean for all cells with SE is shown in green (circles); data from each single cell (“x”) are connected by gray dashed line, control data points (0 mm Asc) are shown in black, 3 mm Asc points are shown in red, wash (0 mm Asc) points are shown in blue. B, Rundown normalization of GABAC currents from cell in A, calculated using the mean time after initial GABA puff, and the linear rundown regressions from Figure 2B, show potentiation of nearly 25% in the presence of 3 mm ascorbic acid, with a nearly complete washout in 0 mm Asc (top). Summary of corrected data (bottom; n = 8) showed a significant increase in puff response amplitude caused by bath application of 3 mm ascorbic acid. Individual repeated-measures data are shown in gray (filled circles), and mean values are shown in black (“x”; bottom). *p < 0.05; **p < 0.01; n.s., p > 0.05; all statistical tests reflect a repeated-measures design; all errors are expressed as ±SE, unless otherwise noted.

  • Figure 4.
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    Figure 4.

    Homomeric ρ1 GABAC receptor function is enhanced by ascorbic acid. Representative traces of ionic (Cl−) currents elicited by different GABA concentrations in oocytes expressing homomeric GABAρ1 receptors. Calibration: current amplitude (y-axis), time (x-axis). For this and the subsequent figures, oocytes were voltage-clamped at −70 mV. A, GABAρ1 responses recorded before (left, control) and after the application of 3 mm ascorbic acid (right). Potentiation of GABAρ1 responses by ascorbic acid was 162 ± 22.6% (n = 3). B, Ionic currents elicited by increasing concentrations of GABA (0.3, 1, and 10 μm) were also enhanced during concurrent applications of 700 μm ascorbic acid.

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    Figure 5.

    Analysis of ascorbic acid effects on GABAρ1 receptor function. A, D–R curves for GABA performed in the presence or absence of ascorbic acid. Response amplitudes were expressed as a fraction of maximal GABA-evoked currents (30 μm GABA). B, Dose–effects curves for ascorbic acid acting on GABAρ1 responses. Data were normalized to control values, obtained in the absence of ascorbic acid, for two different GABA concentrations. All the points tested >100 μm were significantly different from control. C, Degree of potentiation of the GABAρ1 responses by ascorbic acid for increasing concentrations of GABA. D, I–V relationships of the GABAρ1 responses measured in the presence or absence of ascorbic acid.

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    Figure 6.

    Effects of different ascorbic acid analogs on GABAρ1 receptor function. A, Chemical structures of the diverse compounds used. B, Histogram summarizing the values obtained for GABAρ1 responses evoked by 0.3 μm GABA recorded before (control, dotted line) and after exposure to the different ascorbic acid analogs (n = 4).

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    Figure 7.

    Identification of amino acid residues involved in the modulation of homomeric ρ1 GABAC receptors by ascorbic acid. A, The irreversible methylation of C177 and C191 at the Cys-loop, by pretreatment of oocytes with 30 μm NEM, produced similar effects to those shown by ascorbic acid on D–R curves for GABA. B, NEM pretreatment completely abolished the potentiation of GABAρ1 responses induced by 2 mm DTT. C, NEM pretreatment partially prevented the potentiation of GABAρ1 responses by 3 mm ascorbic acid. D, D–R curves for GABA performed on wild-type GABAρ1 and mutant GABAρ1H141D receptors. E, Mutation of H141 to D at the ρ1 subunit partially prevented the potentiating actions of ascorbic acid. Effects of 3 mm ascorbic acid on D–R curves for GABA were performed on GABAρ1H141D receptors. Ascorbic acid significantly enhanced maximal responses and currents evoked by very low GABA concentrations (inset), but failed to induce a leftward shift in the D–R curve (as that observed in C), indicating that the H141 is critical for ascorbic acid modulatory actions. F, NEM pretreatment abolished the potentiation induced by ascorbic acid on maximal responses mediated by GABAρ1H141D receptors, indicating that they are more likely mediated by C177 and C191, but effects at very low GABA concentrations were not prevented by NEM (inset). Response amplitudes were expressed as a fraction of the 30 μm GABA-evoked currents (maximal response). Each point represents the mean and SEM of the responses obtained from four to six oocytes. Notice that many of the SE bars are hidden by the symbols.

  • Figure 8.
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    Figure 8.

    The amplitude of GABAA mIPSCs is enhanced by bath application of ascorbic acid. A, Excerpts (1.3 s each) from continuous records of mIPSCs from a single axotomized Mb1 terminal (at least 120 s per condition) in the presence of 150 μm TPMPA (GABAC antagonist), 10 μm NBQX, 50 μm dl-APV, and 1 μm TTX show that 3 mm ascorbic acid reversibly increased the amplitude of GABAA mIPSCs without significantly increasing mIPSC frequency. B, The amplitude of the mean IPSC waveform of GABAA mIPSCs was reversibly enhanced by nearly 30% (left) following bath application of 3 mm ascorbic acid (same cell as in A). The mIPSC amplitude cumulative probability distribution (bin = 1 pA) for control (0 mm Asc, count = 353), 3 mm ascorbic acid (count = 770), and wash (0 mm Asc, count = 574) conditions (right) showed a distinct, reversible rightward shift (larger amplitudes) in cumulative probability in the presence of 3 mm ascorbic acid. C, Summary of data for all cells (n = 5) shows a significant, reversible increase in GABAA mIPSC amplitude (left), with no significant effect on mIPSC frequency (right), following bath application of 3 mm ascorbic acid. Mean for all cells with SE shown in green (circles), data from each single cell (“x”) connected by gray dashed line, control data points (0 mm Asc) shown in black, 3 mm Asc shown in red, wash (0 mm Asc) shown in blue. *p < 0.05; n.s, p > 0.05; all statistical tests reflect a repeated-measures design; all errors are expressed as ±SE, unless otherwise noted.

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    Figure 9.

    Potentiation of the α1β2 and α1β2γ2 GABAA receptor function by ascorbic acid. A, Representative traces of ionic (Cl−) currents mediated by α1β2 and α1β2γ2 GABAA receptors expressed in HEK cells. Inward GABAA currents were elicited by bath applications of 1 or 5 μm GABA (the corresponding EC50 values for GABA showed by α1β2 and α1β2γ2 GABAA receptors, respectively) and recorded by whole-cell voltage-clamp at −60 mV. GABAA responses mediated by both α1β2 and α1β2γ2 GABAA receptors were significantly enhanced in the presence of 0.5 and 1 mm ascorbic acid. B, Dose–effect curves for ascorbic acid actions on α1β2 and α1β2γ2 GABAA receptors (n = 3; curves performed at the corresponding EC50 values). Data were normalized to control values, obtained in the absence of ascorbic acid.

Tables

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    Table 1.

    Dose-response curve parameters

    EC50 (μm)n (Hill coefficient)n
    Control0.74 ± 0.011.58 ± 0.045–10
    3 mm Asc0.49 ± 0.01*1.88 ± 0.075–10
    Control1.52 ± 0.042.24 ± 0.054
    30 μm NEM1.29 ± 0.022.24 ± 0.104
    30 μm NEM + 3 mm Asc0.75 ± 0.012.44 ± 0.194
    Control0.86 ± 0.102.58 ± 0.303
    30 μm NEM0.78 ± 0.092.32 ± 0.103
    30 μm NEM + 2 mm DTT0.74 ± 0.062.04 ± 0.083
    H141D1.76 ± 0.213.41 ± 0.6110
    H141D + 3 mm Asc2.07 ± 0.322.94 ± 0.385
    H141D + 30 μm NEM1.40 ± 0.163.24 ± 0.164
    H141D + 30 μm NEM + 3 mm Asc1.46 ± 0.243.48 ± 0.284
    • Parameters of the D–R curves for GABA, performed for GABA ρ1 wt and GABA ρ1 H141D in the absence or presence of the different agents.

    • ↵*p < 0.03.

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    Table 2.

    Effects of ascorbic acid and its analogs on GABAρ1 responses

    Compound% Potentiationnτact (s)τdeact (s)n
    Control (GABA 0.3 μm)624.8 ± 1.623.5 ± 3.86
    3 mm Asc139.5 ± 87.5**722.6 ± 2.628.1 ± 3.36
    3 mm D-isoAsc33.5 ± 12.5*428.5 ± 2.533.4 ± 1.96
    3 mm DHA−1.6 ± 4.2326.1 ± 2.832.0 ± 2.86
    1.5 mm Asc(Ca)27.4 ± 4.8622.5 ± 2.527.4 ± 4.34
    • Potentiation (%) produced by ascorbic acid and its different analogs on GABAρ1 responses and values obtained for kinetic parameters in the presence of the different compounds.

    • ↵*p < 0.04,

    • ↵**p < 0.01.

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Journal of Neuroscience
Vol. 31, Issue 26
29 Jun 2011
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Allosteric Modulation of Retinal GABA Receptors by Ascorbic Acid
Cecilia I. Calero, Evan Vickers, Gustavo Moraga Cid, Luis G. Aguayo, Henrique von Gersdorff, Daniel J. Calvo
Journal of Neuroscience 29 June 2011, 31 (26) 9672-9682; DOI: 10.1523/JNEUROSCI.5157-10.2011

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Allosteric Modulation of Retinal GABA Receptors by Ascorbic Acid
Cecilia I. Calero, Evan Vickers, Gustavo Moraga Cid, Luis G. Aguayo, Henrique von Gersdorff, Daniel J. Calvo
Journal of Neuroscience 29 June 2011, 31 (26) 9672-9682; DOI: 10.1523/JNEUROSCI.5157-10.2011
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